Bi 190 2007 Lecture 3 Yeast Genetics cell cycle

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Bi 190 2007 Lecture 3 Yeast Genetics cell cycle Powered By Docstoc
					Bi 190      2007
Lecture 3
Yeast Genetics
cell cycle and mating type


           Paul Sternberg April 2, 2007
Screening for mutants
Dominance
Null phenotype
Complementation
Mapping
Pathways
AWESOME POWER OF YEAST GENETICS
Saccharomyces cerevisiae (budding yeast) Life Cycle

               a                      α

             mating

                                      a/α zygote

          sporulation       -C       -N

                                     Ascus with 4 spores

          germination        +C       +N

                         a
                                 α    haploid cells
                        a
                             α
Saccharomyces cerevisiae
The cells of the baker’s yeast S. cerevisiae alternate between haploid a or α cells
and a/α diploid cells.
The a/α diploids can undergo sporulation if starved for carbon and nitrogen.

The mating type (sex) of the yeast cell is specified by the mating type (MAT) locus.
There are two co-dominant alleles, MATa and MATα.
These specify specialized cell properties involved in mating and for the a/α diploids,
sporulation.
Both a and α cells secrete a mating factor, called a- and α-factor, respectively.
a cells respond to α factor.

Grow as colonies on Petri dishes or in liquid.
One cell generates two cells in 90-120 minutes on rich medium (YEPD;
yeast extract, peptone, dextrose)
Slower on minimal medium (yeast nitrogen base without amino acids).
Most markers are auxotrophies,
e. g., Ura-, no growth without uracil; Leu, no growth without leucine;
His, no growth without histidine.
Leland Hartwell 1970s
    Conditional (Temperature sensitive) mutants
    Defective in the Cell Division Cycle (cdc)
Nomenclature:
Capital for the dominant or co-dominant
URA3 vs ura3 or ura3-52
MATa and MATα



A yeast cross:

a his3 LEU2 x α HIS3 leu2
         select for growth on minimal mdium (Leu+ His+)
a/α diploid
         starve
         dissect tetrads
grow up individual spores (germinate on rich medium)
Tetrad Analysis
In S. cerevisiae all genes are essentially unlinked
since there many chromosomes and lots of recombination.
Tetrad analysis substitutes for linked markers to follow unknown genotypes.
The basic principle is that you recover all four products of meiosis.

Consider segregation of an ade2 mutation that results in a red colony
as opposed to the wild-type ADE2 white colony

In ade2, phosphoribosylaminoimidazole (AIR) -> red pigment

          ADE2           ADE1
AIR               CAIR               SAICAR


Red
Pigment
a           ade2
α           ADE2




                    3




                        ASCUS
                    2
                    1
    A   B   C   D

        SPORE
           a   ade2
           α   ADE2

        Ade phenotype

    tetrad 1 tetrad 2 tetrad 3

a   Ade-       Ade -    Ade +

a   Ade -      Ade +    Ade +

α   Ade +      Ade -    Ade -

α   Ade +      Ade +    Ade -

      PD       TT       NPD
a   ade2
α   ADE2


     PD: parental ditype
    NPD: non-parental ditype
    TT: tetratype

       2:2 segregation implies one locus

       1 PD : 4 TT : 1 NPD is Unlinked
       If PD>1 or NPD<1, there is linkage

       1 : <4 : 1 indicates centromere linkage
     Mackay & Manney 1974 yeast sterile screen

mutagen

                        (1000-fold excess)
           α can1 x     a CAN1



           a CAN1          dies
          a/α can1/CAN1    dies

            α can1         All mated

           α can1 ste
Mackay and Manney (1974)              screened for sterile mutants

                               UV irradiate

                                       α ade6 his6 can1
mix with 1000-fold excess of
                                       a ade2 his2 CAN1

Let mate on rich medium (YEPD) for 24 h

Dilute and plate on canavanine + arginine


[can1 is a recessive drug resistance; CAN1 is the wild-type allele.
arginine is necessary to induce the permease that transports in this arginine analog]

All a and a/α diploids die. The survivors are α steriles.
To analyze these sterile mutants, force a mating
by selecting His+ colonies from the cross:

  α ste his6 x a STE his2.


            His+

      MATa STE HIS6 his2
      MATα ste his6 HIS2



Sporulate the resultant diploids:
                 MATa STE HIS6 his2
                 MATα ste his6 HIS2

Test the spore colonies for mating ability with α and a testers.

Look at non-parental ditypes (NPDs)

spore genotype          mating phenotype

α       +                       α-mater
α       +                       α-mater
a       ste                     ?
a       ste                     ?
A tetratype (TT) such as the following inferred from the
mating phenotype would indicate an α-specific sterile

mating phenotype           spore genotype

a-mater                    a      +
a-mater                    a      ste
non-mater                  α      ste
α-mater                    α      +
A non-parental ditype (NPD) such as the following
inferred from the mating phenotype would also
indicate an α-specific sterile:

mating phenotype         spore genotype

a-mater                   a     ste
a-mater                   a     ste
α -mater                  α     +
α-mater                   α     +
Complementation tests with a Ste mutant

      CEN3 cry1R MATa             ste-x     +
      CEN3 CRY1 MATα              +     ste-y



Select cryptopleurine-resistant     CryR



and thus homozygose MATa to allow an assay of
mating of the MATa/MATa (an a-cell since it is MAT
locus, not ploidy per se that determines sex of yeast).
  α− specific genes
      Production of α− factor
      Agglutination              α mating
      Response to a-factor

non − specific genes
       Response to pheromones
                                 a mating
  a − specific genes
       Production of a- factor
       Agglutination
       Response to α− factor
The α-specific genes include
STE3     receptor (G-protein coupled) for a-factor
STE13    dipeptidyl aminopeptidase (processing of α-factor)
KEX2     protease (processing of α-factor)

The non-specific genes include:
STE4     G protein β subunit
STE5     scaffold for MAP kinase cascade
STE7     MAP kinase kinase
STE11    MAP kinase kinase kinase
STE12    transcription factor
STE18    G protein γ subunit
STE20    protein kinase

The a-specific genes include:
STE2     receptor (G-protein coupled) for α-factor
STE6     secretion of a-factor (mdr or ABC type transporter family)
STE14    protein-S isoprenylcysteine O-methyltransferase (modification of a-factor)
RAM1 farnesyltransferase beta subunit (modification of a-factor)
Saccharomyces cerevisiae (budding yeast) Life Cycle

               a                      α

             mating

                                      a/α zygote

          sporulation       -C       -N

                                     Ascus with 4 spores

          germination        +C       +N

                         a
                                 α    haploid cells
                        a
                             α