Edited, 2018: Mating Type-Dependent Drug Sensitivity

Patterns in Yeast Deletion Libraries: Preliminary

Experiments, Theoretical Interpretation of Gene Expression

and Dose-Response Theory

Richard Pan

Revision dated November 10, 2011.

Talk prepared July-October, 2011.

My original talk was given November 9, 2004 at

Building 5, Room 127, National Institutes of Health, Rockville, Maryland.

1

PowerPoint Presentation, Summary:

I. Preliminary ExperimentsHand screening of ATCC MAT a haploid deletion library mutants, > 2,000 clones screened

Robotics-based, high-throughput screening: carboxypeptidase Y-oversecreting vacuolar processing

gene deletion sublibrary [Bonangelino et al., NICHD]

Drugs and stress agents: hydroxyurea, methyl methanesulfonate, azaserine, 4-nitroquinolonine-n-oxide,

37C temperature, hygromycin B, vanadate, oxidation/reduction

Surprises: 1) High incidence of sensitivity to HU and other drugs, often > 1%

2) ARP5, BDF1, VPS53, MDM20: Artifacts or true mating type-dependent drug sensitivity?

Recurrent (a/, a, ) sensitivity patterns: SRR SRS RRS and numerous SSS

II. Interpretation and Ideas Genomics: Library screening is sampling.

An elementary sampling probability theorem

How to search drug-mutant space: paradigms, strategies, and algorithms

Histograms of library screens are aggregate Dose-Response data.

The power of yeast is genetics.

Crosses which exploit sex-linked drug sensitivities and asymmetries

Super-growth mutants. Overexpression is a variation of established SL, SDL, DS experiments.

The potential of genomics theory is statistics and physics.

Interpreting mating-type sensitivity patterns as differential gene expression, Venn diagrams

Searching SGD gene expression database with WebMiner software

Predicting the future mutants? Mating-type truth tables and Karnaugh maps are possible.

III. Theoretical Research and Further QuestionsIs new statistics possible in “drugs and mating-type” genomics research?

Statistics for cell growth, gene expression, and the mating-type variable

A need for new and better drug research

Additional research questions and problems I may undertake 2

Hydroxyurea Dose-Response Data for

gene deletion clones of mating-type a

haploid*

Solid YPD media, 30C growth > 1.5 days

1. Wild type

2. Δ RAD52

3. Δ RAD57

4. Δ RAD27

5. Δ HSP42

6. Δ YLR235C

7. Δ YKL054C

8. Δ VPS15

9. Δ SEC22

10. Δ VID24

*genotype BY4741=ATCC201388=

MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0

Mutants examined here if anything, show a

monotone decreasing survival percentage

in response to increasing hydroxyurea drug

concentration, i.e., at higher dosages of

hydroxyurea, fewer cells survive. It is an

assertion at higher yet drug doses, even

wild-type cells will die.

1 2 3 4 5 6 7 8 9 10

0 mM HU

5.5 mM HU

22 mM HU

1.38 mM HU

3

What is special about Dose-Response

of microbial growth from the physics

and mathematics standpoint? Must

Dose-Response Curves be monotone?

My basic experimental findings: i) the likelihood a given mutant will survive

decreases and ii) the number of yeast deletion mutants which are growth-sensitive

to a stress agent tends to increase, when the drug concentration is increased.

50 mM 22 mM 5 mM [drug]= 0 mM Hydroxyurea

WildType

4

5

Chemicals and Stress-Axes Agents Tested in Yeast Deletion Library Screening

Class Drug Concentration Structure Formula MW

DNA

Replication/Repair

Stress

Hydroxyurea 22.5 milliM

CH4N2O2 76

Methyl

methanesulfonate 0.01%=

1.2 milliM

C2H6O3S 110

Azaserine

150 microM

C5H5N3O4 173

4-Nitroquinoline

-n-oxide*

167 nanoM

C9H5N2O3 190

Glycosylation

Stress

Hygromycin B 31.6 microM

C20H37N3O13 527.5

Tunicamycin* 297 nanoM

C39H64N4O16 840

Sodium

Orthovanadate*

3 milliM

+ 3Na+

Na3VO4 184

6

Chemicals and Stress-Axes Agents Tested in Yeast Deletion Library Screening, continued

Class Drug Concentration Structure Formula MW

Oxidation/

Reduction Stress

Hydrogen

Peroxide*

1 milliM

H2O2 34

Diamide* 360 microM

C6H12N4O2 172

Paraquat* 600 microM

C12H14N2Cl2 257

Additional

stresses*

Physical Stress Elevated

Temperature

growth

37 C Incubators, microbiological

UV* 0-80 microJoules UV CrossLinker, Stratagene

*Data not presented in this talk.

NB: Elemental stress, eg. Fe, Zn, Cu toxicity, was planned but not tested due to lack of time.

Experimental findings of general interest

• Hydroxyurea, a DNA replication/repair stress agent, yielded surprisingly high percentages (~

3-5%) of growth-sensitive clones from preliminary screening of an ATCC yeast non-essential

gene deletion library of haploid mating-type a. Approximately half of this ~5,000 clone library

was screened for 22.5 mM HU growth sensitivity. Later drug and stress screens were

performed exclusively on a 148-clone library of vacuolar processing gene deletion mutants

selected for carboxypeptidase Y-oversecretion (Bonangelino et al., 2002).

• Dose-response hydroxyurea data for serial dilutions of sample mutants show different gene

deletion mutants can have different LD50 values (Ex. PowerPoint slide #3, this talk). In

principle, a sublibrary of mutants showing absolute sensitivity at 22.5 mM HU may harbor

subpopulations killed by HU at far lower doses. It is asserted for many clones, monotone

dose-response curves or suitable averages of dose-response data which are monotone

decreasing, may be expected though a full theory of pharmacologic dose-response is not yet

known.

• Initial experiments with additional DNA replication-repair drugs (methyl methane sulfonate ,4-

nitroquinoline-n-oxide, azaserine, UV) and other stress modalities such as those specific for

glycosylation (hygromycin B, tunicamycin, ortho-vanadate) and oxidation (paraquat, diamide,

H2O2, vitK) show different stress axes can share common growth-sensitive mutants, making

probability considerations relevant. Planned though unperformed experiments with metal ion

toxicity (Fe2+/3+, Cu+/2+, Zn) may yield invaluable insight. 7

Experimental findings of Special interest

• Surprising mating-type dependence, i.e., “yeast sex-dependence” of growth-

sensitivity to stress agents is observed. SRR, SRS, and SSR (a/α,a,α) growth

sensitivity patterns are common among the gene deletion clones tested though

SSS sensitivity independent of mating-type also is observed. Numerous clones

show mating-type sensitivity patterns for different stresses.

Growth sensitivity photograph data follows on

slides #9-16, i.e.,

Robotics-based library screens of yeast gene deletion mutants, performed in

mating-type triplicate: a/ homozygote diploid, a deletion haploid, and

deletion haploid strains, grown on solid YPD agar media plates with or

without drug or stress, each bearing 12 x 8 = 96 spotted mutant strains

including two corner (LUH=A1, RLH=H12) wild-type control spots. Additional

controls are shown.

8

Experimental Data, Growth-Sensitivity of the (3) Mating-types to Stress Drugs

Rows: a/α homozygote diploid, a haploid, α haploid (top to bottom)

Columns: Control YPD, T37, HU, MMS, AZ, HB (right to left)

8HB 6AZ 5MMS 3HU 2T37 1CTL a/α homozyg dipl

a haploid

α haploid

9

Hydroxyurea challenge versus Mating-types

10

YPD a/α homozygous diploid a haploid α haploid

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

YPD + 22.5 mM Hydroxyurea

37ºC Temperature challenge versus Mating-types

1 2 3 4 5 6 7 8 9 10 11 12

YPD + 37ºC temperature growth

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

YPD a/α homozygous diploid a haploid α haploid

11

Azaserine drug challenge versus Mating-types

YPD a/α homozygous diploid a haploid α haploid

YPD + 150 µMolar Azaserine

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

12

Hygromycin B drug challenge versus Mating-types

YPD + 31.6 µMolar Hygromycin B

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

YPD a/α homozygous diploid a haploid α haploid

13

Most promising findings: I) Sensitive mutants

14

Selected Gene Deletion Mutants show Mating -type Differences of Growth Sensitivity to Drug Stres ses on YPD agar Microliter aliquots of freshly thawed ATCC haploid deletion mutants and Research Genetics homozygous deletion diploid mutants were diluted in liquid YPD containing selected drug at the same concentration later used in solid agar media. A 96-needle programmable Robbins TANGO robot performed dilutions followed by parallel -spotting of ~3 microliters of each sample on YPD

agar media plates containing drug at the following concentrations: HB=Hygromycin B at 31.6 µM; T37=no drug; HU=Hydroxyurea at 22 mM; MMS=Methyl methanesulfonate at 0.01%; AZ=Azaserine at 150 µM. Plates were incubated at 30°C or 37°C and taken out for digital photographs at days 1½, 2½, and 6½. All photos shown are from day 6½ to minimize variable lag phase growth effe cts.

ARP5 A/α A α BDF1 A/α A α VPS53 A/α A α YPT6 A/α A α

YPD

YPD

YPD

YPD

HB

R.R.R

HB S.pS.pS

HB S.R.R

HB

R.R.R

T37

S.S.S

T37 S.S.S

T37 R.R*.R*

T37

S.R.S

HU

pS.R.R

HU S.pS.pS

HU R.R.R

HU

R.R.R

MMS

R.R.R

MMS R.R.R

MMS R.R.R

MMS

R.R.R

AZ

R.pS+.R

AZ S.S.S

AZ R.R.R

AZ

R.R.R

CONTROL

PARENT CELLS

A/α BY4743

A BY4741

α BY4742 COD3 A/α A α PSL10 A/α A α SOS1 A/α A α

YPD

YPD

YPD

YPD

HB R.R.R

HB R.R.S

HB

R.R.S

HB

S.R.R

T37R*.R*.R

* T37 pS.R*.S

T37

R.R.R

T37

S.S.S

HU R.R.R

HU

R.R.R

HU

R.R.R

HU

R.R.R

MMS R.R.R

MMS

R.R.R

MMS

R.R.R

MMS

R.R.R

AZ R.R.R

AZ

R.R.S+

AZ

R.R*.R*

AZ

R.R.R

15

Sensitive mutants continued

VPS65 A/α A α MDM20 A/α A α NPL6 A/α A α VPS33 A/α A α

YPD

YPD

YPD

YPD

HB R.R.R

HB S+.R.R

HB

R.R.R

HB

T37

S.S.S

T37 S.pS.R

T37

R.S.S

T37

HU

R.R.R

HU pS.R.R

HU

R.R.R

HU

MMS

R.R.R

MMS R.R.R

MMS

R.R.R

MMS

AZ

R.R.R

AZ S.S+.S+

AZ

R.R.S

AZ

CONTROL

PARENT CELLS

A/α BY4743

A BY4741

α BY4742 VPS54 A/α A α PER1 A/α A α VPS18 A/α A α

YPD

YPD

YPD

YPD

HB

R.R.R

HB S.S.S

HB

S.S.S

HBS.S+.S.

T37

R*.R*.R

* T37

pS+.S+.S

T37

S.S.S T37

S.S+.S.

HU

R.R.R

HU

R.R.R

HU

pS.R.R

HUR.R.R.

MMS

R.R.R

MMS R.R.R

MS

R.R.R

MMSR.R.R.

AZ

R.R.R

AZ

R.R.R

AZ

S.R.R

AZR.R.R.

Sensitive mutants continued

16

VPS34 A/α A α VPS1 A/α A α IES6 A/α A α GOS1 A/α A α

YPD

YPD

YPD

YPD

HB

HB HB

R.R.R

HB

T37

T37

T37 S.S.S

T37

HU

HU

HU

S+.pS.pS

HU

MMS

MMS

MS R.R.R

MMS

AZ

AZ

AZ pS.pS.S

AZ

Mating-type sensitivity patterns: immediate observationsDiploid versus haploid asymmetries are common: the SRR sensitivity pattern is

frequent for this vps sublibrary across at least the hygromycin B and elevated

temperature screens (VPS53, BDF1, SOS1, MDM20). Though slower growth rates

of diploids than haploids due to metabolic stress is easy to understand, why

glycosylation stress lethality showed a greater incidence among diploids (BDF1,

VPS53, SOS1, MDM20 v. COD3, PSL10,VPS33, VPS34, VPS18) than other

stresses is not certain. NPL6 at T37 is the only RSS sensitivity pattern observed

for this vps sublibrary.

Haploid-asymmetric sensitivities are also observed: YPT6 is SRS for T37, COD3

may be RRS for more than one stress, and also PSL10, VPS34, and VPS33.

NB. Validity of these sensitivity patterns, free of aneuploidy and additional

chromosomal rearrangements, remains a critical experimental issue.

Elevated temperature growth at 37C and hygromycin B stress, yield higher frequency

of sensitivity for the vacuolar processing sublibrary than did DNA replication-repair

and oxidative-reductive stress. Given the critical importance of temperature for

lipids and of receptor modifications on endosome surfaces and associated Golgi

structures, we may have identified two independent and important screening

assays for analyzing vps pathways.

An important genetics question is: are petite mutations present here? BDF1 on

azaserine, COD3 on azaserine, MDM20 on azaserine, VPS18 on hygromycin B

and T37, and IES6 on hydroxyurea may offer special petite mutant opportunities.

17

Most promising findings: II) Super-resistant mutants

18

Super-resistance mutants on YPD with or without MMS or AZ, after 6 days of growth

a/ wildtype VMA1 VMA2 VMA4 VMA6 VMA8 VMA10 VMA11 VMA12 VMA13 VMA21

drug free

0.01% MMS

150 uM AZ

a

drug free

0.01% MMS

150 uM AZ

drug free

0.01% MMS

150 uM AZ

Acknowledgements

My advisors at the National Institutes of Health were• Jay H. Chung, NHLBI, Funding provided by IRTA Fellowship award TEXX002045• Juan Bonifacino and Cecilia Bonangelino, NICHD, collaborators• Alexandra Brown, NHBLI• Kevin O’Connell, NIDDK• Lita Freeman, NHLBI• Orna-Cohen Fix, NIDDK• Bernie Brooks, NHLBI, NHLBI half-time contract #263-MJ-320989.

Additional university and corporate research scientists to whom I am indebted:

David Morris, The George Washington University

Thomas DeChiara, Regeneron Pharmaceuticals

Rodney Rothstein, Columbia University

Thomas Begley and Leona Samson, Harvard Medical School, now at

Massachusetts Institute of Technology

Mike Snyder, Yale University, now at Stanford University

Leonard Guarente, Massachusetts Institute of Technology

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