שיטות מיפוי נוספות

1. שיטות מיפוי נוספות • IA שימוש בכרומוזום Y כ-'בוחן' • IBשימוש במרקרים מולקולרים ותדירות רקומבינציה • IC תדירות רקומבינציה מהכלאה עצמית • II "הגבול" בין תאחיזה להפרדה עצמאית –2C • III התחשבות בשיחלופים שלא רואים – "mapping function”(Haldane) • IVHaplotypes • V טטרדות – מיוזות בודדות, ומיפוי בין גן לצנטרומר. מיפוי משופרת (Perkins)

4. The yeast Saccharomyces cerevisiae is commonly used as a model system =Budding Yeast =Bakers Yeast

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 • Can exist as stable diploid or haploid

7. 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

8. Yeast life cycle

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

12. Haploid Mitosis

13. Diploid Mitosis

14. Haploid Mitosis Diploid Mitosis

15. 1n 1n 1n 2 1 Of diploid Of haploid

17. Animation 3.3

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

19. Centromere mapping

20. Centromere mapping Nonsister chromatids do not cross over First-division segregation pattern or MI pattern

23. Centromere mapping n = 2 -> 4 2 1 1 1 S

24. Centromere mapping Nonsister chromatids do not cross over First-division segregation pattern or MI pattern

25. HALF OF Animation 3.3 – to 37”

26. Centromere mapping Nonsister chromatids cross over Second-division segregation pattern or MII pattern

27. Second-division segregation pattern or MII pattern

28. % of Octads with MII pattern/2 = [cM]

33. The numerical limits of this method . . . ?

37. We’ve learned much more from tetrads/octads. -Let’s go to 2 genes . . . 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

38. Yeast tetrad analysis (classic method) -here UNORDERED tetrads tetrad Step3: let the spores grow into colonies Step1: separate spores by micromanipulation with a glass needle Step2: place the four spores from each tetrad in a row on an agar plate

39. or PDT

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

41. Classical approach (tetrad dissection) Tetrad Dissection NPD PDT TT Tetrad bni1∆ bnr1∆ Only double mutant ( b a ) does not survive BA Ba BA BA Ba Ba ba bA ba ba bA bA

42. Simple RF= 100(NPD + ½ T), but . . . . Perkin formula Corrected map distance=50(T+6NPD) m.u. [cM]

43. Derivation of Perkin’s formula Now, what do these types tell us about linkage? You will notice from examining the three types that only the NPD and T types contain recombinants, so they are key classes in determining recombinant frequency. The NPD class contains only recombinants, whereas half the spores in the T class are recombinant. Therefore, we can write a formula for determining RF by using tetrads, where T and NPD represent the percentages of those classes: Image ch6e14.jpg If this formula gives a frequency of 50 percent, then we know that the loci must be unlinked, and correspondingly, if the RF is less than 50 percent, then the genes must be linked and we could use that value to represent the number of map units between them. However, just as with other linkage analyses studied earlier in the chapter, this value is an underestimate, because it does not consider double recombinants and other higher-level crossovers. Nevertheless, the frequencies of PD, NPD, and T can be used to make a correction for doubles. First, we need to understand how the PD, NPD, and T classes are produced in crosses in which there are linked markers. Let us assume that genes a and b are linked. If we assume that individual meioses can have no crossovers (NCO), a single crossover (SCO), or a double crossover (DCO) in the a-to-b region, then we can represent the classes of unordered asci that emerge from such meioses as shown in Figure 6-14. Triples and higher numbers of crossovers might occur, but we may assume that such crossovers are rare and therefore negligible. SEE Figure 6-14 in the bottom corner. The ascus classes produced by crossovers between linked loci. The ascus classes produced by crossovers between linked loci. NCO, noncrossover meioses; SCO, single-crossover meioses; DCO, double-crossover meioses. The key to the analysis is the NPD class, which arises only from a double crossover between all four chromatids. Because we are assuming that double crossovers occur randomly between the chromatids, we can also assume that the frequencies of the four DCO classes are equal. This assumption means that the NPD class should contain 1/4 of the DCOs, and therefore we can estimate that The single-crossover class also can be calculated by a similar kind of reasoning. Notice that tetratype (T) asci can result from either single-crossover or double-crossover meioses. But we can estimate the component of the T class that comes from DCO meioses to be 2NPD. Hence, the size of the SCO class can be stated as Now that we have estimated the sizes of the SCO and DCO classes, the noncrossover class can be estimated as Thus, we have estimates of the sizes of the NCO, SCO, and DCO classes in this marked region. We can use these values to derive a value for m, the mean number of crossovers per meiosis in this region. We can calculate the value of m simply by taking the sum of the SCO class plus twice the DCO class (because this class contains two crossovers). Hence: In the mapping-function section, we learned that, to convert an m value into map units, m must be multiplied by 50 because each crossover on average produces 50 percent recombinants. So:

44. Derivation of Perkin’s formula – using it, and comparing to RF Let’s assume that, in our hypothetical cross of a+ b+ × a b, the observed frequencies of the ascus classes are 56 percent PD, 41 percent T, and 3 percent NPD. Using the formula, we find the map distance between the a and b loci to be: Let us compare this value with that obtained directly from the RF. Recall that the formula is: In our example, This RF value is 6 m.u., less than the estimate we obtained by using the map-distance formula because we could not correct for double crossovers in the RF analysis.

45. התחשבות בשיחלופים שלא רואים – ”Perkin’s formula”

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

47. What PD, NPD and T values are expected when dealing with unlinked genes? What PD, NPD, and T values are expected when we deal with unlinked genes? The sizes of the PD and NPD classes will be equal as a result of independent assortment. The T class can be produced only from a crossover between either of the two loci and their respective centromeres, and therefore the size of the T class will depend on the total size of these two regions. 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 Mating B b a a a A a a a A a B a Meiosis a B a a b A b A Tetrad Heterozygous diploid BA Ba BA BA Ba Ba Tetrad Dissection ba bA ba ba bA bA NPD PDT TT

48. Classical approach (tetrad dissection) Tetrad Dissection NPD PDT TT Tetrad bni1∆ bnr1∆ Only double mutant ( b a ) does not survive, Synthetic Lethality BA Ba BA BA Ba Ba ba bA ba ba bA bA

49. Synthetic Lethality yfg2 yfg1 yfg2 Viable Viable yfg1 Dead Synthetic (sick) Phenotype – multiple gene causality

50. התחשבות בשיחלופים שלא רואים – ”Perkin’s formula”