Genetics DNA & Protein synthesis A gene is a sequence of Nucleotide bases that code for a specific protein or polypeptide.

Genes are mainly located on the Chromosomes however some can be found in mitochondria. Each gene occupies a specific LOCUS on the chromosome. Genes are involved in the control of all metabolic pathways and the synthesis of all non-protein molecules in cells. The genetic code Triplet code every three amino acids or CODON code for an amino acid There are multiple codons for each amino acid except methionine Some codons code for STOP The codons are widespread but not universal; genes can be used in genetic engineering to place genes in different organisms.

Transcription mRNA is produced The template strand of DNA is used as a template for RNA nucleotides The hydrogen bonds between DNA bases are broken by DNA HELICASE Activated RNA nucleotides, ATP, GTP, CTP and UTP bind to their complementary nucleotides of the template strand of the gene. The extra phosphoryl groups on the activated RNA nucleotides are released, this releases energy for the nucleotides to be bonded by phosphodiester bonds The RNA is released from the DNA gene and passes out of the nucleus through a pore in the nucleur envelope to a ribsome.

Translation Ribosomes Built up of two subunits There is a groove down the centre that mRNA slides down to be translated

Transfer RNA Have 3 exposed bases at the end of their structure where an amino acid can bind At the other end there is an anti-codon which is complementary to the codon which codes for the next amino acid in the polypeptide sequence

How translation occurs 1. The first codon of the gene is always AUG, the tRNAs anticodon then forms hydrogen bonds with the mRNAs codon 2. The amino acid which it has transferred is then bonded (via peptide bond) with the next codon using ATP and an enzyme. 3. The ribosome then moves down the mRNA reading its codons and binding tRNA molecules via hydrogen bonds which carry the amino acid specific to the codon in the mRNA. 4. The amino acids are joined by peptide bonds.

Genetics 5. The polypeptide carries on growing until a stop codon is reached.

Lac operon For E.Coli to respire lactose as a respiratory substrate two enzymes must be produced, these are Beta-Galactosidase - converts lactose into glucose and galactase Lactase permase allows lactase to be brought through the cell membrane

These two enzymes are triggered by the presence of lactose which acts as the INDUCER. The lac operon is a section of DNA which account for the production of these two enzymes, it consists of: The structural genes: these codes for the two enzymes. The operator region: Can switch the structural genes on or off Promoter region: Where RNA polymerase binds to begin the transcription of the structural genes

How the lac operon works When lactose is absent 1. The regulator gene is expressed, this binds to the operator and promoter region 2. This prevents mRNA binding to the promoter region so the structural genes cannot be expressed 3. B-Galactosidase and lactase permase isnt produced When lactose is present 1. Lactose molecules (inducer protein) molecules bind to the repressor protein, changing the shape of the active site, meaning it can no longer bind to the promoter region and operator region. 2. RNA transcribes the promoter region and operator region, these allow RNA to transcribe and translate the structural genes. 3. As a result lactase is allowed into the cell via lactase permase and is broken down into glucose and galactose to be respired by the cell.

Genetics Homeobox genes The development of organisms in relation to differentiation is controlled by HOMEOBOX GENES. These genes perform different functions: Maternal genes affect polarity, anterior and posterior. Segmentation genes affect the polarity of segments. Homeotic selector genes, specify the identity of each segment.

Homeobox genes sequence for specific polypeptides that in turn bind to genes and prevent the expression of them, others act as TRANSCRIPTION FACTORS, which bind to the gene and initiate transcription in protein synthesis, thus specialising the cell. MORPHOGENS such as vitamin A or retinoic acid, can affect the transcription of genes leading to deformities.

Genetics Apoptosis Programmed cell death that occurs in organisms, cells usually undergo 50 mitotic divisions before apoptosis, it is orderly and tidy. How does it occur? Cell signals Cytokines Hormones Growth factors Nitric oxide this induces apoptosis by making the mitochondrial membrane more permeable, this reduces the concentration gradient meaning the cell can no longer respire to produce ATP to survive. Proteins also bind to apoptosis inhibitor proteins and allow the process to take place.

Why does it occur? Reuse of cell components No harmful enzymes can be released Weeds out ineffective or harmful cells Separation of limbs and digits during development

However too large a rate of apoptosis can lead to cell loss and degeneration and too small of a rate of apoptosis can lead to tumours.

Genetics Meiosis Nuclear division where the original number of chromosomes in the organism is halved, this means that when the two cells are combined in sexual reproduction with a different individual the original number of chromosomes is restored. The cells produced are known as HAPLOID (half the number of chromosomes). Ordinary cells produced via mitosis are DIPLOID. Meiosis occurs in two stages. Meiosis produces NON-IDENTICAL CELLS. Meiosis I Prophase Chromosomes supercoil and become visible Chromosomes come together in their pairs, each allele or single chromosome replicates to form two sister chromatids, the two chromosomes with their 2 sister chromatids each (4 in total) form a bivalent. The non-sister chromatids wrap around each other at points called chiasmata; this is where variation occurs due to crossing over, chromatids swap sections of one another leading to different alleles being switched around. Nucleolus disappears Spindle forms

Metaphase Bivalents line up down the equator Spindle fibres attach to Bivalents Chiasmata still present

Anaphase One homologous chromosome gets pulled away to opposite poles Chiasmata separate and different parts of chromatid are attached to different chromatids.

Telophase New membrane forms and cells are split by cytokinesis Most plant cells go from anaphase into Mitosis II

Mitosis II Prophase Chromosomes condense

Metaphase Chromosomes line up down equator Different Chromatids face each pole

Genetics Anaphase Sister chromatids are pulled away to opposite poles as the centromere is pulled apart This is randomly done but each pole is Haploid

Telophase New cell membranes form In animals the two cells divide to form 4 cells In plant the one cell forms 4 cells.

Why is meiosis important? Sexual reproduction produces variation, this increases the chances of evolution which means when there are changes in the environment, and variation can help an organism to survive where other individuals cannot. When meiosis occurs, diploid gametes are produced which produce a haploid zygote when fertilised. How meiosis leads to genetic variation Crossing over of chiasmata during prophase I Random assortment of chromatids during metaphase and anaphase II Random mutation

Crossing over Occurs during prophase one, bivalents cross over at points called Chiasmata, alleles are swapped on chromosomes and this leads to variation. Provides new combinations of alleles on chromosomes.

Genetics Genotype The genetic makeup of an organism, characterised by the alleles they have present in their genes. Those with two identical alleles for a gene are homozygous; those with different alleles for a gene are heterozygous. Phenotype Refers to the characteristics that are expressed through the genes in the organism, features that can be observed, these are determined by genotype and the environment in which it has developed. Dominant allele of a gene will be expressed over one that is recessive if present in the organism Recessive allele of a gene that wont be expressed if a dominant allele is present but instead will be expressed if another recessive allele is present, for a recessive allele to be expressed the organism needs to be homozygous recessive. Co-dominant if two alleles of a gene are present that are both dominant, being that the organism is heterozygous, both alleles will be expressed, this is most easily exampled in the human blood system where O is recessive and A and B are both co-dominant leading to AB blood groups. Linkage refers to two or more genes on a chromosome that are usually inherited together as crossing over doesnt occur frequently between these two genes at chiasmata. For example red hair and freckles. Sex linkage refers to genes on the same sex chromosome.

Genetics Epistasis where one gene masks or supresses the expression of another gene, this occurs in two ways: They work against each other resulting in masking They work together complementary

Agonistic effects Two homozygous recessive genes prevent the expression of a second gene at a different locus on the chromosome, this is known as epistatic and the genes that cannot be expressed are hypostatic. There are two types of epistasis, recessive and dominant. Recessive epistasis homozygous organisms with the same recessive allele on both chromosomes express that gene preventing the expression of another gene at a different locus. Dominant epistasis The expression of the dominant allele masks the expression of another allele at a different location no matter what the second allele expresses for. Complementary genes both genes work together to provide the final pigment, for example the dominant allele must be present in both gene alleles for the final product, such as purple flowers.

Genetics Variation Continuous variation quantitative differences between phenotypes, with a wide range of variation within the population, such as height or mass Discontinuous variation qualitative differences between phenotypes, such as eye colour, gender or blood group

Role of genes and evolution Environmental resistance is a series of factors that can prevent the growth of a large population, it is split into two types: Abiotic non-living factors, such as weather and famine Biotic living factors, such as predation by other species

Selection pressures also determine what individuals within the population will survive, selection pressures are factors within the environment which make one trait advantageous for survival, those with a trait that doesnt help survival are more likely not to reproduce and pass on their trait, meaning the advantageous trait continues to be selected. However the selection pressure may change, this means that the trait is no longer advantageous and those with different traits may be advantaged to reach sexual maturity and pass on their traits to their offspring. What prevents populations from interbreeding? Geographical causes ecological barriers Seasonal causes species may only breed during a specific season which is different to that of another species Reproductive mechanisms species genitals may no longer be compatible.

Genetics What are clones? Clones are genetically identical organisms with the same genetic sequence derived from the same DNA sequence. Clones can be produced by asexual reproduction with the same DNA, this provides advantages because: It is quick, allowing for rapid reproduction Can be conducted if sexual reproduction fails All offspring have the same DNA which allows for survival in the environment.

However there is no genetic variation which means that the organisms can all be susceptible to selection pressures.

Vegetative propagation Cuttings small section of stem is cut from the main plant and treated with plant growth hormones to encourage root growth. Grafting A short stem section is placed in another stem of a plant with desirable roots, and treated with growth hormones to encourage the graft to grow. The plant has genetically different roots however. Tissue culture A small sample tissue is taken from the desired plant, this is known as the explant and this is placed in a growth medium in which a callus is formed (a ball of unspecialised plant cells) one of these cells is then placed in another growth medium which encourages shoot growth and then it is finally placed in another growth medium to encourage root development.

Genetics Cloning animals This can be done in two ways: Splitting embryos: o The embryo is split into smaller segments and placed in surrogate mothers for new calves to be born. o In-vitro fertilisation (in glass) Nuclear transfer: o Cells from mammary gland are taken from valuable individual o Enucleated ovum is taken and is combined with mammary cell via electro fusion o The new cell formed is placed in oviduct of a second sheep till an embryo is formed o This embryo is then placed within surrogate mother.

Genetics Biotechnology All technological processes that make use of living organisms or parts of living organisms to manufacture products or benefit the human population. It is used in four main areas: Healthcare development and production of new drugs Agriculture provide plants with better crop yield and resistance Industry such as enzymes Food science developing food

Why are microorganisms used? Grow rapidly in favourable conditions, with the population doubling in as little as 30 minutes Produce chemicals which are useful for human society, such as antibiotics Produce enzymes that reduce the activation energy and temperature required for industry Can be grown using toxic materials to humans Tend to secrete chemicals in a purer form reduces downstream costs

Metabolites A metabolite is a product formed by an organism through building and constructing molecules (metabolism), they include chemicals such as insulin, glycogen, and proteins. Primary metabolites products built by an organism as part of its normal growth, these are vital in order for the organism to live. Secondary metabolites products which are not part of the organisms normal growth, they are usually produced after the main growth period, for example E.Coli bacteria have been genetically engineered to produce insulin for diabetics during their stationary phase.

Commercial applications of biotechnology Fermenters can be used to coordinate the growth of a microorganism on a large scale, the growing conditions can be manipulated whether the yield wanted is a primary or secondary metabolite. The conditions that can be manipulated are: Temperature: o Too hot and denaturing occurs o Too cold and growth is slower Nutrient added o When and what nutrient is added affects growth and whether or not a primary or secondary metabolite is produced. Oxygen concentration o Many processes use microorganisms that respire aerobically PH changes in pH can affect enzyme activity.

Genetics There are two types of industrial fermentation: Batch Starter population is mixed with a specific amount of nutrient for a set time. No further nutrients are added At the end of the batch period the desired products are removed, e.g. penicillin Batch Continuous

Continuous Nutrients continuously added to the fermentation tank Conditions monitored and manipulated Products removed regularly from the tank Products such as insulin produced.

Asepsis the process of having no unwanted microorganisms present. Aseptic technique the process in which all industrial fermentation is ensured to have no unwanted microorganisms so contamination doesnt occur. This includes: Steam sterilisation at 121 degrees Celsius Equipment made out of stainless steel to prevent MOs sticking to the equipment Sterilising nutrients Filtering

Genetics Enzymes Enzymes are used at industrial scale as a biocatalyst to improve the rate of reaction and use less energy in the form of heat, reducing costs and carbon output. Enzymes are also specific to certain chemicals or substrates and can be used independently to produce products, reducing the amount of by-products used in industrial processes. Immobilising enzymes Immobilised enzymes are used to reduce the amount of mixing of enzymes and products, thus reducing downstream processing costs as the enzymes have to be filtered from the desired products during purification. However this does cost additional time and money initially, however it is more profitable in the long term. Immobilising also protects the enzymes from denaturing and can be used continuously. Ways to immobilise enzymes: Adsorption enzymes are bonded by weak bonds to other particles such as clay. The bonds used are hydrogen, ionic and hydrophobic. However because the bonds are weak, leakage can occur. The active site can also change shape. Covalent bonding enzymes are covalently bonded to particles, these bonds are stronger so leakage is less common, however the covalent bonds can alter the active site shape, reducing the rate of reaction. The covalent bonds can also occupy the active site shape. Entrapment Enzymes are trapped inside gel alginate or cellulose fibres, this prevents the enzyme from leaving but also allows the substrate to enter to be catalysed into a product, this reduces the rate of reaction as the substrate has to diffuse in, however entrapment prevents denaturation of the enzyme. Membrane separation Enzymes are separate from the solution by a partially permeable membrane, the substrate diffuses through and is catalysed into a product before diffusing back through the membrane, this reduces the rate of reaction however.

Genetics Sequencing genomes Firstly the genes to be studied are mapped on the chromosomes, using microsatellites (short runs of repetitive bases). Small sections of DNA called BACs (bacterial artificial chromosomes) are placed in E.Coli bacteria and these reproduce, replicating the DNA segment. The BACs are then treated with various restriction enzymes that cut the BAC into segments of 750 base pairs in length, with over lapping points. The DNA is then heated so that the hydrogen bonds break between the coding and complementary strand, a primer is then added which anneals to the stand of the DNA segment. DNA polymerase then adds nucleotide bases which are complementary to the coding strand as if replication was occurring; however present are dideoxynucleotides which are missing an OH group that allows for the next nucleotide in the sequence to be joined. These dideoxynucleotides are added at various points randomly giving strands of complementary DNA of different lengths. The DNA fragments are then subjected to electrophoresis, during this the smaller parts of DNA move faster than longer ones, the last base of the DNA fragment can then be read to establish the base sequence.

Genetics Recombinant DNA Recombinant DNA is created by first selecting a gene to be added to genetic material or plasmid. Restriction enzymes are then used to cut a small section in the plasmid so the desired gene can be added. Restriction enzymes are enzymes specific to a small sequence of bases on the plasmid, creating two sticky ends which are complementary to the gene which has also been cut by the same restriction enzyme, the gene then fits in the plasmid and DNA ligase binds the phosphodiester bonds of the nucleotides. This is now known as recombinant DNA.

Why do we genetically engineer organisms? To improve the features of an existing organism: Disease resistance Higher yield Larger growth Insect resistance

To engineer organisms to mass synthesise products We can engineer organisms to produce products they normally wouldnt by inserting the gene, this is exampled with insulin in E.Coli, which allows humans to mass produce the hormone. Allowing animals to produce pharmaceutical chemicals in their milk to be easily collected by humans Modifying crops so that the health benefits are greater for humans that eat the crop.

How is this done? Plasmids In bacterial cells plasmids are used as the vector to insert genes that are desired to be expressed. This is done via a restriction enzyme cutting small section of the plasmid and creating sticky ends which are complementary to the sticky ends of the gene. The gene is then inserted and taken up by the plasmid which is then fixed by DNA ligase. This is known as a recombinant plasmid.

Bacterial cells If bacterial cells take up the recombinant plasmid they become transformed or transgenic. They are transformed when they take up the recombinant plasmid and are also known as transgenic as their genetic material is a recombinant, or a combination.

Genetics Bacteria can also swap plasmids and DNA via bacterial conjugation. DNA plasmids are copied and swapped via a conjugation tube and genes such as antibiotic resistance are swapped. Bacteria can also take up genetic material from their surroundings and this too is known as transformation.

How insulin was reverse engineered 1. The mRNA for insulin was found in the pancreatic tissue 2. The mRNA was then treated with reverse transcriptase which provided a copy of the template strand of DNA, which is complementary to the coding strand of DNA. 3. The template strand of DNA is then used to produce the coding strand, DNA Polymerase and free nucleotides are added and the second strand is formed, the resulting DNA is called cDNA (complementary DNA). 4. The cDNA then has free nucleotides which are unpaired added to the ends which form sticky ends which are also complementary to the sticky ends which will be formed in the plasmid. 5. Restriction enzymes are used to cut the plasmid producing sticky ends. 6. The cDNA is then added to the plasmids to form recombinant plasmids. 7. These plasmids are then mixed with bacteria some of which are taken up. 8. The bacteria that take up the plasmid are grown and reproduce forming a colony of identical bacteria on an agar plate. 9. The insulin produced by these bacteria is then harvested.

How are the bacteria with the insulin gene identified? Replica plating is a technique used to do this, the bacteria mixed with the plasmids is grown on a master plate whilst two other plates with tetracycline and ampicillin in the growth medium are used. The plasmids used have resistance to both genes however the restriction enzyme used to insert the insulin gene is specific to the tetracycline gene on the plasmid; this means the gene is nonfunctional when the insulin gene is present in the plasmid as it is broken up. From the master plate copies of the colony are taken using sterilised velvet which is pressed on the two plates. Bacteria that grow on the ampicillin but not the tetracycline have taken up the insulin gene and can be copied and colony formed from.

Genetics Gene therapy If working copies of genes can be inserted into cells that only contain dysfunctional alleles, the functional allele can be expressed thus treating the symptoms of the genetic disorder. There are two types of gene therapy: Germline This is where germline cells (embryos in which each cell can divide to become an individual organism) having a gene engineered into their genetic material that is then copied into every cell of the organism, functioning within every cell of the organism, thus preventing the genetic disease. It can also be passed to the offspring. Somatic Differs from germline as the gene is added to somatic cells, the gene is not then present in every cell in the body, but in the cells it is present the gene can be expressed and treat the genetic disorder. There are two ways in which somatic gene therapy can be used: Augmentation functional alleles of genes can be added to the cell, meaning the polypeptide is expressed and the cells can begin to function normally. Killing specific cells cancers can be treated by adding genes that produce foreign antigens on their cell surface that are killed by the immune system.