BIO 305 1st Edition Lecture 6 Outline of Last Lecture 1. Chromosome Mapping in Eukaryotes2. Three-Point Mapping: A Few Rules and One Big Example3. Interference4. Map Distance Outline of Current Lecture I. Linkage Analysis and Mapping in Haploid EukaryotesA. Mapping the CentromereB. Exchange frequencies – Parents, Non-parents, TetratypesII. Gene Mapping in HumansA. RFLPs – Restriction Fragment Length PolymorphismsB. Assessing Linkage with Likelihood Ratios:III. Vocab and Sample QuestionsCurrent LectureI. Linkage Analysis and Mapping in Haploid EukaryotesWhen two neurospora, a type of fungus, mate: they produce a zygote (with 2n). This zygote undergoes a first meiotic division: (n) and (n) remain in the ascus.Then a second meotic division: there are 2 copies of (n) and 2 copies of the other (n). Each of these are called tetrads. Now there are 8 cells in total. These cells are called acrospores.Why is the ascus important?Asci – sexual spore-bearing cell produced in fungiIn neurospora, the ascus keep the 8 ordered. This makes it the fungus a model organism because we can use this to study how cells divide and crossover in meiosis.A. Mapping the CentromereThis may look intimidating, but looking at things step-by-step will help you.The orange and blue represent the two halves of the zygote (one color for each parent). The black circle indicates the centromere. If there was no crossover in meiosis, the acrospores dividesimply half-and-half in the manner shows in the “acrospores in ascus” box. Familiarize yourself with the notation (ex: aaaa ++++)If there is crossover in the first division of meiosis, half of the products will have changed, but not the other half, right before duplicating, hence your acrosomes look like aa ++ aa ++An alternative version of (b) is shown, one strand crossed over with another further down. In order to map the centromere, aka find the distance between some gene A, you should memorize the equation: where second division segregation loci means “number of sets where there was meiosis II segregation for the particular gene you are comparing with”. Recall that map distance reflects % of recombinants. Note: Mapping is less accurate as distance increases!B. Exchange frequencies – parents, nonparents, and tetratypesNow you have three types of genotypes of tetrads: Parental, Non-parental, and Tetratype.Parental type – usually the highest sample number in your data set, parents have no crossover thus the product looks the same as the parent:Non-Parental – you have crossover that occurred in BOTH genes a and b in EITHER meiosis I or meiosis II. You have to first determine the parent type, than compare to find the non-parentalTetratype - when you either have a single crossover or the crossover of gene a occurred in in a different stage of Meiosis (I or II) than the otherExchange frequency = map distance between genes when doing ascus problemsII. Gene Mapping in HumansThe following topics are less relevant to the exam but are still relevant to the material covered.A. RFLPs – restriction fragment length polymorphisms are techniques that exploit differing locations of restriction enzyme sites. This method uses a probe that cuts a chromosome when ithas been detected a certain genotype: For example, if homozygous for chromosome (A/A), two bands can be seen if the DNA sample is run through a electrophoresis gel because it has been cleaved into two pieces. Three bands willbe seen if it is heterozygous (A/B) and only one large band of 10 kb is seen for homozygous (B/B) DNA. This is useful for pedigrees, in which you can experimentally ensure what genotype the individuals are for a particular gene.B. Assessing Linkage with Likelihood Ratios:The LOD Score Method“Logging the odds favoring linkage” can be done in three steps:1) Compute probability that the two genetic markers are not linked under first hypothesis2) Then compute again, except assuming they are linked with a certain recombination rate3) Ratio of probabilities tells how strong one hypothesis supported by what was observed by comparing it to the other hypothesisLet’s use this pedigree for example:We want to know the probability of observing this particular pattern of segregation of the progeny, assuming d1 and D2 loci are linkedIf they are linked, then probability of the getting the first son as shown is ½ (the likelihood of getting alleles D and 2 from the mother), the first daughter is ½, and also the second daughter is½. Multiply these together and it equals 1/8.But if they are not linked, the son is ¼, first daughter is ¼, and the second daughter is ¼. Multiply these together and it equals 1/64. Plug these in to this equation to get the likelihood ratio, likelihood ratio = (1/8) / (1/64) = 8See if you calculate the ratio for each pedigree shown below:The likelihood ratio for each is 8, 16, and 32.LOD = log10 (likelihood ratio) You take each likelihood ratio and plug it into this equation to get a LOD. You do this for each pedigree, then add up the LODs to get Total LOD. If Total LOD is greater than or equal to 3, it is significant. If the LOD score is less than or equal to 2, linkage is excluded.But what if there is recombination?Observe this example in the case of recombination:In reality: 1) Calculate LOD score at a range of x values2) x with largest LOD is most likely the maximum likelihood estimate 3) When the marker recombines, LOD at x=0 is negative infinitySomatic Cell HybridizationGeorge Barsky – discovered that somatic cells of different species can be induced to fuse into a single hybrid cell. Chromosomes from one species are gradually lost over time.Ex: Human-mouse cells – humans were lost over time while mice chromosomes were entitely retainedAn example of chromosomal banding + translocation:III. Vocabulary and Sample QuestionsElectrophoresis – a lab technique that exploits the negatively charged attributes of DNA in orderto sort the DNA by size (molecular weight) along a visible gelAscospore – eight spores contained in a single ascus produced by meiotic division of fungiOrdered Tetrad Analysis – the genotype of each ascospore and its position in the tetrad Is determined. To do this, individual asci must be dissected and each acospore must be tracked as it germinates Unordered Tetrad Analysis – the genotype of each acospore but not its location in this tetrad is determinedProtoperithcium – one of the organs in fungi that help facilitate