Class Note for ECOL 320 at UA 2
Class Note for ECOL 320 at UA 2
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Date Created: 02/06/15
Linkage and Recombination F T H Morgan Calvin B Bridges Alfred H Sturtevant Herman Joseph Muller Nobel Prize 1933 Nobel Prize 1946 Mendel studied 7 traits and every pair of traits that he reported in his paper segregated independently Interpretation are on different chromosomes Peas have N 7 chromosomes Somewhat unlikely that each trait is on a different chromosome In fact we now know they are not R round vs wrinkled and Gp green vs yellow pod are both on chromosome V syntenic but still segregate independently This we know is because they are so far apart ca 50 cM that there is on average one crossover between them in every meiosis This makes them behave as if they are independent unlinked Le tall long internode vs short internode and V inflated vs constricted pod are both on chromosome III and are so close together that there are very few crossovers between them and they do not segregate independently linked If Mendel had done a cross with these two genes he would have gotten very different results but he didn t Recombination production of new combinations of alleles at two or more PL loci Mechanisms Independent segregation of genes on different chromosomes Crossingover between genes on same chromosome Gene conversion parental genotypes gt recombinant genotypes A B A b a b a B A B A B A b We will study linkage recombination and gene mapping as follows 1 Linkage as it was first seen and understood in Drosophila 2 Definition and mechanisms of recombination 3 Using recombination frequencies to map genes Extend timeline 1866 1900 19023 19101916 basic rules rediscovery chromosome theory Thomas Hunt Morgan Mendel Hugo DeVries Walter Sutton Alfred H Sturtevant Carl Correns Theodore B overi Calvin Bridges H J Muller Drosophilu Linkage was first seen a few years after Mendel39s laws were rediscovered in 1900 First correctly interpreted ca 1912 by Drosophila research group T H Morgan Professor at Cal Tech began working with Drosophila melanogaster Calvin Bridges Alfred H Sturtevant began working in lab as undergrads H J Muller graduate student in another department Morgan got first mutant in 1910 w white eyes next year got two more m miniature wings and y yellow body Drosophila gene nomenclature mutant allele wild type allele mutant recessive white eye w w mutant dominant Bar eye B B Linkage of genes in animals and plants is seen as deviations from independent segregation recombination seen as deviation from complete linakge Found red white trait difference linked to male female sex trait w w red X w white X X female XY male l w w red X w red X X female XY male l all red females 1 red0 white 12 w w amp 12 w w 12redmales 12w 12 white males 12w Frequencies of red and white are different in males and females therefore eye color and sex don39t segregate independently ie sex and eye color are linked Sex already shown probably determined by sex Chromosomes in Drosophila Sex determination in Drosophila Karyotype 1 X 0 sex Chromosomes 1 Y o 2 o autosomes 3 o 4 o X and Y are heteromorphic pair Y has few genes Females X X Males X Y X 0 is sterile male Sex actually determined by interaction of many genes on X and autosomes but for the following we can think of it as a trait determined by whether there is one or two copies of the X Morgan et al hypothesized m and w are both on sex chromosome m m w w X m w no alleles on Y gt F1 female Did testcross to see what kinds of gametes produced Cross to m w male or to male of any genotype and look only at male progeny parentals recombinants Expect female gt eggs m w m w m w m w Expected if independent segreg 025 025 025 025 Expected if complete linkage 05 05 0 0 Observed partial linkage 031 031 019 019 Recombination frequency recombinant gametestotal gametes In this case recomb freq 038 so 38 of chromatids are recombinant for m and w Three Kinds Of Recombination Seen In Tetrads Gene conversion can be distinguished from other kinds of recombination only in tetrads No conversion all tetrads 22 Gene conversion tetrads 31 or 13 In random tetrads conversion genotype a M It can t be distinguished from same recombinant genotype produced by crossingover or independent assortment How distinguish gene conversion from mutation OMuch higher frequency 00ften associated with nearby crossover Firm H Var H l hh 39 quot m H H K xquot 1 0L x K i l B c lt j l I o39I J quotM lrrquot quotJf x39clFrquot39 eff 39 x ff m H a x 39 39 939 0 E39 O I I REM h m HEM g meiosis pmphase Jr d H ix ff Eli EL 3 DP L 134 gene l l 6 E j E 9 E CEIr39I39u39EJISIUh RM h or j h a j 3 HEMquot 5 dquotquot aquot ff n Gm amH laml IlaMH f r 39l IIIla m al Ixx wg r h39l nth1h poiNth 2th Joamh I quotI LK HMJ39 aJif H J f 39x f WSW Iquot We m HIP m H to m HjlicxM H U M X f 39x thlLaMh fam1hamh q l H J gf 39 When does recombination take place 0 Prophase of meiosis I very high frequency Chiasmata are physical manifestation In some organisms required for normal segregation 0 Interphase of mitosis low frequency Genes are linked if show lt 50 recombination Genes are unlinked if show 50 recombination independent segregation Genes are completely linked if show 0 recombination very rare if one looks at very large sample of offspring In organisms with prominent haploid stage and tetrads recombination frequency frequency of recombinant haploid products random spores Also can be calculated from frequency of a certain type of tetrad eg ADE8 trp4 X ade8 TRP4 gt diploid ADE8 trp4ade8 TRP4 meiosisgt many hundreds of asci gt random sample of 100 spores ADE8 trp4 38 parental ade8 TRP4 42 parental ADE8 TRP4 11 recombinant ade8 trp4 i recombinant 100 Recombination frequency 20100 02 Recombinants and parentals are in pairs of equally frequent genotypes because each crossover generates two reciprocal recombinants RECOMBINATION MAPPING Recombination frequencies may be used to map the position of genes loci on linear linkage groups The order of genes and the relative distances between them in a linkage group corresponds to their order and relative distances on a chromosome Recombination is used to map genes Morgan hypothesized that the further apart two genes are the more likely they are to have a crossover between them Sturtevant 1913 worked out method to map genes on chromosomes recombination frequencies between Xlinked genes w v 30 v m 3 gt genetic or linkage map w 30 v 3 m w m 33 Position of gene locus 39W 3939 TI 30 33 Map shows order of loci and relative distance not absolute 1 map unit 1 recombination 1 centimorgan 1 morgan 01 recombination Genes far enough apart on same chromosome can show recombination frequencies of 50 Without additional information one cannot distinguish these cases from loci on independently segregating chromosomes Double crossovers aren39t detected in random gamete samples so recombination frequencies are always underestimated Subsequent studies showed map distances not additive over long distances eg in Sturtevant s data he probably observed w m lt 33 Reason double Xovers Only odd numbers of Xovers between two genes one one pair of chromatids give detectable recombination 39 39 39uI qi I u m quotquot crower i mulw H gt uidI39ll thI II lTl 3quotquot L I39T39l39l39 W ChillIll TilIllin fT39l W m quotN Map function is a mathematical formula that relates the observed number of crossovers to the real number which is a function of the physical distance It assumes a simple model in which crossovers are distributed randomly on the chromosome xquot If If ff ebeerued g39 recombination L5 gx frequency 14 Huff L3 5 I12 I 1 Li gquot I i I in 2 3U 4U SCI 151 RBI physical distance CM One explanation for why maximum recombination frequency 2 50 as distance increases crossovers between any two markers increases but some of these are double and other evennumbered crossovers The number of these increases Number of observed crossovers approaches 05 asymptotically Tetrad analysis in yeast and Chlamydomonas can detect double crossovers Cross a b X a b d1p101ds undergo me10s1s look at chromosomes in me10s1s I detectabl a b K39CIVEIE parental U ampe PD W4 1 NED I a b tetrat39 e 1 a 2311 39 h USED I 1 3 a h 2strantl a b 1 E PU WEI 239 l 3 strand 2 2 3 1E b T 234 I 1 a nonparental 4Str ndf 3 itFla diff I l l 1 39 14 l b Double x overs produce a distinctive type of ascus the NPD detectabl a b xDvers parental n dimpa PEI W l l NED II b a tetrat39ji39pe 1 a 1 a i g b T quoti ESLDJ l 392 2strand a h g E PU Nil 131 l 39 l I 3 3trand 2 a a b l b T 34 a nonparental 4 Strandf a a b 14 I b Double x overs produce a distinctive type of ascus the NPD Consequently NPDs are a way of estimating the number of DCOs which will be 4 X the number of NPDs Genes unlinked PD NPD If every tetrad has a single or double crossover 2 4 50 of crossovers will be detected therefore maximum observable frequency of crossing over is 50 ThreeFactor Crosses In the absence of tetrad analysis some double crossovers can be detected by using three factor crosses Drosophila Xlinked genes yellow body 3 cut wings ct echinus eyes ec Female heterozygous at all three loci y ct ec X This is test cross if look at male progeny because male contributes Y with no genes to male offspring phenotypes gametes males wild type 1080 yellow cut echinus y ct ec 1071 cut ct 293 yellow echinus y ec 282 yellow y 78 echinus cut ct ec 66 echinus ec 6 yellow cut y ct 4 2880 Note that the genes are linked if they weren39t we would have 8 phenotypes and 8 gamete genotypes in approximately equal numbers Arranged in pairs of equal numbers in order of magnitude Which are parental genotypes Which are double crossover genotypes ThreeFactor Crosses pheno pes gametes males wild type 1080 parental yellow cut echinus y ct ec 1071 parental cut ct 293 yellow echinus y ec 282 yellow y 78 echinus cut ct ec 66 echinus ec 6 double xover yellow cut y ct A double xover 2880 Find parental most common and doublecrossover least common types Middle gene is the one that is switched relative to the other two in doubles vs parentals A B C A B C A C gt a b c a b c a B c Which gene is in the middle y ct 0r ec phenotypes wild type yellow cut echinus cut yellow echinus yellow echinus cut echinus yellow cut ThreeFactor Crosses gametes males 1080 parental y ct ec 1071 parental ct 293 y ec 282 y 78 ct ec 66 ec 6 double xover y ct A double xover 2880 Analyze data as three twofactor crosses m 293 282 78 Q 719 0250 ct ec ec 293 78 282 66 6 6 A A 585 154 0203 0053 Longest distance is y ct so these are the outside markers on the map Agrees with previous conclusion that ec is middle marker ThreeFactor Crosses Analyze data as three twofactor crosses m ct ec ec 293 293 78 282 282 66 78 6 6 E A A 719 585 154 0250 0203 0053 Longest distance is y ct so these are the outside markers on the map Agrees with previous conclusion that ec is middle marker y ec ct l i 53 203 distance in map units What is best estimate of distance between y and ct 250 or 203 53 256 ThreeFactor Crosses Analyze data as three twofactor crosses m ct ec ec 293 293 78 282 282 66 78 6 6 E A A 719 585 154 0250 0203 0053 Longest distance is y ct so these are the outside markers on the map Agrees with previous conclusion that ec is middle marker y ec ct 53 203 distance in map units What is best estimate of distance between y and ct 250 or 203 53 256 250 includes only the single crossovers and omits the doubles 6 4 10 doubles x 2 20 crossovers occuring in pairs Interference Morgan39s group first assumed xovers occurred independently Then found out that was wrong expected doubles if independent Pdouble Psingle yecPsingle ecct 00530203 00108 Observed doubles 102880 00035 0r Expected number of doubles 00108 2880 31 observed number of doubles 6 4 10 Can do statistics with numbers not with frequencies Observed lt expected therefore one crossover interferes with occurrence of another but not completely This works in our favor means that problem of double crossovers isn39t quite as bad as it might be Also means that we can39t predict the number of double crossovers exactly from the number of singles without correcting for interference Interference doesn t always happen mapping very large number of markers in rice showed negative interference over short distances Maybe because some sites are hotspots for recombination Recombination frequencies do vary along chromosomes and between chromosomes Drosophila No crossingover in males LINKAGE GROUPS AND CHROMOSOMES Linkage group group of genes each of which is linked r lt 05 to at least one other eg new organism Loci recombination a b 50 Data allow genes to be put in two linkage groups a c 10 a d 50 a 10 c b 20 d b c 50 b d 20 c d 50 We know that a and c are on the same chromosome and that b and d are on the same chromosome Do we know if these two linkage groups are on the same or different chromosomes LINKAGE GROUPS AND CHROMOSOMES Linkage group group of genes each of which is linked r lt 05 to at least one other eg new organism Loci recombination a b 50 Data allow genes to be put in two linkage groups a c 10 a d 50 a 10 c b 20 d b c 50 b d 20 c d 50 We know that a and c are on the same chromosome and that b and d are on the same chromosome Do we know if these two linkage groups are on the same or different chromosomes NO Suppose a new mutation e is found It is linked to both c and d with recombination frequencies ce 45 and de 35 We now have one linkage group a 10 c 45 e 35 d 20 b Note that e will show 50 recombination with a and b even though all of these genes must be on the same chromosome If one begins working with a new organism at first most mutations are unlinked Eventually some linkage groups appear The number increases at first then decreases as mutations are found which combine different linkage groups By Various genetic tricks genes can be assigned to specific chromosomes seen in karyotype Use heteromorphic pairs XY knobbed amp knobless or Variants structure 0 Use molecular methods eg Fluorescent In Sim Hybri zation FISH Cloned DNA segment with Gene of interest is labeled with a uorescent dye Squashed metaphase chromosomes are treated to denature the DNA then the labelled probe is hybridized with the chromosomes When one gene is assigned to a specific chromosome all genes belonging to same linkage group are assigned to that chromosome Predicting the outcome of crosses from linkage maps 1 u U L 0 10 15 45 aa BB XAA bb gt F1 Do testcross What gametes will the F1 produce total crossovers ab 015 010 005 total freq recombinants a b and A B a b 0025 A B 0025 Parentals 1 recombinants 1 005 095 a B 0475 A b 0475 Check 0025 0025 0475 0475 1 Intragenic Recombination Gene is long piece of DNA so recombination can occur within as well as between genes Can have 2 different mutations in same gene gt 2 different mutant alleles Can have recombination between the sites marked by different mutations Recombination between markers in different genes is called intergenic recombination even if the crossover event occurs within a third gene mfg Yfg Recombination between markers in same gene is intragenic recombination E g Drosophila chromosome III gene codes for enzyme xanthine dehydrogenase XDH Mutants that don39t make XDH can39t make isoxanthopterin an eye pigment so eyes are rosy in color instead of brick red Cross ry23 X ry6 gt 131 diagrammed below gt gametes parentals mutant ry23 mutant ry6 recombinants double mutants ry23 ry6 wild type ry mag 520 ebony W1 23 ru 11 E m Eb Intergemc A E E r1453 r95 r1123 mtragemc FQE39 What genotypes and phenotypes would one get if there was a crossover between the two rosy mutants mag 520 ebony W1 23 ru 11 E m Eb Intergemc A E E mtragemc FHE39 What genotypes and phenotypes would one get if there was a crossover between the two rosy mutants Answer ry Wild type and ry23 ry6 double mutant Detecting Intragenic Recombination The frequency of intragenic recombination is usually low so it can only be detected by selecting recombinants from large numbers of progeny Rosy gene is about 0005 cM long ie recombination between markers at the ends of the gene occurs in about 0005 or 000005 5 X 10395 of all gametes from a heterozygous female Would have to look at 105 progeny to be sure of finding one or a few recombnants Only practical because can select for wild type recombinants rear progeny on medium with added purine XDH required to detoxify purine so purine kills rosy mutant larvae but allows wild type larvae to live In above cross these are recombinants Easier in microorganisms yeast bacteria bacterial viruses Section 15 this one is on the web in complete version Homework deadline extended to Wednesday WHAT IS A GENE Initially a gene was a hereditary factor that had two or more alleles which determined the difference between two or more alternative phenotypes 0 Different genes controlled different aspects of phenotype Gene unit of function 0 Mutation changed one allele to another Gene unit of mutation 0 Different genes could be separated by recombination Gene unit of recombination These units usually agreed with each other until genetic analysis was extended to bacteria and viruses in which rare genotypes can be selected and detected Then complications arose 0 Unit of mutation is a single base pair not a whole gene 0 Mutations within a gene even in adjacent base pairs can be separated by recombination 0 Two different genes identified by recombination or mutation may control the same phenotype How can we define a gene How can we define a gene 1 Sequencing Problems Not foolproof 0 Impractical in many cases complete genomes available for very few eukaryotes 0 Most genes in most organisms found initially by finding mutants with different phenotypes 2 Complementation Test Eg Drosophila want to identify all genes required to make eyes All expected to have eyeless phenotype or some kind of abnormal eye shape Select many recessive ey mutants don39t know whether they represent 1 2 or more genes Do complementation test to see which ones are allelic and how many genes they represent Suppose ey and ey2 are in same gene but ey3 is in a different gene ey ey X ey2 ey2 gt ey ey2 heterozygote look at phenotype ey ey X ey3 ey3 gt ey ey3 heterozygote look at phenotype Egi EQZ em EgEE F K x l 99 EM E E E a 392 Eg3 phenntgpe mutant Wild tLIDE no complementation complementation For recessive genes if make double mutant mutations allelic in same gene mutant no complementation mutations nonallelic in different genes Wild type complementation Trans means mutations are on different homologues cis means are on same homologue trans cis trans cis a b a b a b a b a b a b a b a b What will bed the phenotypes of the heterozygotes in the cis configuration Trans means mutations are on different homologues cis means are on same homologue trans cis trans cis b a b a b a b a b a b a b a b Complementation test also called sistrans test because must be done in trans to distinguish mutations in the same or different genes In the cis configuration will get wild type either way Genes defined by complementation are sometimes called cistrons By defining alleles we are also defining genes Mutations that don t complement each other are said to be in same complementation group or rarely cistron Each complementation group one gene Complementation actually means that two genomes complement each other because each one has the wild type allele of one gene and the mutant allele for another Mitotic Recombination Recombination occurs during mitosis as well as meiosis But although there are more than ten mitotic divisions and only one first meiotic divisions in a germ line recombination is more frequent in meiosis when synapsis helps bring homologous sequences together Mitotic recombination occurs between any homologous sequences which may be on the same chromosome sister chromatids or nonhomologous chromosomes as well as on homologues Will look at this during Discussion
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