BT203 - Genetics Unit 2

BT0203 Genetics and Cytogenetics UNIT I Terminologies Karyotype: A Karyotype is an array of chromosomes created by photographing the metaphase chromosomes
s from one cell, cutting out the individual chromosomes from the photograph and lining them up in order from largest to smallest, pairing the appropriate homologous chromosomes. Chromosomes for karyotypes are often stained using special procedures which create banding patterns the chromosomes, thus making pairing easier. Diploid: A diploid nucleus contains two sets of chromosomes (two of each type of chromosome). Haploid: A haploid nucleus contains a single set of chromosomes. Polyploidy: Polyploids contain more than two sets of chromosomes in each nucleus. For example, bananas are triploid. Homologous chromosomes: Homologous chromosomes are the same size, the same shape, and have the same gene map. They are not necessarily genetically identical. Genome; A genome is an individual organisms total array of genetic information. Gene pool: The gene pool is the total genetic diversity of a particular species. Gene: A gene is a segment of DNA which controls the production of a particular characteristic. More precisely, a gene is a recipe for the production of a specific kind of protein. Allele: Alleles are different forms of the same gene. For any gene, an individual may possess only two alleles, and a gamete may possess only one. However, the gene pool of a species may contain many alleles for any gene. Alleles are assigned symbols according Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics to specific rules of convention. All alleles of a particular gene should be given versions of the same symbol. Locus (plural Loci): A genes locus is its position on a chromosome. All genes have individual and unique locations characteristic of the species. What makes two organisms members of the same species is that they have the same assortment of genes, arranged according to the same gene map on their chromosomes. In other words, their various genes have the same loci. Multiple alleles: A gene has multiple alleles if there are more than two different alleles for that gene in the gene pool. For example, there are three different alleles for the A-BO blood type gene in human populations (LA, LB and l). Genotype: The genotype of an organism is the list of the symbols representing that organisms specific genetic constitutionin other words, a list of all the alleles the individual carries for its genes. In actual usage, a stated genotype typically describes only one or two genes at a time. Phenotype: The phenotype is the actual physical expression of an organisms traits. Much of the phenotype is the product of the genotype, but environmental influence can be very important as well. Geneticists discuss the heritability of traits. Heritability is the expression of the degree to which a particular trait is controlled by heredity. Homozygous; A homozygous individual has two identical alleles for the gene in question. For example, BB, bb, AA, PP. Heterozygous: A heterozygous individual has two different alleles for the gene in question. For example, Bb, Aa, Pp. Hemizygous: This term refers to the condition of a gene which is carried on an X or Y chromosome in a male. Since there can be only one copy of such a gene in the cell, the Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics terms homozygous and heterozygous are inappropriate. Essentially, hemizygous means that a gene is present in only one copy. Complete dominance: If two alleles display complete , it is not possible to tell the difference between the homozygous dominant individual and the heterozygous individual. The recessive allele is hidden by the presence of the dominant allele. Dominant: The dominant allele is the one which is displayed in the phenotype of the heterozygote. In assigning allelic symbols, the convention is to assign a capital letter to the dominant allele. Dominant traits can never skip generations in a pedigree, because they can never be present but hiddenthey always show. Recessive: The recessive allele is the one which is hidden in the phenotype of the heterozygote. The recessive allele is generally assigned a lower case letter symbol. Recessive traits can skip generations in a pedigree. Incomplete dominance: If two alleles show incomplete dominance, the phenotype of the heterozygote is intermediate between the phenotypes of the two homozygotes. For example, RR produces red flowers, RR produces white flowers, and RR produces pink flowers. Alleles showing incomplete dominance are typically assigned symbols which are variations of capital letters. Co-dominance: If two alleles show co-dominance, the phenotype of the heterozygote expresses both of the alleles completely. For example, in the A-B-O blood group, the LA and LB alleles show co-dominance. The heterozygote (Type AB) has all of the bloody type characteristics of Type A blood as well as those of Type B blood. Again, codominant alleles are generally assigned different versions of the same capital letter for symbols. Pseudodominance: Pseudodominance results when a particular genotype is lethal. For example, curly sings in Drosophila (fruit flies). The heterozygote has curly wings, the Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics homozygous straight has straight wings (wild type), and the homozygous curly is lethal (the eggs never hatch). Superficially, the effect looks like complete dominance (the curly allele appears to be dominant), but upon closer examination is not. By convention, the homozygous lethal allele is given a capital letter symbol and the pseudo-recessive allele is given a lower case letter symbol. Monohybrid cross: This is a mating between two individuals who are both heterozygous for the one gene which you are following. Eg: Aa x Aa Dihybrid cross: This is a mating between two individuals, both of whom are heterozygous for the two genes you are following. Eg: AaBb x AaBb Test cross: This is the mating between an individual of unknown genotype and a homozygous recessive individual (eg, B- x bb) for the purpose of exposing hidden recessive alleles in the unknown parent. Back cross: Most literally, this is the mating between an offspring and one of its parents. In practice, it is often a mating between an offspring and an individual of the same genotype as one of the offsprings parents. Linkage: Genes which are carried on the same chromosome are considered to be linked. When crosses using two linked genes are made, the two genes do not behave independently, and results do not behave according to simple rules of statistics. Independent Assortment: Genes which are not linked will behave independently of each other, and will assort according to simple rules of probability. Autosomal traits are traits carried on any of the chromosomes other than the sex chromosomes.
Rex Arunraj Assistant Prof.
Department of Genetic Engineering
BT0203 Genetics and Cytogenetics Sex linked traits: This is a special type of linkage. Sex linked genes are carried either on the X or the Y chromosome (in mammals or fruit fliesin birds, it would be the Z or W chromosome). X-linked genes are carried on the X chromosome. Since females have two copies of the X chromosome and males have only one, rare recessive characteristics which are Xlinked will occur more often in males than in females. Also, since an XY zygote always inherits its X from its female parent and its Y from the male parent, males inherit all of their X-linked traits from their mothers. Y-linked (holandric) genes are carried on the Y chromosome. Since Y chromosomes are inherited exclusively through the male line, males inherit all Y-linked traits from their fathers. Y-lined traits found in a father must appear in all of his sons, and all of their sons, etc. In mammals, maleness is carried on the Y chromosome. Sex influenced traits: Sex influenced traits are autosomal traits whose expression is affected by gender. Usually, the alleles are influenced by the presence of certain hormones which either increase or decrease the effects of the alleles. Eg, pattern baldness in humans is sex influenced. The gene for this trait has two alleles, the bald allele and the non-bald allele. The effectiveness of the bald allele is greatly increased in the presence of high levels of the hormone testosterone. Since this hormone is found in much higher levels in males than in females, the bald allele is dominant in males and recessive in females. Sex limited traits are autosomal traits whose expression is possible only in one of the genders. These traits generally affect the primary and secondary sexual characteristics. Eg, cryptorchidism is a condition in males in which one or both testes fail to descend into the scrotum late in gestation. This characteristic is genetically controlled, by an autosomal gene. A female can be genetically cryptorchid, but she cant possibly express the trait.
BT0203 Genetics and Cytogenetics Pliotropy refers to genes with multiple effects. For example, the white spotting gene in gerbils apparently also influences red blood cell count. Another simple example is that genes which influence characteristics of the fingers will also influence characteristics of the toes.
BT0203 Genetics and Cytogenetics Mendelian Genetics Gregor Johann Mendel is called the father of genetics. He was fond of gardening and interested in plant hybridization and he performed a No. of experiments with pea plants. With this experiments he was able to explain the inheritance of characters. His paper experiments in plant hybridization were published is 1866 to 1867 in the proceeding of Natural History Society of Brunn. His work remained unnoticed for 33 years. His work was recognized only in 1900 by Hugo de Veries a Dutch biologist, Carl Correns, a German botanist & Erich Von Tschermak an Austrian Botanist. Mendel was not the first to conduct hybridization experiments, but just the extension of the experiments conducted by earlier worker. Like Knight & Goss. There was another scientist Kolreuter A German Botanist performed experiments is tobacco similar observations were made by a group of workers Garther, Dawis, Naudin etc., but they could not figure out their results numerically as Mendel. Reasons for Mendels Success 1. 2. 3. 4. The flower of pea plants are normally self fertilized. The pea plant has contrasting characters. Cross pollination was not very difficult. The genes for the seven pairs of characters are located on seven separate homologies pairs of chromosomes 5. 6. 7. 8. Pure breeding varieties were available of easy to cultivate. Short growth period & growth cycle. He studied the inheritance of only one character at a time. He maintained statistical records of the results, which helped him to derive numerical ratios of significance.
BT0203 Genetics and Cytogenetics CROSSING TECHNIQUE Garden pea is self fertilizing; the anthers have to be removed and are called as emasculation. This stigma is protected against any foreign pollen grain, and then they are self pollinated. Character selected by Mendel Mendel selected seven characters with contrasting alternatives. No. Character Alternatives Dominant 1. 2. 3. 4. 5. 6. 7. Length of the stem Position of the flower Colour of the pod Shape of the pod Shape of Seed Colour of the Seed coat Colour of the cotyledon Tall Axial Green Inflated Round Coloured Yellow Recessive Dwarf Terminal Yellow Constricted Wrinkled White Green
Plants with one alternative trait were used as female and those with the other alternative as male. Reciprocal crosses were also made. T- tall female plant X t dwarf male plant. Reciprocal Cross T- tall male plant X t dwarf female plant. The population obtained as a result of crossing plants exhibiting contrasting characters is called the first fillal generation or F1 progeny. The progeny of F1 plants obtained due to self pollinations is called second fillial generations or F2. Similarly we can have F3, F4 etc. Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics Results of Mendels experiments When tall plants were crossed with dwarf plants, all plants is the F1, generation were tall. The plants used are the initial crosses are referred to as P1 & P2 (or) parents. When the F1, plants are self fertilized, both tall & dwarf plants were obtained in the F2 generation. The tall and dwarf plants were obtained in the ratio of 3:1 similar patterns were obtained for other six pairs of character also. To summarize the pattern of inheritance in all the seven cases. (i) For any character the F1, individuals from crosses, between two different varieties having alternative characters, showed only one of the traits & never the other. This feature was expressed as dominant of one trait over another. The trait which appeared is the F1 generation was called dominant and the other which did not appear is the F1, population was called recessive. (ii) It did not matter which parent variety provided the pollen which provided the eggs the results were always the same in other words, the reciprocal crosses gave the same results. The determining agent responsible for each trait was called a factor.
BT0203 Genetics and Cytogenetics Monohybrid Cross Single character each controlled by a single pair of genes or alleles were considered such crosses are known as monohybrid crosses the F2 ratio 3 :1 is known as monohybrid ratio. Monohybrid Experiment The crossing of two plants differing in one character is called monohybrid experiment. A pure breeding plant is one that which retains a particular character for any number of generations. A pure breeding tall & dwarf plants were treated as pants & were crossed. These seeds were sown and a group of plants were raised these plants constituted the first filial generations or F1, generation all the F1 plants were tall. The F1, plants crossed. The seeds were collected to the next generation was raised. F2. In the F2 generation two types of plants were found. They were tall and dwarf. He could raise 1064 plants in F2 generation 787 plants were tall and 277 plants were always 75% tall plants 25% dwarf Plants. Parents P Gametes F1 F1 Selfing Gametes F2 Phenotype Tall TT T Tt Tt T TT 3:1 Gametes T t T TT Tall Tt Tall Rex Arunraj Assistant Prof. t Tt Tall Tt Dwarf t X X Tt T t Tt Tt tt X Dwarf. tt t
BT0203 Genetics and Cytogenetics Genotype 1:2:1
The pure tall plant has two dominant alleles for height TT, the pure dwarf plant two receive alleles tt. During gamete formations, the alleles separate to enter two gametes. Hence each gamete will contain only allele. The gametes produced by a homozygous tall plant contain only one type of allele. Dominant - T, recessive t. When a tall plant and dwarf plant are crossed the gametes containing T- allele fuses with t- allele. recessive allele t. The gametes of the F1 generation are 50% T, 50% t. The resulting F1, plant is -Tt. It this plant, the domination allele T masks the effect of the
BT0203 Genetics and Cytogenetics Dihybrid Experiment The crossing of two plants differing in two characters is called dihybrid experiment. Two characters are considered at a time (Colour & Shape). Colour of the cotyledon (Yellow & Green), seed shape (Round & Wrinkled). Mendel selected a pure breeding yellow, round (Dominant) and a Pure breeding green, wrinkled seed producing plant ( Recessive ).The F1 generation plants produced only yellow round seeds. The F1 plants were self fertilized. In F2 generation four kinds of plants were produced. They are plants producing yellow, round seeds Plants producing yellow, wrinkled seeds Plants producing green, round seeds Plants producing green, wrinkled seeds Yellow (Y) is dominant over green (y); round seed shape (R) is dominant over wrinkled (r). The dominant parent produce only one type of gamete and each gamete are carrying one allele for colour (Y) & another allele for seed shape (R). The F1 plants Yellow and Round YyRr When F1 hybrid is selfed Gametes YR YR YYRR yellow round Yr YYRr yellow round yR YyRR Yr YYRr yellow round Yyrr yellow wrinkled YyRr yR YyRR yellow round YyRr yellow round yyRR yr YyRr yellow round Yyrr yellow wrinkled yyRr
BT0203 Genetics and Cytogenetics yellow round Yyrr Yellow wrinkled Dihybrid ratio: 9:3:3:1 Mendels Laws Based on Mendels experiments results certain principles are framed. These are called Mendels laws. 1. 2. 3. Law of dominance Law of segregation or law of purity of gametes. Law of independent assortment yellow round Yyrr Yellow wrinkled green round yyRr green round green round Yyrr green wrinkled
yr
Law dominance Each organism is made of a bundle of character; each character is controlled by factors or alleles or genes. Mendels law of dominance states that one factor in a pair may mask or prevent the expression of the other. He called the variety that appeared in the F1 generation of a monohybrid cross as dominant variety and that which did not appear in F1 generation to be recessive. A recessive factor freely expresses itself with the absence of the dominant allele. Law of segregation Each character is controlled by a pair of alleles. The two alleles of a particular character remain uncontaminated when they are inside the organism during gamete formation the paired alleles segregated & enter different gametes. During gamete formation the alleles of particular character separate and enter different gametes. segregation. formulated based on monohybrid experiments. Mendel made two innovations to the science of genetics: Rex Arunraj Assistant Prof. Department of Genetic Engineering This is the law of These laws were This law is also called law of purity of gametes.
BT0203 Genetics and Cytogenetics developed pure lines counted his results and kept statistical notes
Pure Line - a population that breeds true for a particular trait [this was an important innovation because any non-pure (segregating) generation would and did confuse the results of genetic experiments] Results from Mendel's Experiments Parental Cross Round x Wrinkled Seed Yellow x Green Seeds Red x White Flowers Tall x Dwarf Plants F1 Phenotype Round Yellow Red Tall F2 Phenotypic Ratio 5474 Round:1850 Wrinkled 6022 Yellow:2001 Green 705 Red:224 White l787 Tall:227 Dwarf F2 Ratio 2.96:1 3.01:1 3.15:1 2.84:1
Phenotype - literally means "the form that is shown"; it is the outward, physical appearance of a particular trait Mendel's pea plants exhibited the following phenotypes: - round or wrinkled seed phenotype - yellow or green seed phenotype - red or white flower phenotype - tall or dwarf plant phenotype Dominant - the allele that expresses itself at the expense of an alternate allele; the phenotype that is expressed in the F1 generation from the cross of two pure lines Recessive - an allele whose expression is suppressed in the presence of a dominant allele; the phenotype that disappears in the F1 generation from the cross of two pure lines and reappears in the F2 generation Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics
Mendel's Conclusions The hereditary determinants are of a particulate nature. These determinants are called genes. Each parent has a allele pair in each cell for each trait studied. The F1 from a cross of two pure lines contains one allele for the dominant phenotype and one for the recessive phenotype. These two alleles comprise the allele pair. One member of the allele pair segregates into a gamete, thus each gamete only carries one member of the allele pair. Gametes unite at random and irrespective of the other allele pairs involved. Mendel's First Law - the law of segregation; during gamete formation each member of the allelic pair separates from the other member to form the genetic constitution of the gamete Confirmation of Mendel's First Law Hypothesis With these observations, Mendel could form a hypothesis about segregation. To test this hypothesis, Mendel selfed the F2 plants. If his law was correct he could predict what the results would be. And indeed, the results occurred has he expected.
From these results we can now confirm the genotype of the F2 individuals. Phenotypes F2 Tall Plants Rex Arunraj Assistant Prof. Genotypes Genetic Description 1/3 DD Pure line homozygote dominant Department of Genetic Engineering
BT0203 Genetics and Cytogenetics 2/3 Dd F2 Dwarf Plants all dd Heterozygotes Pure line homozygote recessive
Thus the F2 is genotypically 1/4 Dd : 1/2 Dd : 1/4 dd This data was also available from the Punnett Square using the gametes from the F1 individual. So although the phenotypic ratio is 3:1 the genotypic ratio is 1:2:1 Mendel performed one other cross to confirm the hypothesis of segregation --- the backcross. Remember, the first cross is between two pure line parents to produce an F1 heterozygote.
At this point instead of selfing the F1, Mendel crossed it to a pure line, homozygote dwarf plant. Backcross: Dd x dd Backcross - the cross of an F1 hybrid to one of the homozygous parents; for pea plant height the cross would be Dd x DD or Dd x dd; most often, though a backcross is a cross to a fully recessive parent Testcross - the cross of any individual to a homozygous recessive parent; used to determine if the individual is homozygous dominant or heterozygous So far, all the discussion has concentrated on monohybrid crosses. Monohybrid cross - a cross between parents that differ at a single allele pair (usually AA x aa) Monohybrid - the offspring of two parents that are homozygous for alternate alleles of an allele pair
BT0203 Genetics and Cytogenetics Monohybrids are good for describing the relationship between alleles. When an allele is homozygous it will show its phenotype. It is the phenotype of the heterozygote which permits us to determine the relationship of the alleles. Dominance - the ability of one allele to express its phenotype at the expense of an alternate allele; the major form of interaction between alleles; generally the dominant allele will make a gene product that the recessive can not; therefore the dominant allele will express itself whenever it is present Law of independent assortment This law is based on dihybrid experiments. According to this law the alleles for each pair of character separate independently from those of other character during gametes formation. During gametes formations the allele Y may combine with the dominant allele R or recessive allele -r of the other character. To this point we have followed the expression of only one allele. Mendel also performed crosses in which he followed the segregation of two alleles. These experiments formed the basis of his discovery of his second law, the law of independent assortment. First, a few terms are presented. Dihybrid cross - a cross between two parents that differ by two pairs of alleles (AABB x aabb) Dihybrid- an individual heterozygous for two pairs of alleles (AaBb) Again a dihybrid cross is not a cross between two dihybrids. Now, let's look at a dihybrid cross that Mendel performed. Parental Cross: Yellow, Round Seed x Green, Wrinkled Seed F1 Generation: All yellow, round
BT0203 Genetics and Cytogenetics F2 Generation: 9 Yellow, Round, 3 Yellow, Wrinkled, 3 Green, Round, 1 Green, Wrinkled At this point, let's diagram the cross using specific allele symbols. Choose Symbol Seed Color: Yellow = G; Green = g Seed Shape: Round = W; Wrinkled = w The dominance relationship between alleles for each trait was already known to Mendel when he made this cross. The purpose of the dihybrid cross was to determine if any relationship existed between different allelic pairs. Let's now look at the cross using our allele symbols.
Now set up the Punnett Square for the F2 cross. Female Gametes GW Gw gW gw GGWW GGWw round) Male round) GgWW GgWw round) GgWw round) Ggww
GW (Yellow, (Yellow, (Yellow, (Yellow, Gw GGWw GGww
(Yellow, (Yellow, (Yellow, (Yellow, Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics round) Gametes wrinkled) round) ggWW round) ggWw wrinkled) ggWw (Green,ROUND) ggww (Green, wrinkled)
GgWW GgWw round) GgWw gw round) round) Ggww
gW (Yellow, (Yellow, (Green,
(Yellow, (Yellow, (Green, wrinkled) round)
The phenotypes and general genotypes from this cross can be represented in the following manner: Phenotype 9 Yellow, Round Seed 3 Yellow, Wrinkled Seed 3 Green, Round Seed 1 Green, Wrinkled Seed General Genotype G_W_ G_ww ggW_ ggww
The results of this experiment led Mendel to formulate his second law. Mendel's Second Law - the law of independent assortment; during gamete formation the segregation of the alleles of one allelic pair is independent of the segregation of the alleles of another allelic pair As with the monohybrid crosses, Mendel confirmed the results of his second law by performing a backcross - F1 dihybrid x recessive parent. Let's use the example of the yellow, round seeded F1.
Punnett Square for the Backcross Female Gametes GW Male Gametes Gw gW gw
Ggww ggWw ggww gw GgWw (Yellow, round) (Yellow, wrinkled) (Green, round) (Green, wrinkled)
The phenotypic ratio of the test cross is:

1 Yellow, Round Seed 1 Yellow, Wrinkled Seed 1 Green, Round Seed 1 Green, Wrinkled Seed
Testing for Independent Assortment The Goodness-of-Fit Chi-Square Test Clearly, we need some means of evaluating how likely it is that chance is responsible for the deviation between the observed and the expected numbers. To evaluate the role of chance in producing deviations between observed and expected values, a statistical test called the goodness-of-fit chi-square test is used. This test provides information about how well observed values fit expected values. Before we learn how to calculate the chi square, it is important to understand what this test does and does not indicate about a genetic cross. The chi-square test cannot tell us whether a genetic cross has been correctly carried out, whether the results are correct, or whether we have chosen the correct genetic explanation Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics for the results. What it does indicate is the probability that the difference between the observed and the expected values is due to chance. In other words, it indicates the likelihood that chance alone could produce the deviation between the expected and the observed values. When the probability calculated from the chi-square test is high, we assume that chance alone produced the difference. When the probability is low, we assume that some factor other than chancesome significant factorproduced the deviation. To use the goodness-of-fit chi-square test, we first determine the expected results. The chi-square test must always be applied to numbers of progeny, not to proportions or percentages. The chi-square value is calculated by using the following formula: 2 = (Oserved Expected) 2 / Expected where means the sum of all the squared differences between observed and expected divided by the expected values. The next step is to determine the probability associated with this calculated chi-square value, which is the probability that the deviation between the observed and the expected results could be due to chance. This step requires us to compare the calculated chi-square value with theoretical values that have the same degrees of freedom in a chi-square table. The degrees of freedom represent the number of ways in which the observed classes are free to vary. For a goodness-of-fit chi-square test, the degrees of freedom are equal to n-1, where n is the number of different expected phenotypes. We are ready to obtain the probability from a chi-square table (Table). The degrees of freedom are given in the left hand column of the table and the probabilities are given at the top; within the body of the table are chi-square values associated with these probabilities. First, find the row for the appropriate degrees of freedom. Find where calculated chi-square value lies among the theoretical values in this row. The theoretical chi-square values increase from left to right and the probabilities decrease from left to right. Most scientists use the .05 probability Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics level as their cutoff value: if the probability of chance being responsible for the deviation is greater than or equal to .05, they accept that chance may be responsible for the deviation between the observed and the expected values. When the probability is less than .05, scientists assume that chance is not responsible and a significant difference exists. The expression significant difference means that some factor other than chance is responsible for the observed values being different from the expected values. For example, Suppose we did a testcross for two pairs of genes, such as AaBb X aabb, and observed the following numbers of progeny: 54 AaBb, 56 aabb, 42 Aabb, and 48 aaBb. Is this outcome a 1:1:1:1 ratio? Not exactly, but its pretty close. Perhaps these genes are assorting independently and chance produced the slight deviations between the observed numbers and the expected 1:1:1:1 ratio. Alternatively, the genes might be linked, with considerable crossing over taking place between them, and so the number of nonrecombinants is only slightly greater than the number of recombinants. How do we distinguish between the roles of chance and of linkage in producing deviations from the results expected with independent assortment? Testing for independent assortment between two linked genes requires the calculation of a series of three chi-square tests. To illustrate this analysis, we will examine the data from a cross between German cockroaches, in which yellow body (y) is recessive to brown body (y+) and curved wings (cv) are recessive to straight wings (cv+). A testcross (y+y cv+cv X yy cvcv) produced the following progeny: 63 y+y cv+cv brown body, straight wings 77 yy cvcv yellow body, curved wings 28 y+y cvcv brown body, curved wings 32 yy cv+cv yellow body, straight wings Total = Rex Arunraj Assistant Prof. 200 total progeny Department of Genetic Engineering
Testing ratios at each locus To determine if the genes for body color and wing shape are assorting independently, we must examine each locus separately and determine whether the observed numbers differ from the expected (we will consider why this step is necessary at the end). At the first locus (for body color), the cross between heterozygote and homozygote (y+y X yy) is expected to produce half y+y brown and half yy yellow progeny; so we expect 100 of each. We observe 63 + 28 = 91 brown progeny and 77 + 32 = 109 yellow progeny. Applying the chi-square test to these observed and expected numbers, we obtain:
The degrees of freedom associated with the chi-square test are n- 1, where n equals the number of expected classes. Here, there are two expected phenotypes; so the degree of freedom is 2 - 1 = 1. Looking up our calculated chi-square value in Table, we find that the probability associated with this chi-square value is between .30 and .20. Because the probability is above .05 (our critical probability for rejecting the hypothesis that chance produces the difference between observed and expected values), we conclude that there is no significant difference between the 1:1 ratio that we expect in the progeny of the testcross and the ratio that we observed.
We next compare the observed and expected ratios for the second locus, which determines the type of wing. At this locus, a heterozygote and homozygote also were crossed (cv+ cv X cvcv) and are expected to produce half cv+cv straight-winged progeny and half cvcv curved-wing progeny. We actually observe 63 + 32 = 95 straightwinged progeny and 77 + 28 = 105 curved-wing progeny; so the calculated chi-square value is:
The degree of freedom associated with this chi-square value also is 2 - 1 = 1, and the associated probability is between .5 and .3.We again assume that there is no significant difference between what we observed and what we expected at this locus in the testcross.
Testing ratios for independent assortment
BT0203 Genetics and Cytogenetics We are now ready to test for the independent assortment of genes at the two loci. If the genes are assorting independently, we can use the multiplication rule to obtain the probabilities and numbers of progeny inheriting different combinations of phenotypes:
The observed and expected numbers of progeny can now be compared by using the chisquare test: Here, we have four expected classes of phenotypes; so the degrees of freedom equal 4 - 1 = 3 and the associated probability is considerably less than .001. This very small Probability indicates that the phenotypes are not in the proportions that we would expect if independent assortment were taking place. Our conclusion, then, is that these genes are not assorting independently and must be linked. In summary, testing for linkage between two genes requires a series of chi-square tests: a chi-square test for the segregation of alleles at each individual locus, followed by a test for independent assortment between alleles at the different loci. The chi-square tests for segregation at individual loci should always be carried out before testing for independent assortment, because the probabilities expected with independent assortment are based on the probabilities expected at the separate loci.
BT0203 Genetics and Cytogenetics Gene Interaction The expression of a single character by the interaction of more than one pair of alleles is called gene interaction or interaction of genes. The genes interaction is of two types namely 1. 2. Non allelic gene interaction Allelic gene interaction
The genic interaction occurring between genes located in different rows of the same chromosome or different chromosome is known as non-allelic gene interaction. Allelic gene interaction Incomplete dominance In incomplete dominance both alleles of a character express their character in the F1 generation. So the F1 individual has mixture of character of both the parents. Example : Mirabilis jalapa When a homozygous red flowered (RR) 4 o clock plant is crossed which a homozygous white flowered plant (rr) a pink coloured variety is produced (Rr). This is due to the in complete dominance of the allele R over its allele r. The expression of the two alleles (R and r) in the same individual leads to the production of an individual with mixed characters. Eg: Mirabilis jalapa Parents : Homozygous red flower RR Gametes: R r X Homozygous White flowers rr
BT0203 Genetics and Cytogenetics F1 Generation: Codominance In co-dominance, both alleles of a character an equally dominant and both of them expressed their character in the F1 generation. None is masked. Inheritance of coat colour in short horn cattle is another case of codominance Parents (Red) RR F1 generation (Roan) F1 selfed F2 generation RR Red Rr Rr X rr Roan White Rr X (White) rr Rr Rr (Pink Flower)
In short horn cattle, there are two colour of hair, red and white. Red colour is controlled by R & White by r. When red & white are crossed, the F1 has roan colour having both red & white hairs. generation. This is because r allele also expresses its character in the F1
BT0203 Genetics and Cytogenetics Non allelic gene interaction Epistasis It is the prevention of the expression of one gene by another non-allelic gene. Epistasis means stopping on inhibiting. The inhibiting gene is called epistatic gene, the inhibited gene is called the hypostatic gene. This is counter part of dominance. Dominance Epistasis is is intrallelic or intragenic intergenic
Dominant Epistasis (12:3:1) Dominant Epistasis the prevention of the expression, of a gene by a dominant non-allelic gene.. Inheritance of colour pattern in case of dominant Epistasis. Eg Colour coats of dogs One gene locus has a dominant epistatic inhibitor allele (I) of coat color pigment. The allele I prevents the expression of hypostatic gene locus (B / b) and produces white coat color. The alleles of hypostatic locus (BB, Bb, bb) express only when two recessive alleles (ii) occur on the epistatic loci iiBB / iiBb produce black and iibb produces brown coat color. Parent : Ii Bb (White) X Ii Bb (White)
BT0203 Genetics and Cytogenetics Gamete IB Gameter IB Ib IB IIBB White Ib IIBb White iB IiBB White ib IiBb White iB ib Ib IIBb White Iibb White IiBb White Iibb White iB IiBB White IiBb White iiBB Black iiBb Black ib IiBb White Iibb White iiBb Black iibb Brown
9:3:3:1 has become 12:3:1 Recessive Epistasis - 9:3:4 The prevention of the expression of a gene by a recessive non-allelic gene is called recessive epistasis. Eg- Coat color in mice The common house mouse occurs in a number of coat colors, agouti, black, and albino. The agouti is wild type. When a homozygous agouti (BBAA) is crossed with homozygous albino (bbaa), the F1 all were agouti. When F1 was crossed the ratio was 9:3:4. Parent Agouti (BBAA) F1 Rex Arunraj Assistant Prof. X Albino(bbaa) Agouti(BbAa) Department of Genetic Engineering
BT0203 Genetics and Cytogenetics F1 gametes Gameter BA BA BA BBAA Agouti Ba BBAa Agouti bA BbAA Agouti ba BbAa Agouti Ba bA Ba BBAa Agouti Bbaa Albino BbAa Agouti Bbaa Albino ba bA BbAA Agouti BbAa Agouti bbAA Black bbAa Black ba BbAa Agouti Bbaa Albino bbAa Black bbaa Albino
Hence the ratio of 9:3:3:1 become 9:3:4. Duplicate Recessive genes (Complementary genes) 9:7 Complementary genes may be defined as two or more non-allelic dominant genes interact with one another to produce a character but one gene cannot produce that character in the absence of the other. The action of these independent genes is complementary. IF both loci have homozygous recessive alleles, both of them produce identical phenotypes. Eg: Flower colour is Sweet Pea Inheritance of flower colour is sweet pea, Lathyrutus ordoratus. Two varieties of pea plants, one producing and flower the other white flower. The red Colour of the flower is due to the presence of a pigment called anthocyanin. The anthocyanin is produced from a colourless substance called chromogen by the action of an enzyme or activator. The chromogen cannot be converted in to anthocyanin In the absence of the enzyme. Gene C------ Chromogen
BT0203 Genetics and Cytogenetics Gene A ---- Enzyme Chromogen + Enzyme -- anthocyanin (Red) Red flower is produce by the interaction of both dominant gene C and A. C&A cannot give colour independently. Parent White CCaa Gametes F1 generation F1 Selfing CcAa (Red) Ca CcAa (Red) X CcAa (Red) X White ccAA cA
BT0203 Genetics and Cytogenetics Gametes Gametes CA CA CA CC AA Red Ca CCAa Red cA CcAA Red ca CcAa Red Ca cA ca Ca CC Aa Red CCaa White CcAa Red Ccaa White cA Cc AA Red CcAa Red ccAA White ccAa White ca Cc Aa Red Ccaa White CcAa White ccaa White
Duplicate genes with cumulative effect (Supplementary genes) 9:6:1 Two independent pair of dominant alleles interact in such a way that each dominant gene produces its effect whether the other is present or not, but when the second dominant gene is added to the first, a new character is expressed. Ex: coat color in Duroc jersey breed of pigs Sandy dominant Parent Sandy (SSrr) F1 S_, / R_ X and Sandy (ssRR) Red (SsRr) White ss / rr
F2 gametes Gametes SR SR
SR
Sr Sr SSRr Red SSrr
sR
sr sR SsRR Red SsRr Red ssRR Sandy ssRr Sandy sr SsRr Red Ssrr Sandy ssRr Sandy ssrr White
SSRR Red
Sr
SSRr Red
Sandy SsRr Red Ssrr Sandy
sR
SsRR Red
sr
SsRr Red
Hence the Ratio 9:3:3:1 has become 9:6:1 Duplicate Dominant Genes (15:1) Single character controlled by two or more pair of non-allelic genes independently. The dominant alleles of both gene loci produce the same phenotype without cumulative effect. Eg- Seed case in Capsella Triangular T, or Top/ Oval - D
Any one dominant can produce triangular seed case, both recessive produce oval seed case. Parent TTDD (Triangular) Rex Arunraj Assistant Prof. X ttdd (Top / Oval) Department of Genetic Engineering
BT0203 Genetics and Cytogenetics Gametes F1 Selfing Gametes Gametes TD TD TTDD Triangular Td TTDd Triangular tD TtDD Tringular td TtDd Triangular TD TD td TtDd (Triangular) Td Td TTDd Triangular TTdd Triangular TtDd Tringular Ttdd Triangular tD tD TtDD Triangular TtDd Triangular ttDD Tringular ttDd Triangular td td TtDd Triangular Ttdd Triangular TtDd Tringular ttdd Oval / top
Ratio :
15: (Triangular)
1 (Oval)
Dominant and Recessive epistasis 13:3 The dominant alleles of one gene locus (A) in homozygous (AA) or heterozygous (Aa) condition and the homozygous recessive alleles (bb) of another gene locus (B) produce the same phenotype. Eg- In leghorn fowl the white color of feather is caused by the dominant genotype CCII, similarly the white color of feathers of Plymouth rock is caused by the recessive genotype ccii. When both white are crossed the f1 is white. F2 produces white and colored birds in the ratio 13:3. Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics Parent CCII Gametes F1 F2 gametes Gametes CI CI CI CCII White Ci CCIi White cI CcII White ci CcIi White CI CcIi (White) Ci Ci CCIi White CCii Colored CcIi White Ccii Colored cI ci cI CcII White CcIi White ccII White ccIi White ci CcIi White Ccii colored ccIi White ccii White X ccii ci
Hence the ratio is 13:3 Back Cross : (TT X Tt) The F1 individuals obtained in a cross are usually selfed to get the F2 progeny. They can also be crossed with one of the other two parents from which they were derived. Such a cross of F1 individual with either of the two parents is known as a backcross. Test Cross : (Tt X tt) (1:1) In such back crosses, when F1 is back crossed to the parent with recessive phenotype.
BT0203 Genetics and Cytogenetics Penetrance : The percentage of individuals expressing the character for a particular genotype is called Penetrance. If all the individuals expressing the character for a particular genotype, the Penetrance is called complete Penetrance. If few individuals do not express the character even though they contain the necessary gene, the Penetrance is incomplete Penetrance. BB- Produce blue eyes 90% of human beings. 10% people have white eyes even though they contain the BB genes. Environmental factors influence Penetrance. Expressivity The variation in the degree of expression of a particular gene is called Expressivity.A particular gene may produce varying degrees of expression in different individuals.Expressivity is due to the influence of environmental factor on the genes. Ex : Effect of temperature on length of wings in Drosophila . Lethal Genes A lethal gene kills its possessor. These are genes which not only provide certain phenotypic traits but at the same time influence the viability of the organism. Lethal Genes in mice Yellow coat colour dominant gene Y YY Lethal effect All yellow individuals are heterozygous (Yy) Two yellow individuals are crossed.
BT0203 Genetics and Cytogenetics Off springs 2Yy, 1yy expected 1YY : (Lethal) Pleiotropism The production of many characters by a single pair of genes is called Pleiotropism It is the multiple effect of a pair of genes. Intermediate lethal genes Partial effect in heterozygous condition. Eg- Creepers short, crooked legs - normally they creep. Parent Genotype Gamete: C :creeper Cc c C C CC Normal C Cc Creeper C CC Creeper Cc Dies X creeper Cc 2Yy: 1yy
F1 Selfing Homozygous creeper is missing Balanced Lethal systems Both Homozygotes (Dominant and recessive) die. Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics MULTIPLE ALLELES So for we have observed that a given phenotypic trait is being controlled by two alternate forms of a gene (wild form) and the other being recessive (Mutant form) one bring dominant (Wild form) at the other being recessive (Mutant form). If the wild form mutates to give wise to the mutant form, there why not the wild form should mutate in more than are way to give wise to many mutant forms, on why should not the mutant form, mutate once again to give wise to other mutant forms. So a gene can haw more than two allelomorphs which make a series of multiple alleles. Definition Multiple alleles are a set of three or more allelic form of a gene controlling the same character located on the homologous chromosomes. In other words all the Mutant forms of a single wild type gene constitute a service of multiply alleles. A diploid individual possessor any two alleles of the allelic series and its gametes caries only one allele. Characters of Multiple Alleles. Multiple alleles of a services always occupy the same locus in the chromosome. Because all the alleles of multiple service occupy the seem locus in chromosome, on enclosing over occurs within the same alleles of a multiple service. Multiple alleles always influence the same character. The wild type alleles is nearly always dominant while other mutant alleles may show dominant or not. When two mutant multiple alleles are crosses the phenotype is mutant type all not the wild type Example For Multiple Alleles :
BT0203 Genetics and Cytogenetics 1. ABO blood group 2. Rh blood group 3. Coat colour in Rabbit 4. Nature of wing in Drosophila 5. Self Sterility in tobacco. 6. Skin colour in Mice. ABO Blood Grouping in Humans. On the basis of presence or absense of antigens ABO blood groups have been established by K. Landsteiner. Antigen A A B 2 Antigens 4 Blood groups AB
Antingen B
They are called as ABO blood group or Landsteiner blood group. A B AB O group group group group person person person person contain contain contain contain antigen antigen antigen No A B A&B Antigen on RBC
BT0203 Genetics and Cytogenetics Different ABO Blood groups and the antigens & antitrades associated with them. Blood group Antigen Antibody Antigen for
A B AB O
A B A and B No Antigen
B A No antibody A and B
Antigen
A B
cannot
Co- exist
with
antibody
A B
The three alleles responsible for the inheritance of ABO blood group are. LA LB Responsible For Synthesis of Antigen A B
Lo Absence of Antigen LA LB produces both Antigen A & B. Different ABO blood grupe on thein genotypes Blood group O A Rex Arunraj Assistant Prof. possible genotypes LO LO LA LO or LA LA Department of Genetic Engineering
BT0203 Genetics and Cytogenetics B AB Parent Father LB LO or LB LB LA LB x (A group) Genotype Gamete LA LA LA F1 : LA LB (or) LA LO Ex. 1. 2. 3. 4. AB A group A group B group AB x x x x A B group O group O group O Mother (B group) x LBLO LB LO
( Antigen (or) Agglutinogens present in RBC) (Antibodies (or) Agglutinins present in the serum) A A B AB O + + B + + AB O + + + -
BT0203 Genetics and Cytogenetics RH bood group There are two groups of human brings namely Rh- positive & Rh negative (Rh-) Person Who Donates Blood is called as a Donor Person Who Receives blood is called as a Recipient In transfusion the blood of the donor and the recipient should be compatible while testing the compatibility the reaction between the antigen of the doners RBC and the antibodies of the recipients plasma alone are taken into consideration Antibodies in blood group A will agglutinize RBCs of blood group B on vice versa. AB blood group will not agglutinize any other group science no antibodies are present. O blood group should be able to agglutinize all other three groups. Compatible donor and recipient Blood group of donor A B AB O (Universal Donor) Blood group of recipient A and AB B and AB AB (Universal Reciepient) O, A, B, AB
BT0203 Genetics and Cytogenetics Unit II Introduction E. Strasburger in 1875 discovered thread like structures during cell division. A given region of DNA with its associated proteins is called chromatin. This is true for prokaryotic & Eukaryotic cells & even for viruses. Packing of DNA into chromosome serves several important functions. The chromosome is a compact form of the DNA that readily fits inside the cell. Protects DNA from damage. (Naked DNA is unstable, chromosome DNA is highly stable). Each time a cell divides, only DNA packed can be efficiently transferred. Chromosome provides a definite organization which helps is recombination. Cell cycle Four phases G1 Resting phase / pre DNA synthesis phase S DNA synthesis takes place / DNA synthesis phase inter phase G2 Resting phase following / Post DNA synthesis phase M Mitosis division / Mitotic phase Mitosis Duration of different phases not only varies with the organism but with different tissues in the same organism. Majority of the associated proteins are small basic proteins called histones other proteins are called non-histone proteins. These are DNA binding proteins which regulate the transcriptions, replication recombination of cellular DNA. A human cell contains 3x10 9 bp per haploid set of chromosomes. The thickness is 3.4A. Diameter of a typical human cell nucleus is only 10-15 . Hence DNA has to be compacted by several orders. DNA with histones will form structures called nucleosomes. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics The formation of nucleosomes is the first step in a process that allows the DNA to be folded into much compact structure reducing their linear length by 10,000 times. Prokaryotic cells have comparatively small genomes. Even then the DNA has to be compacted. Bacteria do not have histones or nucleosomes but have small basic proteins which compact DNA. In prokaryotic cell - single circular chromosome In Eukaryotic cell - Multiple linear chromosome. However, there are numerous examples of prokaryotic cells having multiple chromosomes which are linear. But all eukaryotic cells have multiple linear chromosomes. Circular chromosomes require topoisomerases to separate the daughter molecules after they have replicated, without which the few daughter molecules remain inter locked after replication. Ends of linear DNA of the have to be protected from enzyme which will degrade the ends of the DNA. Prokaryotes have one complete copy of their chromosome that is packed into a structure called nucleoid. They also frequently carry one or smaller independent circular DNAs called plasmids. Plasmids like chromosomal DNA are not essential for bacterial growth, but code for desirable traits of bacteria. The majority of eukaryotic cells are diploid i.e. they contain two copies of each chromosome. The two copies of a given chromosome are called homologs, one derived from each parent. Not all cells in eukaryotic organisms are diploid. Some are haploid and some are polyploid. Haploid cells contain a single copy of each chromosome e.g. sperm and egg cells.
BT0203 Genetics and Cytogenetics Size A chromos is normally measured at mitotic metaphase. 0.25u - fungi & birds 30 u plants 3 u Drosophila 5 u Man Shape Chromosome shape is observed at anaphase. Primary constriction or centromere determines the shape of the chromosome. This constriction can be terminal, sub terminal or median in position. This clear zone also called as kinetocore which divides the chromosome into 2 arms each one is called a chromosome arm. The position of the centromere varies from chromosome and chromosome providing different shapes. Telocentric Acrocentric Metacentric Terms Chromonemata Centromere on the proximal end Centromere at one end giving a very short arm and a long arm. Centromere occurs near the center forming two unequal arms. Centromere occurs is the center and forming the equal arms. Polyploid cells have more than 2 copies of each chromosome. In extreme case there can be 100 or 1000 copies of each chromosome. E.g. Megakaryocytes - 128 copies of each chromosome. The segregation of such a large number of chromosomes is difficult. No matter the number eukaryotic chromosomes are already contained with is a membrane bound organelle called nucleus.
Submetacentric-
BT0203 Genetics and Cytogenetics During mitotic prophase the chromosomal material becomes visible as very this filaments, called chromonemata. A chromonema represents a chromatid at the early stages of condensation. Chromonemata form the gene bearing protions of the non-genetic chromosome. The chromonemata is embedded in a achromatic substance called matrix. Matrix is enclosed in a sheath or pellicle both matrix and pellicle are material and appear only at metaphase when the nucleolus disappears. Chromomeres The chromomeres are bead like accumulations of chromatin material that are sometimes visible along interphase chromosomes. At metaphase the chromosomes are highly coiled and the chromomeres are no longer visible. Centromere Consist of granules or spherules. Chromosome remains connected with the spherules of the centromere. It is the region of the chromosome to which the spindle fibers of the mitotic phase are attached. Chromosomes of most organisms contain only one centromere and are known as monocentric chromosomes. Some species have diffuse centromeres with microtubules attached along the length of the chromosome which are called holocentric chromosomes. Chromosomal abnormality Chromosomes may break and fuse forming chromosomes without centromere (acentric chromosomes). Chromosomes with two centromeres are called dicentric chromosomes. Both types of those chromosomal aberations are unstable. The acentric chromosomes remain in the cytoplasm since they cannot attach to mitotic spindle. Telomeres The chromosome extremities or terminal regions on either side are called telomeres. If the chromosome breaks, the broken ends fuse due to lack of telomeres. A chromosome however cannot fuse at the telomeric ends, since a telomere has a polarity which prevents other segments from joining with it. Rex Arunraj Assistant Prof. Department of Genetic Engineering
Secondary Constriction Besides centromere (primary constriction) secondary constrictions can be observed in some chromosomes. Such a constrictions if present is the distal region of the arm would pinch off a small fragment called satellite. The satellite remains attached to rest of the body by a thread of chromatin. Karyotype and Idiogram All the members of a species of plant or the animal are characterized by a set of chromosomes which have certain constant characteristics. These characteristics include the number of chromosomes, their relative size, and position of the centromere, length of the arms, secondary constrictions and satellites. The term karyotype has been given to the group of characteristics that identifies a particular set of chromosomes. A diagrammatic representation of a karyotype (or morphological characteristics of the chromosomes) of a species is called ideogram (Gr., idios = distinctive; gramma = something written). Generally, in an ideogram, the chromosomes of a haploid set of an organism are ordered in series of decreasing size. Sometimes an ideogram is prepared for the diploid set of chromosomes, in which the pairs of homologues are ordered in a series of decreasing size. A karyotype of human metaphase chromosomes is obtained from their microphotographs. The individual chromosomes are cut out of the microphotographs and lined up by size with their respective partners. The technique can be improved by determining the socalled centromeric index, which is the ratio of the lengths of the long and short arms of the chromosome. Some species may have special characteristics in their karyotypes; for example, the mouse has acrocentric chromosomes, many amphibians have only metacentric chromosomes and plants frequently have heterochromatic regions at the telomeres.
BT0203 Genetics and Cytogenetics Uses of karyotypes The karyotypes of different species are sometimes compared and similarities in karyotypes are presumed to represent evolutionary relationship. A karyotype also suggests primitive or advanced features of an organism. Depending on their staining properties, the following two types of chromatin may be distinguished in the interphase nucleus Euchromatin Portions of chromosomes that stain lightly are only partially condensed; this chromatin is termed Euchromatin. It represents most of the chromatin that disperse after mitosis has completed. Euchromatin contains structural genes which replicate and transcribe during G1 and S phase of interphase. The Euchromatin is considered genetically active chromatin, since it has a role in the phenotype expression of the genes. In Euchromatin, DNA is found packed in 3 to 8 nm fibre. Heterochromatin In 1928, Heitz defined heterochromatin as those regions of the chromosome that remain condensed during interphase and early prophase and form the so-called chromocentre. Heterochromatin is characterized by its especially high content of reptititive DNA sequences and contains very few, if any, structural genes (i.e., genes that encode proteins). It is late replicating (i.e., it is replicated when the bulk of DNA has already been replicated) and is not transcribed. It is thought that in heterochromatin the DNA is tightly packed in the 30nm fibre. Types of heterochromatin In an interphase nucleus, usually there is some condensed chromatin around the nucleolus, called perinucleolar chromatin, and some inside the nucleolus, called intranucleolar chromatin. Both types of this heterochromatin appear to be connected and together, they are referred to as nucleolar chromatin.
BT0203 Genetics and Cytogenetics Dense clumps of deeply staining chromatin often occur in close contact with the inner membrane of the nuclear envelope (i.e., with the nuclear lamina) and is called condensed peripheral chromatin. Between the peripheral heterochromatin and the nucleolar heterochromatin are regions of lightly staining chromatin, called dispersed chromatin. In the condensed chromosomes, the heterochromatic regions can be visualized as regions that stain more strongly or more weakly than the euchromatic regions, showing the socalled positive or negative heteropyknosis of the chromosomes (Gr., hetero = different+pyknosis = staining.). Heterochromatin has been further classified into the following types: Constitutive heterochromatin In such a heterochromatin the DNA is permanently inactive and remains in the condensed state throughout the cell cycle. This most common type of heterochromatin occurs around the centromere, in the telomeres and in the C-bands of the chromosomes. In Drosophila virilis, constitutive heterochromatin exists around the centromeres and such Pericentromeric heterochromatin occupies 40per cent of the chromosomes. In many species, entire chromosomes become heterochromatic and are called B chromosome, satellite chromosomes or accessory chromosomes and contain very minor biological roles. Such chromosomes comprising wholly constitutive heterochromatin occur in corn, many phytoparastic insects and salamanders. Constitutive heterochromatin contains short repeated sequence of DNA, called satellite DNA. This DNA is called satellite DNA because upon ultracentrifugation, it separates from the main component of DNA. Satellite DNA may have a higher or lower G + C content than the main fraction. For in the mouse genome, constituting 10 per cent of the total mouse DNA. The exact significance of constitutive heterochromatin is still unexplained. Facultative heterochromatin
BT0203 Genetics and Cytogenetics Such type of heterochromatin is not permanently maintained in the condensed state; in facultative heterochromatin one chromosome of the pair becomes either totally or partially heterochromatic. The best known case is that of the X-chromosomes in the mammalian female, one of which is active and remains euchromatic, whereas the other is inactive and forms at interphase, the sex chromatin or Barr body (Named after its discoverer, Canadian cytologist Murrary L.Barr). Barr body contains DNA which is not transcribed and is not found in males. Indeed, the number of Barr bodies is always one less than the number of X chromosomes (i.e., in humans, XXX female has two Barr bodies and XXXX female has three Barr bodies). Histones Histones are very basic proteins, basic because they are enriched in the amino acids arginine and lysine to a level of about 24 more present. Arginine and lysine at physiological pH are cationic and can interact electrostatically with anionic nucleic acids. Thus, being basic, histones bind tightly to DNA which is an acid. There are five types of histones in the eukaryotic chromosomes, namely H1, H2A, H2B, H3 and H4. One of the important discoveries that come from chemical studies is that the primary structures of histones have been highly conserved during evolutionary history. For example, histone H4 of calf and of garden pea contains only two amino acid differences in a protein of 102 total amino acid residues. These organisms are estimated to have an evolutionary history of at least 600 million years, during which they diverged structurally. This conservation of structure suggests that over the eras, histones have had a very similar and crucial role in maintaining the structural and functional integrity of chromatin. Such an evolutionary conservation suggests that the functions of these two histones involve nearly all of their amino acids so that a change in any position is deleterious to the cell. Histone H1 is the least rigidly conserved histone protein. It contains 210 to 220 amino acids and may be represented by a variety of forms even within a single tissue. H1 is present only once per 200 base pairs of DNA (in contrast to rest of the four types of Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics histones each of which is present twice) and is rather loosely associated with DNA. H1 histone is absent in yeast, Saccharomyces cerevisiae. Histones besides determining the structure of chromatin play a regulatory role in the repression activity of genes. Non-histones In contrast to the modest population of histones in chromatin, non-histone proteins display more diversity. In various organisms, number of non-histones can vary from 12 to 20. Heterogeneity of these proteins is not conserved in evolution as the histones. These non-histones differ even between different tissues of the same organism suggesting that they regulate the activity of specific genes. About 50 per cent non-histones of chromatin have been found to be structural proteins and include such proteins as action, and and tubulins and myosin. Although for sometime these contractile proteins were thought to be contaminants, it is now believed that they are vital ingredients of the chromosome, functioning during chromosome condensation and in the movement of chromosomes during mitosis and meiosis. Many of the remaining 50 per cent of non-histones include all the enzymes and factors that are involved in DNA replication, in transcription and in the regulation of transcription. These proteins are not as highly conserved among organisms, although they must carry out similar enzymatic activities. Apparently they are not as important as the histones in maintaining chromosome integrity.
BT0203 Genetics and Cytogenetics Molecular organization of the eukaryotic genome Three levels of genome organization nuclear genome mitochondrial genome chloroplast genome
Genome size: It refers to the amount of DNA in a haploid cell. Human genome includes 60 ~ 100,000 "genes" occupying <5% of DNA The C value paradox organism complexity does not correlate with genome size Chromosome architecture at the microscopic level DNA packaged into chromosomes. Prokaryote DNA is usually circular, smaller, and less elaborately folded & structured. Eukaryotic DNA is complexed with large amounts of protein to form chromatin, is highly extended & tangled during interphase but is condensed into short thick chromosomes during mitosis. Thus, eukaryotic chromosomes contain enormous amounts of DNA, which must be packed correctly. Primary coiling of DNA is double helix. A. Nucleosomes 1. Histones: small proteins rich in basic amino acid that bind to DNA --> chromatin; similar from one eukaryote to another. H1, H2A, H2B, H3, and H4
BT0203 Genetics and Cytogenetics 2. Nucleosomes: secondary coiling of DNA around histone core. 2o coiling of DNA around histone core basic unit of DNA packing, formed from DNA wound around a protein core of 2 copies each of four types of histone (H2A, H2B, H3, and H4). Resembles beads along a string; may control gene expression by limiting access of transcription.
B. Higher Levels of DNA Packing 1. tertiary coiling of core + linker forms "Solenoid":30-nm chromatin fiber: consists of a tightly wound coil with 6 nucleosomes / turn
2. Looped domain: each loop in the 30-nm fiber contains 20,000 to 100,00 base pairs; looped domains coil and fold further compacting chromosome (as in metaphase)
BT0203 Genetics and Cytogenetics Interphase chromatin is much less condensed than mitotic chromatin a. Heterochromatin: remains highly condensed, not actively transcribed b. Euchromatin: less condensed, is actively transcribed Polytene Chromosome A giant chromosome produced by an endomitotic process in which, following synapsis of the two homologues, multiple rounds of replication produce chromatids that remain synapsed together in a haploid number of chromosomes. Large chromosome consisting of many chromatids formed by rounds of endomitosis following synapsis of the two homologues. Polytene chromosomes have been studied mostly in Drosophila salivary glands, in which chromosomes undergo 10 cycles of replication without separation of the daughter chromosomes. This leads to 1024 identical strands of chromatin aligned side by side. These chromosomes are easy to see with a light microscope because of their large size and precise alignment. The chromosomes is seen as distinct alternating dark and light bands. The dark bands correspond to more condensed regions of the chromatin, and the light (interband) regions are less dense regions. Can be 1000 times thicker than normal meiotic chromosomes. In each polytene chromosome, homologous chromosomes are tightly paired. Polytene chromosomes are joined together at their centromeres by a proteinaceous structure called a chromocenter.
BT0203 Genetics and Cytogenetics When stained, polytene chromosomes show a banding pattern. Each band contains an average of 30,000 bp, and may contain up to seven genes. Interbands also contain genes. In Drosophila, 5,000 bands have been observed.
Chromosome puffs are diffused uncoiled regions of the chromosome that are sites of RNA transcription.
Balbiani ring is a large chromosomal puff.
Lampbrush Chromosomes The lampbrush type of chromosome is characteristic of growing oocytes in the ovaries of most animals with the exception of mammals and certain insects. The chromosomes are greatly elongated diplotene bivalents, sometimes reaching lengths of a millimeter or more. Lampbrush chromosomes are exceedingly delicate structures and no further progress beyond the pioneer studies of Flemming (1882) and Ruckert (1892) was possible until a technique could be devised for dissecting them out of their nuclei and examining them in a life-like condition, separated from the remainder of the nuclear contents. The largest lampbrush chromosomes are to be found in growing oocytes of newts and salamanders. These animals have big genomes, big chromosomes and big cells, so it is scarcely surprising that they have good lampbrushes. The best oocytes for lampbrush studies are the ones that make up the bulk of the ovary of a healthy adult female at the time of year when the eggs are actively growing in preparation for ovulation in the following spring. They are about 1 mm in diameter. They have nuclei that are between a third and a half a millimeter in diameter, big enough
BT0203 Genetics and Cytogenetics to see with the naked eye. It is not difficult to isolate these nuclei by hand and it is not much more difficult to remove their nuclear envelopes and spill out their chromosomes.
A lampbrush chromosome is a meiotic half bivalent. This means that it must consist of two chromatids. The entire lampbrush bivalent will therefore have a total of 4 chromatids. The chromosome appears as a row of granules of deoxyribonucleoprotein (DNP), the chromomeres, connected by an exceedingly thin thread of the same material (figure). Chromomeres are 1/4 to 2um in diameter and spaced 1 2 micrometers along the length of the chromosome axis.
Figure Chromomeres bearing pairs (L) or multiple pairs (LL) of lateral loops: Sister loops of different lengths (L1): polarization of thickness along the lengths of loops (P): Loops consisting of a single unit of polarization (P): Loops consisting of several tandem units of polarization with the same or different directions of polarities (ppp). Each chromomere has 2 or some multiple of 2 loops associated with it. The loops have a thin axis of DNP surrounded by a loose matrix of ribonucleoprotein (RNP). The loops are variable in length, and during the period of oogenesis when they are maximally
BT0203 Genetics and Cytogenetics developed, they extend from 5 to 50 micrometers laterally from the chromosome axis, which means that the longest loops in such a case would be 100 micrometers long.
BT0203 Genetics and Cytogenetics Sex Determination Genetic mechanism Metabolically controlled Hormonally controlled Environmentally controlled
Genetically Controlled a) Heterogametic Males. The XY sex-determination system is the sex-determination system found in humans, most other mammals, some insects (Drosophila) and some plants (Ginkgo). In this system, females have two of the same kind of sex chromosome (XX), and are called the homogametic sex. Males have two distinct sex chromosomes (XY), and are called the heterogametic sex. The XY sex determination system was first described independently by Nettie Stevens and Edmund Beecher Wilson in 1905. Some species (including most mammals) have a gene or genes on the Y chromosome that determine maleness. In the case of humans, a single gene (SRY) on the Y chromosome acts as a signal to set the developmental pathway towards maleness. Other mammals use several genes on the Y chromosome for that same purpose. Not all male-specific genes are located on the Y chromosome. Other species (including most Drosophila species) use the presence of two X chromosomes to determine femaleness. One X chromosome gives putative maleness. The presence of Y chromosome genes are required for normal male development. XX/X0 sex determination
BT0203 Genetics and Cytogenetics The X0 sex-determination system is a system that grasshoppers, crickets, cockroaches, and some other insects use to determine the sex of their offspring. In this system, there is only one sex chromosome, referred to as X. Males only have one X chromosome (X0), while females have two (XX). The zero (sometimes, the letter O) signifies the lack of a second X chromosome. Maternal gametes always contain an X chromosome, so the sex of the animals' offspring is decided by the male. Its sperm normally contain either one X chromosome or no sex chromosomes at all. In this variant of the XY system, females have two copies of the sex chromosome (XX) but males have only one (X0). The 0 denotes the absence of a second sex chromosome. The nematode C. elegans is male with one sex chromosome (X0); with a pair of chromosomes (XX) it is a hermaphrodite. b) Heterogametic females ZW sex chromosomes The ZW sex-determination system is a system that determines the sex of offspring in birds, some fish, and some insects (including butterflies and moths). In the ZW system it is the ovum that determines the sex of the offspring, in contrast to the XY sexdetermination system and the X0 sex-determination system. The letters Z and W are used to distinguish this system from XY system. Males are the homogametic sex (ZZ), while females are heterogametic (ZW). The Z chromosome is larger and has more genes, like the X chromosome in the XY system. It is unknown whether the presence of the W chromosome induces female features or the duplication of the Z chromosome induces male ones; unlike mammals, no birds with a double W chromosome (ZWW) or a single Z (Z0) have been discovered. It is possible that either condition causes embryonic death, and both chromosomes are responsible for gender selection. In Lepidoptera (moths and butterflies), examples of Z0, ZZW and ZZWW females can be found. This suggests that the W chromosome is essential in female determination in some Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics species (ZZW), but not in others (Z0). In Bombyx mori (the commercial silkworm), the W chromosome carries the female-determining genes. The ZW sex-determination system is found in birds and some insects and other organisms. The ZW sex-determination system is reversed compared to the XY system: females have two different kinds of chromosomes (ZW), and males have two of the same kind of chromosomes (ZZ). c) Haploidy The Haplodiploid sex-determination system determines the sex of the offspring of many Hymenopterans (bees, ants, and wasps), and coleopterans (bark beetles). In this system, sex is determined by the number of sets of chromosomes an individual receives. An offspring formed from the union of a sperm and an egg develops as a female, and an unfertilized egg develops as a male. This means that the males have half the number of chromosomes that a female has, and are haploid. This system produces a number of peculiarities; chief among these is that a male has no father and cannot have sons, but he has a grandfather and can have grandsons. Unfertilized eggs develop into haploid individuals, which are the males. Diploid individuals are generally female but may be sterile males. Thus, if a queen bee mates with one drone, her daughters share of their genes with each other, not as in the XY and ZW systems. After mating, fertile Hymenopteran females store the sperm in an internal sac called the spermatheca. The mated female controls the release of stored sperm from within the organ: If she releases sperm as an egg passes down the oviduct, the egg is fertilized. Social bees, wasps, and ants can modify sex ratios within colonies to maximize relatedness among members, and to generate a workforce appropriate to surrounding conditions. d) Sex balance theory or genic balance theory Rex Arunraj Assistant Prof. Department of Genetic Engineering
Sex balance theory or genic balance theory states that the X chromosome determines the sex of the individual and that sex is a dosage phenomena, where the ratio of the amount of the X relative to the autosomes determines the sex. In addition, environmental effects can influence the development of the intersex flies. Further studies have shown that sex is ultimately determined by the locus sex-lethal on the X chromosome, though several other loci on the X chromosome and the autosomes are also needed for sex determination.
Environmental sex determination The same chemical plays a unique role in the worm's sexual differentiation. The planktonic, free-swimming Bonellia larvae are initially sexually undifferentiated. Larvae which land on unoccupied sea-floor mature, over the period of years, into adult females. But most larvae come in contact with the bonellin in the skin of an adult female, bonellinrich proboscis (The adult Bonellia female produces a vivid green pigment in its skin, known as bonellin. This chemical, concentrated mostly in the proboscis) and are masculinised by this exposure. The chemical causes these larvae to develop into the tiny males, which cling to the female's body or are sucked inside it by the feeding tube, to spend the remainder of their lives as parasites inside the female's genital sac, producing sperm to fertilize her eggs and reliant on their host for all other needs. The sex of a Green Spoonworm is thus determined by external, environmental factors (the presence or absence of bonellin), not by internal, genetic factors (chromosomes), as is the case with most other sexually-differentiated organisms. This environmental sex determination helps Green Spoonworm populations respond to the availability of burrows. Hormonally controlled sex determination
BT0203 Genetics and Cytogenetics Sex reversal in Hen In birds only one gonad of a normal female develops into a functional ovary. The other gonad remains rudimentary. If the functional ovary of a hen is destroyed, the rudimentary gonad develops into a testis. Thus the female sex is reversed into male due to the phenomenon called sex reversal. During embryonic development the XY genotype stimulates the pituitary gland to produce female hormones that cause the gonad of the hen to develop into an ovary. After the development of the ovary the pituitary ceases to produce female hormones due to inhibition of the pituitary by hormones produced by the ovary, thus acting as feed-back system. When the ovary of the hen is removed the steroid cells of the adrenal become active and provoke the development of testis.
Sex Linked Inheritance X-linked Inheritance In animals with XY sex determining mechanism, the X chromosome has many loci, many that have nothing to do with sex as such. The Y is usually smaller and possesses fewer loci that are not the same loci as that on the X chromosome. Thus females that have the same allele at a locus on the X chromosome are homozygous. Different alleles would be heterozygous. Males, because they have only one X, are hemizygous and can have only one allele at a locus. Because of this, one copy of a recessive allele will be expressed in the phenotype in males. In sex-linked inheritance, crosses are not reciprocal. The X-linked pattern is called the criss-cross pattern of inheritance because fathers pass the trait to daughters who pass it on to sons. Rex Arunraj Assistant Prof. Department of Genetic Engineering
Transmission of X-linked genes A. Females transmit an X chromosome both to sons and daughters; however, males transmit their X chromosomes only to daughters and their Y chromosomes only to sons. B. For rare X-linked dominant traits: 1. Twice as many females are affected as males. 2. Half the children of an affected female will be affected, regardless of sex. 3. All the daughters of an affected male will be affected but none of the sons. C. For rare X-linked recessive traits: 1. Hemizygous males are affected, but only homozygous females are affected. The ratio of affected males-to-females is >>1. 2. Affected males will transmit the gene to all daughters, who will usually be heterozygous and therefore not affected, but to no sons. 3. Affected males in a pedigree are related to each other through nonaffected females; e.g. grandfathermotherson or maternal unclemotherson. 4. Homozygous recessive females can arise only from matings in which the father is affected and the mother is heterozygous or homozygous. D. Examples of X-linked recessive traits are: 1. Colorblindness. 2. Hemophilia. 3. Duchenne muscular dystrophy. Color blindness: Half of the sons of a woman who is heterozygous for color blindness will be color blind, irrespective of the genotype of their father. If the father is color blind, half of the daughters will also be color blind. If the father has normal vision, none of the daughters will be color blind. A normal vision man and a normal vision woman who is heterozygous for color blindness will produce progeny with a 3:1 ratio of normal vision to color blind, but all of the color blindness will be in male progeny. Note that all of this Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics is an oversimplification. There are actually various types of color blindness involving loss of sensitivity to different colors of light. Hemophilia: This disorder of blood clotting is X-linked and is common in the males of European royal families because of frequent consanguineous matings within this limited breeding pool. Pedigrees suggest that Queen Victoria of England was heterozygous for classic hemophilia (hemophilia A), which involves a deficiency of clotting factor VIII. Hemophilia B, with a deficiency of clotting factor IX, is also X-linked Recessive means that disease only occurs when a person has two copies of the bad gene. For autosomal recessive diseases, this usually means they must inherit the disease from both parents, but this is not the case for X-linked recessive diseases. Men have only a single X chromosome, so they have only one copy of any gene on the X chromosome. Thus, a gene error on X definitely caused disease in men (who are XY), but women are XX, and have two copies of the gene. X-linked recessive disorders are more likely to occur than autosomal recessive disorders, because men have only one X chromosome, whereas all people have 2 copies of each autosome. Recessive diseases often occur in genes that produce an enzyme. In a carrier, who has only one bad copy, there is often no disease, because the second gene can pull up the slack, and maintain health. In some recessive diseases, a carrier gets a mild form of the disease. For example, in X-linked recessive hemophilia, a female carrier has one bad gene on chromosome X, but the good gene on the other X chromosome produces enough of the good clotting enzyme to maintain health. The recessive disease only arises when the male has no good gene on the other chromosome (because they get a Y instead of a second good X).
Holandric or Y-linked Genetic Diseases
BT0203 Genetics and Cytogenetics The Y chromosome is a sex-linked chromosome. Men are XY and women are XX, so only men have a Y chromosome. The Y chromosome is very small and contains few genes. Y-linked transmission- The Y chromosome has a trivially simple inheritance pattern because women are XX and men are XY. Only men have a Y chromosome and so the Y is only passed from father to son. The Y chromosome is small and does not contain many genes. There are few genetic diseases related to genes on Y. Male sex determination: The main Y gene is called the SRY gene, which is the master gene that specifies maleness and male features. It is the single gene that sets off the initial cascade of hormone changes that make a person male. It is not the entire Y chromosome, but just this gene that is necessary for maleness. There is evidence of this in rare diseases where the SRY gene is missing. People who are genetically male with XY chromosomes, but with a mutation or deletion of this SRY gene on the Y chromosome, will be female despite having most of the Y chromosome. And people who are genetically female with XX but also have a tiny piece of the Y chromosome with this gene; will become male despite their female-like XX chromosomes. Transmission of Y-linked genes (holandric inheritance) A. Since males are hemizygous for Y-linked genes on the nonhomologous part of the Y chromosome, all will be expressed. B. Y-linked genes are transmitted from father to son, never to daughters. C. There are no essential genes on the nonhomologous portion of the Y; however, the gene for maleness is there, as is a locus concerned with male fertility. Sex-limited traits Sex-limited traits are traits that are expressed in one sex, but not in the other. Some examples include milk yield in mammals, antlers in deer, and beards in humans.
Sex-influenced traits Sex-influenced traits appear in both sexes but more so in one sex than another. Male pattern baldness in humans is an example. The male hormone testosterone is needed for full expression of baldness. Because of this hormone difference, the allele for baldness behaves as a dominant trait in males (expressed when heterozygous), but behaves as a recessive allele in females (must be homozygous to be expressed).
BT0203 Genetics and Cytogenetics UNIT III LINKAGE AND CROSSING OVER Introduction: Sutton and Bovery proposed that the genes are located on the chromosomes. Later other workers also proved the same with sufficient evidences. During cell division (Mitosis or Meiosis) each chromosome appears to behave as a unit and therefore it would be expected that genes located on the same chromosome would move to the same pole during cell division. As a consequence some genes would fail to show independent assortment (segregation) and tend to be inherited together. Sutton expressed this expectation in 1903 while propounding, the chromosome theory of inheritance. Bateson and Punnet (1905) were the first to report a significant deviation from the expectation due to independent assortment. They studied the flower colour in pea. A dominant gene 'B' produces blue coloured flowers, while its recessive allele 'b' determines red flower. Another dominant gene L governs elongate pollen grains, where as its recessive allele l produces normal round pollen grains. When the F1 from the cross Blue elongate (BBLL) X red round (bbll) was test crossed with red round (bbll), the progenies showed the ratio of 7 1 1 7 blue elongate (BbLI) blue round (Bbll) red elongate (bbLI) red round (bbll)
In the place of expected 1:1:1:1 ratio, it appears that the two dominant gene 'B' (blue colour) and V (elongate pollen grain) have an affinity for each other so that they tend to stay together during the transmission to the progenies. Bateson and Punnet called this situation as "Coupling Phase". But when F1from the cross Blue Round (BBll) ratio of 1 7 blue elongate (BbLI) blue round (Bbll) Department of Genetic Engineering X Red Elongate (bbLL)
Blue Elongate (BbLI) was test crossed with red round (bbll), the progeny exhibited the
BT0203 Genetics and Cytogenetics 7 1 red elongate (bbLI) red round (bbll).
It appears as if the two dominant genes (B and L) repel each other in this case, so that they tend to stay away from each other in the progeny. This situation was referred as "Repulsion Phase". Bateson and Punnet could not give suitable explanation for this situation and suggested that there was a selective multiplication (through mitosis) of some types of gametes i.e. BL and bl in the coupling phase and Bl and bL in the repulsion phase, after meiosis and this gives rise to 7:1:1:7 and 1:7:7:1 ratio instead of 1:1:1:1 test cross ratio. Even though this hypothesis is unstable and later disproved, but the terms 'coupling' and 'repulsion' phase are widely used still to denote the linkage between two dominant genes an recessive genes respectively. Morgans View on Linkage Later in 1919, Morgan worked with gene of Drosophila. and explained linkage. He concluded that 1) 2) Genes located on the same chrome tend to stay together during inheritance this Genes are arranged in a linear fashion on the chromosome. tendency is called linkage. 3) The intensity of the linkage between the two genes on the chromosome is inversely proportional to the distance between the two linked genes on the chromosome. 4) Proposed that coupling and repulsion are the 2 aspects of linkage.
BT0203 Genetics and Cytogenetics LINKAGE: The tendency of two or more genes to stay on the same chromosome is called as linkage. Linkage is the consequence of genes located on the same chromosome. Linked genes do not show independent assortment. KINDS OF LINKAGE T.H. Morgan and his co-workers by their investigation on the Drosophila and other organisms have found two types of linkage, viz., complete linkage and incomplete linkage. 1. Complete Linkage The Complete linkage is the phenomenon in which parental combinations of characters appear together for two or more generations in a continuous and regular fashion. In this type of linkage genes are closely associated and tend to transmit together. Example. The genes for bent wings (bt) and shaven bristles (svn) of the fourth chromosome mutant of Drosophila melanogaster exhibit complete linkage. Complete linkage in male Drosophila. In most of the organisms crossing-over takes place both in males and females. But in male Drosophila and female silkworm, Bombyx mori crossing-over takes place either very rarely or not at all. This becomes clear from Morgans experimental results from Drosophila. In 1919, T.H. Morgan mated gray bodied and vestigial winged (b+vg/b+vg) fruit flies with flies having black bodies and normal wings (bvg+/bvg+). F1 progeny had gray bodies and normal long wings (b+vg/ bvg+), indicating thereby that these characters are dominant. When F 1 males (b+vg/ bvg+), were backcrossed (i.e., test crossed) to double recessive females (bvg/bvg or black vestigial), only two types of progeny (one with gray bodies and vestigial wings, b +vg/bvg and the other with black bodies and normal wings, to bvg +/bvg instead of four types of phenotypes were obtained . Parents: Gametes: Rex Arunraj Assistant Prof. Gray, Vestigial b+vg/b+vg (b+vg) X Black, Long b+vg/b+vg (bvg+) Department of Genetic Engineering
F1: Test Cross : Gametes : F1 male Gray, Long b+vg/bvg+ (b+vg)(bvg+)
All Gray, long b+vg/ bvg+ X Female Black, Vestigial bvg/bvg (bvg)
(Only two types of gametes due to complete linkage and lack of crossing over in male Drosophila) Test cross ratio: 1Gray, vestigial b+vg/ bvg 1Black, Long or 1:1. bvg+/bvg
Here, the use of the testcross is very important. Because one parent (the tester) contributes gametes carrying only recessive alleles, the phenotypes of the offspring represent the gametic contribution of the other double heterozygote parent. So the genetical analyst can concentrate on one meiosis and forget the other. This is in contrast to the situation in an F1 selfing where there are two sets of meiotic divisions to consider one for the F1 male parental gametes and one for the F1 female. 2. Incomplete Linkage The linked genes do not always stay together because homologous non-sister chromatids may exchange segments of varying length with one another during meiotic prophase. This sort of exchange of chromosomal segments in between homologous chromosomes is known as crossing over. The linked genes which are widely located in chromosomes and have chances of separation by crossing over are called incompletely linked genes and the phenomenon of their inheritance is called incomplete linkage. Incomplete Linkage in female Drosophila. When F1 females of the Morgans classical cross in Drosophila between gray, vestigial (b+vg/b+vg) and black, normal or long (bvg+/bvg+) were test-crossed to double-recessive (bvg/bvg) males, all four types of progeny were obtained in following ratio, showing occurrence of crossing-over: Parents: Rex Arunraj Assistant Prof. Gray, Vestigial X Black, Long
BT0203 Genetics and Cytogenetics b+vg/b+vg Gametes: F1 : Test Cross: F1 Female Gray, Long b+vg/bvg+ (b+vg) All Gray, long b+vg/ bvg+ X Male Black, Vestigial bvg/bvg (bvg) 83%parental combination showing linkage 17%recombinants due to crossing over bvg+/bvg+ (bvg+)
Gametes:
(b+vg) (bvg+) = Non-crossovers (b+vg+)(bvg) = Recombinants = 41.5% = 41.5% = 8.5% = 8.5%
Test cross ratio: 1. Gray, Vestigial; b+vg/bvg 2. Black, Long; bvg+/bvg 3. Gray, Long: b+vg+/bvg 4. Black, Vestigial; bvg/bvg Crossing Over The crossing over is a process
that
produces
new
combinations
(recombinations) of genes by interchanging of corresponding segments between nonsister chromatids of homologous chromosomes The chromatins resulting from such interchanges of chromosomal parts are known as cross overs. The term crossing over was coined by T.H.Morgan. In simple, Crossing over is the exchange of segments between non-sister chromatids of homologous chromosomes. Characteristics of Crossing Over 1. Crossing over or recombination occurs at two levels (i) at gross chromosomal level, called chromosomal crossing over and (ii) at DNA level, called genetic recombination. 2. A reciprocal exchange of material between homologous chromosomes in heterozygotes is reflected in crossing over. 3. The crossing over results basically from an exchange of genetic material between non-sister chromatids by break-and-exchange following replication.
BT0203 Genetics and Cytogenetics 4. The frequency of crossing over appears to be closely related to physical distance between genes on chromosome and serves as a tool in constructing genetic maps of chromosomes. Types of Crossing Over 1. Somatic or Mitotic Crossing Over When the process of crossing over occurs in the chromosomes of body or somatic cells of an organism during the mitotic cell division it is known as somatic or mitotic crossing over. The somatic crossing over is rare in its occurrence and it has no genetical significance. The somatic or mitotic crossing over has been reported in the body or somatic cells of Drosophila by Curt Stern. 2. Germinal or Meiotic Crossing Over Usually the crossing over occurs in germinal cells during the gametogenesis in which the meiotic cell division takes place. This type of crossing over is known as germinal or meiotic crossing over. The meiotic crossing over is universal in its occurrence and is of great genetic significance. Mechanism of Meiotic Crossing Over The process of crossing over includes following stages in it, viz., synapsis, duplication chromosomes, crossing over and terminalization. The chromosomes which tend to undergo recombination due to meiotic crossing over necessarily complete two functions: 1.99.7 per cent replication of DNA and 75 per cents synthesis of histones, both of which take place prior to onset of prophase I, and 2. attachment of each chromosome by its both ends (telomeres) to the nuclear envelope (i.e., to nuclear lamina) via the specialized structure, called attachment plaques. This event occurs during the leptotene stage of prophase I and though each chromosome at this stage is visually long and thin thread, but contains material of two sister chromatids (i.e., two DNA molecules plus almost duplicated amount of histones). 1. Synapsis Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics Synapsis or intimate pairing between the two homologous chromosomes (one maternal and another paternal) is initiated during zygotene stage of prophase I of meiosis I. Synapsis often starts when the homologous ends of the two chromosomes are brought together on the nuclear envelope and it continues inward in a zipper-like manner from both ends, aligning the two homologous chromosomes side by side. In other cases, synapsis may begin in internal regions of the chromosomes and proceed towards the ends, producing the same type of alignment. By synapsis each gene is, thus, brought into juxtaposition (=being side by side) with its homologous gene on the opposite chromosome. Thus, synapsis is the phase of prolonged and close contact of homologous chromosomes due to attraction between two exactly identical or homologous regions or chromomeres. The resultant pairs of homologous chromosomes are called bivalents. 2. Duplication of Chromosomes The synapsis is followed by duplication of chromosomes (in pachytene). During this stage, each homologous chromosome of bivalents splits longitudinally and forms two identical sister chromatids which remain held together by an unsplitted centromere. The longitudinal splitting of chromosomes is achieved by the separation of already duplicated DNA molecules along with certain chromosomal proteins. At this stage each bivalent contains four chromatids, so it is known as tetrad. 3. Crossing over by Breakage and Union It is well evident that crossing over occurs in the homologous chromosomes only during the four stranded or tetrad stage. Homologues continue to stay in synapsis for days during pachytene stage and chromosomal crossing over occurs due to exchange of chromosomal material between non-sister chromatids of each tetrad. In pachytene, the recombination nodules become visible between synapsis chromosomes. During the process of crossing over, two non-sister chromatids first break at the corresponding points due to the activity of a nuclear enzyme known as endonuclease. Then a segment on one side of each break connects with a segment on the opposite side of the break, so that the two non-sister chromatids cross each other. At this stage 3 per Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics cent synthesis of DNA occurs to fill the gap. The fusion of chromosomal segments with that of opposite one takes place due to the action of an enzyme known as ligase . The crossing of two chromatids is known as chiasma (Gr., chiasma=cross) formation. The crossing over, thus, includes the breaking of chromatid segments, their transposition and fusion. Chiasma frequency or percentage of crossing over. The crossing over may take place at several points in one tetrad and may result in the formation of several chiasmata. The number of chiasmata depends on the length of the chromosomes because the longer the chromosome the greater the number of chiasmata. In a species each chromosome has a characteristic number of chiasmata. The frequency by which a chiasmata occurs between any two genetic loci has also a characteristic probability. The more apart two genes are located on a chromosome, the greater the opportunity for a chiasma to occur between them. The closer two genes are linked lesser the chances for a chiasma occurring between them. 4. Terminalisation After the occurrence of process of crossing over, the non-sister chromatids start to repel each other because the force of synapsis attraction between them decreases. During diplotene. desynapsis begins, synaptonemal complex dissolves and two homologous chromosomes in a bivalent are pulled away from each other. During diakinesis, chromosomes detaches from the nuclear envelope and each bivalent is clearly seen to contain four separate chromatids with each pair of sister chromatids linked at their centromeres, while non-sister chromatids that have crossed over are linked by chiasmata. The chromatids separate progressively from the centromere towards the chiasma and the chiasma itself moves in a zipper fashion towards the end of the tetrad. The movement of chiasma is known as terminalisation. Due to the terminalisation the homologous chromosomes are separated completely. Kinds of Crossing Over
BT0203 Genetics and Cytogenetics According to the number of chiasma following types of crossing over have been described. 1. Single crossing over. When the chiasma occurs only at one point of the chromosome pair then the crossing over is known as single crossing over. The single crossing over produces two cross over chromatids and two non-crosses over chromatids. 2. Double crossing over. When the chiasmata occur at two points in the same chromosome, the phenomenon is known as double crossing over. In the double crossing over, the formation of each chiasma is independent of the other and in it four possible classes of recombination occur. In the double crossing over following two types of chiasma may be formed: (i) Reciprocal chiasma. In the reciprocal chiasma the same two chromatids are involved in the second chiasma as in the first. Thus, the second chiasma restores the order which was changed by the first chiasma and it produces two non-cross over chromatids. The reciprocal chiasma occurs in two strand double crossing over in which out of four chromatids only two are involved in the double crossing over. (ii) Complimentary chiasma. When both the chromatids taking part in the second chiasma are different from those chromatids involved in the first chiasma is known as complimentary chiasma. The complimentary chiasma produces four single cross overs but no non-cross over. The complimentary chiasma occurs when three or four chromatids of the tetrad undergo the crossing over. 3. Multiple crossing over. When crossing over takes place at more than two places in the same chromosome pair then such crossing over is known as multiple crossing over. The multiple crossing over occurs rarely. Estimation of crossing over-frequency from a test cross: No. of recombinant progeny is given by: Frequency of crossing over = ...................................... X 100 Total no. of progenies Factors affecting the recombination frequency
BT0203 Genetics and Cytogenetics The frequency of recombination between two linked genes is affected by several factors. They are (based on the data from Drosophila melanogaster) Distance between genes: The frequency of cross over between two linked genes is associated with the distance between their location in the chromosome. Thus crossing over between two genes would increase with increase in the distance between them. As location of genes on the chromosome is fixed, the frequency of recombination is expected to be constant with little of variation. However, the frequency of recombination obtained between genes may vary due to sampling error or due to other reasons like: Sex: In Drosophila sp. heterogametic sex individuals show relatively lower frequency than the homogametic individuals (XX). Age of female: In general, the frequency of recombination shows progressive decline with the aging in Drosophila. Temperature: In Drosophila, lowest frequency of recombination is observed when the females are cultured at 22 C and increased when temperature is increased Nutrition: The presence of Ca and Mg in food reduces the recombination frequency in Drosophila, while the removal of metallic ions (through the chelating agents present in the diet) increases the recombination frequency. Increase in the recombination frequency is noticed in the larvae starved during certain stages of development. Chemicals: Injection of females with antibiotics like mytomycin D and actinomycin D promotes recombination. Treatment of female- with alkylating agents such as ethylmethane sulphonate (EMS), also show similar effects. Radiation: Recombination frequency was increased when the females of Drosophila and many organisms when irradiated with X-ray. Even the Drosophila male showed recombination when treated with X-ray. Plasmogenes: Certain genes present in Drosophila also affect the recombination frequency. In Bajra, the gene trifton present in the cytoplasm shows reduced recombination frequency. Genotypes: Many genes are known to affect the occurrence as well as the rate of recombination. Some genes affect the chromosome pairing and some other genes affect Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics the synapsis (after pairing). Genes for asynapsis is known in many crops. The C 3G gene of Drosophila (located on chromosome No. 3) prevents the recombination when present in homozygous condition, while it promotes recombination at heterozygous condition. In addition there are evidences to prove that the recombination is also affected by genetic background, or modifying factors. Chromosomal aberrations: In Drosophila, paracentric inversion reduces recombination between genes located within the inverted segment. Translocations also reduce recombination in the vicinity of breaks. Generally these chromosomal aberrations reduce recombinations in the chromosomes in which they are located; they often increase the recombination in other chromosomes present in the same cell. A similar effect of inversion and translocation on recombination is also known in plants. Distance from centromere: Centromeres tend to suppress recombination. Therefore genes located near the centromere show relatively low recombination frequency than those located away from the centromeres. Crossing over frequency and cross over: Crossing over is responsible for the recombination between linked genes. Crossing over takes place during pachytene stage; each chromosome of a bivalent has two chromatids, so that each bivalent has four chromatids (four-strand stage). During the process (crossing over) a segment of one of the chromatid becomes attached with the homologous segment of a non-sister chromatid and vise versa. It is assumed that breaks occur at homologous points followed by reunion of the acentric segments. This produces X like figure at the point of exchange of the chromatic segments is called as 'Chiasma'. As a result of a cross over, two recombinant chromatids (involved in the cross over) called cross over chromatids and two original chromatids (not involved in cross over) are produced. The gametes carrying the recombinant chromatids (which carry the recombination of linked genes) are called cross over types. Similarly the gametes carrying non cross over chromatids which contain only the parental combination of linked genes called non-cross over types. Therefore the frequency of crossing over Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics between two genes can be estimated as the frequency of recombinant progeny from a test cross. This frequency is expressed as percentage and it is the frequency of cross over between the two genes. Relationship between chiasma and crossing over: Chiasma is the X like configuration observed during diplotene or late pachytene, when a homologous chromosome moves away from each other; it appears to be attached with each other through chiasma. Two divergent theories have been proposed to explain the relationship between chiasma and crossing over. They are: plane theory) ii) Chiasmatype theory. (One plane theory) I. Classical Theory: Proposed by Sharp in 1934 also called as two-plane theory. According to this theory, i) a chiasma is formed when chromatid of a chromosome comes to associate with the non-sister chromatid of its homologous chromosomes, ii) chiasma formation is the cause of crossing over, iii) each chiasma does not lead to crossing over and iv) crossing over occurs during diplotene. The available experimental evidences did not support this hypothesis, and it is only a historical significance. II. Chiasmatype Theory: It is proposed by Janssens in 1909 and further expanded by him in 1924. This is also called as one-plane theory. According to this theory, i) chiasmata are produced due to crossing over, ii) crossing over occurs before diplotene, iii) through out the entire bivalent, only sister chromatids are associated with each other (where as in classical theory the association is only between non-sister chromatids to produce chiasmata), iv) each chromatid is the consequence of crossing over event and v) a 1:1 ratio is expected between frequencies of chiasmata and crossing over. Most of the evidences support only the chiasmata type theory. Further, this theory is able to explain several phenomena which are not explained by the classical theory. For example, as per the classical theory three homologous chromosomes are required for a trivalent configuration, which is not possible. But on the other hand as per the chiasmatatype theory, only two chromosomes are required to pair in the different Rex Arunraj Assistant Prof. Department of Genetic Engineering i) Classical theory (Two
BT0203 Genetics and Cytogenetics segments to yield the same trivalent configuration. In maize, there is a close correlation between the relative length of chromosomes determined on the basis of recombination data and those estimated from the number of chiasmata However, the most important evidence against the chiasmatatype theory comes from the male Drosophila where chiasmata are present but there is no crossing over. Most likely the chiasmata in male Drosophila are produced due to simple association between homologous chromosomes, i.e. they are not chiasmata in real sense. However, chiasmata theory is still universally accepted one. Theories on crossing over: Several models have been proposed to explain the molecular mechanism of crossing over and these models may be grouped into two broad classes: 1. 2. Breakage and reunion Copy choice theory
1. Breakage and reunion: This theory was put forth by Darlington in 1932 and explained in simple words as, "after synapsis, break occurs at identical points in one of the two chromatids of each of the two homologues forming bivalent . The segments of two non-sister chromatids reunite to produce cross over chromatids. This is well proved by 1) by isotope labeling that revealed each cross over chromatids consisted of segments from two non sister chromatids involved in crossing over. 2) as a rule, there is some DNA synthesis associated with crossing over and this DNA synthesis most likely involved in the repairing of chromatid breaks. Based on this theory, several models have been proposed and these models are called as hybrid DNA models. AH these hybrid DNA models are the modifications of either the White House model (1963) or the Holiday model (1964). These two models differ in one important aspect i.e., Whitehouse proposed that single strand breaks occurs in the strands having opposite polarity, While according to Holiday it occurs in the strands having the same polarity. Hence, Holiday model is relatively simpler and more attractive.
BT0203 Genetics and Cytogenetics 2.Duplication theory: This theory is mostly based on the mechanism of crossing over proposed by Belling in 1933. He postulated that i) genes present in a chromosome are the first to be replicated ii) they are subsequently connected with each other through the synthesis of the remaining parts (other than genes of the chromosome) and iii) The homologous chromosome are likely to be coiled with each other so that the newly produced copies of genes present in a segment of one chromosome would be adjacent to those of the neighbouring segment of the homologous chromosome . As a result, the new copies of genes present in a segment of one chromosome may sometimes become joined with those of the neighbouring segment of the homologous chromosome, which gives rise to cross over or recombinant chromatids. According to this theory, chromosome replication, or at least replication of the segment involved in crossing over, must occur after synapsis, which is contrary to the known facts, hence this postulate appears to be unrealistic. 3. Copy-choice Model In 1955 Laderberg proposed a modification in Belling's hypothesis to explain the same unusual features of recombination in bacteria. This modification is commonly known as copy-choice theory. According to this theory, during DNA replication, a DNA molecule serves as a template (for the DNA molecule to synthesis new DNA molecule) upto a certain distance after which the homologous DNA molecule is used as the template. Consequently the newly produced DNA molecule is a recombinant. This theory requires two important pre-requisites viz., i) ii) chromosome replication takes place after synapsis and DNA replication is conservative.
But this is contrary to the fact in eukaryotic chromosome replication occurs before synapsis and DNA replication is universally semi-conservative. Therefore the copychoice model is not acceptable, however, in some prokaryotes the DNA replication appears to be copy-choice type.
BT0203 Genetics and Cytogenetics 4. Cross- over theory: According to this theory, an endonuclease produces single-strand nicks at identical points in the two homologous DNA molecules in strands having the same polarity. The two strands of each DNA molecule separate from each other upto certain distance from the point of 'nick'. The free strands now pair with the intact strands of the homologous DNA molecule during the unwinding of chromatids. This produces a "hybrid DNA molecule". The two nicks present in the molecules are sealed by DNA ligase. Then the hybrid DNA molecule undergoes reorientation to form X-shaped figure; one end of this X rotates to 180. Such hybrid DNA molecules have been actually isolated from bacteria and photographed with the help of electron microscope. An endonuclease now induces nicks in the two intact (not cut earlier) strands of the hybrid molecules; this yields two recombinant DNA molecules. Each recombinant molecule has a nick which is finally sealed by DNA ligase. Most of the evidences are in favour of this model since almost all the enzymes and proteins involved in the process are known to occur in bacteria. LNKAGE GROUPS AND LINKAGE MAPPING All the linked genes form a linkage group. The genes that are linked may be represented on a straight line in the same order in which they are normally present in the concerned chromosome. In such representation, the distance between any two neighbouring genes is proportional to the frequency of recombination between them.Such a diagrammatic representation depicting the linked genes and the recombination frequencies between them is known as linkage map or genetic map or chromosome map. Thus for preparing a chromosome map the following two information are required: i) the frequency of recombination between linked genes, ii) the order or sequence of these genes in the chromosomes. As we have already seen the recombination frequency between any two genes can be estimated from appropriate test crosses or F2 progenies. These percentages of recombination frequencies are used as map units for construction of linkage maps. A map unit is defined as that distance in a chromosome which permits one percent of Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics recombination between the linked genes. A map unit is also referred as Morgan (1 centiMorgan = 1 map unit). It should be noted that a map unit is an imaginary distance and it does not represent the actual distance between two linked genes in the chromosome. Therefore a map unit does not have a unit of measurement like A, u., mm etc. Since the map units are the recombination frequencies between two linked genes, they are likely to be influenced by several factors. DETERMINATION OF THE SEQUENCE OF LINKED GENES: The sequence of linked genes can be determined by studying the test crosses for three genes at a time. The data from a test cross involving three genes will provide the information on the order of the three genes in the chromosome as well as their recombination frequencies between them. To begin with a three point test cross involving any two of these three linked genes and a new gene expected to be linked with them is studied to map the new gene. In this manner each new gene is mapped by ordering it in three-point test cross with two already mapped genes. In such studies it is desirable to include only those genes show less than 20% preferably 10% or less recombination with each other to avoid confusion due to double and triple cross overs. If the crossing over between the genes in test cross is more than 20% the linkage maps would not be very reliable. In addition the number of test cross progeny should be sufficiently large to yield reliable recombination frequencies and to avoid the effects of sampling error. The number of linked group in a species is a rule, equal to its gametic chromosome number (n). Each linkage group of a species is to be assigned to a specific chromosome of that species with the help of chromosomal aberrations. In general the relative length of linkage group of a species correspond closely with the relative lengths of the chromosomes in which they are located. These linkage maps provide the information on i) the genes that are linked together and ii) the frequencies of recombination that may be expected between them. Sometimes the Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics distance between any two genes in a linkage group may exceed 50% or even 100 %, but that does not mean that they show recombination beyond 50%, the recombination between two linked genes cannot exceed 50% which is the frequency expected in independent assortment. However, there is a 1:1 correspondence between map distance and the observed recombinant frequency upto 20 map units. But there is a progressive decline in the frequency of observed recombination for every additional map unit distance beyond 20 map units. . DETERMINATION OF GENE SEQUENCE: Now using this data the gene sequence can be obtained by comparing the genotypes of the parental and double cross over types. In a double crossing over simultaneous cross over occurs on both sides of the genes located in the middle. In the figure the gene 'sh' is located between 'C and 'Wx..In this case the double cross over produced will be between 'C and 'Sh' and 'Sh' and 'Wx'. Hence, parental types are CShWx and cshwx and the double cross over types are CshWx and cShwx. So the comparison between parental and double cross over shows that only the gene located in the middle is shifted. This property of the double cross overs is used for determining the sequences of the genes, which are linked. But in the given table, the parental types are CWxSh and cwxsh and the double cross overs are CWxsh and cwxSh, i.e. the allele 'sh' have been shifted their position in the double cross overs as compared to parental types. Therefore the gene 'sh' must be located between 'c' and 'wx'. ALTERNATE PROCEDURE TO FIND THE ORDER OF THE GENES: An alternate procedure for determining the gene frequency involves the comparison of recombination frequencies. If the genes 'c\ 'sh' and 'wx' are placed in the order of c-sh-wx single cross over between 'c' and 'sh 'which yield the cross over gametes viz., Cshwx and cShWx. In addition to the above frequencies between 'c' and 'Sh' and between 'sh' and 'wx', double cross over viz., CshWx and cShwx are also produced due to simultaneous crossing over between genes 'c' and 'sh' and 'sh' and 'wx', therefore these double cross over frequencies should also be included to estimate recombination frequencies between 'c' and 'sh' as well as between 'sh' and 'wx'. Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics Therefore the frequency of recombination between 'C and 'Sh' will be the sum of the frequencies of Cwxsh cWxSh Total ... ... 1.7 1.8 . 3.5 +freq. of Double cross overs
Similarly single cross over between 'sh' and 'wx' will produce the cross over types viz., CShwx and cshWx and the sum of these frequencies will be their recombination between 'sh' and 'wx' i.e. CShwx cshWx Total ... ... ... 9.2 8.9 18.1 + Double cross overs
Now let us consider that crossing over are between 'c' and 'wx' i.e. two genes located on either sides of the gene 'sh'. In such case the crossing over between *c' and 'sh' and 'sh' and 'wx' would produce recombination between 'c' and 'wx'. Therefore the recombination frequency between C and wx = frequency of c and sh + frequency of sh and wx (It should be noted that double cross over does not lead to recombination between 'c' and 'wx' although two events of crossing over occur between them). A map distance of around 90 units is expected to show to 50% recombination, Thus, i) ii) iii) a map distance is the frequency of recombination upto 20 map units linked genes would show independent segregation if they are more than 80 map units apart and maximum recombination observed between two linked gene is 50%.
COEFFICIENT OF INTERFERRENCE: As a rule the observed frequencies of double cross overs are less than the expected values. This is because" the occurrence of crossing over in one region of a chromosome interferes with its occurrence in the neighbouring segment, this is called as interference. It may be expected that the intensity of interference would decrease as the point of Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics second crossing over becomes farther from that of the first one. Therefore the coefficient of coincidence will be lower when the concerned genes are located close to each other. The intensity of interference may be estimated as coefficient of interference = 1 coefficient of coincidence. Interference=%of observed double cross overs / %of expected cross overs
BT0203 Genetics and Cytogenetics Constructing a Genetic Map with Two-Point Testcrosses Genetic maps can be constructed by conducting a series of testcrosses between pairs of genes and examining the recombination frequencies between them. A testcross between two genes is called a two-point testcross or a two-point cross for short. Suppose that we carried out a series of two-point crosses for four genes, a, b, c, and d, and obtained the following recombination frequencies: Gene loci in testcross a and b a and c a and d b and c b and d c and d Recombination frequency (%) 50 50 50 20 10 28
We can begin constructing a genetic map for these genes by considering the recombination frequencies for each pair of genes. The recombination frequency between a and b is 50%, which is the recombination frequency expected with independent assortment. Genes a and b may therefore either be on different chromosomes or be very far apart on the same chromosome; so we will place them in different linkage groups with the understanding that they may or may not be on the same chromosome:
Linkage group 1
Linkage group 2
The recombination frequency between a and c is 50%, indicating that they, too, are in different linkage groups. The recombination frequency between b and c is 20%; so these genes are linked and separated by 20 map units:
Linkage group 1
Linkage group 2
b 20
mu The recombination frequency between a and d is 50%, indicating that these genes belong to different linkage groups, whereas genes b and d are linked, with a recombination frequency of 10%. To decide whether gene d is 10 map units to the left or right of gene b, we must consult the c-to-d distance. If gene d is 10 map units to the left of gene b, then the distance between d and c should be 20 m.u. + 10 m.u. = 30 m.u. This distance will be only approximate because any double crossovers between the two genes will be missed
BT0203 Genetics and Cytogenetics and the recombination frequency will be underestimated. If, on the other hand, gene d lies to the right of gene b, then the distance between gene d and c will be much shorter, approximately 20 m.u. - 10 m.u. = 10 m.u. By examining the recombination frequency between c and d, we can distinguish between these two possibilities. The recombination frequency between c and d is 28%; so gene d must lie to the left of gene b. Notice that the sum of the recombination between d and b (10%) and between b and c (20%) is greater than the recombination between d and c (28%). (This is what was meant by saying that recombination ratesi.e., map unitsare approximately additive.) This discrepancy arises because double crossovers between the two outer genes go undetected, causing an underestimation of the true recombination frequency. The genetic map of these genes is now complete:
Linkage group 1
a
Linkage group 2
d 10 mu
b 20 mu
BT0203 Genetics and Cytogenetics Linkage and Recombination between Three Genes Genetic maps can be constructed from a series of testcrosses for pairs of genes, but this approach is not particularly efficient, because numerous two-point crosses must be carried out to establish the order of the genes and because double crossovers are missed. A more efficient mapping technique is a testcross for three genes (a three-point testcross, or threepoint cross).With a three-point cross, the order of the three genes can be established in a single set of progeny and some double crossovers can usually be detected, providing more accurate map distances.
Consider what happens when crossing over takes place among three hypothetical linked genes. Figure illustrates a pair of homologous chromosomes from an individual that is heterozygous at three loci (AaBbCc). Notice that the genes are in the coupling configuration; that is, all the dominant alleles are on one chromosome ( A----- B----- C ) and all the recessive alleles are on the other chromosome ( a---- b---- c ). Three types of crossover events can take place between these three genes: two types of single crossovers (Figure ) and a double crossover (Figure). In each type of crossover, two of the resulting chromosomes are recombinants and two are nonrecombinants. Notice that, in the recombinant chromosomes resulting from the double crossover, the outer two alleles are the same as in the nonrecombinants, but the middle allele is different. This result provides us with an important clue about the order of the genes. In progeny that result from a double crossover, only the middle allele should differ from the alleles present in the nonrecombinant progeny. Gene Mapping with the Three-Point Testcross
BT0203 Genetics and Cytogenetics To examine gene mapping with a three-point testcross, we will consider three recessive mutations in the fruit fly Drosophila melanogaster. In this species, scarlet eyes (st) are recessive to red eyes (st+), ebony body color (e) is recessive to gray body color ( e+), and spineless (ss)that is, the presence of small bristlesis recessive to normal bristles (ss+). All three mutations are linked and located on the third chromosome. We will refer to these three loci as st, e, and ss, but keep in mind that either recessive alleles (st, e, and ss) or the dominant alleles (st+, e+, and ss+) may be present at each locus. So, when we say that there are 10 m.u. between st and ss, we mean that there are 10 m.u. between the loci at which these mutations occur; we could just as easily say that there are 10 m.u. between st+ and ss+. To map these genes, we need to determine their order on the chromosome and the genetic distances between them. First, we must set up a three-point testcross, a cross between a fly heterozygous at all three loci and a fly homozygous for recessive alleles at all three loci. To produce flies heterozygous for all three loci, we might cross a stock of flies that are homozygous for normal alleles at all three loci with flies that are homozygous for recessive alleles at all three loci:
st+ ss+ st+ ss+
e+ X e+
st st
e e
ss ss
F 1
st+
e+
ss+
The order of the genes has st e ss been arbitrarily assigned because at this point we do not know which is the middle gene. Additionally, the alleles in these heterozygotes are in coupling configuration (because all the wild-type dominant alleles were inherited from one parent and all the recessive mutations from the other parent), although the testcross can also be done with genes in repulsion. In the three-point testcross, we cross the F1 heterozygotes with flies that are homozygous for all three recessive mutations. In many organisms, it makes no difference whether the heterozygous parent in the testcross is male or female (provided that the genes are autosomal) but, in Drosophila, no crossing over takes place in males. Because crossing over in the heterozygous parent is essential for determining recombination frequencies, the heterozygous flies in our testcross must be female. So we mate female F1 flies that are heterozygous for all three traits with male flies that are homozygous for all the recessive traits: The progeny from this listed in each locus, Rex Arunraj Assistant Prof.
st+ ss+ st ss e+ e fema le X st ss st ss mal e e e
produced cross are FIGURE. For two classes of
BT0203 Genetics and Cytogenetics progeny are produced: progeny that are heterozygous, displaying the dominant trait, and progeny that are homozygous, displaying the recessive trait. With two classes of progeny possible for each of the three loci, there will be 2 3 = 8 classes of phenotypes possible in the progeny. In this example, all eight phenotypic classes are present but, in some threepoint crosses, one or more of the phenotypes may be missing if the number of progeny is limited. Nevertheless, the absence of a particular class can provide important information about which combination of traits is least frequent and ultimately the order of the genes, as we will see. To map the genes, we need information about where and how often crossing over has occurred. In the homozygous recessive parent, the two alleles at each locus are the same; and so crossing over will have no effect on the types of gametes produced; with or without crossing over, all gametes from this parent have a chromosome with three recessive alleles ( --st--- e---- ss-- ). In contrast, the heterozygous parent has different alleles on its two chromosomes; so crossing over can be detected. The information that we need for mapping, therefore, comes entirely from the gametes produced by the heterozygous parent. Because chromosomes contributed by the homozygous parent carry only recessive alleles, whatever alleles are present on the chromosome contributed by the heterozygous parent will be expressed in the progeny. As a shortcut, we usually do not write out the complete genotypes of the testcross progeny, listing instead only the alleles expressed in the phenotype (as shown in Figure), which are the alleles inherited from the heterozygous parent. Determining the gene order The first task in mapping the genes is to determine their order on the chromosome. In Figure aove, we arbitrarily listed the loci in the order st, e, ss, but we had no way of knowing which of the three loci was between the other two. We can now identify the middle locus by examining the double-crossover progeny. First, determine which progeny are the nonrecombinants they will be the two most-numerous classes of progeny. (Even if crossing over takes place in every meiosis, the nonrecombinants will comprise at least 50% of the progeny.) Among the progeny of the testcross in Figure, the most numerous are those with all three dominant traits
st+
e+
ss+
and those with all three recessive traits
st
ss
Next, identify the double-crossover progeny. These should always be the two leastnumerous phenotypes, because the probability of a double crossover is always less than the probability of a single crossover. The least-common progeny among those listed in Figure are progeny with red eyes, gray body, and spineless bristles( st+ e+ ss ) and progeny with scarlet eyes, ebony body, and normal bristles ( st e ss+ ); so they are the double-crossover progeny. Three orders of genes are possible: the eye-color locus could be in the middle ( e st ss ), the body-color locus could be in the middle ( st e ss ), or the bristle locus could be in the middle ( st ss e ). To determine which gene is in the middle, we can draw the chromosomes of the heterozygous parent with all three possible gene orders and then see if a double crossover produces the combination of genes observed in the doublecrossover progeny. The three possible gene orders and the types of progeny produced by their double crossovers are: The only gene order that produces chromosomes with alleles for the traits observed in the double crossovers (st+ e+ ss and st e ss+) is the third one, where the locus for bristle shape lies in the middle. Therefore, this order (-- st--- ss--- e-- ) must be the correct sequence of genes on the chromosome. With a little practice, its possible to quickly determine which locus is in the middle without writing out all the gene orders. The phenotypes of the progeny are expressions of the alleles inherited from the heterozygous parent. Recall that, when we looked at the results of double crossovers, only the alleles at the middle locus differed from the nonrecombinants. If we compare the nonrecombinant progeny with double-crossover progeny, they should differ only in alleles of the middle locus. Lets compare the alleles in the double-crossover progeny st+ e+ ss with those in the nonrecombinant progeny st+ e+ ss+ . We see that both have an allele for red eyes ( st+) and both have an allele for gray body (e+), but the nonrecombinants have an allele for normal bristles (ss+), whereas the double crossovers have an allele for spineless bristles (ss). Because the bristle locus is the only one that differs, it must lie in the middle.We would obtain the same results if we compared the other class of double-crossover progeny ( --st ---e ss+ --) with other nonrecombinant progeny ( -- stess-- ). Again the only trait that differs is the one for bristles. Dont forget that the nonrecombinants and the double crossovers should differ only at one locus; if they differ in two loci, the wrong classes of progeny are being compared. Determining the locations of crossovers
BT0203 Genetics and Cytogenetics When we know the correct order of the loci on the chromosome, we should rewrite the phenotypes of the testcross progeny with the loci in the correct order so that we can determine where crossovers have taken place ( FIGURE ). Among the eight classes of progeny, we have already identified two classes as nonrecombinants ( st+ ss+ e+ and st ss e ) and two classes as double crossovers ( st+ ss e+ and st ss+ e ). The other four classes include progeny that resulted from a chromosome that underwent a single crossover: two underwent single crossovers between st and ss, and two underwent single crossovers between ss and e. To determine where the crossovers took place in these progeny, compare the alleles found in the single-crossover progeny with those found in the nonrecombinants, just as we did for the double crossovers. Some of the alleles in the singlecrossover progeny are derived from one of the original (nonrecombinant) chromosomes of the heterozygous parent, but at some place there is a switch (due to crossing over) and the remaining alleles are derived from the homologous nonrecombinant chromosome. The position of the switch indicates where the crossover event took place. For example, consider progeny with chromosome st+ ss e . The first allele (st+) came from the nonrecombinant chromosome st+ ss+ e+ and the other two alleles (ss and e) must have come from the other nonrecombinant chromosome st ss e through crossing over(figure). This same crossover also produces the st ss+ e+ progeny. This same method can be used to determine the location of crossing over in the other two types of singlecrossover progeny. Crossing between ss and e produces st+ ss+ e and st ss e+ chromosomes (figure). Calculating the recombination frequencies Next, we can determine the map distances, which are based on the frequencies of recombination. Recombination frequency is calculated by adding up all of the recombinant progeny, dividing this number by the total number of progeny from the cross, and multiplying the number obtained by 100%. To determine the map distances accurately, we must include all crossovers (both single and double) that take place between two genes. Recombinant progeny that possess a chromosome that underwent crossing over between the eye-color locus ( st) and the bristle locus (ss) include the single crossovers ( st+ / ss e and st / ss+ e+ ) and the two double crossovers ( st+ / ss / e+ and Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics st / ss+ / e ). There are a total of 755 progeny; so the recombination frequency between ss and st is: stss recombination frequency = (50+52+5+ 3)/755 X 100 = 14.6% The distance between the st and ss loci can be expressed as 14.6 m.u. The map distance between the bristle locus (ss) and the body locus (e) is determined in the same manner. The recombinant progeny that possess a crossover between ss and e are the single crossovers st+ ss+ / e and st ss / e+ , and the double crossovers st+ / ss / e+ and st / ss+ / e . The recombination frequency is: sse recombination frequency =
(43+41+5+3)/755 X 100 = 12.2%
Thus, the genetic distance between ss and e is 12.2 m.u. Finally, calculate the genetic distance between the outer two loci, st and e. Add up all the progeny with crossovers between the two loci. These progeny include those with a single crossover between st and ss, those with a single crossover between ss and e, and the double crossovers ( st+ / ss / e+ and st / ss+ / e ). Because the double crossovers have two crossovers between st and e, we must add the double crossovers twice to determine the recombination frequency between these two loci: st e recombination frequency = (50+52+43+41+(2X5)+(2X3)) / 755 X100 = 26.8% Notice that the distances between st and ss (14.6 m.u.) and between ss and e (12.2 m.u.) add up to the distance between st and e (26.8 m.u.). We can now use the map distances to draw a map of the three genes on the chromosome.
26.8 mu
st
ss
12.2 mu Interference 14.6 mu The degree to which one crossover interferes with additional crossovers in the same region is termed the interference.
Coefficient of coincidence, which is the ratio of observed double crossovers to expected double crossovers.
Unit-IV Mutation
A Gene mutation is abrupt inheritable qualitative or quantitative change in the genetic material of an organism. Since in most organisms genes are segments of DNA molecule, so a mutation can be regarded as a change in the DNA sequence which is reflected in the change of sequence of corresponding RNA or protein molecules. Such a change may involve only one base/base pair or more than one base pair of DNA. Mutation occurs in a random manner, ie.., they are not directed according to the requirements of the organism. Most mutations occur spontaneously by the environmental effect; however, they can be induced in the laboratory either by radiations, physical factors or chemicals (called mutagens.) A unicellular organism is more subjected to environmental onslaughts since it is at the same time a somatic or germ cell. In multicellular organisms the germ cells are distinct cells, and are relatively protected from the environment. Mutation has a significant role to play in the origin of species or evolution. HISTORICAL BACKGROUND Hugo de vries was the first hybridist who used the term mutation to describe the heritable phenotypic changes of the evening primrose, Oenothera lamarckiana. Many mutations described by de vries in Oenothera lamarckiana, are known to be due to variation in chromosome number or ploidy and chromosomal aberrations (viz gross mutations). The first scientific study of mutations was started in 1910, when Morgan and his work was in fruit fly, Drosophila melanogaster and reported white eyed male individuals among red eyed male individuals. The discovery of white eyed mutants in Drosophila was followed by Morgan and his co-workers and other geneticists. Consequently about 500 mutants of Drosophila have been reported by geneticists all over the world. Mutants occur frequently in the nature and have been reported in many organisms, e.g., drosophila, mice and other rodents, rats, rabbits, guinea pigs and man. In the drosophila, mutation causes white and pink eyes, black and yellow body colours, and vestigial wings. Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics In rodents the mutations are responsible for black, white and brown coats. In man the mutations cause variation in hair colour, eye colour, skin pigmentation and several somatic malformations. Various genetical diseases of human beings such as haemophilia, colour blindness, phenylketonuria etc., form other examples for mutation in human beings. How does a mutation act? Any change in sequence of nucleotides in the DNA will result in the corresponding change in the nucleotide sequence of mRNA. This may result in alignment of different RNA molecules on mRNA (during protein synthesis). Thus the amino acid sequence and hence the structure and properties of the enzyme formed will be changed. This defective enzyme structural protein may adversely affect the trait controlled by the protein. In consequence a mutant phenotype makes its expression. KINDS OF MUTATIONS 1. Classification of Mutation According to Type of Cells According to their occurrence in somatic and germinal cells following types of mutations have been classified: Somatic Mutations. The mutations occurring in non-reproductive body cells are known as somatic mutations. The genetical and evolutionary consequences body cells are insignificant, since only single cells and their daughter cells are involved. If, however, a somatic mutation occurs early during embryonic life, the mutant cells may constitute a large proportion of body cells and the animal body may be a mosaic for different type of cells. Somatic mutations have been often related with malignant (cancerous) growth. Examples of somatic mutation have been reported in Oenothera lamarckiana (Hugo de Vries) and several other cases including man. In man somatic mutation causes several fatal diseases such as paraoxysmal nocturnal hemoglobinura, unilateral retinoblastoma and heterochromia of the iris.
BT0203 Genetics and Cytogenetics Gametic mutations. The mutations occurring in gamete cells (e.g., sperms and Ova) are called gametic mutations. Such mutations are heritable and of immense genetical significance. The gametic mutations only form the base for the natural selection. 2. Classification of Mutations According to the Size and Quality According to size following two types of mutations have been recognized: Point mutation. When heritable alterations occur in a very small segment of DNA molecule, i.e., a single nucleotide or nucleotide pair, then this type of mutations are called point mutations. The point mutations may occur due to following types of subnucleotide change in the DNA and RNA. 1. Deletion mutations. The point mutation which is caused due to loss or deletion of some portion (single nucleotide pair) in a triplet codon of a cistron or gene is called deletion mutation. Deletion mutations have been frequently reported in some bacteriophages. 2. Insertion or addition mutation. The point mutations which occur due to addition of one or more extra nucleotides to a gene or cistron are called insertion mutations. The insertion mutations can be artificially induced by certain chemical substances called mutagens such as acridine dye and proflavin. A proflavin molecule, it is believed, insert between two successive bases of a DNA strand, thereby stretching the strand lengthwise. At replication, this situation would allow the insertion of an extra nucleotide in the complementary chain at the position occupied by the proflavin molecule. The mutations which arise from the insertion or deletion of individual nucleotides and cause the rest of the message downstream of the mutation to be read out phase, are called frameshift mutations. They result in the production of an incorrect, hence, inactive protein, due to which the death of the cell may occur. 3. Substitution mutation. A point mutation in which a nucleotide of a triplet is Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics replaced by another nucleotide, is called substitution mutation. The substitution mutation affects only a particular triplet codon. Such an altered code word (triplet codon) may designate a different amino acid and may result in the production of a protein with a single amino acid substitution. The substitution mutations alter the phenotype of an organism variously are of great genetical significance. They may be of following: (i) Transition. When a purine (e.g., ademine) base of a triplet codon of a cistron is substituted by another purine base (e.g., guanine) or a pyrimidine (e.g., thymine) is substituted by another pyrimidine base, (e.g., cytosine) then such kind of substitution is called transition. The transitional substitution mutations occur due to tuatomerization. Tautomerization In a DNA molecule, normally, the purine, adenine (A) is linked to the pyrimidine, thymine (T), by two hydrogen bonds, while the purine guanine (G) is linked to the pyrimidine, cytosine (C) by three hydrogen bonds. Besides the common molecular configurations, each DNA base may have some altered uncommon molecular configuration. Such uncommon forms of DNA bases are generated by single proton shifts and are called rare states of tautomers. A tautomeric shift is believed to occur when the amino (NH 2) form of adenine is changed to an imino (NH) form. Similarly, a tautomeric shift may occur in thymine changing it form the keto (C = 0) form to the rare enol (COH) form. When a base occurs in its rare or tatuomeric state, it cannot be linked to its normal partner. However, a purine, such as adenine can in its rare state forms a bond with cytosine (besides thymine), provided the cytosine is in its normal state. . 3. Transversion. The substitution mutation when involves the substitution or replacement of a purine with a pyrimidines of vice versa then type of substitution Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics mutation is called transversion mutation. The existence of transversion mutation was first of all postulated by E. Freese in 1959. We have still poor information about the mechanism of induction, identification and charectirization of trnsversion mutations. Moreover, it is extremely difficult to recognize transversion mutations genetically. However, they can be recognized only by amino acid substitution in proteins. Effects of physical Conditions on Nucleotide Sequence High temperature and low pH value are known to affect depurination or loss of purine bases. The removal of a purine from a strand of DNA leaves a gap at that. At the time of replication, it would be possible for any of the four bases to insert in the complementary strand would contain a transversion. B. Multiple mutations or gross mutations. When changes involving more than one nucleotide pair, or entire gene, then such mutations are called gross mutations, the gross mutations occur due to rearrangements of genes within the genome and may be of the following types: 1. The rearrangements of genes may occur within a gene. Two mutations within the same functional gene can produce different effects depending on gene whether they occur in the cis or trans position. 2. The rearrangements of gene may occur in number of genes per chromosome, they may cause different types of phenotypic effects over the organisms. 3. Due to movement of a gene locus new type of phenotypes may be created, especially when the gene loci may take place due to following method: (i) Translocation: Movement of a gene may take place to a non-homologous chromosome and this is known as translocation. (ii) Inversion. The movement of a gene within the same chromosome is called inversion. 3. Classification of Mutation According to the Origin According to the mode of origin, following two kinds of mutations have been recognized. Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics (1) Spontaneous mutations. The spontaneous mutations occur suddenly in the nature and their origin is unknown. They are also called background mutation and have been reported in many organisms such as, Oenothera, maize, bread molds, microorganisms (bacteria and viruses), Drosophila, mice, man, etc.
(2) Induced mutations. Besides naturally occurring spontaneous mutations, the mutations can be induced artificially in the living organisms by exposing them to abnormal environment such as radiation, certain physical conditions (i.e., temperature) and chemicals. The substances or agents which induce artificial mutations are called mutagens or mutagenic agents. Mutagenic agents. The mutagenic agents are of the following kinds: A. Radiations. The radiations which are important in mutagenesis are of two categories: one type is ionizing radiations such as X-rays and gamma rays; alpha and beta rays; electrons, neutrons, protons and other fast moving particles. The second type is non-ionizing radiations such as ultraviolet and visible light. Both types of radiations induce mutations by following methods: (i) Ionizing radiations as mutagens. Relatively little is known about the mechanism by which ionizing radiations cause mutation. As, familiar that matter composed of atoms and atoms, in their turn, are made up of a positively charged atomic nucleus (with neutrons, protons) and a surrounding constellation of negatively charged electrons. The charges of atomic particles remain so balanced that normal is electrically neutral. When ionizing radiations pass through matter, they dissipate their energy in part through the ejection of electrons from the outer shell of atoms and the loss of these balancing, negatively charged particles (electrons) leaves atoms which are no longer neutral but are positively charged. The positively-charged atom is called ion. The ejected electrons move at high speed, knock other electrons free from their respective atoms and when their energy is dissipated, become attach to other atoms and convert the ion into negatively charged ions. To achieve their stable configuration (i.e., neutral charge) ions undergo many chemical
BT0203 Genetics and Cytogenetics reactions and during these chemical reactions ionizing radiation is thought to cause mutation. Further, ionizing radiations cause breaks in poly-sugar phosphate backbone of DNA and, thus, causing chromosomal mutations such as break, deletion, addition, inversion and translocation. During breakage of DNA molecule due to ionizing radiation the active role of oxygen is predicted. Because, oxygen is important in the formation of H 2O2 and HO2 in irradiated water and these products may induce breaks in DNA molecule. (ii) Non-ionized radiations as mutagens: The ultraviolet (UV) light is a non-ionizing radiation which may cause mutation. The most effective wave length of ultraviolet for inducing mutations is about 2,600 Ao. This is a wave length that is best absorbed by DNA and a wave length at which proteins absorb little energy. When a substance absorbs sufficient energy from the ultraviolet light, some of their electrons are raised to higher energy levels, a state called excitation. The excited molecule becomes reactive and mutated and is called photoproducts. Dimerization: The ultraviolet radiation produces several effects on DNA, one being the formation of chemical bonds between two adjacent pyrimidines molecules in a polynucleotide and particularly, between adjacent thymine residues. As the two thymine residues associate, or dimerize to form a dimer, their position in the DNA helix becomes so displaced that they can no longer from hydrogen bonds with the opposing purines and thus regularity of the helix becomes resorted. Thus, dimerization interferes with the proper base pairing of thymine with adenine, may result in thymines pairing with guanine. This will produce a T-A to C-G transition. B.Temperature as mutagen. The rate of all chemical reactions is influenced by temperature. It is reported that the rate of mutation is increased due to increase in temperature. For example, an increase of 10o C temperature increases the mutation rate two or three fold. Temperature probably affects both thermal stability of DNA and the rate of reaction of other substances with DNA. Rex Arunraj Assistant Prof. Department of Genetic Engineering
C. Chemical mutagens. Many chemical substances have been responsible to increase the mutability of genes. The ability of chemicals to induce mutation was first of all demonstrated by Auerbach and Robson in 1947 using mustard gas and related compounds as the nitrogen and sulphur mustards, mustard oil and chloracetone in experiments with male Drosophilae melanogaster. Since then many chemical compound which are ordinarily considered to be non-toxic have been found to be mutagenic in certain specific situations. Any chemical substance that affects the chemical environment of chromosomes is likely to influence, at least indirectly, the stability of DNA and its ability to replicate without error. A chemical mutation can cause mutation only when it enters in the nucleus of the cell. It can affect the chromosomal DNA by following two ways: (1) Direct gene change. Certain chemical mutagens affect DNA directly. They affect the constituents of DNA only when DNA is not replicating. For example, nitrous acid converts adenine into hypoxanthine and cytosine to uracil by deamination. Like the nitrous acid, nitrogen mustard, formaldehyde, epoxides, dimethyl and diethyl sulphonate, methyl and ethyl methanesulphonate (MMS and EMS) and nitrosoguadine (NG) also have direct mutagenic effect on the DNA molecule. (2) Copy error. Certain chemical compounds, called base analogues (e.g., 5bromouracil, 2-aminopurine, etc.)Closely resemble with certain DNA bases and are, therefore, act as mutagens. During analogues such as urethane triazine, caffeine (in coffee, tea and soft drinks), phenol and carcinogens, chloride is mutagenic for many organisms, as they are the compounds which bind calcium and, thus, interfere with the integrity of the chromosome structure. Effect of Chemical Mutagens on Nucleotide Sequence (a) Alteration in Resting Nucleic Acid Deamination: Some chemical substances such as nitrous acid causes transitional mutation due to oxidative deamination of DNA base, where the amino group is replaced Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics by hydroxyl (OH) group by the chemical mutagen. Thus, adenine is deaminated into hypoxanthine by nitrous acid. Similarly, deamination converts cytosine to uracil, which has pairing properties similar to thymine and in such a case G: C pair would be changed into A: T pair. Alkylating agents: Some alkylating agents carry one, two or more alkyl groups in a reactive form and act as strong mutagens. Example of some most extensively studied alkylating agents include diethylsulphate (DES), dimethyl sulphate (DMS), methyl methane sulphonate (MMS), ethylethane sulphonate (EMS). These mutagens produce mutations in the following ways: (1) They add ethyl methyl groups to guanine. This makes guanine the base analogue to adenine. (2) They remove the alkylated guanine. This is known as depurination. The loss of the base produces gaps in the DNA chain which may be filled with a wrong base, thus, producing mutation. (3) The gap may also produce a deletion, causing mutation. (b) Alteration during Replication of Nucleic Acid 1. Base analogues. Certain chemical substances have molecular structure similar to the usual DNA bases that, if they are available, such analogues may be incorporated into a replicating DNA stand. For example, 5-bromouracil (5BU) or its nucleoside 5bromodeoxyuridine (5-BUdR) in its usual (keto) form is a structural analogue of thymine (5-methyl uracil) and it will substitute for thymine. Thus, an A-T pair becomes and remains A-BU. There is some in vitro evidence to indicate the BU immediately adjacent to an adenine in one of DNA strands causes the latter to pair with guanine. But, in its rare (enol) state, 5BU behaves similar to the tautomer of thymine and pairs with guanine. This converts A: T to G: C. 2-Aminopurine (2-AP) is another base analogue which is relatively undifferentiated purine that apparently can pair with cytosine and thymine. It is thought that 2-AP acts by switching pyrimidines: for example, it may be incorporated opposite thymine during Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics one round of replication and then pair with a cytosine at the next round to produce an AT GC transition. 2. Inhibition of precursors of nucleic acid: There are some mutagents which interfere with the synthesis of nitrogen bases of nucleic acids such as purines or pyrimidines. Often lack of one base either causes breaks or causes pairing mistakes. For example, azaserine (a potent alkylating agent) is an inhibitor of pyrimidine synthesis. However, urethane induced chromosome breaks are inhibited by thymine
4. Classification of Mutation According to the Direction According to their mode of direction following types of mutations have been recognized: (A) Forward mutations. In an organism when mutations create change from wild type to abnormal phenotype, then that type of mutations are known as forward mutations. Most mutations are forward type. (B) Reverse or back mutations. The forward mutations are often corrected by error correcting mechanism, so that an abnormal phenotype changes into wild type phenotype. They may be of the following types: (i) Single site mutation. Some reverse mutations change only one nucleotide in the gene and are called single site mutations. For example, due to forward mutation the adenine is changed into guanine and backward mutation change guanine into adenine: Forward Adenine reverse Guanine Adenine
(ii) Mutation suppressor. When a mutation occurs at a different site from the site where already primary mutation occurred and that mutated gene reverse the effects of primarily mutated gene, then such (secondary) mutations are called mutation suppressors. They may be of following types: Rex Arunraj Assistant Prof. Department of Genetic Engineering
(a) Extragenic suppressor. The extragenic suppressor mutation occurs in a different gene from that of the mutant gene. In E.coli, a gene called rec A (rec for recombination) is known which is necessary for recombination and is found to repair ultraviolet-induced thymine dimmers of a gene by a process called post replication recombinational repair . (b) Intergenic suppressor. The intergenic suppessor mutation occurs in a different nucleotide within the same gene and shifts the reading frame back into register. (c) Photoreactivation. In photoreactivation type reverse mutation reversal of ultraviolet induced thymine dimers takes place by specific enzymes in the presence of visible light waves. During ultraviolet radiation a particular enzyme is selectively bound to the bacterial DNA. During photoreactivation the enzyme is activated by visible light and that cleaves the pyrimidine or purine dimers into monomers and restores their original forms. (d) Excision repair or Dark reactivation. In an ultraviolet (UV) induced mutations, the reverse mutation may also occur in the absence of light. According to Howard Flanders and Boyce (1964) dark reactivation includes following stages: (i) An enzyme possibly endonuclease makes a cut in the polynucleotide strand, on either side of the dimer which may be formed due to ultraviolet radiation and excises a short, single strand segment of the DNA. (ii) Another enzyme, possibly exonuclease widens the gap produced by the action of the endonuclease. (iii) DNA polymerase resynthesizes the missing segment, using the remaining opposite strand as a template; and (iv) the final gap is closed by some enzymatic rejoining process, (i.e., DNA ligase). 5. Classification of mutation According to Magnitude of Phenotypic Effect According to their phenotypic effects following kinds of mutations may occur: 1. Dominant mutations. The mutations which have dominant phenotypic expression are called dominant mutations. For example, in man the mutation disease aniridia (absence of iris of eyes) occurs due to dominant mutant gene. Rex Arunraj Assistant Prof. Department of Genetic Engineering
2. Recessive mutations. Most types of mutations are recessive in nature and so they are not expressed phenotypically immediately. The phenotypic effects of mutations of a recessive gene are seen only after one or more generations, when the mutant gene is able to recombine with another similar recessive gene. 3. Isoalleles. Some mutations alter the phenotype of an organism so slightly that they can be detected only by special techniques. Mutant genes that give slightly modified phenotypes are called isoalleles. They produce identical phenotypes in homozygous or heterozygous combinations. 4. Lethal mutations. According to their effects on the phenotype mutations may be classified as lethals, subvitals and supervitals. Lethal mutations result in the death of the cells or organisms in which they occur. Subvital mutations reduce the chances of survival of the organism in which they occur. Supervital mutations, in contrast, cause the improvement of biological fitness under certain conditions. 6. Classification of Mutation According to Consequent Change in Amino Acid Sequence. 1. Missence mutations. They change the meaning of a codon, changing one amino acid into another. 2. Temperature sensitive mutations or Ts mutations. If the substitution produces a protein that is active at one temperature (typically 30 o C) and inactive at a higher temperature (usually 40- 42o C). 3. Nonsense or chain termination mutations. They arise when a codon for an amino acid is mutated into a termination codon (UAG, UAA or UGA), resulting in the production of a shorter protein.
BT0203 Genetics and Cytogenetics Since, temperature-sensitive and chain termination mutations exhibit the mutant phenotype only under certain conditions, they are called conditional mutations; they are the most versatile and useful mutations. 4. Silent mutations. They change nucleotide but not the amino acid sequence because they affect the third position of the codon, which is usually less important in coding. This is a silent mutation because it leaves the protein sequence unchanged. 7. Classification of Mutation According to the types of Chromosomes. According to the types of chromosomes, the mutations may be of following two kinds: 1. Autosomal mutations. This type of mutation occurs in autosomal chromosomes. 2. Sex chromosomal mutations. This type of mutation occurs in sex chromosomes. Mutation rate The frequency with which genes mutate spontaneously is called mutation rate. Most genes are relatively stable and mutation is a rate. The great majority of genes have mutation rate of 1X10-5 to 1 X 10-5, viz, one gamete in 100,000 to one gamete in million would contain a mutation at a given locus. Mutations occur much more frequently in certain regions of the gene than in others. The favoured regions are called hot spots.
BT0203 Genetics and Cytogenetics CHROMOSOME ABERRATIONS Changes in Structure of Chromosomes The changes in the genome involving chromosome parts, whole chromosomes, or whole chromosome sets are called chromosome aberrations or chromosome mutations. Chromosome mutations have proved to be of great significance in applied biologyagriculture (including horticulture), animal husbandry and medicine. Chromosome mutations are inherited once they occur and are of the following types: A. Structural changes in chromosomes: 1. Changes in number of genes (a) (b) 2. (a) inversion (b) translocation. B. Changes in number of chromosomes: 1. 2. Loss, or gain, of a part of the chromosome set (aneuploidy) Loss, or gain, of whole chromosome set (euploidy) (a) (b) Loss of an entire set of chromosomes (haploidy) Addition of one or more sets of chromosomes (polyploidy) Exchange of parts between chromosomes of different pairs: Loss: deletion Addition: Duplication Rotation of as group of genes 180 within one chromosome:
Changes in gene arrangement:
Both types of changes (structural and numerical) in chromosomes can be detected not only with a microscope (i.e., cytologically) but also by standard genetic analysis. This gave birth to a hybrid science, called Cytogenetics which attempts to correlate cellular events, especially those of chromosomes, with genetic phenomena. A. Structural Changes in Chromosomes For better understanding of the abnormalities of chromosome structure, let us consider two important features of chromosome behaviour : (1) During prophase I of meiosis, Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics homologous regions of chromosomes show a great affinity for pairing and they often go through considerable contortions in order to pair. This property results in many curious structures observed in cells containing one normal chromosome set plus an aberrant set. (2) structural changes usually involve chromosome breakage; the broken chromosome ends are highly reactive or stickly, showing strong tendency to join with broken ends. Types of Structural Changes in Chromosome Structural changes in chromosome may be of following types: 1. deficiency or deletion which involves loss of a broken part of a chromosome; 2. duplication involves addition of a part of chromosome (i.e., broken segment becomes attached to a homolog which, thus, bears one block of genes in duplicate); 3. inversion in which broken segment reattached to original chromosome in reverse order, and 4. translocation in which the broken segment becomes attached to a non-homologous chromosome resulting in new linkage relations. Further, structural abnormalities can occur in both homologous chromosomes of a pair in only one of them. When both homologous chromosomes are involved, these are called structural homozygotes, e.g., deletion homozygote, duplication homozygote, etc. When only one homologous chromosome is involved, it is called structural heterozygote. 1. Deletion (or Deficiency) The simplest result of breakage is the loss of a chromosome. Portions of chromosomes without a centromore (called acentric fragments) lag in anaphase movement and are lost from reorganizing nuclei or digested by nucleases. Such loss of a portion of a chromosome (and of some genes) is called deletion. The chromosomes with deletions can never revert to a normal condition. If gametes arise from the cells having a deleted chromosome, this deletion is transmitted to the next generation. Further, a deletion can be terminal or intercalary (insterstitial). In terminal deletion a terminal section of a chromosome is absent and it is resulted by only one break. While in the intercalary deletion, an intermediate section or portion of chromosome is lost and it is caused by two Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics breaks one on either end of the deleted region. Thus, in the latter case, the chromosome is broken into three pieces, the middle one of which is lost and the remaining two pieces get joined again. In general, if a homozygous deletion is made, it is lethal. Even individuals heterozygous for deletion (deletion in one of the homologous chromosomes) may not survive. However, smaller deletion in heterozygous condition can be tolerated by the organisms. If meiotic chromosomes in such heterozygotes are examined, the region of deletion can be detected by the failure of the corresponding segment on the normal chromosome to pair properly; so a deletion-loop results. The cytological studies of pairing between normal and deleted chromosomes have helped a lot in finding out the relative position of genes in chromosomes.
a b c d e f a b c d e f c d e f g h g h g h g h g h
a b c d e f a b e f
TERMINAL DELETION Genetical effects of deletion.
INTERCALARY DELETION
Deletion of some chromosome regions produce their own unique phenotypes. A good example of this is a dominant notch wing mutation in Drosophila. In fact, this is a small deletion and acts as a recessive lethal in this regard. Further, in the presence of a deletion, a recessive allele of the normal homologous chromosome will behave like a dominant allele, i.e., it will be phenotypically expressed, this phenomenon is called pseudodominance. The phenomenon of pseudodominance exhibited by deficiency heterozygotes has been utilized for the location of genes on specific chromosomes and in preparing cytological maps in Drosophila, maize, bacteriophage and other organisms. Such cytological maps are often used to verify the genetic maps (based on linkage analysis) of these organisms. Rex Arunraj Assistant Prof. Department of Genetic Engineering
Examples of pseudodominance (deletion) Human babies missing a portion of the short arm of chromosome 5 (autosome) have a distinctive cat-like cry; hence, the French name cridu chat (cry of the cat) syndrome (first described by Lejeune et al., 1963). They are also mentally retarded (IQ below 20), have malformation in the larynx, moon faces, saddle noses, small mandibles (micrognathia), malformed low-set ears and microcephally (small head). 2. Duplication The presence of a part of a chromosome in excess of the normal complement is known as duplication. Thus, due to duplication some genes are present in a cell in more than two does. If duplication is present only on one of two homologous chromosomes, at meiosis the chromosome bearing the duplicated segment forms a loop to maximize the juxtaposition (during pairing) of homologous regions. Extra segments in a chromosome may arise in a varsity of ways such as follows: 1. Tandem duplication. In this case the duplicated regions are situated just by the side of the normal corresponding section of the chromosome and the sequences of genes are the same in normal and duplicated region. For example, if the sequence of genes in a chromosome is ABC. DEFGH (The full stop depicts the Centro mere) and if the chromosomal segment containing the genes DEF is duplicated, the sequence of genes in tandem duplication will be ABC. DEF DEFGH. 2. Reverse tandem duplication. Here, the sequence of genes in the duplicated region of a chromosome is just the reverse of a normal sequence. In the above mentioned example, therefore, the sequence of genes due to reverse tandem duplication will be ABC. DEF FEDGF. 3. Displaced duplication. In this case the duplicated region is not situated adjacent to the normal section. Depending on whether the duplicated portion is on the same side of the Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics centromere as the original section or on the other side, the displaced duplication can be termed either homobranchial or heterobranchial. Example. Homobranchial duplication =ABC.DEFG. DEFH Heterobranchial duplication = A.DEFB.C.DEFGH
4. Transposed duplication. Here, the duplicated portion of chromosome becomes attached to a non-homologous chromosome. For example, if ABC.DEFGH and LMNOPQ.RST represent the gene sequences of two nonhomologous chromosomes, a transposed duplication will result into chromosomes with gene sequence ABC.GH and LMN DEF.OPQ.RST. Such a transposed duplication may be either interstitial (e.g., LMN. DEFOPQ.RST) or terminal (i.e., LMN OPQ.RST DEF). 5. Extra-chromosomal duplication. In the presence of centromere the duplicated part of a chromosome act independent chromosome. Genetical effects of duplication. Due to duplication, there occur unequal crossing over which results in deletion and reduplication which produce distinct phenotypes as shown by the following examples : Duplications of Drosophila lead to following phenotypic effects : (1) a reverse repeat in chromosome 4 causes eyeless dominant (Ey); (2) a tandem duplication in chromosome 3 causes confluens (Co) resulting in thickened veins, and (3) another duplication causes hairy wing (Hw) Genetic redundancy, of which duplication is one type, may protect the organism from the effects of a deleterious recessive gene or from an otherwise lethal deletion. 3. Inversion Inversion involves a rotation of a part of a chromosome or a set of genes by 180 on its own axis. It essentially involves occurrence of breakage and reunion. The net result of inversion is neither a gain nor a loss in the genetic material but simply a rearrangement of Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics the gene sequence. An inversion can occur in the following way: suppose that the normal order of segments within a chromosome is 1-2-3-4-5-6; breaks occur in regions 2-3 and 5-6 and broken piece is reinserted in reverse order. This results in an inverted chromosome having segments 1-2-5-4-3-6. An inversion heterozygote has one chromosome in the inverted order and its homologue in the normal order. The location of the inverted segment can be detected cytologically in the meiotic nuclei of such heterozygotes by the presence of an inversion loop in the paired homologs. The location of the centromere relative to inverted segment determines the genetic behaviour of the chromosomes. If the centromere is not included in the inversion it is called paracentric inversion and when inversion includes the centromere it is called pericentric inversion. Homologous chromosomes, with identical in meiosis. However, crossing over in inversion heterozygotes produce deletions, duplications and other curious configurations. Advantage of inversions. Fertility of inversion homozygotes and sterility of inversion heterozygotes lead to establishment of two group (or varieties) which are mutually fertile but do not breed well with the rest of the species. Both varieties evolve in different directions and later become reproductively isolated species. There is plenty of cytological evidence to prove that such evolutionary mechanisms have and are operating in Drosophila and a number of other orgasisms. 4. Translocation The shifting or transfer of a part of a chromosome or a set of genes to a nonhomologous one, is called translocation. There is no addition or loss of genes during translocations, only a rearrangement (i.e., change in the sequence and position of a gene). Translocations may be of following three types. 1. Simple translocation. They involve a single break in a chromosome. The broken piece gets attached to one end of a nonhomologous chromosome. 2. Shift translocation. In this type of translocation, the broken segment of one chromosome gets inserted interstitially in a nonhomologous chromosome. Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics 3. Reciprocal translocations. In this case, a segment from one chromosome is exchanged with a segment from another nonhomologous one, so that in reality two translocation chromosomes are simultaneously achieved. Outcomes of reciprocal translocation. The exchange of chromosome parts between nonhomologous chromosomes creates new linkage relationships. Such translocations also drastically change the size of a chromosome as well as the position of its centromere. For example, a large metacentric chromosome is shortened by one-half in length to an acrocentric one, where as the small chromosome becomes a large one. Two types of translocations have been recognized: homozygous and heterozygous. The translocation homozygotes may have normal meiosis and in fact, are difficult to detect cytologically unless morphologically dissimilar chromosomes are involved, or banding patterns differ markedly. The translocation heterozygotes produce both translocated and normal chromosomes and exhibit characteristic cytological and genetical effects. Thus, translocation heterozygotes are marked by considerable degree of meiotic irregularity. B.Changes in number of chromosomes (ploidy) Polyploidy Any organism with more than more than two genomes (2x) is called a polyploidy. Many plant genera include species whose chromosome numbers constitute a euploid series. For example the rose genus Rosa includes species with the somatic numbers 14, 21, 28, 35 and 56. These numbers are the multiples of 7. Therefore, this is a euploid series of the basic monoploid number 7, which gives diploid, triploid, tetraploid, pentaploid, hexaploid and octaploid species. Except diploids, rest of these belongs to polyploidy category. Ploidy levels higher than tetraploid are not commonly encountered in natural populations, but our most important crops and ornamental flowers are polyploids, e.g., wheat (hexaploid 6x), strawberries (octaploid, 8x), many commercial fruits and ornamental plants. Generally, polyploidy is common in plants (more common in monocots) but rare in animals. Types of polyploidy Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics These are the three different kinds of polyploids: (i) autopolyploids, (ii) allopolyploids, (iii) autoallopolyploids. (a) Autopolyploids The autopolyploids are those, polyploids, which consist of same basic set of chromosomes multiplied. For example, if a diploid species has two similar sets of chromosomes or genomes (AA), an autotriploid will have three similar genomes (AAA), and an autotetraploid, will have four such genomes (AAAA). (i) Origin and production of autopolyploids The autopolyploids may occur in nature or may be produced artificially. When they are found in nature, their autopolyploidy is deduced by their meiotic behaviour. Some of common examples of autotriploid crop plants, which are mainly produced by artificial methods, are seedless varities of watermelons, sugar beet, tomato, grapes and banana. Similarly, many important crop plants include autotetraploids such as rye (Secale cereale), corn (Zea mays), red clover (Trifolium pretense), berseem (Trifolium alexandrium), marigolds (Tagetes), snapdragons (Antirrhinum), Phlops, grapes, apples, Oenothera lamarckiana (which was recognized as mutation by Hugo de vries). Induced autopolyploidy The autopolyploidy have been induced in many plant and animal cells by artificial means such as chemical (e.g., chloral hydrate, colchicines, sulphanil amide, mercury chloride, hexachlocyclohexane, etc,), radioactive substances, e.g., radium and X-ray) and temperature shocks. These inducers usually disturb the mitotic spindle and cause nonsegregation of already duplicated chromosomes, during cell divisions. Colchicine Colchicine is a drug (i.e., an alkaloid obtained from the corms of plants- Colchicum autmunale and C.luteum) and its aqueous solution is found to prevent the formation and organization of spindle fibres, so the metaphase chromosomes of the affected cells (called C-metaphase or colchicine metaphase) do not move to a metaphase plate and remain Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics scattered in the cytoplasm. Even the process of cytokinesis is prevented by colchicines and with duplications of chromosomes the number goes on increasing. As colchicine interferes with spindle formation, its effects are limited to divided and meristematic cells. (ii) Effects of autopolyploidy Autopolyploidy results in gigantism of plant cells, i.e., leaves, flowers and fruits of an polyploid are larger in size than a diploid plant. For example, the size of lower epidermis of leaf of a tetraploid Saxifraga pencylvanica was found greater than the diploids. Some of the significant effects of autopolyploidy are as follows : (1) With the increase of cell size, the water content increases which leads to a decrease in osmotic pressure. This results into loss of resistance against frost, etc, (2) Due to lower rate of cell division, the plants growth rate in decreases. This leads to a decrease in auxin supply and a decrease in respiration. (3) Due to slow growth rate, the time of blooming of an autopolyploid is delayed. (4) At higher ploidy level, such as autooctoploids, the adverse effects become highly pronounced and lead to the death of the plants. Polyploid varieties with an even number of genomes (e.g., tetraploids) are often fully fertile whereas those with an odd number (e.g., triploids) are highly sterline. Uses of induced polyploidy Since in the induced polyploids, the fertility level and seed set are low, so seedless fruits can be produced by using triploids as in case of seedless watermelons which were produced by using triploids as in case of seedless watermelon which were produced by a Japanese scientist, Dr. Hitoshi Kihara. These triploids are obtained from seeds by colchicines treatment. Allopolyploids When the polyploidy results due the doubling of chromosome number in a F 1 hybrid which is derived from two distinctly different species, then it is called allopolyploidy and the resultant species is called an allopolyploid. Let A represent a set of chromosomes (genome) in species X; and let B represent another genome in a species Y. The F1 hybrids of these species then would have one A genome and another B genome. The Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics doubling of chromosomes in the F1 hybrids will give rise to allotetraploids with two A and B genomes. Raphanobrassica is a classical example of allopolyploidy or amphipolyploidy. In 1927, a Russian geneticist, G.D.Karpechenko performed a cross between radish (Raphanus sativum, 2n = 18) and cabbage (Brassica oleracea, 2n = 18) and in F 1 got sterile (diploid) hybrids. Among these sterile F1 hybrids, he found certain fertile plants which were found to contain 36 chromosomes. These fertile tetraploids were called Raphanobrassica. Synthesized Allopolyploids To find out the origin of naturally occurring allopolyploids some cytogeneticists produced certain allopolyploids in laboratory by employing artificial means. Common hexaploid wheat and tetraploid cotton furnish two such examples. Triticum spelta Triticum spelta is a hexaploid wheat which was artificially synthesized in 1946 by E.S.McFadden and E.R.Sears and also by H.Kihara. They crossed an emmer wheat, Triticum dicoccoides, (tetraploid : 2n = 28) with goat grass, Aegilops squarrosa (diploid ; 2n = 14) and doubled the chromosome number in the F 1 hybrid. This artificially synthesized hexaploid wheat was found to be similar to the primitive wheat T.spelta. When the synthesized hexaploid wheat was crossed with naturally occurring T.spelta, the F1 hybrid was completely fertile. This suggested that hexaploid wheat must have originated in the past due to natural hybridization between tetraploid wheat and goat grass followed by subsequent chromosome doubling. Triticale Triticale (Triticosecale Wittmack) is the first man made cereal which has been developed in recent years and is cultivated on about one million hectares of land throughout the Globe for the commercial use. Triticale is an artificial allopolyploid which has been derived by crossing wheat (Triticum) and rye (Secale). Depending upon whether Triticum is a tetraploid (2n = 4x =28) or hexaploid (2n = 4x = 42), one would get hexaploid Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics triticale (2n = 6x = 42) or octaploid triticale (2n = 8x = 56), respectively. In each case, only diploid rye (2n = 4x = 14) was used. Segmental allopolyploids Different genomes of some allopolyploids are not quite different from each other. Consequently in these polyploids chromosomes belonging to different genomes do pair together to some extent. This indicates that segments of chromosomes and not the whole chromosomes are homologous. Therefore, such allopolyploids are called segmental allopolyploids .The segmental allopolyploids are intermediate between autopolyploids and allopolyploids and can be identified by their peculiar meiotic behaviour. Phenotypic Effects of Polyploidy The increase in the genomes size beyond diploid level is often caused following detectable phenotypic characteristics in a polyploidy organism: (i) Morphological effect of polyploidy. The polyploidy is invariably related with gigantism. The polyploidy plants have been found to contain large-sized pollen grains, cells, leaves, stomata, xylem, etc. The polyploid plants are more vigorous than diploids. (ii) Physiological effect of polyploidy. The ascorbic acid content has been reported to be higher in tetraploid cabbages and tomatoes than in corresponding diploids. Likewise corn meal of a tetraploid maize seed contains 40 per cent more than vitamin A than cornmeal from a diploid plant. (iii) Effect on fertility of polyploidy. The most important effect of polyploidy is that it reduces the fertility of polyploid plants in variable degrees. (iv) Evolution through polyploidy. Interspecific hybridization combined with polyploidy offers a mechanism whereby new species may arise suddenly in natural populations. Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics Extra-nuclear inheritance by cellular Organelles Cytoplasmic extra nuclear genes or DNA molecules of plastids, mitochondria, chloroplasts have a characteristic pattern of inheritance which does not resembles genes of nuclear chromosomes and are hence called as non-Mendelian, non-chromosomal, extra chromosomal, cytoplasmic and extra nuclear inheritance.. Chloroplasts and mitochondria are organelles that contain their own DNA and protein synthesizing apparatus. A widely held theory concerning their origin proposes that they were once infectious endosymbiotic prokaryotes that involved such as dependence on the gene products of the host that they are no longer able to function autonomously. (a) Chloroplast inheritance in variegated four o clock plant. The cytoplasmic or extra nuclear inheritance of colour in plant by plasticides was first of all described by C. Correns in 1908 in the four o clock plant, Mirabilis Jalapa. In contrast to other higher plants, Miabilis contains three types of leaves and parts: (1) Full green leaves or branches having chloroplast, (2) White (pale) leaves and branches having no chloroplast, (3) Variegated branches having leucoplast in white (pale) areas and chloroplast in green patches. Because the chlorophyll pigment of chloroplast is related with photosynthesis of food and leucoplasts are incapable to perform photosynthesis, so the white or pale parts of plant survive by receiving nourishments from green parts. Correns reported that flowers in green branches produced only green offsprings, regardless of the genotype and phenotype of pollen parent and likewise, flowers from the white or pale branches produced only white or pale seedings regardless of genotype and phenotype of pollen parent. The plants from the white or pale seedings die because the lack chlorophyll and cannot carry photosynthesis. Correns further reported that flowers from the variegated branches yielded mixed progeny of green, white (pale) and variegated plants in widely varying ratios. The irregularly of transmission from variegated branches could be understood by considering cytoplasmic genes (plasmagenes) of plastids. A study of the egg during oogenesis in Mirabilis reveals that the ooplasm contains plastids like cytoplasm of other Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics plant cells. If the egg cell is derived from green plant tissues, its ooplasm will contain coloured plastids; if derived from white plant tissues, its ooplasm will contain white plastids; if derived from variegated tissues, its cytoplasm may contain coloured plastids only, white plastids only or a mixture of coloured and white plastids. A study of the pollenogenesis, however, reveals that pollen contains very little cytoplasm which in most cases is devoid of plastids. Without the plastids, pollen cannot affect this aspect of the offsprings phenotype. Mitotic segregation. Variegated branches of Mirabilis Jalapa produce three types of eggs: some contain only white chloroplasts, some contain only green chloroplasts and some contain both types of chloroplasts. In the subsequent mitotic divisions, some form of cytoplasmic segregation occurs that segregate the chloroplast types into pure cell lines, thus, producing the variegated phenotype in the progency individual. This process of sorting might be described as mitotic segregation of this is a pure extra nuclear phenomenon. In mitotic segregation since both segregation and recombination of organelle genotype takes place, so it is called cytoplasmic segregation and recombination (its acronym is CSAR). (b) Cytoplasmic male sterlity (CMS). In maize and many other plants, cytoplasmic control of male sterility is known. In such cases, if the female parent is male sterile (having plasmagene for male sterility), the F1 progeny would always be male sterile, because the cytoplasm is mainly derived from the egg which is obtained from the male sterile female parent. (c) Cytoplasmic genetic male sterility. In certain plants, though the male sterility is fully controlled by the cytoplasm, but a restorer gene if present in the female parent in the nucleus, will restore fertility. For example, if the female parent is male sterile (due to plasmagene of male sterility) then the nuclear genotype of the male parent will determine the phenotype of F1 progeny. Thus, if male sterile female parent contains recessive nuclear genotype rr of restorer gene and male parent is RR, having homozygous dominant restorer genes. Their F1 progeny would be male fertile Rr. However, if the male Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics parent is male fertile rr, the F 1 progeny would be male sterile rr. If the F 1 male fertile heterozygote (Rr) is test crossed with male fertile progeny with 50 per cent male fertile and 50 per cent male sterile will be obtained. Since, in maize expression of male sterility depends on an interaction between nuclear and extra chromosomal genes. Male sterile lines can bear seeds only after crosspollination. For this reason they are useful in raising hybrid seeds, especially on large scale. Later on, in maize the following four types of cytoplasms have been recognized: the normal (N) cytoplasm and three types of male sterile cytoplasms (T, C and S). The recent studies of mitochondria in these cytoplasm revealed that the factors responsible for cytoplasmic male sterility are located in mitochondrial DNA (mt DNA) and mt DNA of N, T, C and S cytoplasms are found to be different. The cytoplasmic male sterility (CMS) of C and S type can be reversed by nuclear storer genes, however, the CMS-T cannot.
Parents
,,
Male sterile F1 (O) Intercross or selfing Male fertile Male fertile
Male fertile
(O)
Male fertile
BT0203 Genetics and Cytogenetics F2
25% Male fertile -Pure
50% 25% Male fertile- hybrid Male sterile -pure Inheritance pattern of genetic male sterility
UNIT V GENETIC TRANSFER

Recombination, however, is undoubtedly important in the evolution of bacteria just as it is in the evolution of eukaryotes. Three different processes have evolved that mediate transfer of genetic material from one bacterium to another, making possible the subsequent recombination events. The most obvious difference between these three processes is the most of transfer of DNA from one cell to another. (1) Transformation involves the uptake of naked DNA molecules from one molecule from one bacterium (the donor cell) by another bacterium (the recipient cell). (2) Transduction occurs when bacterial genes are carried from a donor cell to a recipient cell by a bacteriophoge. (3) Conjugation is the process during which DNA from a donor or male cell is transferred to a recipient or female cell through a specialized sex plus or conjugation tube. TRANSFORMATION Transformation was discovered in pathogenic strains of Diplococcus pneumoniae by Griffith in 1928. The details of Avery, Macleod, and McCartys (1944) proof that the transforming principle (the cellular component mediating transformation) is DNA. The uptake of DNA molecules by recipient bacteria is an active, energy-requiring process. It does not involve passive entry of DNA molecules through permeable cell walls and membranes (although this type of uptake of DNA molecules may be induced by experimental manipulations of bacteria in the laboratory, such as in the Escherichia coli recombinant DNA cloning experiments). Thus, transformation does not occur naturally in all species of bacteria, only in those species possessing the enzymatic machinery involved in the active uptake and recombination processes. Most of the studies on transformation have been done with three species, D. pneumoniae, Bacillus subtilis, and Haemophilus influenzae. Even in the species, all cells in the given population are not capable of active uptake of DNA. Only Competent cells, which possess a so-called competence factor (probably a cell-surface protein or enzyme involved in binding or Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics in taking up DNA), are capable of serving as recipients in transformation. The proportion of bacteria in a culture that are in the physiologically competent state varies with growth conditions and the stage of the growth curve (becoming maximal in late log-phase). Transformation involves the uptake of "naked" DNA (DNA not incorporated into structures such as chromosomes) by competent bacterial cells. Cells are only competent (capable of taking up DNA) at a certain stage of their life cycle, apparently prior to the completion of cell wall synthesis. Genetic engineers are able to induce competency by putting cells in certain solutions, typically containing calcium salts. At the entry site, endonucleases cut the DNA into fragments of 7,000-10,000 nucleotides, and the doublestranded DNA separates into single strands. The single-stranded DNA may recombine with the host's chromosome once inside the cell. This recombination replaces the gene in the host with a variant - albeit homologous - gene. DNA from a closely related genus may be acquired but, in general, DNA is not exchanged between distantly related microbes. Not all bacteria can become competent. While transformation occurs in nature, the extent to which it contributes to genetic diversity is not known.
The process of transformation can be divided into several stages: (1) reversible binding of doublestranded DNA molecules to receptor sites on the cell surface; (2) irreversible uptake of the donor DNA (at which time the donor DNA becomes resistant to DNase in Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics the medium); (3) conversion of the doublestranded donor DNA molecules to singlestranded molecules by nucleotic degradation of one strand; (4) integration (covalent insertion) of all or part of the single strand of donor DNA into the chromosome of the recipient; and (5) the segregation and phenotypic expression of the integrated donor gene or genes in the recombinant (transformed) cell. Steps (2) and (3) may well be coincident effects of a single process. One attractive model, for which there is supporting evidence in the case of Pneumococcus, proposes that a specific exonuclease (or DNA translocase) pulls one strand of donor DNA into the cell using energy derived from the degradation of the complementary strand. Whether degradation of the complementary strand or DNA actually occurs during uptake or immediately after uptake is uncertain. Moreover, considerable evidence suggests that these processes may vary in different species. The first three steps in transformation-binding, uptake, and degradation of one strand of the double stranded DNA are not specific for homologous DNA. In fact, competent bacteria will carry out these three processes equally with other foreign DNAs. However, the integration, or DNA recombination step, is specific for homologous DNA. This is not to say that the integration of segments of heterologous (foreign) DNA never occur, however, it does so at frequencies very much lower than the frequencies observed using homologous DNA. Although very small fragments of DNA taken up by competent cells, a minimum length of about 500 nucleotide-pairs appears to be required for integration to occur. During integration, a single strand (either strand, the previous degradation of one strand is at random) of donor DNA is physically inserted into the recipient chromosome, replacing a segment of one strand of the recipient chromosome). In most transformation experiments, donor DNA fragments are about 20,000 nucleotide-pairs (or about 1/200 of the total chromosome) in length. This means that mapping experiments can be done using transformation only if the genetic markers employed are located close together on the host chromosome. Genetic mapping by transformation
BT0203 Genetics and Cytogenetics If two genes are far apart on the chromosome, they will never be carried on the same molecule of transforming DNA. Thus, double transformants for the two genes (say a to a+ and b to b+, using an a + b+ donor and an a b recipient) will require two independent transformation events (uptake and integration of one DNA molecule carrying a+ and another molecule carrying b+). The probability of two such independent events occurring together will equal the product of the probability of each occurring alone. Since transformation of any single marker occurs with allow frequency, double independent transformation events of this type will be extremely rare. If, on the other hand, two genes are closely linked, they may be carried on a single molecule of transforming DNA. In this case, double transformants can be formed by the uptake and integration of one molecule of donor DNA carrying both genes. Thus, if two genes or genetic markers are very closely linked, double transformants may be formed at a frequency approaching the frequency of single transformants in comparable single-marker experiments. The frequency with which two genetic markers are cotransformed can thus be used as a crude estimate of the linkage distance between them. Genetic markers can also be ordered be means of three-factor transformation experiments using the same rationale as in threefactor transduction, conjugation, or sexduction experiments. TRANSDUCTION Principle Transduction is another method for transferring genes from one bacterium to another; this time the transfer is mediated by bacteriophages (bacterial viruses, also called phages). A bacteriophage infection starts when the virus injects its DNA into a bacterial cell. The bacteriophage DNA may then direct the synthesis of new viral components assembled in the bacterium. Bacteriophage DNA is replicated and then packaged within the phage particles. Early in the infective cycle the phage encodes an enzyme that degrades the DNA of the host cell. Some of these fragments of bacterial DNA are packaged within the bacteriophage particles, taking the place of phage DNA. The phage can then break open (lyse) the cell. When released from the infected cell, a phage that contains bacterial genes can continue to infect a new bacterial cell, transferring the bacterial genes. Sometimes genes transferred in this manner become integrated into the genome of their new bacterial Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics host by homologous recombination. Such transduced bacteria are not lysed because they do not contain adequate phage DNA for viral synthesis. Transduction occurs in a wide variety of bacteria and is a common mechanism of gene transfer.
Transduction, discovered by N.Zinder and J. Lederberg in 1952, occurs when a bacteriophage particle carries a segment of the chromosome from one bacterium (the donor) to another bacterium (the recipient), facilitating subsequent recombination of the genetic markers of the two cells. Two very different types of transduction are known. (1) In generalized transduction, a random segment of bacterial DNA is wrapped up during phage maturation or along with phage chromosome in a few progeny particles, called transducing particles. Generalized transducing phages can therefore transport any gene of the donor cell to the recipient cell. Since all the genes of the donor are represented in a population of these transducing particles (although any one transducing phage contains only one segment of host DNA, representing 1/100 to 1/50 of the total donor of the chromosome), this type of transduction was named generalized transduction. (2) In specialized transduction (also called restricted transduction), a recombination event involving the host chromosome and the phage chromosome occurs, producing a phage chromosome containing a segment a bacterial DNA. Specialized transducing particles thus always contain both phage and bacterial DNA. Specialized transduction is so named because a given virus transduces only genetic markers of the host that are located Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics in one small region of the bacterial chromosome. Bacteriophage lambda, the best-known specialized transducing phage, for example, usually mediates transduction of the gal and bio genes of E.coli. GENERALIZED TRANSDUCTION Bacteriophages have been classified into two types on the basis of their interactions with the bacterial cell. Virulent phages always multiply and lyse the host cell after infection. Temperate phages have a choice between two life-styles after infections. They can either (1) enter the lytic cycle, during which they reproduce and lyse their host cells just like virulent phages, or, alternately, they can (2) enter the lysogenetic pathway during which their chromosomes are integrated into the chromosomes of the host and replicate like any other segments of the host chromosomes. Generalized transduction is mediated by some virulent bacteriophages whose chromosomes are not integrated at specified attachment sites on the host chromosome. Generalized transducting particles are produced during the lytic cycles of these phages. Of the generalized transducing phages, E.coli phage P1, Salmonella phage P22, and Bacillus subtilis phages PBS1 and SP10 have been extensively used for genetic fine structure maping. After a transducing phage injects a fragment of DNA into a recipient cell, that DNA may either (1) be integrated into the host chromosome in a manner similar to the integration of transforming DNA, except that the integrated segment is double-stranded, or (2) remain free in the cytoplasm. If it is not integrated, it will not replicate and will be transmitted to only one progeny cell during each cell division. The genes located on the transduced chromosome fragments may be expressed, even they are not integrated. Cells carrying nonintegrated transducing fragments are called abortive transductants. Gene mapping by transduction Transducing particles are produced at a low frequency. Only one out of 10 5-107 of the progeny particles present in a lysate contains bacterial DNA. Thus, the probably of the cell being doubly transduced for markers carried in two different transducing particles in negligible. (If cells are simultaneously infected with 100 or more phage particles, they are Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics rapidly killed by a process called lysis-from-without, which apparently results from simply punching too many holes in the cell membrane.) The cotransduction of two or more genetic markers therefore indicates that the markers are relatively closely linked, and the frequency of cotransduction of any two markers is indicative of the degree of linkage between them. Occasionally, genetic markers can be ordered by cotransduction patterns. If (1) markers a+ and b+ are cotransduced, (2) markers b+ and c+ are cotransduced, but (3) markers a+ and c+ are not cotranduced, then the order of the three markers must be a+-b+-c+. More frequently, however, three-factor transduction experiments must be used to unambiguously order genetic markers. SPECIALIZED TRANSDUCTION Specialized transduction is mediated by temperate bacteriophages whose chromosomes are able to integrate at one or a few specified attachment sites on the host chromosome. The chromosomes of temperate phages of this type are thus capable of both (1) autonomous replication (replication independent of the replication of the host chromosome) and (2) integrated replication (replication as a segment of the host chromosome). As such, they are examples of genetic elements called episomes. Integration of the chromosome of a specialized transducing phage, such as the coliphage lambda, involves a recombination event between the circular intercellular form of the phage chromosome and the circular bacterial chromosome at specific attachments sites on the two chromosomes. This site specific recombination event results in the covalent linear insertion of the phage chromosome and the circular bacterium chromosome at specific attachment sites on the two chromosomes. This site specific recombination event results in the covalent linear insertion of the page chromosome into the chromosome bacterium. In its integrated state, the phage chromosomes are called a prophage. The lytic genes of the virus, those involved in viral reproduction and lysis of the host, are repressed (turned off) when the chromosome is in the prophage state. (The mechanism by which the prophased genes is repressed). A bacterium harboring a prophage is said to be lysogenic, the prophage-host relationship is called lysogeny. A lysogenic cell is immune to secondary infections by the same virus (homologous to the prophage), because the
BT0203 Genetics and Cytogenetics lytic genes of the infecting virus will be repressed just as those of the prophage are repressed.
Temperate phages undergo rare (about one in105 cell divisions) spontaneous transitions from the lysogenic or prophage state to the lytic state. Such transitions can be also induced, for example, by irradiation with ultraviolet light. During the switch from the lysogenic state to lytic growth, the prophage is excised from the host chromosome and commences replicating autonomously. The excision process is site specific, like the integration process. The site-specific integration and excision processes are catalyzed by enzymes encoded by phase genes. The excision process is usually very precise in cutting out the phage chromosome in exactly the form in which it existed prior to its integration. Occasionally, however, the excision event occurs at a site other than the original attachment site. When this happens, a portion of the phage chromosome is left in the host chromosome and a portion of the bacterial chromosome is excised with the phage DNA. Such mistakes during prophage excision are responsible for the formation of specialized transducing particles. Only host genes located close to the site of prophage insertion can be excised with the phage DNA and packaged in phage particles. Thus specialized transduction is restricted to the transfer of genes located within a short distance on each side of the prophage attachment site. Phage lambda integrates between the gal genes (required for the utilization of Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics galactose as an energy source) and the bio genes (essential for the synthesis of biotin) on the E.coli chromosome; lambda thus usually only transduces gal and bio markers. Specialized tansducing phage O 80, on the other hand, integrates near the E.coli trp genes (required for the synthesis of the amino acid trytophan) and transduces trp markers. If specialized transducing particles are formed during prophage excision, only phage lysates produced by induction of lysogenic cells should have transducing activity. This is indeed the case. If bacteria are infected by specialized transducing phages under conditions where only lytic infections occur, no transducing particles are present in the phage lysates. The frequency of transducing particles in lysates produced by induction of induction of lysogenic cells is about one in 106 progency particles. CONJUGATION Conjugation was discovered in 1946 by J. Lederberg and E.L. Tatum (1958 Nobel Prize co recipients). During conjugation, DNA is transferred from a donor cell to a recipient cell through a specialized intercellular connection, or conjugation tube, that forms between them. The donor and recipient cells are sometimes referred to as male and female cells, respectively). The transfer of genetic information is thus a one-way transfer during conjugation, just as in transformation and transduction, rather than a reciprocal exchange of genetic material. Cells that have the capacity to serve as donors during conjugation are differentiated by the presence of specialized cell-surface appendages called F pili. The synthesis of these F pili is controlled by several (nine, based on current data) genes that are carried by a small circular molecule of DNA or minichromosome (about 94,500 nucleotide-pairs long) called on F factor (for fertility factor; also called sex factor and F plasmid). Cells carrying an F factor (donor cells) form conjugation tubes and initiate DNA transfer making contact with cells not carrying an F factor, called F - cells (recipient cells). The F factor can exist in two different states: (1) the autonomous state, in which it replicates independently of the chromosome, and (2) the integrated state, in which it is Rex Arunraj Assistant Prof. Department of Genetic Engineering
BT0203 Genetics and Cytogenetics covalently inserted into the host chromosome like any other set of chromosomal genes. The F factor is thus, like the chromosomes of specialized transducing phages, an example of a class of genetic elements called episomes.
A donor cell containing the F factor in the autonomous state is called an F+ cell. When an F+ donor cell conjugates with an F- recipient cell, only the autonomous F factor is transferred. Both exconjugants (cells that have been involved in conjugation) become F + because the F factor replicates during transfer. Thus, mixing a population of F+ cells with a population of F - cells results in virtually all the cells in the new population becoming F+. The F factor can integrate into the host chromosome at any one of many sites by a mechanism that appears analogous to the integration of the chromosomke of a specialized transducing phage, namely, a site-specific recombination event. The integration of the F factor is believed to be mediated by short DNA sequences called IS elements. A cell carrying a integrated F factor is called an Hfr. In the integrated state, the F factor mediates the transfer of a chromosome of the Hfr cell to a recipient (F -) cell. Usually, only a portion of the Hfr chromosome is transferred before the cells separate, thus breaking the chromosome is transferred.
BT0203 Genetics and Cytogenetics The mechanism of transfer of DNA from a donor cell to a recipient cell during conjugation appears to be the same whether just the F factor is being transferred, as in F + by F matings, or the chromosome is being transferred, as in Hfr by F matings. Transfer is believed to be initiated by an endonucleolytic nick in one strand at a specific site (the origin of transfer) on the F factor. The 5 end of the nicked strand is then transferred through the conjugation tube into the recipient cell. Transfer is believed to be coupled to rolling circle replication, with the circular strand being replicated in the donor cell and the displaced stand being replicated in the recipient cell as it is transferred. Because the origin of transfer is within the integrated F factor, one portion of the F factor is transferred from an Hfr cell to an F - cell prior to the sequential transfer of chromosomal genes. The remaining part of the F factor, however, is the last segment of DNA to be transferred. Thus, in Hfr by F matings, the recipient F cell acquires a complete F factor (thus becoming an Hfr donor) only in those rare cases when an entire Hfr chromosome, with its integrated F factor, is transferred. Gene mapping by conjugation Subsequent studies with different Hfr strains revealed similar fixed transfer sequences, although different Hfrs initiated transfer from different sites on the chromosome. It is now clear that the F factor can integrate at many different sites in the circular E .coli chromosome, and the site of integration determines the origin of transfer characteristic of a given Hfr. The transfer of a complete chromosome from an Hfr to an F cell takes from 90 to 100 minutes, depending on the strain. Chromosome transfer appears to proceed at a fairly constant rate. Thus, the time interval between the transfer of any two markers (easily determined by interrupted mating experiments) is a good estimate of the physical distance separating the markers on the chromosome. It has therefore proven convenient to use the minute, representing the time interval between the transfer of markers in interrupted mating experiments, as the standard unit for measuring linkage in E. coli. A map distance of 1 minute corresponds to the length of the segment of the chromosome transferred in 1 minute during conjugation. The standard E. coli linkage map is thus divided into minute intervals from 0 (arbitrarily set at the thrA gene) to 100 minutes on the basis of interrupted mating experiments. Rex Arunraj Assistant Prof. Department of Genetic Engineering
Linkage relationships can also be determined from uninterrupted mating experiments. Consider, for example, the Hfr H by F cross, with the matings being allowed to proceed uninterrupted for 1-2 hours. If thr+ leu+ str r recombinants are selected and scored for the presence of other segregating markers by replicating, what will the frequencies of the donor (say azi-s, T1-s, lac+, and gal+ markers be among the recombinants? Will these donor markers all be present with the same frequency? The frequencies of these donor markers are observed to vary, with the frequency of the marker decreasing as a function of its distance from the selected (thr+ and leu+) donor markers. The frequency will, in fact, be identical to the plateau frequencies observed in the interrupted mating experiment. Donor markers azi-s, T1-s,lac+, and gal+ will occur among thr+ leu+ str-r recombinants with percentage frequencies of 90, 80, 40, and 25, respectively. The farther a marker is from the selected donor marker (in the HfrH experiment, thr+ and leu+), the lower its frequency among the recombinants. The marker frequency gradient is caused by two major factors: (1) the approximately constant probability per unit time of spontaneous rupture of the conjugation tube and the chromosome and (2) the decreasing probability that any two donor markers will be incorporated into the recipient chromosome by a single pair of recombination events (incorporation of a donor fragment into a recipient chromosome always requires two recombination events) as the distance separating the two markers increases. Although uninterrupted conjugation experiments of this type can be used to determine linkage relationships, interrupted mating experiments are simpler and more direct. Thus, when a new mutation is identified, its approximate location is usually first determined by interrupted conjugation mapping. Its exact location is then usually determined by transduction mapping.

BT203 - Genetics Unit 2

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BT0203 Genetics and Cytogenetics UNIT I Terminologies Karyotype: A Karyotype is an array of chromosomes created by photographing the metaphase chromosomes

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Department of Genetic Engineering

BT0203 Genetics and Cytogenetics Genotype 1:2:1

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

BT0203 Genetics and Cytogenetics

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

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Rex Arunraj Assistant Prof.

Department of Genetic Engineering

BT0203 Genetics and Cytogenetics

Punnett Square for the Backcross Female Gametes GW Male Gametes Gw gW gw

The phenotypic ratio of the test cross is:

BT0203 Genetics and Cytogenetics

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

BT0203 Genetics and Cytogenetics

Testing ratios for independent assortment

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

BT0203 Genetics and Cytogenetics

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Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.

Department of Genetic Engineering

Rex Arunraj Assistant Prof.