Purpose The object of this lab is to demonstrate the relationship between the genotype of a yeast strain (its genetic makeup), and its phenotype (the way the strain behaves under varied conditions).  Students will take a series of strains of known genotype and challenge them in different ways to determine what effect the particular mutations have on strain growth and viability.

Day 1

Part 1 We will have prepared log cultures of the required yeast strains which will be growing in rich media (YPD) at the permissive temperature of 25°C.  We will give you 6 mls from each culture, and you should aliquot them into two 3 ml duplicate samples in apprpriately labelled sterile test tubes.  One sample will be incubated at 25 °C (permissive temperature for the control) and the other sample will be the experimental culture.

Treat the experimental samples as indicated for the following strains:

 YMM16  incubate at 18 °C for 6 hrs
 YMM54  incubate at 18 °C for 6 hrs

 YMM63  incubate at 36°C for 3-4 hrs
 YMM64  incubate at 36°C for 3-4 hrs
 YMM68  incubate at 36°C for 3-4 hrs
 YMM65   incubate at 36°C for 3-4 hrs

 At the end of the incubation, take off 1 ml of cells and fix in formaldehyde (add 100 ul of 37% formaldehyde to 1 ml of cell culture in an eppi tube).  Sonicate samples (setting “2.5” for 10-15 sec.)  and score morphologies of 100 cells each from the experimental and control cultures.  Determine what effect the experimental conditions had on the cell morphology.

Part 2 Take a sterile frog pond and separately add 100 ul of each growing culture to one of the wells (be sure to use sterile technique and record which strain went into which well).  After adding all the cultures to the pond, use a sterile frogger to innoculate the following plates.  Incubate the plates as directed, and score growth after 2 days (36°C), 3 days (25°C) or 6 days (18°C).  Determine how cell growth correlates with cell genotype.

Plates needed:

3 YPD plates:  18, 25, and 35°C
1 plate each of SC, SC - Ura, SC - Leu, SC - His, SC - Lys, SC - Ade      all at 25°C

Strains to use :

 YMM28  Mat a  ade2  his4  ura3-52
 YMM10  Mat a  ade2  leu2-3,112  ura3-52
 YMM16  Mat a  his4  lys2-801  ura3-52  cdc44-1.URA3
 YMM54  Mat a  his4  lys2  ura3-52  cdc44-1.URA3  pol30-37
 YMM55  Mat a  ebp2::HIS3  ura3-52  leu2  lys2  trp1  his3   (ebp2-1  LEU2)
 YMM59  Mat a  leu2-3,112  ura3-52  rad9::LEU2
 YMM63   Mat a   his7  ura1  cdc4
 YMM64   Mat a  his7  ura1  cdc28
 YMM68   Mat a  cdc24-2   ade1  ade2  gal1  his7  lys2  tyr1  ura1
 YMM65   Mat a  cdc34-1   ade1  ade2  gal1  his7  lys2  tyr1  ura1

Day 8

Alpha factor arrest experiment

 In this experiment, we will demonstrate that haploid yeast cells are only responsive to the mating pheremone that is secreted by cells of the opposite mating type.  We will take both  Mat a and a cells and challenge them with the peptide pheremone a factor.  Only Mat a cells will respond to this treatment by arresting in the cell cycle, and forming specialized mating structures called schmoos.

 Take 2.5 mls from an early log culture of strains YMM28 and YMM10 that are growing in acidic YPD.  Split cultures into two duplicate 1.25 ml samples, and incubate one set at 25°C on a rollerdrum.  To the other tubes add 13 ul of alpha factor (0.5 mg/ml) to get a final concentration of 5 ug/ml and then incubate at 25°C on the rollerdrum.  After 3 hours, take off 1 ml of each culture, fix in formaldehyde, sonicate and score morphologies of 100 cells.  Determine how the cells respond to the pheremone treatment.

 YMM28  Mat a  ade2  his4  ura3-52
 YMM10  Mat a  ade2  leu2-3,112  ura3-52
 

Test for sensitivity to ultraviolet irradiation

 One way that we can determine the role that specific genes play in cells is to investigate the properties of cells that carry mutations in such genes.  For example, if gene X plays a role in DNA replication or DNA repair, one might expect that cells carrying a mutation in gene X may not be able replicate or repair its DNA very well.  Such a defect could be tested by challenging the mutant cells with a DNA damaging agent, and then determining how well the cells survive.  In this experiment, we will investigate the UV sensitivity of strains carrying mutations in two known DNA replication proteins Cdc44p/Rfc1p (Replication factor C) and Pol30p (Proliferating cell nuclear antigen or PCNA).

 Take a sample of early log cultures for each strain listed below, dilute culture 10x in sterile water (i.e. add 100ul of cells to 900 ul of water) and then sonicate.  Count cell number with a hemacytometer and then dilute cells (serial 10 x dilutions) as necessary to a density of about 2,000 cells/ml in sterile water.  You will need a total of 2 mls from each strain at this cell density.  Plate 150 ul of the diluted cells onto each of 12 YPD plates (total of about 300 cells per plate).  Label and irradiate duplicate plates with UV light at 0, 40, 80, 120, 160, 200 J/m2.  Incubate plates at 30°C and count colonies after 2-3 days.  Determine the viability of the strains after UV irradiation and plot on semi log curve (% viability vs dose UV).
 
strains to use:

 YMM10  Mat a  ade2  leu2-3,112  ura3-52
 YMM16  Mat a  his4  lys2-801  ura3-52  cdc44-1.URA3
 YMM54  Mat a  his4  lys2  ura3-52   cdc44-1.URA3  pol30-37
 YMM59  Mat a  leu2-3,112  ura3-52  rad9::LEU2

 To use UV irradiator, take plates with cells spreaded on them (make sure you label plates on the bottom) and place (maximum 8 at a time) right side up in the Stratalinker 2400.  Remove covers of plates and close door.  Turn Stratalinker on, and set to desired UV dose as follows.  Push ‘energy’ button, set energy value with numerical keypad (i.e. 20 = 20 J/m2), press ‘start’.  Your samples will begin to be irradiated.  When the dose of UV has been reached, the machine will beep and you should then push ‘reset’.  You may now remove samples, replace the tops to the petri dishes, and irradiate another set at a new energy dose.