Molecular Genetics EXAM 3 (copy)

Recombination CH13

Introduction

• Site specific recombination involves specific DNA sequences

• Somatic Recombination- recombination that occurs in nongerm cells (not during meiosis); most commonly used to refer to recombination in the immune system

• recombination systems have been adapted for experimental use

Homologous Recombination Occurs Between Synapsed Chromosomes in Meiosis

• Chromosomes must synapse (pair) in order for chiasmata to form where crossing over occurs

• the stages of meiosis can be correlated with the molecular events at the DNA level

• sister chromatid- each of two identical copies of a replicated chromosome; this term is used as long as the two copies remain linked at the centromere

  • sister chromatids separate during anaphase in mitosis or anaphase II in meiosis

• bivalent- the structure containing all four chromatids (two representing each homolog) at the start of meiosis

• a sister chromatid from mom and sister chromatid from dad makes a bivalent

• synaptonemal complex- the morphological structure of synapsed chromosomes

• joint molecule- a pair of DNA duplexes are connected together through a reciprocal exchange of genetic material

Double Strand Breaks Initiate Recombination

• The double strand break repair (DSBR) model of recombination is initiated by making a double strand break in one (recipient) DNA duplex and is relevant for meiotic and mitotic homologous recombination

• in 5’ end resection, exonuclease action generates 3’-single-stranded ends that invade the other (donor) duplex

• when a singe strand from one duplex displaces its counterpart in the other duplex (single-strand invasion), it creates a branched structure called a D-loop

• strand exchange generates a stretch of heteroduplex DNA consisting of one strand from each parent

• New DNA synthesis replaces the material that has been degraded

• branch migration- the ability of DNA strand partially paired with its complement in a duplex to extend its pairing by displacing the resident strand with which it is homologous

• Capture of the second double-strand break end by annealing generates a recombinant joint molecule in which the two DNA duplexes are connected by heteroduplex DNA and two Holliday junctions.

• The joint molecule is resolved into two separate duplex molecules by nicking two of the connecting strands.

• Whether recombinants are formed depends on if the strands involved in the original exchange or the other pair of strands is nicked during resolution

Gene Conversion Accounts for Interallelic Recombination

• Heteroduplex DNA that is created by recombination can have mismatched sequences where the recombining alleles are not identical

• Repair systems may remove mismatches by changing one of the strands so its sequence is complementary to the other

• Mismatch (gap) repair of heteroduplex DNA involves a process that recognizes the incorrect base pairing and facilitates the correction of these mismatches through various repair mechanisms, ultimately leading to gene conversion and increased genetic diversity.

Break-Induced Replication Can Repair Double-Strand Breaks

• Break-induced replication (BIR) is initiated by a one-ended double-strand break

• BIR at repeated sequences can result in translocations

Recombining Meiotic Chromosomes Are Connected by
the Synaptonemal Complex

• During the early part of meiosis, homologous chromosomes are paired in the synaptonemal complex

• the mass of chromatin of each homolog is separated from the other by a proteinaceous complex

• axial element- a proteinaceous structure around which the chromosomes condense at the start of synapsis

• lateral element- a structure in the synaptonemal complex that forms when a pair of sister chromatids condenses on to an axial element

• central element- a structure that lies in the middle of the synatonemal complex, along which the lateral elements of homologous chromosomes align

  • it is formed from zip proteins

• recombination nodules (nodes)- dense objects present on the synaptonemal complex; they may represent protein complexes involved in crossing over

Specialized Recombination Involves Specific Sites

• specialized recombination involves reaction between specific sites that are not necessarily homologous

• recombinase- enzyme that catalyzes site-specific recombination

• phage lambda integrates into the bacterial chromosome by recombination btwn the attP site on the phage and the attB site on the E. coli chromosome

• core sequence- the segment of DNA that is common to the attachment sites on both the phage lambda and bacterial genomes

  • it is the location of the recombination event that allows phage lambda to integrate

• The phage is excised from the chromosome by recombination between the sites at the end of the linear prophage.

• Phage lambda int encodes an integrase that catalyzes the integration reaction.

• Integrases are related to topoisomerases, and the recombination rxn resembles topoisomerase action except that nicked strands from different duplexes are sealed together

Recombination Pathways Adapted for Experimental Systems

• homologous recombination allows for targeted transformation

• The Cre/lox and Flp/FRT systems allow for targeted recombination and gene knockout construction.

Linkage Mapping CH12

Linkage and Crossing over

• crossing over may produce recombinant genotypes

  • crossing over may alter the linkage of genes

  • bivalent chromosomes consist of two homologous chromosomes with a pair of sister chromatids each

  • genetic recombination by crossing over can produce new combinations of alleles on chromosomes

  • the process of this leading to a new combination of alleles

  • the cells that contain the new allelic combinations are called nonparental or recombinant cells

  • the cells that contain the original combination of alleles are called parental

• Bateson and Punnett discovered two traits that did not assort independently

  • demonstrated that not all traits assort independently

• Morgan- the linkage of X-linked genes and proposed that crossing over between X chromosomes can occur

• A Chi square analysis- distinguish between linkage and independent assortment

• Creighton and McClintock- crossing over produced new combinations of alleles and resulted in the exchange of segments between homologous chromosomes

• Crossing over occasionally occurs during mitosis

• Chromosomes consist of more than one gene, typically in the hundreds to thousands.

• The term linkage is use to indicate:

  • two genes that are located on the same chromosome.

  • Genes that are close together tend to be transmitted from parent to offspring as a group

• Chromosomes are often called linkage groups, since the genes on a chromosome are physically connected to one another.

• Genes that are far apart on a chromosome may assort independently due to crossing over.

• In humans there are 22 autosomal linkage groups, the X linkage group, and the Y linkage group.

• When geneticists follow different traits in a cross they rely upon dihybrid (two-factor) and trihybrid (three- factor) crosses and so on.

• The outcome of the cross depends on whether the genes are linked on the same chromosome or not

Genetic Mapping in Plants and Animals

Background

• the frequency of recombination between two genes can be correlated with their map distance along a chromosomes.

• Alfred Sturtevant used the frequency of crossing over in dihybrid crosses to produce the first genetic map.

• Trihybrid Crosses can be used to determine the order of distance between linked genes.

• Interference can influence the number of double crosses over that occur in a short region

Genetic Mapping in Haploid Eukaryotes

Background

• Ordered tetrad analysis can be used to map the distance between a gene and the centromere.

• Unordered tetrad analysis can be used to map genes in dihybrid crosses

Molecular Evolution

• orthologous genes (orthologs) – Related genes in different species.

• The minimum size of the proteome can be estimated from the number of types of genes.

• Only 1% of the human genome consists of exons.

• The exons comprise about 5% of each gene, so genes (exons plus introns) comprise about 25% of the genome.

• The human genome has about 20,000 genes

• There is no clear correlation between genome size and genetic complexity.

• C-value – The total amount of DNA in the genome (per haploid set of chromosomes)

• C-value paradox – The lack of relationship between the DNA content (C-value) of an organism and its coding potential.

• junk DNA– Non-coding regions of DNA that do not encode proteins but may play roles in regulation and genome stability.

• There is an increase in the minimum genome size associated with organisms of increasing complexity.

• There are wide variations in the genome sizes of organisms within many taxonomic groups

• Roughly 60% of human genes are alternatively spliced.

• Up to 80% of the alternative splices change protein sequence, so the human proteome has 50,000 to 60,000 members

• monocistronic mRNA – mRNA that encodes one polypeptide.

• polycistronic mRNA – mRNA that includes coding regions representing more than one gene

• Repeated sequences (present in more than one copy) account for more than 50% of the human genome.

• The great bulk of repeated sequences consist of copies of nonfunctional transposons.

• There are many duplications of large chromosome regions

• Orthologous sequences are genes in different species that evolved from a common ancestral gene through speciation, often retaining similar functions across species.

• Paralogous sequences, on the other hand, arise from gene duplication events within the same species, leading to genes that may evolve new functions or acquire different expressions.

• Not all genes are essential. In yeast and flies, deletions of less than 50% of the genes have detectable effects.

• When two or more genes are redundant, a mutation in any one of them might not have detectable effects

• In any particular cell, most genes are expressed at a low level.

• scarce (complex) mRNA – mRNA that consists of a large number of individual mRNA species, each present in very few copies per cell.

  • This accounts for most of the sequence complexity in RNA.

• Only a small number of genes, whose products are specialized for the cell type, are highly expressed.

  • abundance – The average number of mRNA molecules per cell.

  • abundant mRNA – Consists of a small number of individual species, each present in a large number of copies per cell

• mRNAs expressed at low levels overlap extensively when different cell types are compared.

  • housekeeping gene – A gene that is (theoretically) expressed in all cells because it provides basic functions needed for sustenance of all cell types

• The abundantly expressed mRNAs are usually specific for the cell type.

  • luxury gene – A gene encoding a specialized function (usually) synthesized in large amounts in particular cell types.

• About 10,000 expressed genes might be common to most cell types of a multicellular eukaryote

• The probability of a mutation is influenced by the likelihood that the particular error will occur and the likelihood that it will be repaired.

• synonymous mutation – A change in DNA sequence in a coding region that does not alter the amino acid that is encoded.

• nonsynonymous mutation – A change in DNA sequence in a coding region that alters the amino acid that is encoded

• In small populations, the frequency of a mutation will change randomly and new mutations are likely to be eliminated by chance.

• fixation – The process by which a new allele replaces the allele that was previously predominant in a population

• The frequency of a neutral mutation largely depends on genetic drift, the strength of which depends on the size of the population.

• The frequency of a mutation that affects phenotype will be influenced by negative or positive selection