Transcript 18.11
שיטות מיפוי נוספות • • • • • תדירות רקומבינציה מהכלאה עצמית שימוש בכרומוזום Yכ'-בוחן' "הגבול" בין תאחיזה להפרדה עצמית – C2 התחשבות בשיחלופים שלא רואים – ”"mapping function טטרדות – מיוזות בודדות ,ומיפוי בין גן לצנטרומר 1 2 3 The yeast Saccharomyces cerevisiae is commonly used as a model system =Budding Yeast =Bakers Yeast 4 The yeast Saccharomyces cerevisiae is clearly the most ideal eukaryotic microorganism for biological studies. The "awesome power of yeast genetics" has become legendary and is the envy of those who work with higher eukaryotes. The complete sequence of its genome has proved to be extremely useful as a reference towards the sequences of human and other higher eukaryotic genes. Furthermore, the ease of genetic manipulation of yeast allows its use for conveniently analyzing and functionally dissecting gene products from other eukaryotes. --Fred Sherman 5 Major advantages of the budding yeast as a genetic system • Grows fast • Cheap • Compact genome, fully sequenced since 1996. • Easy to handle • Superb Genetics, Biochemistry, Molecular Biology • Easy to transform, high efficiency of gene targeting 6 Major characteristic of the budding yeast • Unicellular Eukaryote • Grows by budding • 16 linear chromosomes • Generation Time: ~100 min •7 Can exist as stable diploid or haploid "for their discoveries of key regulators of the cell cycle" Yeast: A model eukaryote The Nobel Prize in Physiology or Medicine 2001 Yeasts – the ultimate model eukaryote for unicellular issues and some basic cell-cell interactions Yeast studies have broken new ground in: Tim Hunt Lee Hartwaell Cytoskeleton functions Paul Nurse transcription mechanisms** cell cycle** transcriptional regulation organelle biogenesis chromatin modification secretion* signal transduction Randy W. Schekman protein James E. degradation* Rothman Thomas C. Südhof protein targeting mechanisms chromosome replication DNA repair The Nobel Prize in Physiology dynamics retroviral packaging or genome Medicine 2013 prions recombination mechanisms ageing function of new genes metabolism protein modification for their discoveries of machinery regulating vesicle traffic, 8 a major transport system in our cells *Lasker Award **Nobel Prize What is yeast? • • • • • • Yeast - a fungus that divides to yield individual separated cells (as opposed to molds- mycelium) Saccharomyces cerevisiae (budding yeast) baker’s yeast closely related to brewer’s yeasts grows on rotting fruits Schizosaccharomyces pombe (fission yeast) African brewer’s yeast Saccharomyces relatives (S. bayanus, S. paradoxus, etc.) Candida albicans Cryptococcus neoformans 9 Yeast life cycle 10 Yeast cell cycle (mitosis) Major control point is at G1/S morphology • reflects cell cycle position same in haploids • and diploids major control • point is ‘start’-cells can choose – mitosis, meiosis or mating depends on ploidy, – env. & presence of partner 11 Morphology + nuclear localization and MT localization indicates the precise stage of the cell cycle 12 13 • Mendel’s rules are relevant for organisms that sexually reproduce: diploid/haploid Plants, Animals, many Mora… – Those that ‘do’ meiosis 14 15 Haploid Mitosis 16 Diploid Mitosis 17 Haploid Mitosis 18 Diploid Mitosis Of diploid Of haploid 1n 1n 1n 2 1 19 Centromere mapping 20 Centromere mapping Nonsister chromatids do not cross over 21 First-division segregation pattern or MI pattern 22 23 n = 2 -> 4 S 24 2 1 1 1 Centromere mapping Nonsister chromatids do not cross over 25 First-division segregation pattern or MI pattern Centromere mapping Nonsister chromatids cross over 26 Second-division segregation pattern or MII pattern 27 28 29 30 31 32 33 34 35 36 1. The loci are on separate chromosomes 2. The loci are on opposite sides of the centromere on the same chromosome 3. The loci are on the same side of the centromere on the37same chromosome Unordered tetrads B a Mating b B a b A A Heterozygous diploid Meiosis B B b b a a A A Tetrad Tetrad Dissection 38 BA BA ba ba NPD Ba Ba bA bA PDT BA Ba ba bA TT Yeast tetrad analysis (classic method) Step1: separate spores by micromanipulation with tetrad a glass needle Step2: place the four spores from each tetrad in a row on an agar plate Step3: let the spores grow into colonies 39 Classical approach (tetrad dissection) Tetrad Dissection Tetrad bni1∆ bnr1∆ 40 BA BA ba ba Ba Ba bA bA BA Ba ba bA NPD PDT TT 41 MESSAGE Linear and unordered tetrads can be used to calculate the frequencies of single and double crossovers, which can be used to calculate accurate map distances. Perkin formula 42 Map distance=50(T+6NPD) m.u What PD, NPD and T values are expected when dealing with unlinked genes? B B b b Mating aa aa Meiosis aA aA B B aa aa b b aA A Tetrad Heterozygous diploid Tetrad Dissection 43 BA BA ba ba NPD Ba Ba bA bA PDT BA Ba ba bA TT What PD, NPD and T values are expected when dealing with unlinked genes? B B aa aa Meiosis The sizes of the PD and NPD classes will be equal as a result b of independent assortment. Mating b The T class can be produced only form a crossover between the aA aA specific loci and the and their respective centromeres B B b b aa aa a A A Tetrad Heterozygous diploid Tetrad Dissection 44 BA BA ba ba NPD Ba Ba bA bA PDT BA Ba ba bA TT שיטות מיפוי נוספות • • • • • תדירות רקומבינציה מהכלאה עצמית שימוש בכרומוזום Yכ'-בוחן' "הגבול" בין תאחיזה להפרדה עצמית – C2 התחשבות בשיחלופים שלא רואים – ”"mapping function טטרדות – מיוזות בודדות ,ומיפוי בין גן לצנטרומר 45 Haldane’s mapping function The true determinant of RF is the relative sizes of the classes with no crossovers, versus classes with any nonzero Number of cross overs 46 "mapping function” – התחשבות בשיחלופים שלא רואים The larger m get e-m tends to 0 and RF tends to 1/2 , or 50m.u A formula that relates RF values to “real” physical distance m-is the mean number of cross overs that occur in a segment per meiosis 47 48 49 התחשבות בשיחלופים שלא רואים – ”"mapping function 50 MESSAGE The inherent tendency of multiple crossovers to lead to an underestimate of map distance can be circumvented by the use of map function (in any organism), and by the Perkin formula (in tetrad-producing organisms such as fungi) 51