2581 final

synaptonemal complex

forms during prophase I of meiosis

proteinaceous complex that holds two homologous chromosomes together

meiosis steps

Meiosis I: DNA replicated, alignment (synapsis), monopolar attachment of MT to centromeres of homologous chromosomes, anaphase two homologous chromosomes separated

meiosis II: separation of sister chromatids leaving 4 gametes

meiosis I exchange of information

homologous chromosomes physically exchanged, mixture of information

what processes give rise to recombinants?

recombination

law of independent assortment (Mendel’s second law)

law of independent assortment (Mendel’s second law)

two parents one with dominant alleles on separate chromosomes and one with recessive alleles cross to form a progeny with all four alleles, cross F1 progeny with individual homozygous with recessive alleles, progeny are either dominant or recessive

parentals

combinations are the ones that occurred in the parental generation

non recombined

recombinants

product of the mixture of alleles

undergone recombination

recombination and gene mapping

recombination can separate alleles even on same chromosome, ability of recombination to move alleles allows us to map genes

number of recombinations is proportional to

distance between two genes on the same chromosome

two-point test cross

* cross to create a diploid heterozygous for alleles at two genes
* cross back to a test strain homozygous for the recessive alleles in the two genes
* follow what occurred in meiosis of F1 heterozygotes in creating gametes for test cross

number of parental or recombinant progeny of two point test cross

number of individuals with non-recombined chromosomes is higher than recombinants because genes are linked close to one another in chromosome

distance between genes of parental recombinant equation

number of recombinants/ total x 100 = distance between genes (percentage)

if gene is 50 mu (for example) away from rest of genes that means

not linked, could be on the same chromosome but too far away form loci (high number of recombinations)

three point test cross

* efficient method to determine gene map
* provides evidence that one crossover can suppress the occurrence of a nearby crossover
* interested in meiosis I of heterozygotes (F1 progeny)
* cross F1 with recessive homozygous

progeny types of three point cross

2 parentals, 4 single cross overs, 2 double cross overs

double crossovers

allele of middle gene is switched

chance of a double crossover

produce of 2 single crossovers

frequency of double crossing over

lower than single crossing overs

frequency of recombination when genes are linked

lower than 50% / 50mu

parental frequency in three point test cross

highest

how to find recombination frequency between two genes

\# of recombinants x 100% / total

how to map genes in three point test cross

find recombination frequency between ordered genes (a**b*c- between a and b, between b and c)* and add total

what happens when you remove double crossing over phenotype

looks like parental

what happens when you remove the middle gene in a three point test cross

number of parentals increase

double recombinations affect on map distance

shrink map distance between two genes

do the theoretical number of double crossovers equal observed?

observed = number given for frequency of double crossovers

theoretical = 2 single crossover events multiplied = double crossing over event proportion multiply this by total

coefficient of coincidence

\#observed/#expected

interference

1- coefficient of coincidence (#observed/#expected)

interference definition

when a crossover suppresses the occurrence of a second nearby crossing over spreading out crossing overs across the genome

primary oocyte

meiosis arrested at meiosis I

first polar body

holds one homolog

second polar body

holds on sister chromatid

what happens when you have access to info of a single meiosis?

conclude that recombination occurs after DNA replication, map position of centromeres

advantage of studying yeast

viable haploid stages that can be brought together to form a diploid through mating

creates ascospore (4 haploid spores)

ascospore

4 haploid spores of yeast

tetrad analysis

* dissolve ascus cell wall to release spores
* glass needle picks up spores and place on rich media in rows of four spores

URA3

dominant functional allele for a gene that encodes an enzyme for the biosynthesis of uracil

ura3

recessive loss of function allele in the URA3 locus, results in cells requiring the addition of uracil to the medium for growth because the cell can no longer synthesize uracil

tetrad analysis result

mendel’s first law of segregation (2+ alleles 2- alleles)

what happens when tetrad analysis of unlinked genes

create diploid heterozygous for both loci on distinct chromosomes (each spore has one sister chromatid from each chromosome)

mendel’s second law of independent assortment

parental ditype PD of tetrad

two genotypes of parents in the spores (ex. TRP his4, TRP his4, trp1 HIS4, trp1 HIS4)

non parental ditype

genotypes distinct from parents (ex. trp1 his4, trp1 his4, TRP1 HIS4, TRP HIS4)

in unlinked genes number of parental ditypes and non parental ditypes

are equal due to random alignment of chromosomes on the metaphase plate

tetratype T

all four spores have a different genotype of which two have the same as the parents and two do not

result of recombination between centromere and locus

linked genes on the same chromosome tetrad analysis

what can this tell us

when recombination occurs

no crossing over results in (tetrad analysis of linked genes)

parental ditype, parental ditype >> NPD

single crossover results in (tetrad analysis of linked genes)

tetratype

two strand double crossing over event results in (tetrad analysis of linked genes)

parental ditype

3 strand double crossover event results in (tetrad analysis of linked genes)

tetratype

4 strand double cross over event results in (tetrad analysis of linked genes)

nonparental ditype

recombination frequency in (tetrad analysis of linked genes)

NPD + 1/2 T x 100% or mu / total tetrads

ordered tetrad

4 cells undergo mitosis resulting in octad (tetrad undergone mitosis) resulting in two sets of cells that are genetically identical

centromere linked gene no recombination in ordered tetrad

4 identical gametes on top and 4 identical gametes on bottom

segregation across metaphase plate of meiosis I

centromere linked gene recombination in ordered tetrad

second divisions are distinct from one another

identical gametes in groups of 2

recombination between a gene and a centromere equation

1/2 x (#of tetrad with 2nd division events) / total tetrads

double strand break model for recombination

* induce double strand break
* resect at 5’ ends to expose single stranded 3’ ends
* one single strand break finds sequences on homologous chromosome
* 3’ end turns into primer
* formation of double holiday junction
* homologous chromosomes connected through base pairing
* mismatches during heteroduplex formation (single stranded breaks are paired with homologous chromosome)
* branch migration (large stretches of heteroduplex dna)
* holiday intermediate and resolution

how did he propose heteroduplex formation

3:1 segregation pattern

gene conversion

1. breaks mendel’s law of segregation
2. replacement of one piece of information with another
3. 3:1 segregation in a tetrad analysis
4. first evidence of heteroduplex formation

mismatches in a heteroduplex

dna sequence variation between homologous chromosomes

repair of mismatch in heteroduplex results in

3:1 segregation

role of recombination

exchange of information between homologous chromosome

synaptonemal complex contains

zipper proteins

random cross overs

do not regulate where crossing over occurs in a genome during meiosis

random cross overs and size of chromosome

more crossing over events will occur on larger chromosome (assuming no interference) simply due to the fact that they contain more genomic DNA than a smaller chromosome

what is required for segregation during meioiss

spreading of crossing overs

negative interference

process where crossing over events are regulated so crossing over event can inhibit a nearby crossing over event

why larger chromosomes have more crossing over events

classes of genome sequence variation

substitution, indels, inversion, translocation

detection of larger scale genome changes

cytogenetics, pcr, dna sequencing

cytogenetics

what you can see down a microscope

karyotype, salivary gland giant chromosomes

how many chromosomes in humans

46

karyotype

cells arrested at metaphase, content of nucleus spread on glass slide and observed in a microscope

organized by size, 1 = largest

metacentric chromosome

centromere in middle

submetacentric chromosome

centromere a little off centre

acrocentric chromosome

centromere way towards one end

telocentric chromosome

centromere at the end

what type of chromosome do human karyotype do not have

telocentric

giant polytene chromosomes

large chromosomes with high degree of detail to map genes cytogenetically

drosophila giant polytene chromosomes

* during salivary gland development cell division stops, but not DNA replication
* homologs are aligned
* protein associate differentially along the chromosomes creating a specific banding pattern when stained
* each band 20-100kb of data

PCR detection of genome variation

analyze microsatellites or short tandem repeats to find areas where there are dinucleotide repeats due to slippage or unequal crossing over

PCR detection of genome variation experiment

design primers on either side of repeat run band in gel

longer length repeats run slower

CODIS

short tandem repeat basis for forensic identification of individuals because of 13 core STR and high degree of variation in loci

DNA sequence analysis

modern method, direct dna sequence analysis

align to reference genome to classify variation

chromosomal deletions

multiple genes deleted

lethal when homozygous

effect of chromosomal deletions on recombination frequency

suppress recombination frequency, no recombination in deleted region, distance between genes decreases

chromosomal deletion effect on recessive alleles on the homolog

lf allele in genes deleted, genes lost in deletion become null lf alleles

chromosomal deletion effect on recessive alleles on the homolog during a cross

dominant allele can complement the loss of function allele, if both recessive, loss of function

chromosomal deletions on polytene chromosomes

bubble in chromosome, wt homolog bulges out

chromosomal deletions in PCR analysis

use multiple primers along the chromosome to detect deficiency, product from deletion chromosome since wt is too long to detect

tandem duplication

duplication inserted in direct orientation

displaced duplication

duplication inserted far away from original loci

reverse duplication

inverted orientation

chromosomal duplications in polytene chromosome

duplicated homolog bulges out

use of a duplication

looking for haploinsufficiency

chromosomal duplications and gene dosage

in duplicated chromosome, copy number of duplicated region is 3

increase in gene dosage can affect development of chromosome and physical phenotype

how much of human genomic sequence are copy number variants

5-10%

consequence of transposable element in direct orientation on same chromosome

deletion

consequence of transposable element in direct orientation on same chromosome misaligned

deletion and duplication

paracentric inversion

inversion next to centromere

pericentric inversion

centromere is within the region inverted

effect of inversions

gene that is split becomes loss of function allele

gene next to new DNA can become a gain of function allele