Meiosis: Recombination and Chromosome Behavior

• Chromosomes already have entered the S phase and have already replicated
• Centromeres is AT rich
• Sister chromatids are held together by the cohesin complex

Meiosis

• Meiosis is essential for gametogenesis
• Chromosomes replicate once, like mitosis
• The cell with its DNA content divides twice, unlike mitosis
• Meiosis increases genetic diversity through independent assortment, also known as alignment
• Genetic diversity is also increased through crossing-over between homologous chromosomes
• Mitosis is cell division of somatic cells
• Meiosis is unique to the gonads
• Through 2 mechanisms, 4 new haploid cells are genetically unique
• Chromosomes come together and pair very tightly in Meiosis 1
• Homologous chromosomes randomly pair together
• Both chromosomes will go to one pole, or split to opposite poles
• Recombination also happens during Meiosis 1
• Homologous chromosomes exchange material between NON-sister chromatids
• The chromosomes then go to opposite poles
• New, unique chromosomes become part mom, part dad
• It is statistically impossible to make 2 of the same gamete

Testing the "Law of Independent Assortment”

• Drosophila genetics were used
• A large number of mutations were identified
• Efforts were made to identify the genes that made the flies different, and try to map the genes
• Vestigial is in purple

Law of Independent Assortment - Crossing Over

• In Drosophila, gene names are italicized and if recessive, lower case
• vg stands for vestigal, pr stands for purple, where "+" indicates a wild-type allele
• absence of "+" indicates a mutant allele
• When the flies were crossed, all heterozygotes were expected, and to be all phenotypically wild-type
• In the second progeny, there was an expectation of 50% wild-type and 50% double mutant
• Parentals are what is expected
• The 2 new classes are the recombinant classes, which are not expected
• The percentage of recombinants can be used to determine location of the genes
• If there are 2 genes that are closer together, there will way fewer recombinants than farther apart
• Crossing over during meiosis allows new combinations of alleles for genes on a single chromosome

Crossing Over During Meiosis

• In prophase I, chromosomes condense and homologous sets pair, followed by crossing-over among all four chromatids
• Zygotene is the onset of pairing between homologs at synaptonemal junctions
• Pachytene is pairing completion and assembly of recombination nodules
• Diplotene is recombination between chromosomes at sites of crossing over, chiasmata
• Scientists could identify structures above the cells, which led them to believe that there was an exchange of information
• Homologous chromosomes form loop-like structures
• The hypothesis was that there must be an exchange of genetic material
• Synaptonemal junction is made of many proteins
• At the end of prophase 1 after recombination, crosses/chiasmata can be seen
• Kinetochore microtubule forms on top of the microtubule
• Tetrad is a four-part structure that forms in prophase 1, of 2 homologous chromosomes of 2 identical chromatids

Molecular Mechanisms of Recombination

• Spo11 cleaves phosphodiester bond between nucleotides to induce double strand break
• An exonuclease degrades 5' ends, exposing 3' single-stranded tails
• Synapsis must occur for chromosomes to pair
• Spo11 enzyme induces double stranded breaks, express only in meiosis, NOT mitosis
• If broken, a cell will try to repair damage
• Exonucleus degrades 5' ends, exposing 3' stranded tails, and there is now free strand of dna
• Dmc1 facilitates one of the tails to invade and open up the other chromatid's double helix
• First Strand Invasion is when strand invasion of non-sister chromatid results in heteroduplex (X-shape is "Holliday junction")
• These mechanisms can be thought of as a nucleophilic attack
• The free strand will invade, base complementary
• DMC1 will help one of the tail to invade, and open up the other chromatid's double helix
• RPA will bind to the sequence and stabilize it because DNA doesn't like to be single stranded
• The Holliday junction is a reciprocal second strand invasion of original chromatid
• Ligase joins the the 5' end of one nucleotide to the other 3' end
• This requires ATP to covalently bond the two

Branch Migration

• In branch migration, the two Holliday junctions move away from each other to lengthen the heteroduplex region
• After this process has come to completion, you get a holiday junction
• Holiday junctions are how homologous chromosomes are physically kept together
• Cohesin is how sister chromatids are kept together
• Holliday junctions are resolved when cut by resolvase in one of two directions
• In order to complete meiosis, holiday junctions need to resolve and can result good recombination

Recombination, Linkage, and Gene Mapping

• Non-disjunction is the failure of chromosomes to separate during cell division
• This can result in abnormal numbers of chromosomes in daughter cells
• Resolvase can cut in either one of 2 directions
• If you synaptonemal complex connects homologous chromosomes together during meiosis, then Spo11 creates DNA breaks

Chromosome structure

• During DNA replication, Cohesin complex keeps sister chromatids together
• Cells must enter S phase, whether they're going through mitosis or meiosis
• ALL chromosomes have it
• During Meiosis, in prophase 1, homologous chromosomes will come together and pair, synapsis
• Synaptonemal complex holds them together, and there can be exchange of genetic material in this step
• When the break happens, the cells will want to fix themselves/starts the process of recombination
• After the double-stranded break, exonuclease appears
• Formation of the holiday junction resects the DNA ends to create single-stranded overhangs
• This facilitates the recruitment of recombination proteins like RAD51 for homologous recombination repair
• Formation leads to RAD51 facilitating homologous recombination repair

Holiday Junction Mechanisms

• If the cell tries to divide, DNA can get breakage
• Holiday junctions get resolved when cut by an endonuclease within
• Asymmetric can result in a productive crossover effect
• Holiday junctions can be resolved to produce non-crossover products
• You aren;t getting a chiasmata= a point where paired chromosomes remains in contact during the first metaphase of meiosis, and at which crossing over and exchange of genetic material occur between the strands
• If you resolve the holiday junctions asymmetrically, you can get a crossover
• If dessolution, it is also non-crossover
• Meiotic recombination begins with a double-strand break (DSB) made by Spo11
• The 5' ends of this break are resected, leaving stretches of 3' ssDNA, which is probably used in a homology search
• Once the appropriate homologue is identified, Dmc1 begins a crossing-over event by stable invasion
• Follows by DNA synthesis that extends the end of the invading strand, and recapture of this strand generates double Holliday junction, which resolves to a cross-over
• The connection between two homologous chromosomes through a double Holliday junction is crucial for accurate segregation during meiosis

Genetic processes during meiosis

• During chromosome pairing, local distortion might allow homology to be sensed (alignment is independent of DSB formation)
• During DSB-dependent homolog interactions, 3' single-stranded regions engage in the homologous chromosome
• The synaptonemal complex is a proteinaceous structure forms between homologous chromosomes during synapsis

Recombination and Genetic Classes

• Parental phenotypes represent the original combination of traits, while recombinant phenotypes result from new combinations of alleles
• A higher proportion of parental phenotypes compared to recombinants suggests the genes are genetically linked
• The closer two genes are, the more they tend to be inherited together
• Recombination events only occur when genes are linked
• With linked genes, the presence of some recombinant phenotypes indicates crossing over has occurred during meiosis
• Greater physical distance means greater likelihood of crossing over
• Recombination frequency estimates genetic distance in centiMorgans(CM), so 10% recom. frequency= 10 CM
• Class data points will be unexpected when observed, meaning a genetic echange occured
• Top is mom, bottom is dad
• The key to determining gene positions and genetic distance is looking at recombinations vs crossovers in chromosomes

Mapping Genes - Recombination Analysis

• Four classes of progeny can be produced
• The most frequent pair has parental configuration of alleles
• Which recombination allele combinations show the greatest range
• It is key looking at recombinants between phenotypes in an experiment
• Must count and assess the allele difference in a population

Sturtevant and 3-Point Crosses

• 3-point test crosses can be used to map linked genes
• Recombination frequency must be adjusted for double cross overs: an adjustment is made by adding double crossover distance twice since the data point represents two exchanges

Genetic Vocabulary

• chiasma (plural chiasmata): X-shaped connection during meiosis between paired homologous chromosomes. Represents a site of chromosomal crossing-over,
• Bivalent: A four-chromatid structure formed during meiosis, consisting of a duplicated chromosome tightly paired with its homologous duplicated chromosome.
• a pair of synapsed homologous chromosomes during prophase of meiosis I, otherwise know as bivalent
• Nondisjunction: Event occurring occasionally during meiosis in which a pair of homologous chromosomes fails to separate; gives a gamate either too many or too few chromosomes.
• Tetrad and Bivalent same for our purposes

Aneuploidy

• results from nondisjunction of chromosomes or chromatids during meiosis to effect gamates
• Affects both somatic cells and germ cells
• If it normal meiosis can result in gametes with an number of chromosome numbers
• Most often during Meiosis I _ Depending on number of chromosomes affected, complications can arise like Down's/ Robert's Syndrome

Human Chromosome Defects

• Karyotypes reveal chromosomal abnormalities, used in genetic counselling
• We have 23 chromosomes ranked by size with characteristic bonding patterns
• Translocation, inversions, intra-chromosome deletions, aneuploidy are the most common
• Inversions have to be inverted from their locus
• Philadelphia translocation is example
• Trisomy 18 is lethal phenotype common
• Microcephaly, cleft palate and rocker feet are symptoms
• Large chromosomes have high likelihood for disease state
• Aneuploidy is the major driver of early miscarriage because not tolerateable in early life
• Incidence is dependent on maternal age because meiotic arrest is common
• Chromosomal aberrations also increases linearly with age

Human Aneuploidy

• Correlates with embryonic lethality (monosomy) and chromosome density
• Down Syndrome, Trisomy 13/18, Edwards most common
• Sex chromosome expression can effect development differently

DNA Replication

• DNA replication is semi-conservative
• DNA can be replicated three different semi-conservatively, conservatively and dispersively
• Watson and Crick proposed semiconservative replication in 1953
• Amount of A is the same as T, G always the same as c and the two DNA strands are complements
• Semi conservatively separated from each other to synthesize a new DNA strand
• Requires the nitrogenous based nucleotides to be labeled and synthesized
• Nitrogen had two isotopes that were tracked

DNA Replication Enzymatics

• The process must start with specific components and the template is read be polymerase in one direction ONLY (5' to 3')
• Requires nucleotide additions to hydroxyl to replicate
• Origin must be recognized
• Helix must also be prepared for template addition with enzymes

DNA replication process

• Replication orginis are activated per cell division, requiring a replicarot to recognize origins
• Chromosomes a licensed for replication so that the process occurs end to end
• At rich islands are what most DNA sequences utilize

Necessary elements for DNA synthesis

• DNA Helicases (MCM) or DNA topoisomerases: unwind DNA, relieve stress ahead of fork
• Single stranded DNA binding protein: stabililze single strands
• Primase: initiates synthesis with RNA primer (10-12nt) with RNA
• Polymerase: elongates the RNA primers
• Sliding clamp: hold polymerase to template
• RNA endonucleases, RNase: removes RNA primer
• DNA ligase: closes DNA, joining fragments

DNA Replication Detail

• DNA must coordinate all enzymatic activity
• In order for new DNA polymerase the addition must have hydroxyl
• Pol a and ligase join okazaki fragments
• Direction relative to DNA will affect synthesis
• Thermal replication utilizes all these process with different temperature gradients to express specific enzymes
• Single strand breaks require a helicase with temperature conditions

Problems with Chromosome Ends in Replication

• Priming is necessary for DNA polymerase, which involves RNA that is degraded
• Chromosomes are shortened with each cycle of machinery to start lost
• Cell cycle arrest and replacation takes time and cycles to initiate
• Problems with replication process due to end degradation effect expression significantly and prevent genetic propogation

Explore the intricacies of meiosis, focusing on homologous recombination, enzyme functions like DMC1 and resolvase, and the crucial role of the synaptonemal complex. Understand the differences between meiosis and mitosis, and the impact of genetic distance on recombinant frequency.