Gene Inheritance And Interactions PDF

GENE INHERITANCE AND INTERACTIONS Mendel’s Laws 1. Law of segregation: alleles segregate during meiosis so that one allele is present in each gamete 2. Law of independent assortment: different traits assort independently of each other Chi-square (X2) - X2 = (O-E)2/E Calculate...

GENE INHERITANCE AND INTERACTIONS Mendel’s Laws 1. Law of segregation: alleles segregate during meiosis so that one allele is present in each gamete 2. Law of independent assortment: different traits assort independently of each other Chi-square (X2) - X2 = (O-E)2/E Calculate expected number from observed. Calculate chi square values for each class. Each different phenotype is one class. Add these values together. Determine the degrees of freedom (n -1) then probability Dominant and Recessive genes Co-dominance - heterozygote expresses phenotype of both alleles simultaneously Incomplete dominance - a heterozygote is intermediate between the two homozygous phenotypes Loss of function mutations: decrease or complete loss of functional gene product, usually recessive. ◦ hypomorphic mutation: partial loss of gene function. Null maturation: complete loss of gene function ◦ Haplo-sufficient: one WT allele provides enough normal gene product to produce WT phenotype ◦ Haplo-insufficient: single WT allele in heterozygote cannot provide amount of gene product needed for WT phenotype Gain of function mutations: increase in functional gene product or new cellular function, usually dominant ◦ Hypermorphic: increase in gene activity. Neomorphic mutation: new function Mutants Conditional mutants: mutation causes a phenotype only under certain environmental conditions ◦ Restrictive condition = mutant phenotype Permissive phenotype = WT phenotype Incomplete Penetrance: not every individual with the mutant genotype displays a mutant phenotype Penetrance: the proportion of individuals of a specific genotype that exhibit the corresponding genotype Dominant lethal alleles: homozygous mutant, Recessive lethal alleles: F1 intercross of heterozygotes, 1/4 offspring will die (1) Determining if mutations are in the same or different genes with complementation mutants with the same phenotype could be alleles of the same gene, or mutations in 2 different genes The complement test: a functional test between the recessive mutants with the same phenotype. 2 homozygous mutants with the same phenotype are mated. F1 offspring are analysed. If the F1 offspring exhibit the wild-type phenotype, it indicates that the two mutations affect different genes. This is because the wild-type allele from one parent can complement the mutant allele from the other parent, restoring the wild- type function. If the F1 offspring exhibit the mutant phenotype, it suggests that the two mutations affect the same gene. In this case, both parents lack a functional copy of the gene, so the F1 offspring inherit two mutant alleles and display the mutant phenotype Gene interaction inheritance ratios monohybrid cross = 3:1 dihybrid cross = 9:3:3:1 (no gene interaction but can be modified by different types of interactions and epistasis)  Complementary gene interaction: activity of both genes needed for final phenotype (9:7) Duplicate gene interaction: either gene can carry out the biological process (15:1) Epistasis: when expression of one gene is modified by the expression of one or more other genes. EG: Labradors - Gene E (for pigment deposition) is epistatic to Gene B (pigment synthesis) Recessive epistasis: recessive genotype of one gene blocks the phenotype controlled by another gene (9:3:4) Dominant epistasis: dominant genotype of one gene blocks the phenotype controlled by another gene (12:3:1 - rare) Dominant suppression epistasis: Dominant allele of one gene suppresses the expression of the dominant allele gene (13:3 - rare) SEX LINKED INHERITANCE AND DETERMINATION Mitosis: Somatic cells - chromosomes replicate and divide; chromosome number is maintained Meiosis: Germline cells - Chromosomes replicate and undergo 2 rounds of division; chromosome number is halved The Chromosome theory of inheritance: from both genes and chromosomes, one member of the pair comes from the mother and the other from the father. The members of a homologous pair seperate in meiosis, so each sperm or egg receives one. Different chromosome pairs sort into gametes independently of one another in meiosis. First experimental proof identified in Drosophila (male with white eyes) Human Sex Chromosome: X and Y greatly differ in size and morphology. X has 165 megabases and Y has 59. Heterogametic sex: sex with different sex chromosomes. Homogametic sex: sex with homologous sex chromosomes. Human sex chromosomes: X and Y (Biological males heterogametic (XY) Biological females homogametic (XX)) Hemizygote: only one copy of an allele is present at a locus instead of 2. Reciprocal crosses: used to determine sex linkage - two crosses are performed, when genotypes of male and female parents are swapped. If offspring ratios differ, then it indicates the trait is sex linked Traits of inheritance Autosomal dominant: one mutant allele is sufficient for trait or disease Autosomal recessive: two mutant alleles result in trait or disease X-linked: hemizygous males express the trait, regardless of dominance. More X sex-linked traits than Y Y linked inheritance only occurs in males, occurs in all sons of affected males X-linked dominant: trait is present in males hemizygous for the allele, males transmit to all of their daughters. Trait is present in females homozygous or heterozygous for the allele. Each individual who has the disease has at least one affected parent. X-linked recessive: trait is present in males hemizygous for the allele, is present in females homozygous for the allele. Most affected individuals are  male. The disease may skip generations Gamete size Defines which gametes is 'male' or 'female', male gametes: small and numerous, female gametes: large and few. Anisogamy: fusion of gametes that differ in size or form. Isogamy: fusion of gametes of similar morphology Genetic and Environmental sex determination mechanism Heteromorphic sex chromosomes: presence of specific sex chromosomes that are different from each other and distinct from autosomes. Other X-based sex mechanisms: XO systems - the number if X chromosomes determines in Drosophila and many other invertebrates. 2X chromosomes = female, 1X chromosome male (XO). XO is biologically male but sterile Sex chromosomes in Drosophila: have 4 chromosomes, sex determination depends on the number of X chromosomes, Sex lethal is the master sex- determining locus in Drosophila. XX embryo: high Slx expression, XY or XO is low Slx expression, ZW sex determination all birds, some reptiles and some insects, males are the homogametic sex (ZZ) while demales are the heterogametic sex (ZW). Sex chromosome imbalance: Dosage compensation mechanisms have evolved that balance the level of X-linked gene products between the sexes: 1. up regulation of expression of X linked genes in XY individuals 2. Down-reglation of X linked genes in XX individuals 3. Complete inactivation of one of the two X chromosomes in XX individuals X chromosome inactivation in mammals , one of the two X chromosomes becomes condensed and localised to the nuclear membrane where it is not transcribed. Barr Body: the inactive X chromosome. once X chromosome is inactivated, it remains inactive through subsequent cell divisions. X inactivation specific transcript (Xist) gene is a RNA gene on the X chromosome. Xist is transcribed from X inactivation centre of the future inactive X to produce long non coding RNA. Xist RNA coats the inactive X chromosome and recruits proteins that silence gene expression and result in chromosome condensation POPULATION GENETICS Haplotype: set of DNA variants along single chromosome that are inherited together, because they are close to each other and recombination is rare Allele frequency: p + q = 1, Genotype frequency: p2 + 2pq +q2 = 1 Question: if blood type frequencies are A = 0.504, B = 0.175, O = 0.185, what are allele IA, IB and i frequencies? Solution: p = IA q = IB r = i. r2 = O = 0.185 thus r = 0.43. 'A' = p2 + 2pr. A+O = p2 +2pr +r2 +0.504 +0.185. (p+r)2 = 0.689 thus p+r =0.83. SInce r = 0.43, p = 0.4 and thus q = 0.17 Assumptions: Large population, no genetic drift, random mating, no mutations, no natural selection. frequencies of different alleles at a locus should not change over time if there is completely random mating. In negative assortative mating - opposites attract so homozygotes decrease and heterozygotes will increase but frequencies still stay the same. In positive assortative mating the homozygotes increase and heterozygotes decrease but frequencies stay the same. But if non mating the genotype frequencies will change --> outside HWE Genetic Diversity the environment is not constant, so a favourable allele in one environment at one time may not always be the favourable allele Frequency dependent selection: a type of balancing selection as two alleles are maintained over time. The favourable allele or genotype in the host depends on which allele is more common.  Detecting selection: directly from genomes. Two genomes from a population are compared, if we se a number of 1 its a neutral evolution, if above 1 positive selection if below 1 its negative selection. Haplotype length can be used to detect positive selection, haplotypes become shorter each generation due to recombination, a long haplotype that is widespread in a population indicates recent origin, or large selective advantage Lactase Persistence: Lactase is the product of the LCT gene, breaks down lactose into glucose and galactose. Most people shut off LCT transcription at 2 years. People from some populations continue to transcribe the LCT gene. Lactase persistence is common among populations that herded cattle, goats, camels, etc. It can be advantageous. Lactase persistence is found in upstream regulatory region of the LCT gene. Different pastoralist populations have differentSNPs but all allow for transcription of the LCT gene. Mutations occurred independently in each population but selective advantage allowed them to become common EDAR variant among the Han Chinese: EDAR affects many processes including sweat gland production. A variant in EDAR gene is nearly fixed in Han Chinese but absent elsewhere. This variant is present on very long haplotype on chromosome 2, causes increased sweat gland production Inbreeding and F coefficient of inbreeding Inbreeding leads to high homozygosity and alleles identical by descent. A homozygote who arises from parents with a common ancestor will also be homozygous for the founder's haplotype. Coefficient of inbreeding: F - probability that two alleles in an individual are identical because they descend from same copy in an ancestor. If F = 1, all individuals in population are homozygous for the same allele; If F. = 0, no individuals have two alleles derived from the same ancestral copy Allele Frequencies 4 factors affecting allele frequencies: some individuals leave more offspring than others, small population sizes result in random changes, new alleles arise from mutation, individuals move into or out of the population When migration between 2 populations is prevented --> pop diverges. When population is allowed --> pop becomes similar Drift: alleles present at a low frequency, only a small number of individuals survive so extremely likely that alleles are lost. Every population is a sample of the previous generation so allele frequencies drift randomly because of the size of populations. Founder effect: the origination of a population from a small number of individuals. Typically involves strong genetic drift: founders carry a small subset of alleles from original population. Speciation Speciation: if gene flow ceases or reduces between populations, they may diverge to the point members of each population cant interbreed (reproductive isolating mechanisms). May be pre or postzygotic LINKAGE AND MAPPING Structure of chromosomes: Heterochromatin, Euchromatin, Telomere, centromere, short (p) and long (q) arms. Occur in karotypes, DNA is packaged into chromosomal proteins and histones to form compact chromatin. Chromatin is folded to form higher order structures (chromosome). Chromosomal DNA wraps around histone proteins to form nucleosomes. Histone proteins are positive, DNA backbone is negative. Euchromatin contains most of the active genes. Heterochromatin is more condensed and densely staining. Dark stain is densely packed heterochromatin, light stain is less dense euchromatin. Ploidy: number of chromosome sets in organism genome. Sister chromatids: DNA replication has occured. Homologous pair: one inherited from each parent. Meiosis I - Separation of homologous: Includes PMAT I, Prophase I: Chromosomes condense: Chromatin condenses into visible chromosomes. Homologous chromosomes pair up: Each chromosome finds its homologous partner and pairs up. Crossing over: Non-sister chromatids from homologous chromosomes exchange genetic material, leading to genetic recombination. Nuclear envelope breaks down: The nuclear envelope disintegrates. 2. Metaphase I: Homologous pairs align at the metaphase plate: The tetrads line up along the equator of the cell, with each homologous pair oriented randomly. 3. Anaphase I: Homologous chromosomes separate: Homologous chromosomes are pulled apart by spindle fibers towards opposite poles of the cell. Sister chromatids remain attached: Sister chromatids stay connected at the centromere. 4. Telophase I and Cytokinesis: Nuclear envelopes re-form: Two new nuclear envelopes form around the separated chromosomes. Cytokinesis occurs: The cell divides into two daughter cells, each with a haploid number of chromosomes. Meiosis II - Includes PMAT II, sister chromatids separate to produce 4 haploid products. Recombination frequencies to calculate map distances between linked genetic markers: use genetic mapping to link phenotypes back to sequence changes in the genome. Linkage analysis and genetic mapping leads to: identifying genes and biological processes underlying inherited traits, Functional genomics to discover why the gene causes the phenotype, genetic tests for diseases and potential cures, a scaffold or map to inform the assembly of whole genome sequences. Genes that are close together on a chromosome are often inherited together. The further 2 genes are apart, the more likely there will be recombination between them. Genes > 50 map units apart have so much recombination between them that they are effectively unlinked and assort independently. Recombination maxes out at 50%. Recombination: separates alleles of linked genes, increases genetic variation by producing new allelic combinations, occurs during prophase I of meiosis, crossing over occurs when chromatids between two homologous chromosomes exchange DNA, crossing over creates parental (P) and recombinant (R) gametes after segregation How to tell when genes are linked: linked genes dont assort independently, dont have 9:3:3:1 or 1:1:1:1 ratio, linked genes usually result in higher ratio of parental and lower ratio of recombinant phenotypes --> more equal in unlinked. Positional cloning: technique to locate position of a gene along the chromosome. Uses genetic mapping to connect between the gene that affects the phenotype and the DNA sequence. Map the phenotype by recombination between known molecular markers --> analyse the DNA sequence of the interval between the two markers --> identify candidate gene. GWAS (Genome Wide Association Studies): compare genome-wide SNP variation in of thousands of 'affected' vs 'unaffected' individuals. The relative frequencies of each SNP and haplotype are calculated and plotted among affected and unaffected individuals. Linkage maps recombination frequency of 0.01 = 1 map unit, can be used to determine the arrangement of elements along a chromosome. —> recombination frequency: r = (# of recombinant offspring)/(total # of offspring) x 100 Dihybrid Test Cross: done by crossing a heterozygote with a double-recessive. —> Step 1 (generate a heterozygote): Ab/Ab x aB/aB --> F1 Ab/aB. Step 2 (perform a test cross): Ab/aB x ab/ab --> F2. Step 3 (identify parental & recombinant offspring), Step 4 (calculate recombination frequency) Trihybrid Test Cross: determine if 3 genes are linked, the gene order and map distance. —> Step 1: determine is genes are linked. Outcome of recombination: no recombination most frequent, single recombination intermediate, double recombination least frequent. Step 2: determine the gene order (look for double crossover - will be middle marker). Step 3: consider markers in pairwise combination and do recombination frequencies. Step 4: put the map together. Step 5: factor in the double recombination (add in DR twice) Meiotic Error Variation in chromosome number: Polyploidy occurs when there is a variation in the number of sets of chromosomes. (Autopolyploid: multiple copies of the same gene, occurs when first division of meiosis fails and gives gametes without reduction in chromsoome number. Allopolyploid: contain two different genomes after a hybridisation event.) Aneuploidy occurs when a number of chromosomes of a particular pair is unbalanced, occurs at meiosis I or II as A and a normally segregate to different poles, but error makes them go to the same pole.. (monosomy: missing one member of a pair. trisomy: with one extra chromosome in a pair) Non-disjunction is the failure of chromosomes to segregate normally, can result in trisomy disorders (trisomy 21 - down syndrome. during oogenesis, chromosome 21 usually has a single cross over, occasionally chiasma at extreme ends don't provide enough resistance to tension from spindle.) The incidence of some trisomies increase with maternal age Translocation: when segments of chromosome break off and attach to exchange with other non-homologous chromosomes. (Reciprocal translocations: segment of one chromosome is exchanged with a segment from another non-homologous chromosome creating two translocation chromosomes, Meiosis in translocation heterozygotes: if translocation has happened between chromosomes and can no longer cleanly pair up when meiosis occurs then an inviable and viable gamete will be produced, Robsertsonian translocation: long arms of 2 telocentric chromosomers fuse, forming a single meta or acrocentric chromsome) Inversion: a type of chromosomal rearrangement where a segment of a chromosome breaks off, flips 180 degrees, and then reattaches to the same chromosome. This inversion changes the order of genes within that chromosomal segment Molecular Markers sites of silent DNA variation not associated with any measurable phenotypic variation and are detected by molecular methods. PCR: begins with sample of chromosomal DNA, extracted from organisms, specific portions of DNA are amplified into larger quantities using target specific primers, DNA polymerase, free nucleotides. Involves heating and cooling reagents in a three step cycle: dsDNA denatured --> primers are complementary to the sequences flanking the region to be copied anneal to their target sequence --> DNA polymerase amplify the region, extending from the primers by adding free nucleotides to developing strand. Cycle repeats with each subsequent round more target sequences for the primers are created so 'chain reaction' results, synthesising large amounts of the target DNA product. Cycle repeats 30-40 times to exponentially amplify the target region of DNA. Product of PCR can be analysed using gel electrophoresis, cut within restriction enzymes, cloned into vectors, etc SNPs: most common type of molecular change between individuals, differences in DNA sequence may or may not be associated with differences in protein structure or phenotypes, can be identified by sequencing or microarray chip. SNP array: microarray chip containing spots of DNA oligonucleotide probes, to detect sites of known SNP variation in the genome. RFLPs (restriction fragment length polymorphisms): nucleotide differences that can generate a change in a restriction enzyme cutting site. SNP variation can destroy or create restriction enzyme recognition sequences, difference in DNA sequence results in different sized bands after a restriction diges Microsatellites (short tandem nucleotide repeats): 2-5 nucleotides tandemly repeated in a DNA sequence, individuals have variable number of repeats, leading to different sizes Recombination rates: recombination hotspots - high levels of recombination = greater genetic distance relative to physical distance. Recombination coldspots - low levels of recombination = shorter genetic distance relative to physical distance. May be due to feasibility of DNA in those regions to initiate crossing over. Recombination rates differ between males and females for most animals. Chromosome inversions: inversion - a type of chromosome aberration, a structural variation in a chromosome leading to rearrangement of chromosome segments. individuals may be phenotypically normal but have partial sterility. Can be pericentric (with chromatin) or paracentric (without chromatin). QUANTITATIVE GENETICS Nilsson-Ehle experiment inheritance of traits might not be one single gene pair but multiple gene alleles with additive effects which influence the final genotype. Multiple Genes: A and B, each with two alleles: A and a, B and b. Each dominant allele contributes to a darker colour, while recessive alleles contribute to a lighter colour. Additive Effect: The more dominant alleles present, the darker the kernel color. For example: AABB: Darkest red. AAbb or aaBB: Medium red Aabb or aaBb: Light red. aabb: White. Continuous Variation: Due to the combination of different genotypes, a continuous range of kernel colours is observed, from dark red to white. This is because each additional dominant allele adds a small increment of colour. Edward East Experiment in Nicotiana longiflora 1. Generate two pure-breeding lines: one with short and one with long corolla. 2. Crossed these to generate F1. Result: lengths between that of the parents, and with similar variation in phenotypes 3. Crossed the F1 to generate the F2. Result: average length was about the same as F1, but greater variation in phenotypes. 4. Selected the plants from the upper and lower ends for three generations (F5). Result: lengths similar to the parental. Again, greater variation in phenotypes. Phenotypic Variance (VP) Phenotypic variation is dependent on genetic and environmental variation. VP = VG+VE where VP = phenotypic variance, VG = genetic variance, VE = environmental variance. Effect of environment can differ depending on genotype so: VP = VG + VE + VGxE Qualitative Traits: simple inheritance, one or few number of loci, discrete phenotypes, independent environment, populations differ from genotype and phenotype frequencies, eg: blood groups, coat colour. Quantitative traits: complex inheritance, many number of loci, continuous phenotypes, dependent environment, population differs by means and variances, eg: body size, milk production, plant yield. When studying quantitative traits we cant count allele frequencies, need quantitative measurements such as height/length, stats required: mean, standard deviation, analysis of variance, correlation Heritability determines what proportion of the phenotypic variation is due to genetic variation. Heritability is in reference to a particular population in a particular environment. Heritability can be used described using: Broad-sense Heritability (H2), Narrow-sense (h2). Value range from 0 (entirely due to environmental variation) to 1 (entirely due to genetic variation) Broad sense heritability - estimate of proportion of the phenotypic variation due to total genetic variation: H2 = VG/VP = VG/ VG+VE. Narrow sense heritability - estimate of proportion of phenotypic variation due to additive genetic variation: h2 = VA/VP. Additive genetic variance is mainly what determines resemblance. if total os close to 0, you could not use elective breeding (if h2 = 0 then VA = 0) Genetic Variation: can be broken down into - additive genetic variance (VA - additive effect of the allele), dominance genetic variance (VD - where Aa does not give an intermediate between AA and aa) gene interaction variance (VI - epistatic effects). Selective breeding is performed by homozygotes. Response to selection: how much the trait changes from the mean, response to selection (R), is dependent on heritability (h2), selection differential (S). Breeder's Equation: R=h2S Other Quantitative Trait Loci (QTLs): regions of the chromosome containing loci that correlate to a trait. Mapping QTLs: 1. Start with 2 strains with large differences in phenotype (eg tall plant and short plant). 2. Cross to generate F1 heterozygotes. 3. Cross F1 to generate F2. 4. Measure the trait to identify markers that are associated. Estimated Breeding Values (EBVs): important to get the right individuals to get desired traits, estimate the genetic merit for traits that affect profitability. Heterosis: mean of F1 higher or lower than the parents. Transgressive Segregation: range of F2  and later generations greater than the range of the parents. MUTATIONS + HORIZONTAL GENE TRANSFER Types of repair proofreading during DNA replication: polymerase removes most mispaired bases during replication, measures distance between nucleotides, removes wrong ones via 3' exonuclease activity, slows down replication Post DNA replication repair: not part of DNA replication, similar in most organisms. Tautomers & Non-standard base pairing: tautomers - base pairs can go through a tautomeric shift to create rare forms (rare forms), when in rare forms they have different pairing characteristics. If tautomeric shift occurs during DNA replication problems can arise --> mutation. Mismatch repair: catches mismatches missed by DNA replication proofreading or that have occurred at another time, Proteins detect incorrect pairings, stretch of newly synthesised ssDNA removed, gap filled by polymerase, ligase. UV damage: UV light has high energy, join between two adjacent pyrimidines, usually thymine dimer, bulge in helix, triggers repair Photoreactivation: pyrimidine dimers repaired Types of Mutations Silent mutations: mostly phenotypically neutral, bp change, no aa sequence change, mRNA structure which may modulate mRNA stability. Missense mutation: bp change, aa change in protein. Nonsense mutation: bp change, early stop codon frameshift mutation: insertion of a single base pair or deletion of a single base pair. results in altered aa sequence Consequences of mutation if mutation is advantageous it will be selected by the population. Fluctuation test: Key experiment in understanding the relationship between mutation and selection. Investigates if mutations develop in the rise of selection or absence of selection, Demonstrates theres a constant level of mutation in the absence of selection, Different populations added into each flask, grown up for a certain time period. Resistant cells appear due to mutation rates (when they appear is random). Plate out from each flask and count the population concentration of the resistant cells. Gene transfer VGT: transmission of DNA from one generation to the next generation, sexually or asexually reproducing organisms, new alleles now and then. HGT: all organisms, transfer between cells within a generation, transfer from environment, can become stably integrated Transformation, Conjunction, Transduction Transformation: Direct uptake of DNA molecules from environment, Requires energy, ssDNA taken up, Double recombination for integration, Low efficiency (selection), DNA often degraded and lost. crossovers between transforming DNA and host chromosome lead to transforming DNA  replacing the corresponding region in the host chromosome. Conjugation: 1. Pilus Formation: An F-donor bacterium forms a pilus to connect with an F-recipient bacterium. 2. F Plasmid Transfer: The F plasmid, a circular DNA molecule, is replicated and transferred from the F-donor to the F-recipient through the pilus. 3. Gene Transfer by Hfr Cells: In Hfr cells, the F plasmid is integrated into the bacterial chromosome. During conjugation, parts of the bacterial chromosome can be transferred along with the F plasmid. Transduction: 1. Phage Attachment and Injection: A bacteriophage attaches to a bacterial cell and injects its DNA into the cell. 2. Phage Replication and Assembly: The phage DNA replicates and new phage particles are assembled inside the bacterial cell. 3. Bacterial Lysis and Phage Release: The bacterial cell lyses, releasing the new phage particles.4. Phage-Mediated Gene Transfer: During the assembly process, some bacterial DNA may be accidentally packaged into phage particles. When these phage particles infect new bacterial cells, they can transfer the bacterial DNA to the recipient cells. Transposable elements DNA sequences that can move within a genome, Encode their own movement mechanisms, Have defined ends, Relatively small in size Retrotransposons: Copy-and-paste mechanism involving an RNA intermediate, Long Terminal Repeats (LTRs) at both ends, Common in eukaryotes: Particularly abundant in mammals, contributing to a significant portion of the human genome, Disrupting gene function, Altering the expression of nearby genes, Some retroviral proteins, like gag-like proteins, influence synaptic traffic of cellular RNA. DNA Transposons: Cut-and-paste mechanism involving a DNA intermediate, Inverted repeats at both ends. Insertion Sequences (IS): Small, simple transposons that encode a transposase enzyme. Complex Transposons: Larger transposons that carry additional genes, such as antibiotic resistance genes GENETIC ANALYSIS OF DEVELOPMENT Mutant Screen Process - Random mutagenesis: The organism's genome is subjected to random mutations using mutagens like chemicals or radiation. Selection: The mutagenized population is screened for individuals with the desired mutant phenotype. Analysis: The identified mutants are further analyzed to determine the underlying genetic changes Testing intermediates: This technique helps identify the specific step in a metabolic pathway that is affected by a mutation. Mutant strains are grown on minimal media supplemented with different intermediates. If a mutant strain can grow when a specific intermediate is added, it means the mutation affects a step before the addition of that intermediate. Gene-Enzyme Relationship: Mutations in specific genes can lead to defects in the corresponding enzymes. This can disrupt the metabolic pathway and result in a mutant phenotype. Complementation Testing: Used to determine whether two mutations affecting the same phenotype are in the same gene or different genes. Mutant strains are crossed to create diploid individuals. If the mutations are in different genes, the wild-type allele from one mutant can complement the mutant allele of the other, allowing for normal growth. Mutagenesis: Inducing mutations to identify genes involved in early development. Identifying Lethal Mutations: Mutations affecting segmentation genes are often lethal, so researchers maintain them in heterozygous form to study their effects on embryonic development. Gap Genes: These genes establish broad regions of gene expression along the anterior-posterior axis of the embryo. Mutations in gap genes result in the loss of entire segments. Genes in space location of gene activity saturation in genetic screens: a state where a sufficient number of mutations identified to cover all genes involved in a particular biological process or phenotype. Efficiency of screen depends on mutagenesis method and ability to identify mutants: genetic tools, transposable elements, chemical and X-ray mutagenesis. Can also target every gene in a genome with molecular tool. Flower organ development: 4 concentric whorls of organs: Sepal, petal, stamens (pollen), carpels (ovules). Floral A-class mutants: 2 outer whorls affected, Sepals -> carpels, Petals -> stamens. Floral B-class, C-class mutants: B-class 2 middle whorls affected, Petals -> sepals, Stamens -> carpels, C-class 2 inner whorls affected, Stamens -> petals, Carpels -> sepals (repeat). Floral double mutants: BC double mutants - Additive, All sepals, AC double mutants - Novel organs, Repeat. Floral triple mutant: All leaf- like, Flower organs may be derived from leaves  A, B, C gene activity modifies leaves into specialised organs. ABC model: Different combinations of homeotic gene activity. Each whorl has a different combination of gene activity. Whorl 1 – A-class gene activity, Whorl 2 – A+B-class gene activity, Whorl 3 – B+C-class gene activity, Whorl 4 – C-class gene activity. A and C-class gene activity is mutually antagonistic Temperature sensitive mutants + Yeast cell cycle: A type of conditional mutation, Protein product has full function at regular (permissive) temperatures, Protein product destabilises with heat and has no functional ability (restrictive temperature). Permissive Temperature: At the permissive temperature, the mutant protein can fold correctly and function normally, allowing for normal cell growth and division. Restrictive Temperature: At the restrictive temperature, the mutant protein is unable to fold correctly or function properly, leading to a mutant phenotype. This could be a complete arrest of cell division or a specific defect in a particular cellular process. Temperature shift experiments: Experiment: Population of yeast cells all with the same mutation, grow and divide at the permissive temperature. They are all at different cell cycle stages. Move to restrictive temperature. Observation: Cells eventually all arrest at same stage of cycle. Conclusions: Mutation is in a gene encoding a product that is needed for that stage of the cell cycle. Gene product is not needed for other stages. Cdc mutants: Single, connected pathway (cycle) not several mini cycles, Genes could be place in order in cycle, No alternative pathways (redundancy) GENOMICS complete DNA sequence of an individual, structure and size varies between organisms, generally the more complex the organisms the larger the genome. Genome size mRNA - code for proteins. rRNA - form the core of the ribosome's structure and catalyse protein synthesis microRNA (miRNA) - regulate gene espression. tRNA - severe as adaptors between mRNA and amino acids during protein synthesis other noncoding RNAs - used in RNA splicing, gene regulation, telomere maintenance and many other processes. Applying genomics Biology: species are threatened and cryptic. Sequencing the life on earth can help understand eukaryotic life on earth Agriculture: enhance productivity, consistency and progeny quality, speed up selective breeding, identify disease-causing genetic variants Medicine: understand biology of non-model organisms for human medicine, answer fundamental questions, infectious diseases (antibiotic targets, rapid disease ID), useful in first response to SARS-CoV-2 (found where it was spread from), standard human health (gut microbiome, detect genetic variants that increase disease risk, identify cancer mutations, gene therapies) DNA sequencing methods Sanger sequencing: older method still in use for short segments of the genome. Step 1: make many copies of the piece of DNA of interest by PCR or cloning. Step 2: dye termination sequencing of product. Machine size separate the labelled DNA --> produces a chromatogram. Sequence short stretches of DNA (cheap if only a few genes). For whole genomes: Slow, Expensive. Next generation DNA sequencing: Massively parallel genome sequencing, works by exploiting DNA synthesis, several types ◦ Semiconductor sequencing: DNA fragments are bound to a chip: chip has sensors that can detect pH change. DNA synthesis add dNTPs one type at a time, detects H+ released each time a dNTP is incorporated. Reversible chain-termination sequencing: similar chemistry to Sanger sequencing but uses reversible chain terminators, Modified dNTPs have a removable chemical 'block'. DNA fragments are bound to a solid plate in clusters: Mixture of all 4 modified dNTPs with a different fluorescent tag added, Fluorescent flash of specific colour each time dNTP is incorporated. Third generation sequencing: massively parallel, several types, sequences single molecules, very sensitive, no amplification of DNA needed. PacBIO (Pacific Biosciences): start with double stranded high quality DNA --> add in adapters to DNA sequence to allow strands to be associated together when separated --> add DNA polymerases --> anneal primers and bind DNA polymerases --> Circularised DNA is sequenced in repeated passes --> the polymerase reads are trimmed of adapters to yield subreads --> consensus and methylation statues are called form subreads. Genome assembly output of genome sequencing: many small fragments of DNA are termed reads but we ideally want the whole genome in one piece. now requires bioinformatic analysis, assemble into contigs Assembly of reads: reads are the fragments of DNA sequenced, find the overlaps --> lay out the reads, make a consensus sequence. Genomic data fragments of DNA error rate in DNA sequencing technology, Read length, repeat sequences or repetitive DNA, size of genome, etc. Read length: shorter reads are more difficult to place uniquely, overlaps that are acceptable for assembly are dependent on the error rate, the longer the read the easier and more accurate the assembly. the larger the genome the more reads are required to cover it. Number of reads: increasing read depth increases genome coverage. Genome coverage is the average number of  reads that cover the same region of the genome. Genome annotation Identify genes - Experimental approach: 1. Extract RNA from tissue, 2. Artificially create cDNA from RNA transcript. Reverse transcriptase copies RNA into cDNA, 3. Sequence cDNA using Next Generation sequencing, Sequence includes exons but not introns Identify genes - Computational approach - algorithms predict gene structure: predict protein coding gene structure based on recognition of certain sequence motifs, such as open reading frames (ORFs) sequence that could not encode polypeptides. Continuous stretch of codons that contain a start codon and a stop codon. Eukaryotes – ORFs span intron/ exon regions (Exons spliced together -> mRNA -> protein). Most algorithms search for ORFs (Specify some minimum ORF size). 50-90% accurate. Predicting protein domains: Can be identified by searching for sequence similarity against a database of known domains. Conserved protein domains provide clues about biochemical properties. Eukaryotic proteins are often modular: Distinct domains joined together, Some domains are found in many genes, Others are specific to gene families, Domains can be shuffled via duplication, translation and inversion. Predicting non-protein coding genes: non-coding RNA (ncRNA) = functional RNAs that don’t code for proteins, but are involved in housekeeping and regulating gene expression. Identify genes - Computational approach - Sequence similarity predicts gene structure: Homology approach: Search for stretches of DNA that are similar to known gene sequences, such as those in closely related species. Sequences conserved among species are more likely to be functional than non- conserved sequences, Can identify gene locus (“address”) AND predict biological function. Genes with similar sequences are assumed to encode products with similar biochemical functions BLAST: A common tool to search for similar sequences in genome assemblies is BLAST (Basic Local Alignment Search Tool). Cuts database into “words” of customisable length, Searches for an exact match between your query sequence and the “words” in the database, If there is a match, the algorithm extends the search in both directions, Allows for some mismatches, Matching sequences are called hits, Scored based on number of identical and non-identical amino acids/nucleotides, Each hit is assigned a measure of statistical significance = e-value, Smaller e-value = better match Predict their location within the genome: Use bioinformatic software that utilise the approaches, neatly packaged into programs we can run on super computers, Typical bioinformatic workflow for eukaryote genome annotation , Run on high performance computer Assign predicted biological function (protein-coding genes): ~50% of gene predictions within a genome are based on sequence similarity to known proteins. These need to be confirmed experimentally, but that’s not always possible. Uses comparisons of genomes and their annotation to understand genome evolution and function, Help us to answer important questions. Organisms differ due to: Different gene sequences, gene copy numbers, Variation in gene regulation, Variation in splicing, Novel genes. E.g. what makes us human? ~200 genes found in humans and not chimps, Many are expressed in the brain. Sequences that are different between closely related species might explain phenotypic differences