Download 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