תדירות רקומבינציה מהכלאה עצמית שימוש בכרומוזום Y כ -' בוחן ' " הגבול " בין תאחיזה להפרדה עצמית – 2  התחשבות בשיחלופים שלא רואים – "mapping function” טטרדות – מיוזות בודדות, ומיפוי בין גן לצנטרומר שיטות מיפוי נוספות 1

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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 fastGrows fast CheapCheap Compact genome, fully sequenced since 1996.Compact genome, fully sequenced since Easy to handleEasy to handle Superb Genetics, Biochemistry, Molecular BiologySuperb Genetics, Biochemistry, Molecular Biology Easy to transform, high efficiency of gene targetingEasy to transform, high efficiency of gene targeting 6

Major characteristic of the budding yeast Unicellular EukaryoteUnicellular Eukaryote Grows by buddingGrows by budding 16 linear chromosomes16 linear chromosomes Generation Time: ~100 minGeneration Time: ~100 min Can exist as stable diploid or haploidCan exist as stable diploid or haploid 7

Yeast: A model eukaryote Yeasts – the ultimate model eukaryote for unicellular issues and some basic cell-cell interactions Yeast studies have broken new ground in: Cytoskeleton functionstranscription mechanisms** cell cycle**transcriptional regulation organelle biogenesischromatin modification secretion*signal transduction protein targeting mechanismsprotein degradation* chromosome replicationDNA repair genome dynamicsretroviral packaging prionsrecombination mechanisms ageingfunction of new genes metabolismprotein modification *Lasker Award **Nobel Prize "for their discoveries of key regulators of the cell cycle" Lee HartwaellPaul NurseTim Hunt The Nobel Prize in Physiology or Medicine 2001 for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells James E. RothmanThomas C. SüdhofRandy W. Schekman The Nobel Prize in Physiology or Medicine

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) 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 Morphology + nuclear localization and MT localization indicates the precise stage of the cell cycle Major control point is at G1/S 11

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Mendel’s rules are relevant for organisms that sexually reproduce: diploid/haploid –Plants, Animals, many Mora… Those that ‘do’ meiosis 14

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Haploid Mitosis 16

Diploid Mitosis 17

Diploid Mitosis Haploid Mitosis 18

Of diploid 1n 2121 Of haploid 19

Centromere mapping 20

Centromere mapping Nonsister chromatids do not cross over First-division segregation pattern or M I pattern 21

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n = 2 -> S 24

Centromere mapping Nonsister chromatids do not cross over First-division segregation pattern or M I pattern 25

Centromere mapping Nonsister chromatids cross over Second-division segregation pattern or M II pattern 26

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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 the same chromosome 37

B Mating Heterozygous diploid Tetrad BA ba Ba bA BA Ba ba bA NPD PDT TT Tetrad Dissection Meiosis a b A B a b A B a b A B a b A Unordered tetrads 38

Yeast tetrad analysis (classic method) tetrad Step1: separate spores by micromanipulation with 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) BA ba Ba bA BA Ba ba bA NPD PDT TT Tetrad Tetrad Dissection bni1∆ bnr1∆ 40

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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 Map distance=50(T+6NPD) m.u 42

Meiosis Mating Heterozygous diploid Tetrad BA ba Ba bA BA Ba ba bA NPD PDT TT Tetrad Dissection What PD, NPD and T values are expected when dealing with unlinked genes? B B a a a a b b a a A A B B a a a a a A A b b 43

Meiosis Mating Heterozygous diploid Tetrad BA ba Ba bA BA Ba ba bA NPD PDT TT Tetrad Dissection What PD, NPD and T values are expected when dealing with unlinked genes? B B a a a a b b a a A A B B a a a a a A A b b The sizes of the PD and NPD classes will be equal as a result of independent assortment. The T class can be produced only form a crossover between the specific loci and the and their respective centromeres 44

תדירות רקומבינציה מהכלאה עצמית שימוש בכרומוזום Y כ -' בוחן ' " הגבול " בין תאחיזה להפרדה עצמית – 2  התחשבות בשיחלופים שלא רואים – "mapping function” טטרדות – מיוזות בודדות, ומיפוי בין גן לצנטרומר שיטות מיפוי נוספות 45

Haldane’s mapping function 46 The true determinant of RF is the relative sizes of the classes with no crossovers, versus classes with any nonzero Number of cross overs

התחשבות בשיחלופים שלא רואים – "mapping function” 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 The larger m get e -m tends to 0 and RF tends to 1/2, or 50m.u

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התחשבות בשיחלופים שלא רואים – "mapping function” 50

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) MESSAGE 51