Gene Inheritance and Interactions PDF

GEGE ~ WHAT’S LEFT Gene inheritance and interactions - Dominant lethal alleles: homozygous mutant, Recessive lethal alleles: F1 intercross of heterozygotes, 1/4 o spring will die (1) - monohybrid cross = 3:1 - dihybrid cross = 9:3:3:1...

GEGE ~ WHAT’S LEFT Gene inheritance and interactions - Dominant lethal alleles: homozygous mutant, Recessive lethal alleles: F1 intercross of heterozygotes, 1/4 o spring will die (1) - monohybrid cross = 3:1 - dihybrid cross = 9:3:3:1 (no gene interaction but can be modi ed by di erent types of interactions and epistasis) - Complementary gene interaction: activity of both genes needed for nal phenotype (9:7) - Duplicate gene interaction: either gene can carry out the biological process (15:1) - Epistasis: when expression of one gene is modi ed 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 - 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 - 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 speci c 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 - 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 - 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 - Coe cient 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 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. Euchromatin contains most of the active genes. Heterochromatin is more condensed and densely staining. Dark stain = heterochromatin, light stain = euchromatin. Ploidy: number of chromosome sets in organism genome. Sister chromatids: DNA replication has occurred. Homologous pair: one inherited from each parent. - Meiosis I - Separation of homologous: Prophase I: Chromatin condenses into visible chromosomes, Each chromosome nds its homologous partner and pairs up, Crossing over: Non-sister chromatids from homologous chromosomes exchange genetic material, leading to genetic recombination, The nuclear envelope disintegrates. 2. Metaphase I: Homologous pairs align at the metaphase plate along the equator ff ffi fi fi fi fi fi ff  of the cell, with each homologous pair oriented randomly. 3. Anaphase I: Homologous chromosomes are pulled apart by spindle bres towards opposite poles of the cell. Sister chromatids remain attached: Sister chromatids stay connected at the centromere. 4. Telophase I: Two new nuclear envelopes form around the separated chromosomes. - Autopolyploid: multiple copies of the same gene, occurs when rst division of meiosis fails and gives gametes without reduction in chromsome number. - Allopolyploid: contain two di erent 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 di erent 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 - Translocation: when segments of chromosome break o 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 acrocentric chromsome - Inversion: a type of chromosomal rearrangement where a segment of a chromosome breaks o , ips 180 degrees, and then reattaches to the same chromosome. - Molecular markers are 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, speci c portions of DNA are ampli ed into larger quantities using target speci c primers. Involves heating and cooling reagents in a three step cycle: dsDNA denatured --> primers are complementary to the sequences anking 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 - 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 di erences that can generate a change in a restriction enzyme cutting site. SNP variation can destroy or create restriction enzyme recognition sequences, di erence in DNA sequence results in di erent sized bands after a restriction digest - Microsatellites (short tandem nucleotide repeats): 2-5 nucleotides tandemly repeated in a DNA sequence, individuals have variable number of repeats, leading to di erent sizes Quantitative Genetics Qualitative Quantitative Simple inheritance Complex inheritance One or few loci Many number of loci Discrete phenotypes Continuous phenotypes Independent environment Dependent environment Populations di er from genotype and phenotype Population di ers by means and variances frequencies - Quantitative Trait Loci (QTLs): Mapping QTLs: 1. 2 strains with large di erences 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. Mutations + Horizontal Gene Transfer - Transformation: Direct uptake of DNA molecules from environment, Requires energy, ssDNA taken up, Double recombination for integration, Low selection. Crossovers between transforming DNA and host chromosome lead to transforming DNA replacing the corresponding region in the host chromosome. ff ff ff ff fi fi ff ff fi ff ff fi ff fl ff fi ff fl  - 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. - DNA sequences that can move within a genome, Encode their own movement mechanisms, Have de ned 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 signi cant portion of the human genome, Disrupting gene function, Altering the expression of nearby genes, Some retroviral proteins, like gag-like proteins, in uence synaptic tra c 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 Genomics - 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, Modi ed dNTPs have a removable chemical 'block'. DNA fragments are bound to a solid plate in clusters: Mixture of all 4 modi ed dNTPs with a di erent uorescent tag added, Fluorescent ash of speci c colour each time dNTP is incorporated. - Third generation sequencing: massively parallel, several types, sequences single molecules, very sensitive, no ampli cation of DNA needed. - PacBIO (Paci c 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. - Identify genes - Experimental approach: 1. Extract RNA from tissue, 2. Arti cially 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 identi ed 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 speci c to gene families, Domains can be shu ed 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 fi fi fi fi fl fi fi fi fi fl fi ffl fi ffi ff fl  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 signi cance = 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 work ow 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 con rmed 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 di er due to: Di erent 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 di erent between closely related species might explain phenotypic di erences Genetic Analysis of Development - Testing intermediates: This technique helps identify the speci c step in a metabolic pathway that is a ected by a mutation. Mutant strains are grown on minimal media supplemented with di erent intermediates. If a mutant strain can grow when a speci c intermediate is added, it means the mutation a ects a step before the addition of that intermediate. Gene-Enzyme Relationship: Mutations in speci c 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 a ecting the same phenotype are in the same gene or di erent genes. Mutant strains are crossed to create diploid individuals. If the mutations are in di erent 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 a ecting segmentation genes are often lethal, so researchers maintain them in heterozygous form to study their e ects 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. - saturation in genetic screens: a state where a su cient number of mutations identi ed to cover all genes involved in a particular biological process or phenotype. E ciency 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 a ected, Sepals -> carpels, Petals -> stamens. Floral B- class, C-class mutants: B-class 2 middle whorls a ected, Petals -> sepals, Stamens -> carpels, C-class 2 inner whorls a ected, 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 modi es leaves into specialised organs. ABC model: Di erent combinations of homeotic gene activity. Each whorl has a di erent 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 speci c 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 di erent 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) ff ff ff ff ff ff fi fi ff fi ff ffi ff ff ff fi ff ffi fi fi ff ff fi fl ff ff ff ff fi