In genetics, a mutation is a change of the nucleotide sequence of the genome of an organism, virus, or extrachromosomal genetic element.

Mutations result from unrepaired damage to DNA or to RNA genomes (typically caused by radiation or chemical mutagens), errors in the process of replication, or from the insertion or deletion of segments of DNA by mobile genetic elements.[1][2][3] Mutations may or may not produce discernible changes in the observable characteristics (phenotype) of an organism. Mutations play a part in both normal and abnormal biological processes including: evolution, cancer, and the development of the immune system. Mutation can result in several different types of change in sequences. Mutations in genes can either have no effect, alter the product of a gene, or prevent the gene from functioning properly or completely. Mutations can also occur in nongenic regions. One study on genetic variations between different species of Drosophilasuggests that if a mutation changes a protein produced by a gene, the result is likely to be harmful, with an estimated 70 percent of amino acid polymorphismswhich have damaging effects, and the remainder being either neutral or weakly beneficial.[4] Due to the damaging effects that mutations can have on genes, organisms have mechanisms such as DNA repair to prevent or correct (revert the mutated sequence back to its original state) mutations.[

Description[edit]

Mutations can involve large sections of DNA becoming duplicated, usually through genetic recombination.[5] These duplications are a major source of raw material for evolving new genes, with tens to hundreds of genes duplicated in animal genomes every million years.[6] Most genes belong to larger families of genes of shared ancestry.[7] Novel genes are produced by several methods, commonly through the duplication and mutation of an ancestral gene, or by recombining parts of different genes to form new combinations with new functions.[8][9] Here, domains act as modules, each with a particular and independent function, that can be mixed together to produce genes encoding new proteins with novel properties.[10] For example, the human eye uses four genes to make structures that sense light: three for color vision and one for night vision; all four arose from a single ancestral gene.[11] Another advantage of duplicating a gene (or even an entire genome) is that this increases redundancy; this allows one gene in the pair to acquire a new function while the other copy performs the original function.[12][13] Other types of mutation occasionally create new genes from previously noncoding DNA.[14][15] Changes in chromosome number may involve even larger mutations, where segments of the DNA within chromosomes break and then rearrange. For example, in the Homininae, two chromosomes fused to produce human chromosome 2; this fusion did not occur in the lineage of the other apes, and they retain these separate chromosomes.[16] In evolution, the most

important role of such chromosomal rearrangements may be to accelerate the divergence of a population into new species by making populations less likely to interbreed, and thereby preserving genetic differences between these populations.[17] Sequences of DNA that can move about the genome, such as transposons, make up a major fraction of the genetic material of plants and animals, and may have been important in the evolution of genomes.[18] For example, more than a million copies of the Alu sequence are present in the human genome, and these sequences have now been recruited to perform functions such as regulating gene expression.[19] Another effect of these mobile DNA sequences is that when they move within a genome, they can mutate or delete existing genes and thereby produce genetic diversity.[2] Nonlethal mutations accumulate within the gene pool and increase the amount of genetic variation.[20] The abundance of some genetic changes within the gene pool can be reduced by natural selection, while other "more favorable" mutations may accumulate and result in adaptive changes. For example, a butterfly may produce offspring with new mutations. The majority of these mutations will have no effect; but one might change the color of one of the butterfly's offspring, making it harder (or easier) for predators to see. If this color change is advantageous, the chance of this butterfly surviving and producing its own offspring are a little better, and over time the number of

butterflies with this mutation may form a larger percentage of the population. Neutral mutations are defined as mutations whose effects do not influence the fitness of an individual. These can accumulate over time due to genetic drift. It is believed that the overwhelming majority of mutations have no significant effect on an organism's fitness. Also, DNA repair mechanisms are able to mend most changes before they become permanent mutations, and many organisms have mechanisms for eliminating otherwise permanently mutated somatic cells. Beneficial mutations can improve reproductive success.

Causes[edit] Main article: Mutagenesis Four classes of mutations are (1) spontaneous mutations (molecular decay), (2) mutations due to error prone replication by-pass of naturally occurring DNA damage (also called error pronetranslesion synthesis), (3) errors introduced during DNA repair, and (4) induced mutations caused by mutagens. Spontaneous mutation[edit] Spontaneous mutations on the molecular level can be caused by:[21]

Tautomerism A base is changed by the repositioning of a hydrogen atom, altering the hydrogen bonding

pattern of that base resulting in incorrect base pairing during replication. Depurination Loss of a purine base (A or G) to form an apurinic site (AP site). Deamination Hydrolysis changes a normal base to an atypical base containing a keto group in place of the original amine group. Examples include C U and A HX (hypoxanthine), which can be corrected by DNA repair mechanisms; and 5MeC (5-methylcytosine) T, which is less likely to be detected as a mutation because thymine is a normal DNA base. Slipped strand mispairing Denaturation of the new strand from the template during replication, followed by renaturation in a different spot ("slipping"). This can lead to insertions or deletions. Error prone replication by-pass[edit] There is increasing evidence that the majority of spontaneously arising mutations are due to error prone replication (translesion synthesis) past a DNA damage in the template strand. As described in the article DNA damage (naturally occurring), naturally occurring DNA damages arise about 10,000 to 100,000 times per day per mammalian cell. In mice, the majority of mutations are caused by translesion synthesis.[22] Similarly, in yeast, Kunz et al.[23] found that more than 60% of the spontaneous single base pair substitutions and deletions were caused by translesion synthesis. Errors introduced during DNA repair[edit] Although naturally occurring double-strand breaks occur at a relatively low frequency in DNA (see DNA damage

(naturally occurring)) their repair often causes mutation. Non-homologous end joining (NHEJ) is a major pathway for repairing double-strand breaks. NHEJ involves removal of a few nucleotides to allow somewhat inaccurate alignment of the two ends for rejoining followed by addition of nucleotides to fill in gaps. Consequently, NHEJ often introduces mutations.[24]

Induced mutation[edit] Induced mutations on the molecular level can be caused by:

Chemicals Hydroxylamine NH2OH Base analogs (e.g. BrdU) Alkylating agents (e.g. N-ethyl-N-nitrosourea) These agents can mutate both replicating and nonreplicating DNA. In contrast, a base analog can only mutate the DNA when the analog is incorporated in replicating the DNA. Each of these classes of chemical mutagens has certain effects that then lead to transitions, transversions, or deletions. Agents that form DNA adducts (e.g. ochratoxin A metabolites)[26] DNA intercalating agents (e.g. ethidium bromide) DNA crosslinkers Oxidative damage Nitrous acid converts amine groups on A and C to diazo groups, altering their hydrogen bonding

patterns which leads to incorrect base pairing during replication. Radiation Ultraviolet radiation (nonionizing radiation). Two nucleotide bases in DNA cytosine and thymine are most vulnerable to radiation that can change their properties. UV light can induce adjacent pyrimidine bases in a DNA strand to become covalently joined as a pyrimidine dimer. UV radiation, particularly longer-wave UVA, can also cause oxidative damage to DNA.[27] Classification of mutation types[edit]

Illustrations of five types of chromosomal mutations.

Selection of disease-causing mutations, in a standard table of the genetic code of amino acids.[28] By effect on structure[edit] The sequence of a gene can be altered in a number of ways. Gene mutations have varying effects on health depending on where they occur and whether they alter the function of essential proteins. Mutations in the structure of genes can be classified as:

Small-scale mutations, such as those affecting a small gene in one or a few nucleotides, including: Point mutations, often caused by chemicals or malfunction of DNA replication, exchange a single nucleotide for another.[29]These changes are classified as transitions or transversions.[30] Most common is the transition that exchanges a purine for a purine (A G) or a pyrimidine for a pyrimidine, (C T). A transition can be caused by nitrous acid, base mis-pairing, or mutagenic base analogs such as 5-bromo-2-deoxyuridine (BrdU). Less common is a transversion, which exchanges a purine for a pyrimidine or a pyrimidine for a purine (C/T A/G). An example of a transversion is adenine (A) being converted into a cytosine(C). A point mutation can be reversed by another point mutation, in which the nucleotide is changed back to its original state (true reversion) or by second-site reversion (a complementary mutation elsewhere that results in regained gene functionality). Point mutations that

occur within the protein coding region of a gene may be classified into three kinds, depending upon what the erroneous codon codes for: Silent mutations: which code for the same (or a sufficiently similar) amino acid. Missense mutations: which code for a different amino acid. Nonsense mutations: which code for a stop and can truncate the protein. Insertions add one or more extra nucleotides into the DNA. They are usually caused by transposable elements, or errors during replication of repeating elements (e.g. AT repeats[citation needed]). Insertions in the coding region of a gene may alter splicing of themRNA (splice site mutation), or cause a shift in the reading frame (frameshift), both of which can significantly alter the gene product. Insertions can be reversed by excision of the transposable element. Deletions remove one or more nucleotides from the DNA. Like insertions, these mutations can alter the reading frame of the gene. They are generally irreversible: though exactly the same sequence might theoretically be restored by an insertion, transposable elements able to revert a very short deletion (say 12 bases) in any location are either highly unlikely to exist or do not exist at all. Note that a deletion is not the exact opposite of an insertion: the former is quite random while the latter consists of a specific sequence inserting at locations that are not entirely random or even quite narrowly defined.

Large-scale mutations in chromosomal structure, including: Amplifications (or gene duplications) leading to multiple copies of all chromosomal regions, increasing the dosage of the genes located within them. Deletions of large chromosomal regions, leading to loss of the genes within those regions. Mutations whose effect is to juxtapose previously separate pieces of DNA, potentially bringing together separate genes to form functionally distinct fusion genes (e.g. bcr-abl). These include: Chromosomal translocations: interchange of genetic parts from nonhomologous chromosomes. Interstitial deletions: an intra-chromosomal deletion that removes a segment of DNA from a single chromosome, thereby apposing previously distant genes. For example, cells isolated from a human astrocytoma, a type of brain tumor, were found to have a chromosomal deletion removing sequences between the "fused in glioblastoma" (fig) gene and the receptor tyrosine kinase "ros", producing a fusion protein (FIG-ROS). The abnormal FIG-ROS fusion protein has constitutively active kinase activity that causes oncogenic transformation (a transformation from normal cells to cancer cells). Chromosomal inversions: reversing the orientation of a chromosomal segment.

Loss of heterozygosity: loss of one allele, either by a deletion or recombination event, in an organism that previously had two different alleles. By effect on function[edit]

Loss-of-function mutations result in the gene product having less or no function. When the allele has a complete loss of function (null allele) it is often called an amorphic mutation. Phenotypes associated with such mutations are most often recessive. Exceptions are when the organism is haploid, or when the reduced dosage of a normal gene product is not enough for a normal phenotype (this is called haploinsufficiency). Gain-of-function mutations change the gene product such that it gains a new and abnormal function. These mutations usually have dominant phenotypes. Often called a neomorphicmutation. Dominant negative mutations (also called antimorphic mutations) have an altered gene product that acts antagonistically to the wild-type allele. These mutations usually result in an altered molecular function (often inactive) and are characterized by a dominant or semi-dominant phenotype. In humans, dominant negative mutations have been implicated in cancer (e.g. mutations in genes p53,[31] ATM,[32] CEBPA[33] and PPARgamma.[34] I t was once thought that Marfan syndrome is an example of a dominant negative mutation occurring in an autosomal dominant disease where the defective glycoprotein product of the fibrillin gene (FBN1) antagonizes the product of the normal allele. However,

it may appear this is not that case and that Marfan's may be a result of Haploinsufficiency due to the absence of one normal allele that causes the disease not the presence of an abnormal allele (i.e. Dominant negative).[citation needed] Lethal mutations are mutations that lead to the death of the organisms which carry the mutations. A back mutation or reversion is a point mutation that restores the original sequence and hence the original phenotype.[35] See also Behavior mutation. By effect on fitness[edit] In applied genetics it is usual to speak of mutations as either harmful or beneficial.

A harmful, or deleterious, mutation decreases the fitness of the organism. A beneficial, or advantageous mutation increases the fitness of the organism. Mutations which promotes traits that are desirable, are also called beneficial. In theoretical population genetics, it is more usual to speak of mutations as deleterious or advantageous than harmful or beneficial. A neutral mutation has no harmful or beneficial effect on the organism. Such mutations occur at a steady rate, forming the basis for the molecular clock. In the neutral theory of molecular evolution, neutral mutations provide genetic drift is the basis for most variation at the molecular level.

A nearly neutral mutation is a mutation that may be slightly deleterious or advantageous, although most nearly neutral mutations are slightly deleterious. Distribution of fitness effects[edit] In reality, viewing the fitness effects of mutations in these discrete categories is an oversimplification. Attempts have been made to infer the distribution of fitness effects (DFE) using mutagenesisexperiments and theoretical models applied to molecular sequence data. Distribution of fitness effects, as used to determine the relative abundance of different types of mutations (i.e. strongly deleterious, nearly neutral or advantageous), is relevant to many evolutionary questions, such as the maintenance of genetic variation,[36] the rate of genomic decay,[37] the maintenance of outcrossing sexual reproduction as opposed to inbreeding[38] and the evolution of sex and recombination.[39] In summary, DFE plays an important role in predicting evolutionary dynamics.[40][41] A variety of approaches have been used to study the distribution of fitness effects, including theoretical, experimental and analytical methods.

Mutagenesis experiment: The direct method to investigate DFE is to induce mutations and then measure the mutational fitness effects, which has already been done in viruses, bacteria, yeast, and Drosophila. For example, most studies of DFE in viruses used site-directed mutagenesis to create point mutations and measure relative fitness of each mutant.[42][43][44][45] InEscherichia coli, one study used transposon mutagenesis to directly measure the

fitness of a random insertion of a derivative of Tn10.[46] In yeast, a combined mutagenesis and deep sequencing approach has been developed to generate high-quality systematic mutant libraries and measure fitness in high throughput.[47] However, given that many mutations have effects too small to be detected[48] and that mutagenesis experiments can only detect mutations of moderately large effect, DNA sequence data analysis can provide valuable information about these mutations.

The distribution of fitness effects of mutations in vesicular stomatitis virus. In this experiment, random mutations were introduced into the virus by site-directed mutagenesis, and the fitness of each mutant was compared with the ancestral type. A fitness of zero, less than one, one, more than one, respectively, indicates that mutations are lethal, deleterious, neutral and advantageous. Data from.[42]

Molecular sequence analysis: With rapid development of DNA sequencing technology, an enormous amount of DNA sequence data is available and even more is

forthcoming in the future. Various methods have been developed to infer DFE from DNA sequence data.[49][50][51][52] By examining DNA sequence differences within and between species, we are able to infer various characteristics of the DFE for neutral, deleterious and advantageous mutations.[53] Specifically, the DNA sequence analysis approach allows us to estimate the effects of mutations with very small effects, which are hardly detectable through mutagenesis experiments. One of the earliest theoretical studies of the distribution of fitness effects was done by Motoo Kimura, an influential theoretical population geneticist. His neutral theory of molecular evolution proposes that most novel mutations will be highly deleterious, with a small fraction being neutral.[54][55] Hiroshi Akashi more recently proposed a bimodal model for DFE, with modes centered around highly deleterious and neutral mutations.[56] Both theories agree that the vast majority of novel mutations are neutral or deleterious and that advantageous mutations are rare, which has been supported by experimental results. One example is a study done on the distribution of fitness effects of random mutations in vesicular stomatitis virus.[42] Out of all mutations, 39.6% were lethal, 31.2% were non-lethal deleterious, and 27.1% were neutral. Another example comes from a high throughput mutagenesis experiment with yeast.[47] In this experiment it was shown that the overall distribution of fitness effects is bimodal, with a cluster of neutral mutations, and a broad distribution of deleterious mutations.

Though relatively few mutations are advantageous, those that are play an important role in evolutionary changes.[57] Like neutral mutations, weakly selected advantageous mutations can be lost due to random genetic drift, but strongly selected advantageous mutations are more likely to be fixed. Knowing the distribution of fitness effects of advantageous mutations may lead to increased ability to predict the evolutionary dynamics. Theoretical work on the DFE for advantageous mutations has been done by John H. Gillespie[58] and H. Allen Orr.[59] They proposed that the distribution for advantageous mutations should be exponential under a wide range of conditions, which has generally been supported by experimental studies, at least for strongly selected advantageous mutations.[60][61][62] In summary, it is generally accepted that the majority of mutations are neutral or deleterious, with rare mutations being advantageous; however, the proportion of types of mutations varies between species. This indicates two important points: first, the proportion of effectively neutral mutations is likely to vary between species, resulting from dependence on effective population size; second, the average effect of deleterious mutations varies dramatically between species.[53] In addition, the DFE also differs between coding regions and non-coding regions, with the DFE of non-coding DNA containing more weakly selected mutations.[53] By impact on protein sequence[edit]

A frameshift mutation is a mutation caused by insertion or deletion of a number of nucleotides that

is not evenly divisible by three from a DNA sequence. Due to the triplet nature of gene expression by codons, the insertion or deletion can disrupt the reading frame, or the grouping of the codons, resulting in a completely different translation from the original.[63] The earlier in the sequence the deletion or insertion occurs, the more altered the protein produced is. In contrast, any insertion or deletion that is evenly divisible by three is termed an in-frame mutation

A nonsense mutation is a point mutation in a sequence of DNA that results in a premature stop codon, or a nonsense codon in the transcribed mRNA, and possibly a truncated, and often nonfunctional protein product. (See Stop codon.) Missense mutations or nonsynonymous mutations are types of point mutations where a single nucleotide is changed to cause substitution of a different amino acid. This in turn can render the resulting protein nonfunctional. Such mutations are responsible for diseases such as Epidermolysis bullosa, sickle-cell disease, and SOD1 mediated ALS (Boille 2006, p. 39). A neutral mutation is a mutation that occurs in an amino acid codon which results in the use of a different, but chemically similar, amino acid. The similarity between the two is enough that little or no change is often rendered in the protein. For example, a change from AAA to AGA will encode arginine, a chemically similar molecule to the intended lysine.

Silent mutations are mutations that do not result in a change to the amino acid sequence of a protein, unless the changed amino acid is sufficiently similar to the original. They may occur in a region that does not code for a protein, or they may occur within a codon in a manner that does not alter the final amino acid sequence. The phrase silent mutation is often used interchangeably with the phrase synonymous mutation; however, synonymous mutations are a subcategory of the former, occurring only within exons (and necessarily exactly preserving the amino acid sequence of the protein). Synonymous mutations occur due to the degenerate nature of the genetic code. (See Genetic code.) By inheritance[edit]

A mutation has caused this garden moss rose to produce flowers of different colors. This is a somatic mutation that may also be passed on in the germ line.

In multicellular organisms with dedicated reproductive cells, mutations can be subdivided into germ line mutations, which can be passed on to descendants through their reproductive cells, and somatic mutations (also called acquired mutations),[64] which involve cells outside the dedicated reproductive group and which are not usually transmitted to descendants. A germline mutation gives rise to a constitutional mutation in the offspring, that is, a mutation that is present in every cell. A constitutional mutation can also occur very soon after fertilisation, or continue from a previous constitutional mutation in a parent.[65] The distinction between germline and somatic mutations is important in animals that have a dedicated germ line to produce reproductive cells. However, it is of little value in understanding the effects of mutations in plants, which lack dedicated germ line. The distinction is also blurred in those animals that reproduce asexually through mechanisms such as budding, because the cells that give rise to the daughter organisms also give rise to that organisms germ line. A new mutation that was not inherited from either parent is called a de novo mutation. Diploid organisms (e.g. humans) contain two copies of each gene a paternal and a maternal allele. Based on the occurrence of mutation on each chromosome, we may classify mutations into three types.

A heterozygous mutation is a mutation of only one allele.

A homozygous mutation is an identical mutation of both the paternal and maternal alleles. Compound heterozygous mutations or a genetic compound comprises two different mutations in the paternal and maternal alleles.[66] A wildtype or homozygous non-mutated organism is one in which neither allele is mutated. Special classes[edit]

Conditional mutation is a mutation that has wildtype (or less severe) phenotype under certain "permissive" environmental conditions and a mutant phenotype under certain "restrictive" conditions. For example, a temperature-sensitive mutation can cause cell death at high temperature (restrictive condition), but might have no deleterious consequences at a lower temperature (permissive condition). Nomenclature[edit] A committee of the Human Genome Variation Society (HGVS) has developed the standard human sequence variant nomenclature,[67] which should be used by researchers and DNA diagnosticcenters to generate unambiguous mutation descriptions. In principle, this nomenclature can also be used to describe mutations in other organisms. The nomenclature specifies the type of mutation and base or amino acid changes.

Nucleotide substitution (e.g. 76A>T) The number is the position of the nucleotide from the 5' end, the first letter represents the wild type nucleotide, and the second letter represents the nucleotide which

replaced the wild type. In the given example, the adenine at the 76th position was replaced by a thymine. If it becomes necessary to differentiate between mutations in genomic DNA, mitochondrial DNA, and RNA, a simple convention is used. For example, if the 100th base of a nucleotide sequence mutated from G to C, then it would be written as g.100G>C if the mutation occurred in genomic DNA, m.100G>C if the mutation occurred in mitochondrial DNA, or r.100g>c if the mutation occurred in RNA. Note that for mutations in RNA, the nucleotide code is written in lower case. Amino acid substitution (e.g. D111E) The first letter is the one letter code of the wild type amino acid, the number is the position of the amino acid from the Nterminus, and the second letter is the one letter code of the amino acid present in the mutation. Nonsense mutations are represented with an X for the second amino acid (e.g. D111X). Amino acid deletion (e.g. F508) The Greek letter (delta) indicates a deletion. The letter refers to the amino acid present in the wild type and the number is the position from the N terminus of the amino acid were it to be present as in the wild type. Mutation rates[edit] Further information: Mutation rate Mutation rates vary across species. Evolutionary biologists[citation needed] have theorized that higher mutation rates are beneficial in some situations, because they

allow organisms to evolve and therefore adapt more quickly to their environments. For example, repeated exposure of bacteria to antibiotics, and selection of resistant mutants, can result in the selection of bacteria that have a much higher mutation rate than the original population (mutator strains). According to one study, two children of different parents had 35 and 49 new mutations. Of them, in one case 92% were from the paternal germline, in another case, 64% were from the maternal germline.[68] Harmful mutations[edit] Changes in DNA caused by mutation can cause errors in protein sequence, creating partially or completely non-functional proteins. Each cell, in order to function correctly, depends on thousands of proteins to function in the right places at the right times. When a mutation alters a protein that plays a critical role in the body, a medical condition can result. A condition caused by mutations in one or more genes is called a genetic disorder. Some mutations alter a gene's DNA base sequence but do not change the function of the protein made by the gene. One study on the comparison of genes between different species of Drosophila suggests that if a mutation does change a protein, this will probably be harmful, with an estimated 70 percent of amino acid polymorphisms having damaging effects, and the remainder being either neutral or weakly beneficial.[4] Studies have shown that only 7% of point mutations in non-coding DNA of yeast are deleterious

and 12% in coding DNA are deleterious. The rest of the mutations are either neutral or slightly beneficial. [69] If a mutation is present in a germ cell, it can give rise to offspring that carries the mutation in all of its cells. This is the case in hereditary diseases. In particular, if there is a mutation in a DNA repair gene within a germ cell, humans carrying such germ-line mutations may have an increased risk of cancer. A list of 34 such germ-line mutations is given in the article DNA repair-deficiency disorder. On the other hand, a mutation may occur in a somatic cell of an organism. Such mutations will be present in all descendants of this cell within the same organism, and certain mutations can cause the cell to become malignant, and thus cause cancer.[70] A DNA damage can cause an error when the DNA is replicated, and this error of replication can cause a gene mutation that, in turn, could cause a genetic disorder. DNA damages are repaired by the DNA repair system of the cell. Each cell has a number of pathways through which enzymes recognize and repair damages in DNA. Because DNA can be damaged in many ways, the process of DNA repair is an important way in which the body protects itself from disease. Once a DNA damage has given rise to a mutation, the mutation cannot be repaired. DNA repair pathways can only recognize and act on "abnormal" structures in the DNA. Once a mutation occurs in a gene sequence it then has normal DNA structure and cannot be repaired. Beneficial mutations[edit]

Although mutations that change in protein sequences can be harmful to an organism; on occasions, the effect may be positive in a given environment. In this case, the mutation may enable the mutant organism to withstand particular environmental stresses better than wild-type organisms, or reproduce more quickly. In these cases a mutation will tend to become more common in a population through natural selection. For example, a specific 32 base pair deletion in human CCR5 (CCR5-32) confers HIV resistance to homozygotes and delays AIDS onset in heterozygotes.[71] The CCR5 mutation is more common in those of European descent. One possible explanation of the etiology of the relatively high frequency of CCR5-32 in the European population is that it conferred resistance to thebubonic plague in mid14th century Europe. People with this mutation were more likely to survive infection; thus its frequency in the population increased.[72] This theory could explain why this mutation is not found in southern Africa, which remained untouched by bubonic plague. A newer theory suggests that the selective pressure on the CCR5 Delta 32 mutation was caused bysmallpox instead of the bubonic plague.[73] Another example is Sickle cell disease, a blood disorder in which the body produces an abnormal type of the oxygen-carrying substance hemoglobin in the red blood cells. One-third of allindigenous inhabitants of SubSaharan Africa carry the gene,[74] because in areas where malaria is common, there is a survival value in

carrying only a single sickle-cell gene (sickle cell trait).[75]Those with only one of the two alleles of the sickle-cell disease are more resistant to malaria, since the infestation of the malaria plasmodium is halted by the sickling of the cells which it infests. Prion mutations[edit] Prions are proteins and do not contain genetic material. However, prion replication has been shown to be subject to mutation and natural selection just like other forms of replication.[76] Somatic mutations[edit] Main article: Loss of heterozygosity See also: Carcinogenesis A change in the genetic structure that is not inherited from a parent, and also not passed to offspring, is called a somatic cell genetic mutation or acquired mutation.[77] Cells with heterozygous mutations (one good copy of gene and one mutated copy) may function normally with the unmutated copy until the good copy has been spontaneously somatically mutated. This kind of mutation happens all the time in living organisms, but it is difficult to measure the rate. Measuring this rate is important in predicting the rate at which people may develop cancer.[78] Point mutations may arise from spontaneous mutations that occur during DNA replication. The rate of mutation may be increased by mutagens. Mutagens can be physical, such as radiation fromUV rays, X-rays or

extreme heat, or chemical (molecules that misplace base pairs or disrupt the helical shape of DNA). Mutagens associated with cancers are often studied to learn about cancer and its prevention.

A Mutation occurs when a DNA gene is damaged or changed in such a way as to alter the genetic message carried by that gene. A Mutagen is an agent of substance that can bring about a permanent alteration to the physical composition of a DNA gene such that the genetic message is changed. Once the gene has been damaged or changed the mRNA transcribed from that gene will now carry an altered message. The polypeptide made by translating the altered mRNA will now contain a different sequence of amino acids. The function of the protein made by folding this polypeptide will probably be changed or

lost. In this example, the enzyme that is catalyzing the production of flower color pigment has been altered in such a way it no longer catalyzes the production of the red pigment. No product (red pigment) is produced by the altered protein. In subtle or very obvious ways, the phenotype of the organism carrying the mutation will be changed. In this case the flower, without the pigment is no longer red. Mutagens Chemical Mutagens change the sequence of bases in a DNA gene in a number of ways;

mimic the correct nucleotide bases in a DNA molecule, but fail to base pair correctly during DNA replication. remove parts of the nucleotide (such as the amino group on adenine), again causing improper base

pairing during DNA replication. add hydrocarbon groups to various nucleotides, also causing incorrect base pairing during DNA replication.

Radiation High energy radiation from a radioactive material or from X-rays is absorbed by the atoms in water molecules surrounding the DNA. This energy is transferred to the electrons which then fly away from the atom. Left behind is a free radical, which is a highly dangerous and highly reactive molecule that attacks the DNA molecule and alters it in many ways. Radiation can also cause double strand breaks in the DNA molecule, which the cell's repair mechanisms cannot put right. Sunlight contains ultraviolet radiation (the component that causes a suntan) which, when absorbed by the DNA causes a cross link to form between certain

adjacent bases. In most normal cases the cells can repair this damage, but unrepaired dimers of this sort cause the replicating system to skip over the mistake leaving a gap, which is supposed to be filled in later. Unprotected exposure to UV radiation by the human skin can cause serious damage and may lead to skin cancer and extensive skin tumors. Spontaneous mutations occur without exposure to any obvious mutagenic agent. Sometimes DNA nucleotides shift without warning to a different chemical form (know as an isomer) which in turn will form a different series of hydrogen bonds with it's partner. This leads to mistakes at the time of DNA replication. Mutagen From Wikipedia, the free encyclopedia In genetics, a mutagen is a physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations above the natural background level. As many

mutations cause cancer, mutagens are therefore also likely to be carcinogens. Not all mutations are caused by mutagens: so-called "spontaneous mutations" occur due to spontaneous hydrolysis, errors in DNA replication, repair and recombination.

Discovery of mutagens[edit] The first mutagens to be identified were carcinogens, substances that were shown to be linked to cancer. Tumors were described more than 2,000 years before the discovery of chromosomes andDNA; in 500 B.C., the Greek physician Hippocrates named tumors resembling a crab karkinos (from which the word "cancer" is derived via Latin), meaning crab.[1] In 1567, Swiss physicianParacelsus suggested that an unidentified substance in mined ore (identified as radon gas in modern times) caused a wasting disease in miners,[2] and in England, in 1761, John Hill made the first direct link of cancer to chemical substances by noting that excessive use of snuff may cause nasal cancer.[3] In 1775, Sir Percivall Pott wrote a paper on the high incidence of scrotal cancer in chimney sweeps, and suggested chimney soot as the cause of scrotal cancer.[4] In 1915, Yamagawa and Ichikawa showed that repeated application of coal tar to rabbit's ears produced malignant cancer.[5] Subsequently in the 1930s the carcinogen component in coal tar was identified as a polyaromatic hydrocarbon (PAH), benzo[a]pyrene.[2][6] Polyaromatic hydrocarbons are also present in soot, which was

suggested to be a causative agent of cancer over 150 years earlier. The mutagenic property of mutagens was first demonstrated in 1927, when Hermann Muller discovered that x-rays can cause genetic mutations in fruit flies, producing phenotypic mutants as well as observable changes to the chromosomes.[7] His collaborator Edgar Altenburg also demonstrated the mutational effect of UV radiation in 1928.[8] Muller went on to use x-rays to createDrosophila mutants that he used in his studies of genetics.[9] He also found that X-rays not only mutate genes in fruit flies but also have effects on the genetic makeup of humans.[10] Similar work by Lewis Stadler also showed the mutational effect of X-ray on barley in 1928,[11] and ultraviolet (UV) radiation on maize in 1936.[12] The effect of sunlight had previously been noted in the nineteenth century where rural outdoor workers and sailors were found to be more prone to skin cancer.[13] Chemical mutagens were not demonstrated to cause mutation until the 1940s, when Charlotte Auerbach and J. M. Robson found that mustard gas can cause mutations in fruit flies.[14] A large number of chemical mutagens have since been identified, especially after the development of the Ames test in 1970s by Bruce Ames that screens for mutagens and allows for preliminary identification of carcinogens.[15][16] Early studies by Ames showed around 90% of known carcinogens can be identified in Ames test as mutagenic (later studies however gave lower figures),[17][18][19] and ~80% of the mutagens identified through Ames test may also be

carcinogens.[19][20] Mutagens are not necessarily carcinogens, and vice versa. Sodium Azide for example may be mutagenic (and highly toxic), but it has not been shown to be carcinogenic.[21] Effects of mutagens[edit] Main article: Mutagenesis Mutagens cause changes to the DNA that can affect the transcription and replication of the DNA, which in severe cases can lead to cell death. The mutagen produces mutations in the DNA, and deleterious mutation can result in aberrant, impaired or loss of function for a particular gene, and accumulation of mutations may lead to cancer. Different mutagens act on the DNA differently. Powerful mutagens may result in chromosomal instability,[22] causing chromosomal breakages and rearrangement of the chromosomes such astranslocation, deletion, and inversion. Such mutagens are called clastogens. Mutagens may also modify the DNA sequence; the changes in nucleic acid sequences by mutations include substitution of nucleotide basepairs and insertions and deletions of one or more nucleotides in DNA sequences. Although some of these mutations are lethal or cause serious disease, many have minor effects as they do not result in residue changes that have significant effect on the structure and function of the proteins. Many mutations are silent mutations, causing no visible effects at all, either because they occur in noncoding or non-functional sequences, or they do not

change the amino-acid sequence due to the redundancy of codons. Some mutagens can cause aneuploidy and change the number of chromosomes in the cell. In Ames test, where the varying concentrations of the chemical are used in the test, the dose response curve obtained is nearly always linear, suggesting that there is no threshold for mutagenesis. Similar results are also obtained in studies with radiations, indicating that there may be no safe threshold for mutagens. However, some proposed that low level of some mutagens may stimulate the DNA repair processes and therefore may not necessarily be harmful. Types of mutagens[edit] Mutagens may be of physical, chemical or biological origin. They may act directly on the DNA, causing direct damage to the DNA, and most often result in replication error. Some however may act on the replication mechanism and chromosomal partition. Many mutagens are not mutagenic by themselves, but can form mutagenic metabolites through cellular processes. Such mutagens are called promutagens. Physical mutagens[edit]

Ionizing radiations such as X-rays, gamma rays and alpha particles may cause DNA breakage and other damages. The most common sources include cobalt-60 and cesium-137.

Ultraviolet radiations with wavelength above 260 nm are absorbed strongly by bases, producing pyrimidine dimers, which can cause error in replication if left uncorrected. 14 Radioactive decay, such as C in DNA which decays into nitrogen. DNA reactive chemicals[edit]

A DNA adduct (at center) ofbenzo[a]pyrene, the major mutagen intobacco smoke. A large number of chemicals may interact directly with DNA. However, many such as PAHs, aromatic amines, benzene are not necessarily mutagenic by themselves, but through metabolic processes in cells they produce mutagenic compounds.

Reactive oxygen species (ROS) - These may be superoxide, hydroxyl radicals and hydrogen peroxide, and large number of these highly reactive species are generated by normal cellular processes, for

example as a by-products of mitochondrial electron transport, or lipid peroxidation. A number of mutagens may also generate these ROS. These ROS may result in the production of many base adducts, as well as DNA strand breaks and crosslinks. Deaminating agents, for example nitrous acid which can cause transition mutations by converting cytosine to uracil. Polycyclic aromatic hydrocarbon (PAH), when activated to diol-epoxides can bind to DNA and form adducts. Alkylating agents such as ethylnitrosourea. The compounds transfer methyl or ethyl group to bases or the backbone phosphate groups. Guanine when alkylated may be mispaired with thymine. Some may cause DNA crosslinking and breakages. Nitrosamines are an important group of mutagens found in tobacco, and may also be formed in smoked meats and fish via the interaction of amines in food with nitrites added as preservatives. Other alkylating agents include mustard gas and vinyl chloride. Aromatic amines and amides have been associated with carcinogenesis since 1895 when German physician Ludwig Rehn observed high incidence of bladder cancer among workers in German synthetic aromatic amine dye industry. 2-Acetylaminofluorene, originally used as a pesticide but may also be found in cooked meat, may cause cancer of the bladder, liver, ear, intestine, thyroid and breast. Alkaloid from plants, such as those from Vinca species[citation needed], may be converted by

metabolic processes into the active mutagen or carcinogen. Bromine and some compounds that contain bromine in their chemical structure. Sodium azide, an azide salt that is a common reagent in organic synthesis and a component in many car airbag systems Psoralen combined with ultraviolet radiation causes DNA cross-linking and hence chromosome breakage. Benzene, an industrial solvent and precursor in the production of drugs, plastics, synthetic rubber and dyes. Base analogs[edit] Base analog, which can substitute for DNA bases during replication and cause transition mutations. Intercalating agents[edit]

Intercalating agents, such as ethidium bromide and proflavine, are molecules that may insert between bases in DNA, causing frameshift mutation during replication. Some such asdaunorubicin may block transcription and replication, making them highly toxic to proliferating cells. Metals[edit] Many metals, such as arsenic, cadmium, chromium, nickel and their compounds may be mutagenic, they may however act via a number of different mechanisms.[23] Arsenic, chromium, iron, and nickel may be associated with the production of ROS, and some of these may also alter the fidelity of DNA replication. Nickel may also be linked to DNA

hypermethylation and histonedeacetylation, while some metals such as cobalt, arsenic, nickel and cadmium may also affect DNA repair processes such as DNA mismatch repair, and base and nucleotide excision repair.[24] Biological agents[edit] Transposon, a section of DNA that undergoes autonomous fragment relocation/multiplication. Its insertion into chromosomal DNA disrupt functional elements of the genes. Virus - Virus DNA may be inserted into the genome and disrupts genetic function. Infectious agents have been suggested to cause cancer as early as 1908 by Vilhelm Ellermann and Oluf Bang,[25] and 1911 by Peyton Rous who discovered the Rous sarcoma virus.[26] Bacteria - some bacteria such as Helicobacter pylori cause inflammation during which oxidative species are produced, causing DNA damage and reducing efficiency of DNA repair systems, thereby increasing mutation. Protection against mutagens[edit]

Fruits and vegetables are rich in antioxidants.

Antioxidants are an important group of anticarcinogenic compounds that may help remove ROS or potentially harmful chemicals. These may be found naturally in fruits and vegetables.[27] Examples of antioxidants are vitamin A and its carotenoid precursors, vitamin C, vitamin E, polyphenols, and various other compounds. Carotene is the red-orange colored compounds found in vegetables like carrots and tomatoes. Vitamin C may prevent some cancers by inhibiting the formation of mutagenic N-nitroso compounds (nitrosamine). Flavonoids, such as EGCG in green tea, have also been shown to be effective antioxidants and may have anti-cancer properties. Epidemiological studies indicate that a diet rich in fruits and vegetables is associated with lower incidence of some cancers and longer life expectancy,[28] however, the effectiveness of antioxidant supplements in cancer prevention in general is still the subject of some debate.[28][29] Other chemicals may reduce mutagenesis or prevent cancer via other mechanisms, although for some the precise mechanism for their protective property may not be certain. Selenium, which is present as a micronutrient in vegetables, is a component of important antioxidant enzymes such as gluthathione peroxidase. Many phytonutrients may counter the effect of mutagens; for example, sulforaphane in vegetables such as broccoli has been shown to be protective against prostate cancer.[30] Others that may be effective against cancer include indole-3-carbinol from cruciferous vegetables andresveratrol from red wine.[31]

An effective precautionary measure an individual can undertake to protect themselves is by limiting exposure to mutagens such as UV radiations and tobacco smoke. In Australia, where people with pale skin are often exposed to strong sunlight, melanoma is the most common cancer diagnosed in people aged 1544 years.[32][33] In 1981, human epidemiological analysis by Richard Doll and Richard Peto indicated that smoking caused 30% of cancers in the US.[34] Doll and Peto also estimated that diet may cause around 35% of cancers. Mutagens identified in food include mycotoxins from food contaminated with fungal growths, such as aflatoxins which may be present in contaminated peanuts (prevalent in Southern China) and corn; heterocyclic amines generated in meat when cooked at high temperature; PAHs in charred meat and smoked fish, as well as in oils, fats, bread, and cereal;[35] and nitrosamines generated from nitrites used as food preservatives in cured meat such as bacon (ascobate, which is added to cured meat, however, reduces nitrosamine formation).[27] Excessivealcohol consumption has also been linked to cancer; the possible mechanisms for its carcinogenicity include formation of the possible mutagen acetaldehyde, and the induction of the cytochrome P450 system which is known to produce mutagenic compounds from promutagens.[36] For certain mutagens, such as dangerous chemicals and radiations, as well as infectious agents known to cause cancer, government legislations and regulatory bodies are necessary for their control.

Molecular Mechanisms of Spontaneous Mutations O. L. Klamerth Institut ffir Anthropologie und Humangenetik der Universit~t, Heidelberg The molecular mechanisms leading to spontaneous mutations have so far not yet been clarified experimentally. We mns~ point out that the concept of "spontaneous mutation" is in itself problematic, for we have no conclusive evidence as to whether these genetic changes are in fact spontaneous, in other words, whether they follow the statistical rules of reduplication errors, or whether they are brought about by the influence of cell metabolism, the absence or alteration of enzymes such as polymerases, of mutagenic substances, or by the environment as such. It might be possible, for instance, that under certain circumstances changes in temperature or ionic strength may activate or inactivate important enzymes or even convert one isozymic form to another. Peroxides or intermediately formed

radicals or methylating enzymes might attack nucleic acid bases in a manner which resembles the action of radiation or alkyating agents. The small content of UV rays in ordinary hght might also play a role in spontaneous mutations, at least in bacteria at the so-called hot spots. Here the well-known formation of thymidine dimers, or in small amounts of cytosine dimers, followed by deamination to uracil, should be mentioned. In addition it is conceivable that the very small amount of beta or gamma rays from isotopes incorporated into the cell might be important. This hypothesis is supported by the fact that mutation frequency increases with the time of storage even when no nucleic acid metabohsm is observed, as is the ease with viruses, phage, or resting bacteria. It is perhaps

worth mentioning that, for example, thyminehydroxyhydroperoxide formed after irradiation of nucleic acids remains attached as such to the nueleotide chain. Reduplication or transcription of DNA with such an altered base could probably not proceed normally. Finally the integration into the genome of the cell of some as yet unknown viruses could be the cause of mutation. We must always take into consideration that the initiation of mutation can occur in nondividing cells. Re/erence8 Freese, E.: Molecular Mechanism of Mutations. In: Molecular Genetics, g. Herbert Taylor, Ed., Part I, p. 207 ft. New York-London: Academic Press 1963. Kaplan, R. W.: Molekulare Mechanismen der Mutationsprozesse. In: Molekularbiologie, T. Wieland and G. Pfleiderer, Eds., 13. 79 ft. Frankfurt: UmsehauVerlag 1967.

Van Peenen, H. J. : Pathology of Protein Structure and Function. In: Biochemical Genetics, H. van Peenen, Ed., p. 194 ft. Springfield (Ill.): Thomas 1966. Weiss, J. g. : Effects of Radiation on Nucleic Acid. In: Progress in Nucleic Acid Research and Molecular Biology, J. N. Davidson and W. E. Co]an (Eds.), Vol. III, p, 135. New YorkLondon: Academic Press 1964. Priv.-Doz. Dr. Olaf L. Klamerth Institut fiir Anthropologie und Humangenetik der Universit[t D-6900 Heidelberg, MSnchhofstral]e 15A Germany 60 Discussion Discussion Vogel: All models of mispairing of bases during replication only lead to transitions. Is there any other system which would lead to transversions ? This is needed, because we do know that many transversions occur spontaneously. Winkler: It has been shown by physical means that A forms a complex with A and T

forms a complex with T. However, the mean lifetime is about 10 -6, compared to the mean lifetime of 10 -6 of an A -- T complex. The other possible explanation of the origin of transversions is that they arise by unequal crossing-over. Recently, it was found that there exist hot spots for frameshfft mutations. These were partly identified as repeating units. As a consequence of frameshffts, it is possible that transversions arise. Kaplan: The work on base pairing is done with free nucleotides and free bases. Is mispairing still possible when the DNA helix is formed ? Klamerth: It is possible because the variations of the angles of the bindings are very small. Furthermore, the backbone of the DNA helix is flexible in some way and it can stand small deviatious at normal temperature, because the distances also alter when the angles change.

Site-directed mutagenesis From Wikipedia, the free encyclopedia Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotidedirected mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering. With decreasing costs of oligonucleotide synthesis, artificial gene synthesis is now occasionally used as an alternative to site-directed mutagenesis.

History[edit] Early attempts at mutagenesis were non-site specific using radiation or chemical mutagens.[1] Analogs of nucleotides and other chemicals were later used to generate localized point mutations,[2]examples of such chemicals are aminopurine,[3] nitrosoguanidine,[4] and bisulfite.[5] Sitedirected mutagenesis was achieved in 1973 in the laboratory of Charles Weissmann using a nucleotide analogue N4-hydroxycytidine which induces transition of GC to AT.[6][7] These methods of mutagenesis however are limited by the kind of mutation they can achieve and they are not as specific as later site-directed mutagenesis methods. In 1971, Clyde Hutchison and Marshall Edgell showed that it is possible to produce mutants with small fragments of phage X174 and restriction nucleases.[8][9] Hutchison later produced with his collaborator Michael Smith in 1978 a more flexible approach to site-directed mutagenesis by using oligonucleotides in a primer extension method with DNA polymerase.[10] For his part in the development of this process, Michael Smith later shared the Nobel Prize in Chemistry in October 1993 with Kary B. Mullis, who invented polymerase chain reaction. Basic mechanism[edit] The basic procedure requires the synthesis of a short DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene

of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion or insertion. The single-stranded primer is then extended using a DNA polymerase, which copies the rest of the gene. The gene thus copied contains the mutated site, and is then introduced into a host cell as a vector and cloned. Finally, mutants are selected. The original method using single-primer extension was inefficient due to a lower yield of mutants. The resulting mixture contains both the original unmutated template as well as the mutant strand, producing a mix population of mutant and non-mutant progenies. The mutants may also be counter-selected due to presence of mismatch repair system which favors the methylated template DNA, resulting in fewer mutants. Many approaches have since been developed to improve the efficiency of mutagenesis. Approaches in site-directed mutagenesis[edit] A large number of methods are available to effect sitedirected mutagenesis,[11] although most of them are now rarely used in laboratories since the early 2000s as newer techniques allow for simpler and easier ways of introducing site-specific mutation into genes. Kunkel's method[edit] In 1987 Thomas Kunkel introduced a technique which reduces the need to select for the mutants.[12] The DNA fragment to be mutated is inserted into a phagemid such as M13mp18/19 and is then transformed into an E. coli strain deficient in two enzymes, dUTPase (dut) and uracil deglycosidase (ung). Both enzymes are part of

a DNA repair pathway that protects the bacterial chromosome from mutations by the spontaneous deamination of dCTP to dUTP. The dUTPase deficiency prevents the breakdown of dUTP, resulting in a high level of dUTP in the cell. The uracil deglycosidase deficiency prevents the removal of uracil from newly synthesized DNA. As the double-mutant E. coli replicates the phage DNA, its enzymatic machinery may therefore misincorporate dUTP instead of dTTP, resulting in single stranded DNA which contains some uracils (ssUDNA). The ssUDNA is extracted from the bacteriophage that is released into the medium, and then used as template for mutagenesis. An oligonucleotide containing the desired mutation is used for primer extension. The heteroduplex DNA that forms consists of one parental non-mutated strand containing dUTP and a mutated strand containing dTTP. The DNA is then transformed into an E. coli strain carrying the wildtype dut and ung genes. Here, the uracilcontaining parental DNA strand is degraded, so that nearly all of the resulting DNA consists of the mutated strand. Cassette mutagenesis[edit] Unlike other methods, cassette mutagenesis need not involve primer extension using DNA polymerase. In this method, a fragment of DNA is synthesized, and then inserted into a plasmid.[13] It involves the cleavage by a restriction enzyme at a site in the plasmid and subsequent ligation of a pair of complementary oligonucleotides containing the mutation in the gene of interest to the plasmid. Usually the restriction enzymes that cuts at the plasmid and the oligonucleotide are the

same, permitting sticky ends of the plasmid and insert to ligate to one another. This method can generate mutants at close to 100% efficiency, but is limited by the availability of suitable restriction sites flanking the site that is to be mutated. PCR site-directed mutagenesis[edit] The limitation of restriction sites in cassette mutagenesis may be overcome using polymerase chain reaction with oligonucleotide "primers", such that a larger fragment may be generated covering two convenient restriction sites. The exponential amplification in PCR produces a fragment containing the desired mutation in sufficient quantity to be separate from the original, unmutated plasmid by gel electrophoresis, which may then be inserted in the original context using standard recombinant molecular biology techniques. There are many variations of the same technique. The simplest method places the mutation site towards one of the ends of the fragment whereby one of two oligonucleotides used for generating the fragment contains the mutation. This involves a single step of PCR, but still has the inherent problem of requiring a suitable restriction site near the mutation site unless a very long primer is used. Other variations therefore employ three or four oligionucleotides, two of which may be non-mutagenic oligonucleotides that cover two convenient restriction sites and generate a fragment that can be digested and ligated into a plasmid, while the mutagenic oligonucleotide may be complementary to a location within that fragment well away from any convenient restriction site. These methods

require multiple steps of PCR so that the final fragment to be ligated can contain the desired mutation. Whole plasmid mutagenesis[edit] For plasmid manipulations, other site-directed mutagenesis techniques have been largely supplanted by techniques which are highly efficient but relatively simple, easy to use, and commercially available as a kit. An example of these techniques is the Quikchange method,[14] where a pair of complementary mutagenic primers are used to amplify the entire plasmid in a thermocyclingreaction using a high-fidelity non-stranddisplacing DNA polymerase such as pfu polymerase. The reaction generates a nicked, circular DNA which is relaxed. The template DNA must be eliminated by enzymatic digestion with a restriction enzyme such as DpnI which is specific for methylated DNA. All DNA produced from most Escherichia coli strains would be methylated; the template plasmid which is biosynthesized in E. coli will therefore be digested, while the mutated plasmid, which is generated in vitro and is therefore unmethylated, would be left undigested. Note that in these double-stranded plasmid mutagenesis methods, while the thermocycling reaction may be used, the DNA need not be exponentially amplified as in a PCR, instead the amplification is linear, and it is therefore inaccurate to describe them as a PCR reaction since there is no chain reaction. In some applications this method has been observed to lead to insertion of multiple copies of primers.[15] A variation of this method, called SPRINP, prevents this

artifact and has been used in different types of site directed mutagenesis.[15] In vivo site-directed mutagenesis methods[edit] Delitto perfetto[16] Transplacement "pop-in pop-out" Direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker Direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker using long homologous regions In vivo site-directed mutagenesis with synthetic oligonucleotides[17] Applications[edit]

Site-directed mutagenesis is used to generate mutations that may produce rationally designed protein that has improved or special properties. Investigative tools - specific mutations in DNA allow the function and properties of a DNA sequence or a protein to be investigated in a rational approach. Commercial applications - proteins may be engineered to produce proteins that are tailored for a specific application. For example, commonly used laundry detergents may contain subtilisinwhose wild-type form has a methionine that can be oxidized by bleach, inactivating the protein in the process. This methionine may be replaced by alanine, thereby making the protein active in the presence of bleach.

Separation and detection[edit source] In separation and detection DNA and mRNA are isolated from cells (the separation) and then detected simply by the isolation. Cell cultures are also grown to provide a constant supply of cells ready for isolation. Cell cultures[edit source] A cell culture for molecular genetics is a culture that is grown in artificial conditions. Some cell types grow well in cultures such a skin cells, but other cells are not as productive in cultures. There are different techniques for each type of cell, some only recently being found to foster growth in stem and nerve cells. Cultures for molecular genetics are frozen in order to preserve all copies of the gene specimen and thawed only when needed. This allows for a steady supply of cells. DNA isolation[edit source] DNA isolation extracts DNA from a cell in a pure form. First, the DNA is separated from cellular components such as proteins, RNA, and lipids. This is done by placing the chosen cells in a tube with a solution that mechanically, chemically, breaks the cells open. This solution contains enzymes, chemicals, and salts that breaks down the cells except for the DNA. It contains enzymes to dissolve proteins, chemicals to destroy all RNA present, and salts to help pull DNA out of the solution. Next, the DNA is separated from the solution by being spun in a centrifuge, which allows the DNA to collect in the bottom of the tube. After this cycle in the centrifuge the

solution is poured off and the DNA is resuspended in a second solution that makes the DNA easy to work with in the future. This results in a concentrated DNA sample that contains thousands of copies of each gene. For large scale projects such as sequencing the human genome, all this work is done by robots. mRNA isolation[edit source] Expressed DNA that codes for the synthesis of a protein is the final goal for scientists and this expressed DNA is obtained by isolating mRNA (Messenger RNA). First, laboratories use a normal cellular modification of mRNA that adds up to 200 adenine nucleotides to the end of the molecule (poly(A) tail). Once this has been added, the cell is ruptured and its cell contents are exposed to synthetic beads that are coated with thymine string nucleotides. Because Adenine and Thymine pair together in DNA, the poly(A) tail and synthetic beads are attracted to one another, and once they bind in this process the cell components can be washed away without removing the mRNA. Once the mRNA has been isolated, reverse transcriptase is employed to convert it to single-stranded DNA, from which a stable double-stranded DNA is produced using DNA polymerase. Complementary DNA (cDNA) is much more stable than mRNA and so, once the double-stranded DNA has been produced it represents the expressed DNA sequence scientists look for.[4] The Human Genome Project[edit source]

The Human Genome Project is a molecular genetics project that began in the 1990s and was projected to take fifteen years to complete. However, because of technological advances the progress of the project was advanced and the project finished in 2003, taking only thirteen years. The project was started by the U.S. Department of Energy and the National Institutes of Health in an effort to reach six set goals. These goals included: 1. identifying 20,000 to 25,000 genes in human DNA (although initial estimates were approximately 100,000 genes), 2. determining sequences of chemical base pairs in human DNA, 3. storing all found information into databases, 4. improving the tools used for data analysis, 5. transferring technologies to private sectors, and 6. addressing the ethical, legal, and social issues (ELSI) that may arise from the projects.[5] The project was worked on by eighteen different countries including the United States, Japan, France, Germany, and the United Kingdom. The collaborative effort resulted in the discovery of the many benefits of molecular genetics. Discoveries such as molecular medicine, new energy sources and environmental applications, DNA forensics, and livestock breeding, are only a few of the benefits that molecular genetics can provide.

Section 8.2Isolation and Analysis of Mutants Many different types of mutants have been identified in organisms ranging from bacteria to humans. Mutants can differ from their normal counterparts in a variety of ways. Some mutations cause only subtle changes; for example, certain mutations in the fruit fly,Drosophila melanogaster, result in failure of a single type of neuronal cell to develop, but mutant flies otherwise are normal. Other mutations lead to significant changes in development, cellular function, appearance (Figure 8-8), and behavior of an individual. Many mutations are nonlethal, but some result in organismal death.

Figure 8-8 Mutants in Drosophila.. (a) White-eyed mutants lack the pigment that is present in the normal bright red eyes of fruit flies. The white mutant, the first known Drosophila mutant, was identified in (more...) The procedures used to identify and isolate mutants, referred to as genetic screens, depend on whether the experimental organism is haploid or diploid and, if the latter, whether the mutation is recessive or dominant. Usually, mutations are induced by treatment with a mutagen, and the mutagenized population subjected to a genetic screen designed to identify and isolate individuals with mutations affecting a particular process of interest. Genes that encode proteins essential for life are among the most interesting and important ones to study.

Since phenotypic expression of mutations in essential genes leads to death of the individual, ingenious screens are needed to isolate and maintain organisms with a lethal mutation. Characterization of mutants in a variety of experimental organisms has been used to investigate many different fundamental biological processes. Genetic analyses of mutants defective in a particular process can reveal: (a) the number of genes required for the process to occur; (b) the order in which gene products act in the process; and (c) whether the proteins encoded by different genes interact with one another. Genetic studies of this type have helped unravel various metabolic pathways, regulatory mechanisms, and developmental processes. Go to: Temperature-Sensitive Screens Can Isolate Lethal Mutations in Haploids In haploid organisms (e.g., prokaryotes and yeast), all mutations are in effect dominant so that the mutant phenotype is exhibited immediately in the progeny of the mutagenized population. For instance, mutations that disrupt arginine synthesis lead to cells that require arginine for growth. Such mutations are easily detected by growing mutagenized populations in the presence and absence of arginine (see Figure 6-2). In prokaryotes and haploid eukaryotes such as yeast, essential genes can be studied through the use of conditional mutations. For instance, a mutant protein may be fully functional at 30 C but

completely inactive at 37 C, whereas the normal protein would be fully functional at both temperatures. A temperature at which the mutant phenotype is observed is called nonpermissive; apermissive temperature is one at which the phenotype is not observed. Mutant strains can be maintained at a permissive temperature; then, for analysis, a subculture can be set up at a nonpermissive temperature. Such temperature-sensitive (ts) mutants can also be generated in Drosophila and C. elegans, but cannot be isolated in warm-blooded animals. An example of a particularly important temperaturesensitive screen in the yeast S. cerevisiae comes from the studies of L. H. Hartwell and colleagues in the late 1960s and early 1970s. They set out to identify genes important in regulation of the cell cycle. Cell division in this yeast occurs through a budding process, and the size of the bud, which is easily visualized by light microscopy, is an indication of the cells position in the cell cycle. In these studies, the researchers first identified mutagenized yeast cells that did not grow at 36 C (Figure 8-9a). Then they used video microscopy to analyze the identified mutants for cell-division defects at the nonpermissive temperature (Figure 8- 9b). These yeast mutants were not simply slow growing as they might be if they carried amutation affecting general cellular metabolism; rather, they grew normally but showed a stage-specific block in growth at the nonpermissive temperature. The cell-cycle stage at which cell growth was arrested at the nonpermissive temperature indicated when the protein encoded by the

mutated gene was required. Cloning and analysis of various genes defined by cell-cycle mutations are described in detail in Chapter 13. This work has provided important insights about the regulation of cell division in organisms ranging from yeast to humans.

Figure 8-9 Two-step genetic screen used to identify cell-cycle mutants in yeast. (a) Yeast cells were grown in a large liquid culture, treated with a chemical mutagen, and then subcultured into smaller (more...) Go to: Recessive Lethal Mutations in Diploids Can Be Screened by Use of Visible Markers In diploid organisms, phenotypes resulting from recessive mutations can be observed only in individuals homozygous for the mutant alleles. Figure 810 outlines a procedure for inducing, identifying, and maintaining recessive lethal mutations in Drosophila, a diploid organism. Male fruit flies are treated with a mutagen and then mated with females, yielding F1 progeny that are heterozygous for any induced mutations. Because these mutations are recessive, the mutant phenotype is not observed in the F1 generation, and two additional crosses are needed to reveal the mutant phenotype. By using fly strains carrying known mutations (called markers) that give rise to visible phenotypes, researchers can distinguish heterozygous

F2 progeny carrying one mutagenized chromosomeand one normal chromosome from siblings with other genotypes. Mating of these F2 heterozygous siblings produces an F3generation in which one-fourth of the flies will be homozygous for any mutation induced on the mutagenized chromosome, and if the mutation is in a gene essential for viability, they will not survive; onefourth will be homozygous for the normal allele; and half will be heterozygous. The effects of the mutation can then be assessed in the homozygous class that does not survive, and the mutation can be maintained in the flies that are heterozygous for the mutation.

Figure 8-10 Procedure used to identify and maintain recessive lethal mutations on chromosome 3 (an autosome) in Drosophila, a diploid organism. This approach requires three sequential crosses. First, (more...) Current understanding of the molecular mechanisms regulating development of multicellular organisms is based, in large part, on this type of genetic screen in Drosophila. C. Nsslein-Volhard, E. Wiechaus, and their colleagues systematically screened forrecessive lethal mutations affecting embryogenesis in Drosophila using a scheme similar to that shown in Figure 8-10. Deadhomozygous embryos carrying lethal recessive mutations identified by this screen were analyzed for specific defects in the cuticular structures on the embryo surface (Chapter 14). A detailed picture of embryonic

development has emerged from the characterization of these defects and the analysis of both the structure of the encoded proteins and their patterns of expressionduring embryogenesis. We will discuss some of the fundamental discoveries based on these genetic studies in Chapters 14 and 23. Go to: Complementation Analysis Determines If Different Mutations Are in the Same Gene A common type of genetic analysis can reveal whether different recessive mutations associated with the same phenotype are in the same gene or in different genes. This analysis depends on the phenomenon of genetic complementation, that is, the restoration of the wild-type phenotype by mating of two different mutants. If two mutations, A and B, are in the same gene, then a diploidorganism heterozygous for both mutations (i.e., carrying one A allele and one B allele) will exhibit the mutant phenotype. In contrast, if mutation A and B are in separate genes, then heterozygotes carrying a single copy of each mutant allele will exhibit the wild-type (normal) phenotype. In this case, the mutations are said to complement each other. Complementation analysis of a set of mutants exhibiting the same phenotype can distinguish the individual genes in a set of functionally related genes, all of which must function to produce a given phenotypic trait. In the yeast S. cerevisiae, for example, four enzymes are required for growth on galactose (Figure 8-11a). If any one

of these enzymes is absent or defective, yeast cells cannot grow on galactose. Because haploid yeast cells exist in one of two different mating types, a or , which can be mated to yield a/ diploids, yeast can be subjected to complementation analysis like other diploid organisms. Figure 8-11b illustrates complementation analysis of Gal yeast strains defective for growth on galactose. When Gal strains with mutations in differentGAL genes are mated, the resulting diploid cells will grow on galactose, because the wild-type gene in each strain will compensate for the genetic defect in the other. In contrast, diploids formed from Gal strains that are mutated in the same gene will not grow on galactose.

Figure 8-11 Complementation analysis in S. cerevisiae.. (a) Pathway used by yeast cells to metabolize galactose to glucose, which then enters the glycolytic pathway. Yeast cells must produce (more...) Go to: Metabolic and Other Pathways Can Be Genetically Dissected Various types of analysis can order the genes involved in biochemical pathways and other cellular processes. A fairly straightforward example involves the genetic dissection of the biochemical pathway for synthesis of arginine in the bread moldNeurospora crassa. Four different mutant strains that are unable to synthesize

arginine and require arginine for growth (calledarginine auxotrophs) were identified years ago. Each of the steps in biosynthesis of arginine is catalyzed by an enzyme encoded by a separate gene. The order of action of the different genes, hence the order of the biochemical reactions in the pathway, was determined by assessing which mutants could grow on different intermediates (Figure 8-12). Numerous biochemical pathways have been dissected by this type of study.

Figure 8-12 Genetic dissection of the arginine biosynthetic pathway. Mutants in the bread mold N. crassa that required arginine for growth were identified many years ago; analysis of these mutants provided (more...) Other types of cellular processes also are amenable to genetic analysis. For example, the maturation pathway for secretory proteins in yeast has been dissected and ordered by analysis of a set of conditional temperaturesensitive secretion-defective (sec)mutants. In these mutant strains, the secretion of all proteins is blocked at the higher (nonpermissive) temperature but is normal at the lower (permissive) temperature. At the higher temperature, sec mutants accumulate proteins in the rough endoplasmic reticulum (ER), Golgi complex, or secretory vesicles (see Figure 17-14). At least 60 gene products are required to complete the maturation pathway as defined by the number of genes in which mutations give rise to a secretion defect. The genes can

be ordered in a pathway by analyzing double-mutant combinations of sec genes. For instance, when ER and Golgi accumulating mutants are combined, proteins accumulate in the ER. These types of studies have shown that the pathway must be ordered in the following sequence: rough ER Golgi secretory vesicles. This maturation pathway is believed to apply to all secretory proteins in all eukaryotic organisms, including plants. Go to: Suppressor Mutations Can Identify Genes Encoding Interacting Proteins The phenomenon of genetic suppression can be used to identify proteins that specifically interact with one another in the living cell. The underlying logic is as follows: point mutations may lead to structural changes in protein A that disrupt its ability to associate with another protein (protein B) involved in the same cellular process. Similarly, mutations in protein B might lead to small structural changes that would inhibit its ability to interact with protein A. In rare cases small structural changes in protein A may be suppressed by compensatory changes in protein B. In these rare cases, strains carrying a specific mutant allele of protein A or B would be mutant, but strains carrying both would be normal. This is analogous to changes made in a lock and key. Identification of such suppressor mutations has been elegantly applied in studies of the cytoskeletal protein actin in yeast (Figure 8-13). A strain of yeast that was temperature-sensitive for growth and

carried a mutant actin allele called act1-1 was plated at the nonpermissive temperature. A few cells were capable of growth at this temperature; these revertants were shown to have a secondmutation in another gene, called SAC6, that allowed the act1-1 mutants to grow. This sac6 mutation acted as a dominantsuppressor of the act1-1 mutation, so that the double mutants (act1-1 sac6) exhibited the wild-type phenotype. This suppression was found to be allele-specific; that is, the sac6 mutation suppressed the act1-1 mutation but not other act1 mutations. Single mutants carrying any one of several different sac6 mutations were, like act1-1 mutants, temperature-sensitive for growth. Remarkably, some act1 mutations were found to be dominant suppressors of the recessive temperaturesensitive lethality of varioussac6 mutations.

Figure 8-13 Genetic suppression in yeast involving temperaturesensitive (Ts) mutations in the actin gene (ACT1) and in the SAC6 gene, which encodes an (more...) In summary, then, mutations in either the SAC6 or ACT1 gene confer the same recessive lethal phenotype, and specific lethal alleles of each gene can act as dominant suppressors of specific lethal alleles of the other gene, resulting in a viable organism. This reciprocal suppression argues strongly for a direct interaction in vivo between the proteins encoded by the two genes (seeFigure 8-

13, bottom). Indeed, biochemical studies have shown that these two proteins ACT1 and SAC6 do interact, and immunolocalization studies indicate that the two proteins are present in the same part of the cell. Gradient Plate Technique:
An excellent way to determine the ability of organisms to produce mutants that are resistant to antibiotic is to grow them on a gradient plate of a particular antibiotic. The gradient plate consists of two wedges like layers of media: a bottom layer of plain nutrient agar and top layer of antibiotic with nutrient agar. The antibiotic in the top layer, diffuse into the bottom layer producing a gradient of antibiotic concentration from low to high. A gradient plate is made by using Streptomycin in the medium. E. coli, which is normally sensitive to Streptomycin, will be spread over the surface of the plate and incubated for 24 to 72 hours. After incubation colonies will appear on both the gradients. The colonies develop in the high concentration are resistant to the action of Streptomycin, and are considered as Streptomycin resistant mutants. For isolation of antibiotic resistant of gram negative enteric bacteria, the antibiotics commonly used are Rifampicin, Streptomycin, and Erythromycin etc.

Fig 1: Gradient plate preparation steps

Replica Plating Method:

If an organism has the ability to produce mutant strains resistant to antibiotics, the nature of mutation, whether it is spontaneous or induced have to be tested. It would be a difficult task to identify a few mutant colonies from a vast population of 100-500 colonies. This can be

accomplished by a replica plating technique. The technique was developed by Joshua and Esther Lederberg in 1952 for providing the direct evidence for the existence of pre-existing mutations. This technique isolates both nutritional mutants and antibiotic resistant mutants. Their actual experiment concerned with replicating master plates of sensitive cells to two or more plates containing either streptomycin or T1 phage. Replica plating allows the observation of microbes under a series of growth conditions. The bacteria are grown in an environment that is not selective for given mutation. This technique is used to transfer the members of each colony to a selective environment. A simple velveteen covered colony transfer device is used to transfer the colonies in nutrient agar medium supplemented with or without a particular antibiotic or nutrient. The fibers of velvet act as fine inoculating needles, picking up the bacterial cells from the surface of this master plate. The velvet with its attached microbes is then touched to the surface of a sterile agar plate, inoculating it. In this manner, microbes can be repeatedly stamped onto media of differing composition. By comparing the presence of colonies following incubation we can indirectly determine the mutant colonies by their absence in the selective environment. A colony that develops on a complete medium fail to develop on a minimal medium that lacks a specific growth factor, the occurrence of a nutritional mutant is indicated. The microbes that do not grow on the minimal medium represent auxotrophic strains. A simple velveteen covered colony transfer device is used to transfer the colonies in nutrient agar medium supplemented with or without a particular antibiotic or nutrient. A colony that develops on a complete medium

fail to develop on a minimal medium that lacks a specific growth factor, the occurrence of a nutritional mutant is indicated. The microbes that do not grow on the minimal medium represent auxotrophic strains. This method has been applied in numerous experiments to identify the occurrence of mutations. Many of the biochemical pathways in microbes were elucidated in this way by using nutritional mutants (Fig 2).

Fig 2: Replica plating technique

Screening is a method of mutation isolation where two cultures are compared to see which colonies grew on enriched media, but not on minimal. Replica plating is a method of screening to isolate mutant auxotrophs from wild-type bacteria. First, the master plate is imprinted upon a velveteen-covered wood block. Then, using the block+velveteen, replicas of the original plate are made, one on enriched media, and one on minimal media (lacking the factor that the auxotrophs need to grow). When incubated, the plates should grow with colonies at the same places; the spots that are missing on the minimal media correspond to auxotrophic colonies on the enriched media, and thus the auxotrophs can be identified. Enrichment is an isolation technique that involves streaking on a plate that has minimal media (so auxotrophs cant

grow) and adding a bacteriocidal agent that is only effective against dividing bacteria (such as penicillin). The prototrophs will begin growing, and suicide themselves upon the penicillin, while the auxotrophs will stay dormant because they are lacking the nutrient they need to start growing. Restreaking whats left on the plate onto enriched media should let only the auxotrophs that survived the penicillin suicide selection to grow.

Selection is an isolation technique that checks for mutations that allow the bacteria to grow in adverse conditions (notably, in the presence of antibiotics). By plating the bacteria upon a plate with nalidixic acid or Rifampacin, the bacteria can be checked for mutations in gyrase or B-RNA polymerase, respectively, that allow them to resist the effects of the antibiotics.

Scoring is the usage of (usually) a chromogenic substrate, degraded by an enzyme produced by the bacterium. It allows detection of the presence of active enzyme; conversely, if there is a mutation that inactivates the enzyme, it can be identified. In our lab, we used blue/white scoring with IPTG/X-Gal. X-Gal is a chromogenic substrate that is cleaved by B-galactosidase, giving a characteristic blue color. Because our experiment focuses on interrupting the lac Z a fragment, the enzyme produced will not be functional, and thus we check the white (no active Bgalactosidase) colonies for transformation.

Go to: SUMMARY

Treating experimental organisms with mutagens can produce mutations disrupting organismal function, development, and viability. With appropriate genetic screens, lethal mutations can be isolated and maintained in both haploid organisms (seeFigure 8-9a) and diploid organisms (see Figure 8-10). The number of functionally related genes involved in a process can be defined by complementation analysis (seeFigure 8-11). The order in which their encoded proteins act can be deduced from other types of genetic analysis. The identification of allele-specific suppressor mutations in two genes suggests that the encoded proteins interact in vivo.

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Transposons are segments of DNA that can move around to different positions in the genome of a single cell. In the process, they may

cause mutations increase (or decrease) the amount of DNA in the genome of the cell, and if the cell is the precursor of a gamete, in the genomes of any descendants.

These mobile segments of DNA are sometimes called "jumping genes". There are two distinct types:

Class II transposons. These consist of DNA that moves directly from place to place. Class I transposons. These are retrotransposons that o first transcribe the DNA into RNA and then o use reverse transcriptase to make a DNA copy of the RNA to insert in a new location.

Class II Transposons Class II transposons move by a "cut and paste" process: the transposon is cut out of its location (like command/controlX on your computer) and inserted into a new location (command/control-V). This process requires an enzyme a transposase that is encoded within some of these transposons.

Transposns binds to:

both ends of the transp oson, which consis t of inv erted repeats; that is, identical sequences reading in opposite directions. a sequence of DNA that makes up the target site. Some transposases require a specific sequence as their target site; others can insert the transposon anywhere in the genome.

The DNA at the target site is cut in an offset manner (like the "sticky ends" produced by some restriction enzymes [Examples]). After the transposon is ligated to the host DNA, the gaps are filled in by Watson-Crick base pairing. This creates identical direct repeats at each end of the transposon. Often transposons lose their gene for transposase. But as long as somewhere in the cell there is a transposon that can synthesize

the enzyme, their inverted repeats are recognized and they, too, can be moved to a new location. Miniature Inverted-repeat Transposable Elements (MITEs) The recent completion of the genome sequence of rice and C. elegans has revealed that their genomes contain thousands of copies of a recurring motif consisting of

almost identical sequences of about 400 base pairs flanked by characteristic inverted repeats of about 15 base pairs such as 5' GGCCAGTCACAATGG..~400 nt..CCATTGTGACTGGCC 3' 3' CCGGTCAGTGTTACC..~400 nt..GGTAACACTGACCGG 5'

MITEs are too small to encode any protein. Just how they are copied and moved to new locations is still uncertain. Probably larger transposons that

do encode the necessary enzyme and recognize the same inverted repeats

are responsible. There are over 100,000 MITEs in the rice genome (representing some 6% of the total genome). Some of the mutations found in certain strains of rice are caused by the insertion of a MITE in the gene.

MITEs have also been found in the genomes of humans, Xenopus, and apples. Transposons in maize The first transposons were discovered in the 1940s by Barbara McClintock who worked with maize (Zea mays, called "corn" in the U.S.). She found that they were responsible for a variety of types of gene mutations, usually

insertions and deletions (indels) translocations

Some of the mutations (c, bz) used as examples of how gene loci are mapped on the chromosome were caused by transposons. [Link] In developing somatic tissues like corn kernels, a mutation (e.g., c) that alters color will be passed on to all the descendant cells. This produces the variegated pattern which is so prized in "Indian corn". (Photo courtesy of Whalls Farms.) It took about 40 years for other scientists to fully appreciate the significance of Barbara McClintock's discoveries. She was finally awarded a Nobel Prize in 1983. Transposons in Drosophila

P elements are Class II transposons found in Drosophila. They do little harm because expression of their transposase gene is usually repressed. However, when male flies with P elements mate with female flies lacking them, the transposase becomes active in the germline producing so many mutations that their offspring are sterile. In nature this is no longer a problem. P elements seem to have first appeared in Drosophila melanogaster about 50 years ago. Since then, they have spread through every population of the species. Today flies lacking P elements can only be found in old strains maintained in the laboratory. P elements have provided valuable tools for Drosophila geneticists. Transgenic flies containing any desired gene can be produced by injecting the early embryo with an engineered P element containing that gene. Other transposons are being studied for their ability to create transgenic insects of agricultural and public health importance. Transposons in bacteria Some transposons in bacteria carry in addition to the gene for transposase genes for one or more (usually more) proteins imparting resistance to antibiotics. When such a transposon is incorporated in a plasmid, it can leave the host cell and move to another. This is the way that the alarming phenomenon of multidrug antibiotic resistance spreads so rapidly.

Transposition in these cases occurs by a "copy and paste" (command/control-C -> command/control-V) mechanism. This requires an additional enzyme a resolvase that is also encoded in the transposon itself. The original transposon remains at the original site while its copy is inserted at a new site. Retrotransposons Retrotransposons also move by a "copy and paste" mechanism but in contrast to the transposons described above, the copy is made of RNA, not DNA. The RNA copies are then transcribed back into DNA using a reverse transcriptase and these are inserted into new locations in the genome. Many retrotransposons have long terminal repeats (LTRs) at their ends that may contain over 1000 base pairs in each. Like DNA transposons, retrotransposons generate direct repeats at their new sites of insertion. In fact, it is the presence of these direct repeats that often is the clue that the intervening stretch of DNA arrived there by retrotransposition. Some 50% of the entire human genome consists of retrotransposons. LINEs (Long interspersed elements)

The human genome contains over one million LINEs (representing 19% of the genome).

The most abundant of these belong to a family called LINE1 (L1). These L1 elements are DNA sequences that range in length from a few hundred to as many as 9,000 base pairs. Only about 50 L1 elements are functional "genes"; that is, can be transcribed and translated. The functional L1 elements are about 6,500 bp in length and encode three proteins, including o an endonuclease that cuts DNA and a o reverse transcriptase that makes a DNA copy of an RNA transcript. L1 activity proceeds as follows: o RNA polymerase II transcribes the L1 DNA into RNA. o The RNA is translated by ribosomes in the cytoplasm into the proteins. o The proteins and RNA join together and reenter the nucleus. o The endonuclease cuts a strand of "target" DNA, often in the intron of a gene. o The reverse transcriptase copies the L1 RNA into L1 DNA which is inserted into the target DNA forming a new L1 element there.

Through this copy-paste mechanism, the number of LINEs can increase in the genome. The diversity of LINEs between individual human genomes make them useful markers for DNA "fingerprinting". Variation occurs in the length of L1 elements:

Transcription of an active L1 element sometimes continues downstream into additional DNA producing a longer transposed element. Reverse transcription of L1 RNA often concludes prematurely and produces a shortened transposed element.

While L1 elements are not functional, they may play a role in regulating the efficiency of transcription of the gene in which they reside (see below). Occasionally, L1 activity makes and inserts a copy of a cellular mRNA (thus a natural cDNA). Lacking introns as well as the necessary control elements like promoters, these genes are not expressed. They represent one category of pseudogene. SINEs (Short interspersed elements) SINEs are short DNA sequences (100400 base pairs) that represent reverse-transcribed RNA molecules originally transcribed by RNA polymerase III; that is, molecules of tRNA, 5S rRNA, and some other small nuclear RNAs. The most abundant SINEs are the Alu elements. There are over one million copies in the human genome (representing 9% of our total DNA). Alu elements consist of a sequence averaging 260 base pairs that contains a site that is recognized by the restriction enzyme AluI. They appear to be reverse transcripts of 7S RNA, part of the signal recognition particle.

Most SINEs do not encode any functional molecules and depend on the machinery of active L1 elements to be transposed; that is, copied and pasted in new locations. HIV-1 HIV-1 the cause of AIDS and other human retroviruses (e.g., HTLV-1, the human T-cell leukemia/lymphoma virus) behave like retrotransposons. The RNA genome of HIV-1 contains a gene for

reverse transcriptase and one for integrase. The integrase serves the same function as the transposases of DNA transposons. The DNA copies can be inserted anywhere in the genome.

Molecules of both enzymes are incorporated in the virus particle. Link to an illustration and further discussion.

Transposons and Mutations Transposons are mutagens. They can cause mutations in several ways:

If a transposon inserts itself into a functional gene, it will probably damage it. Insertion into exons, introns, and even into DNA flanking the genes (which may contain promoters and enhancers) can destroy or alter the gene's activity. The insertion of a retrotransposon in the DNA flanking a gene for pigment synthesis is thought to have produced white grapes from a black-skinned ancestor. Later, the loss of that retrotransposon produced the red-skinned grape varieties cultivated today.

Faulty repair of the gap left at the old site (in cut and paste transposition) can lead to mutation there. The presence of a string of identical repeated sequences presents a problem for precise pairing during meiosis. How is the third, say, of a string of five Alu sequences on the "invading strand" of one chromatid going to ensure that it pairs with the third sequence in the other strand? If it accidentally pairs with one of the other Alu sequences, the result will be an unequal crossover one of the commonest causes of duplications. Link to an example of a mutation caused by unequal

crossing over. SINEs (mostly Alu sequences) and LINEs cause only a small percentage of human mutations. (There may even be a mechanism by which they avoid inserting themselves into functional genes.) However, they have been found to be the cause of the mutations responsible for some cases of human genetic diseases, including:

Hemophilia A (Factor VIII gene) and Hemophilia B [Factor IX gene] X-linked severe combined immunodeficiency (SCID) [gene for part of the IL-2 receptor] porphyria predisposition to colon polyps and cancer [APC gene] Duchenne muscular dystrophy [dystrophin gene] What good are transposons?

Transposons have been called "junk" DNA and "selfish" DNA.

"selfish" because their only function seems to make more copies of themselves and "junk" because there is no obvious benefit to their host.

Because of the sequence similarities of all the LINEs and SINEs, they also make up a large portion of the "repetitive DNA" of the cell. Retrotransposons cannot be so selfish that they reduce the survival of their host. And it now appears that many, at least,

confer some benefit. The ENCODE project found that some 75% of our repetitive DNA occurs within, or overlaps with, sequences, like enhancers, that regulate gene expression. Some other possibilities:

Retrotransposons often carry some additional sequences at their 3' end as they insert into a new location. Perhaps these occasionally create new combinations of exons, promoters, and enhancers that benefit the host. Example:
o

Thousan ds of our Alu element s occur in the introns of genes. Some of these contain sequences that when transcribed into the primary transcript are recognized by the spliceosome. These can then be spliced into the mature mRNA creating a new exon, which will be transcribed into a new protein product. Alternative splicing can provide not only the new mRNA (and thus protein) but also the old. In this way, nature can try out new proteins without the risk of abandoning the tried-and-true old one.

L1 elements inserted into the introns of functional genes reduce the transcription of those genes without harming the gene product the longer the L1 element, the lower the level of gene expression. Some 79% of our genes contain L1 elements, and perhaps they are a mechanism for establishing the baseline level of gene activity. Telomerase, the enzyme essential for maintaining chromosome length, is closely related to the reverse transcriptase of LINEs and may have evolved from it. RAG-1 and RAG-2. The proteins encoded by these genes are needed to assemble the repertoire of antibodies and T-cells receptors (TCRs) used by the adaptive immune system [Link]. The mechanism [Link] resembles that of the cut and paste method of Class II transposons , and the RAG genes may have evolved from them. If so, the event occurred some 450 million years ago when the jawed vertebrates evolved from jawless ancestors [Link]. Only jawed vertebrates have the RAG-1 and RAG-2 genes. In Drosophila, the insertion of transposons into genes has been linked to the development of resistance to DDT and organophosphate insecticides. Transposons and the C-value Paradox

The genome of Arabidopsis thaliana contains ~1.2 x 108 base pairs (bp) of DNA. About 14% of this consists of transposons; the rest functional genes (25,498 of them).

The maize (corn) genome contains 20 times more DNA (2.4 x 109 bp) but surely has no need for 20 times as many genes. In fact, 60% of the corn genome is made up of transposons. (The figure for humans is 42%.)

So it seems likely that the lack of an association between size of genome and number of functional genes the C-value paradox is caused by the amount of transposon DNA accumulated in the genome. 16. Mechanisms of Gene Mutation Key Concepts Mutations can occur spontaneously owing to several different mechanisms, including errors of DNA replication and spontaneous damage to the DNA. Mutagens are agents that increase the frequency of mutagenesis, usually by altering the DNA. Potentially mutagenic and carcinogenic compounds can be detected easily by mutagenesis tests with bacterial systems. Biological repair systems eliminate many potentially mutagenic alterations in the DNA. Cells lacking certain repair systems have higher than normal mutation rates.

Introduction This chapter describes the main types of molecular processes that give rise to the mutant alleles discussed in the previous chapter. Such processes are relevant not only to experimental genetics but also have direct bearing on human health. Molecular basis of gene mutations Gene mutations can arise spontaneously or they can be induced. Spontaneous mutations are naturally occurring mutations and arise in all cells. Induced mutations are produced when an organism is exposed to a mutagenic agent, or mutagen; such mutations typically occur at much higher frequencies than spontaneous mutations do. To understand the mechanisms of gene mutation requires analysis at the level of DNA and protein molecules. Preceding chapters have described models for DNA and protein structure and have considered the nature of mutations that alter these structures (see, for instance, Table 15-1). Molecular genetics techniques can be used to determine the sequence of large segments of DNA and the sequence changes resulting from mutations. Sequencing has greatly increased our understanding of the pathways that lead to mutagenesis and has even helped to unravel the mysteries of mutational hot spotsgenetic sites with a penchant for mutating.

Much work on the molecular basis of mutation has been carried out in single-celled bacteria and their viruses. However, many mutations leading to inherited diseases in humans also have been analyzed. We shall review some of the findings of these studies. We shall also consider biological repair mechanisms, because repair systems play a key role in mutagenesis, operating to lower the final observed mutation rates. For example, in Escherichia coli, with all repair systems functioning, base substitutions occur at rates of 1010 to 109per base pair per cell per generation. As a general principle, base substitutions arise by a perturbation of the normal pairing of complementary bases.