Yeast Genetics and Molecular Biology. Lecture I. Yeast basics and classical yeast genetics

Июль 25, 2022

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Yeast Genetics and Molecular Biology. Lecture I. Yeast basics and classical yeast genetics

Содержание

2. What is Yeast Genetics? Definition of Genetics in Wikipedia: “Genetics (from Ancient Greek γενετικός genetikos, “genitive”
3. This slide was nicked from internet lecture notes of a course held at the Universität München
4. Pioneers of yeast genetics Øjvind Winge (1886-1964), Carlsberg laboratory, Kopenhagen: http://www.genetics.org/cgi/content/full/158/1/1 Discovery of alternation of Haplo
5. Baker’s Yeast Saccharomyces cerevisiae: - Also “Budding yeast” - Ascomycete (ascus as fruiting body) - Oldest
6. Requirements for Model Organisms:
7. Yeast similarity to human cells
8. Yeast as a Model Organism David Botstein, Steven A. Chervitz, and J. Michael Cherry Science 1997
9. “Bacterial” aspects of yeast: Single cell organism Haploid growth phase -> phenotype of recessive mutations shows
10. Processes that can be studied in yeast Cell cycle (mitosis, meiosis) (Principles of) gene regulation Metabolic
11. Growth requirements of Baker’s Yeast Wild type S. cerevisiae: prototrophic as long as there is a
12. Crabtree effect and oxygen requirements of S. cerevisiae Preferred carbon source: glucose, but many other carbon
13. Examples of Fermentable and Non-Fermentable Carbon Sources
14. Diauxic shift Yeast prefers alcoholic fermentation if the carbon source allows for it, until the fermentable
15. Growth Media “Favorite” Media (RICH media): YP (Yeast extract and Peptone=peptic digest of meat) + carbon
16. Synthetic complete media Contain all the amino acids, some nucleic acid precursors and some vitamins and
17. Minimal media Carbon source and Nitrogen source (YNB) Only wild type yeast can grow
18. Yeast Gene and Gene Product Nomenclature Dominant alleles are written in italicised capital letters: LEU2, ADE3,
20. In most cases the wild type allele is denoted in upper case italics: LEU2, the mutant
21. Classical yeast genetics Pre-molecular biology
23. The Life Cycle of Saccharomyces cerevisiae Wild type strains Most laboratory strains (ho-) 2n 1n 2n
24. Yeast has a haploid growth phase Phenotype of mutation apparent immediately Every haploid strain is a
25. Genetic Manipulation Ability to mate yeast cells allows combining of mutations Meiotic products (spores) are packed
26. Genetic analysis of a simple mutation “Wild type” strain (Leu+) “mate”
27. Segregation of two alleles involved in Leucine biosynthesis Cells are Leu+, as the functional copy of
28. Meiosis 1: separation of the homologous chromosomes Meiosis 2: separation of the chromatids
29. Digest off cell wall Tetrad with 3 spores visible in one focal plane and 4th spore
31. Line up on grid Non-selective media (e.g. YPD) Selective media (SC- Leu) Leu- Leu-
32. Original Dissection on Non-selective plate Replica on selective plate (e.g. Leu- strain on SC – Leucine)
33. Segregation of two unlinked genes Example: TRP1, LEU2 Haploid, Leu+, Trp- Haploid, Leu-, Trp+ Diploid, Trp+,
34. Possible distribution of chromosomes during meiosis Resulting tetrads after sporulation parental ditype (Trp+, Leu- : Leu+,Trp-)
35. or trp1 trp1 TRP1 TRP1 Tetratype
36. Ratios of different types of tetrads! (NOT spores)
37. Distances between linked genes can be calculated by counting the different tetrad types; Formula: ½ T
38. Dissecting Metabolic Pathways in Yeast Question: What enzymes are involved in the Biosynthesis of Uracil? Approach:
39. Replica plating:
40. YPD SC - Ura Most colonies still wild type – can grow on synthetic media lacking
41. Sorting of mutations In our hypothetical screen, we have identified several haploid mutants in the uracil
42. Complementation analysis Scenario 1: mutations are in the same gene Ura- Ura- Ura- Diploid cannot grow
43. Complementation analysis Scenario 2: mutations are in different genes Ura- Ura- Ura+ Mutant A, α mating
44. Complementation of mutants in the uracil biosynthesis pathway (+) = mutants complement each other ; (-)
45. Epistatic Analysis Epistasis - the interaction between two or more genes to control a single phenotype
46. Example of Epistatic analysis Example: Adenine biosynthesis mutants ade2 and ade3 (unlinked genes): ade2 mutants are
47. Scenario I Scenario II Two possibilities of order of action of Ade2p and Ade3p in this
48. ade2 mutant, α mating type Ade -, RED ade3 mutant, a mating type Ade-, creamy white
49. Possible distribution of chromosomes during meiosis Parental Ditype - uninformative All Ade-
50. Nonparental Ditype – informative (two spores carry both mutations) Two Scenarios
51. 2 x Ade+, white Double mutant, Ade-, RED A -------> B (red pigment) ------> C ------>
52. 2 x Ade+, white Double mutant 2x Ade-, WHITE The ADE3 gene product catalyzes a reaction
53. or ade 3 ade3 ADE3 ADE 3 ADE2 ade 2 ade2 ADE 2 Tetratype
54. Tetratype Ade-, white Ade+, white Ade-, red Ade-, white Ade-, white Scenario1 Scenario2 A -----> B
56. Скачать презентацию

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What is Yeast Genetics?
Definition of Genetics in Wikipedia: “Genetics (from Ancient

Greek γενετικός genetikos, “genitive” and that from γένεσις genesis, “origin”), a discipline of biology, is the science of heredity and variation in living organisms”
Classical yeast genetics:
Desireable traits of naturally occuring yeast strain variants were combined by mating of the strains to generate hybrids and selection of offspring carrying combinations of these traits
Modern yeast genetics:
the cells are manipulated to generate mutants in pathways and processes of interest (generation of heritable variation)
Mutants with interesting phenotypes are selected or screened for and subsequently analyzed with molecular biology and biochemical methods to determine their function in the cell

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This slide was nicked from internet lecture notes of a course

held at the Universität München (Prof. Horst Feldman)

http://biochemie.web.med.uni-muenchen.de/Yeast_Biol/

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Pioneers of yeast genetics
Øjvind Winge (1886-1964), Carlsberg laboratory, Kopenhagen: http://www.genetics.org/cgi/content/full/158/1/1
Discovery

of alternation of Haplo – and Diplophase in Saccharomyces sp. –”Yeast Sex”; development of mechanical yeast manipulation and dissection methods
Carl C. Lindegren (1896-1987), Washington University, St. Louis; University of Southern Illinois, Carbondale, USA Isolation of heterothallic yeast strains (= mutant strains with a stable haploid growth phase)
Boris Ephrussi (1901-1979), Institutes Pasteur, Paris; Centre national de la recherche scientifique, Gif-sur-Yvette, France
Cytoplasmic inheritance (= mitochondrial genetics)

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Baker’s Yeast
Saccharomyces cerevisiae:
- Also “Budding yeast”
- Ascomycete (ascus as fruiting body)
-

Oldest domesticated organism?
Used in brewing and baking for millennia
Favorite organism for molecular biologists
First eukaryotic genome to be sequenced in its entirety (1996)!

Yeast is a molecular biology model organism

http://biochemie.web.med.uni-muenchen.de/Yeast_Biol/

Yeast ascus with spore tertad

Source: wikimedia

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Requirements for Model Organisms:

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Yeast similarity to human cells

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Yeast as a Model Organism David Botstein, Steven A. Chervitz, and J.

Michael Cherry Science 1997 August 29; 277: 1259-1260. (in Perspectives)

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“Bacterial” aspects of yeast:
Single cell organism
Haploid growth phase -> phenotype of

recessive mutations shows up in the first mutant generation
Fast growing (doubling every 1.5 hours on rich media)
Moderate growth media requirements
Transformation, gene replacement “easy”

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Processes that can be studied in yeast
Cell cycle (mitosis, meiosis)
(Principles of)

gene regulation
Metabolic processes
Cell-to-cell signaling
Cell specialization
Cytoskeletal organization
Intracellular transport mechanisms
Compartmentalization
Mechanisms of retroviral activity

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Growth requirements of Baker’s Yeast
Wild type S. cerevisiae: prototrophic as long

as there is a useable carbon source and nitrogen source as well as trace salts available
required molecules (amino acids, nucleic acids, polysaccharides, vitamins etc.) can be synthesized by the organism itself (there are, however, mutants available that are auxotroph for certain amino acids or nucleic acid precursors)

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Crabtree effect and oxygen requirements of S. cerevisiae
Preferred carbon source: glucose,

but many other carbon sources can be used
If the carbon source allows, S.cerevisiae prefers to generate energy mainly by alcoholic fermentation
When glucose is in abundance, baker’s yeast turns off all other pathways utilizing other carbon sources and grows solely by fermenting glucose to ethanol (“Crabtree effect”)
S. cerevisiae is a facultative anaerobe: can grow by fermentation in the complete absence of oxygen, as long as the growth media is substituted with sterols and unsaturated fatty acids
On non-fermentable carbon sources energy generated solely by respiration, and oxygen in the environment becomes essential (required for survival)

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Examples of Fermentable and Non-Fermentable Carbon Sources

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Diauxic shift
Yeast prefers alcoholic fermentation if the carbon source allows for

it, until the fermentable carbon source is exhausted
When there is no more fermentable carbon source in the media, the metabolism switches from fermentative to respiratory
This process requires the upregulation of genes involved in respiratory breakdown of ethanol, downregulation of genes involved in fermentation
Growth slows down after the diauxic shift

Time (hrs)

OD600= optical density at the wavelength of 600 nm;
Not Absorbance!; only linear between 0.3 and 0.7
The corresponding cell count differs from strain to strain (cell size!)

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Growth Media
“Favorite” Media (RICH media):
YP (Yeast extract and Peptone=peptic digest of

meat) + carbon source
YPD= YP+ dextrose
YPR= YP+ raffinose
YPG= YP+glycerol
YPGal= YP+ galactose
These are “complex media” (exact composition not known)
Non-selective! Mutants in amino acid or nucleic acid biosynthetic pathways can grow (unless mutant cannot metabolize carbon source)

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Synthetic complete media
Contain all the amino acids, some nucleic acid precursors

and some vitamins and trace elements
Nitrogen source: Ammonium sulfate (usually as Yeast Nitrogen Base (YNB) – containg also vitamins and trace salts)
Carbon source can be varied (SCD, SCR, SCD, SCGal..)
Non-selective if all amino acids/nucleic acid precursors are included
Certain amino acids or nucleic acid precursors can be omitted => selective media
Select against mutations in biosynthetic pathways! (Select for plasmids that carry the wild type copy of a mutated gene ? plasmid marker)

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Minimal media
Carbon source and Nitrogen source (YNB)
Only wild type yeast can

grow

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Yeast Gene and Gene Product Nomenclature
Dominant alleles are written in italicised

capital letters: LEU2, ADE3, ARG2
Attn:The number of the gene does not necessarily denote the place of the gene in a metabolic pathway. The numbering is often historical due to the order in which mutant alleles of the gene were obtained
Recessive alleles are written in italicised lower case letters: leu2, ade3, arg2
Sometimes mutant allele variants are indicated with a dash and an additional number: leu2-1, leu2-3….
Dominant gene products (=proteins) are written in regular letters, with the first letter capitalized: Leu2, sometimes followed by a lower case p: Leu2p
Recessive gene products are written in lower case: leu2 (leu2p)

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In most cases the wild type allele is denoted in upper

case italics: LEU2,
the mutant allele in lower case italics: leu2
!!!!
Special nomenclature for mutations involving mitochondrial genes – will not be talked about in this lecure

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Classical yeast genetics
Pre-molecular biology

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The Life Cycle of Saccharomyces cerevisiae
Wild type strains
Most laboratory strains (ho-)
2n
1n
2n
1n
Starvation
(no

sugar,
no NH4+)

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Yeast has a haploid growth phase
Phenotype of mutation apparent immediately
Every

haploid strain is a “pure bred” strain for its genetic traits
Haploids are “Gametes”
Sporulation = Meiosis; products of the same meiotic event can be examined!

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Genetic Manipulation
Ability to mate yeast cells allows combining of mutations
Meiotic

products (spores) are packed in a spore sac (Ascus) and can be physically separated -> dissection of spores allows for dissection of pathways

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Genetic analysis of a simple mutation
“Wild type” strain (Leu+)
“mate”

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Segregation of two alleles involved in Leucine biosynthesis
Cells are Leu+,

as the functional copy of LEU2 is sufficient to support growth on media lacking the amino acid Leucine

Sporulate on acetate medium
(Meiosis)

Diploid = Zygote

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Meiosis 1: separation of the homologous chromosomes
Meiosis 2: separation of the

chromatids

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Digest off cell wall
Tetrad with 3 spores visible in one focal

plane and 4th spore visible in a second focal plane

4 spores of a tetrad

Dissect ascospores!

Spores = Gametes!

Ascus = spore sac

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Line up on grid
Non-selective media (e.g. YPD)
Selective media (SC- Leu)
Leu-
Leu-

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Original Dissection on Non-selective plate
Replica on selective plate (e.g. Leu- strain

on SC – Leucine)

2 : 2 segregation ratio
(Leu+ vs. Leu- spores)

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Segregation of two unlinked genes
Example: TRP1, LEU2
Haploid, Leu+, Trp-
Haploid, Leu-, Trp+
Diploid,

Trp+, Leu+,

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Possible distribution of chromosomes during meiosis
Resulting tetrads after sporulation
parental ditype
(Trp+,

Leu- : Leu+,Trp-)

TRP1

nonparental ditype
(Trp+, Leu+ :Trp- Leu-)

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or
trp1
trp1
TRP1
TRP1
Tetratype

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Ratios of different types of tetrads! (NOT spores)

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Distances between linked genes can be calculated by counting the different

tetrad types;
Formula:

½ T + NPD (recombinants)

Total tetrads

Distance is expressed as recombination frequency in %
1% recombination = 1cM (centimorgan, after the famous fruit fly geneticist Thomas Hunt Morgan)
Recombination frequencies can never be > 50%
(= random assortment; genes behave unlinked)

X 100

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Dissecting Metabolic Pathways in Yeast
Question: What enzymes are involved in the

Biosynthesis of Uracil?
Approach: Screening for mutants dependent on uracil in the growth media
Mutagenize a healthy yeast strain (UV light, alkylating agents)
Plate mutagenized cells on non-selective media
Replica plate onto synthetic media lacking uracil (SC – Ura)

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Replica plating:

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YPD
SC - Ura
Most colonies still wild type – can grow on

synthetic media lacking uracil, but a, b and v are uracil auxotrophs – they have a new growth requirement (presence of uracil in the media) – and can’t grow on synthetic media lacking uracil

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Sorting of mutations
In our hypothetical screen, we have identified several haploid

mutants in the uracil biosynthesis pathway in both mating types
To test if the mutations are in the same pathway, we carry out Complementation analysis
Mutants are mated against each other
If the mutants are in the same gene, they will not complement each other an the diploid will be a uracil auxotroph
If the mutants are in different genes, they will complement each other, and the diploids will be able to grow on media lacking uracil

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Complementation analysis Scenario 1: mutations are in the same gene
Ura-
Ura-
Ura-
Diploid cannot

grow on SC – ura => Mutant A (α) and mutant 1 (a) cannot complement each other and are therefore in the same complementation group
Conclusion: Mutant A (α) and mutant 1 (a) are in the same gene uraX; as there is no functional copy of uraX in the cells, they are uable to synthesize uracil;

Mutant A, α mating type

Mutant 1, a mating type

Diploid a/α

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Complementation analysis Scenario 2: mutations are in different genes
Ura-
Ura-
Ura+
Mutant A, α

mating type

Mutant 2, a mating type

The diploid is able to grow on SC – ura => Mutant A (α) and mutant 1 (a) are able to complement each other and are in different complementation groups
Conclusion: Mutant A (α) and mutant 2 (a) are in different genes uraX and uraY; as there is one functional copy of each URAX and URAY in the cells, they are able to synthesize uracil;

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Complementation of mutants in the uracil biosynthesis pathway
(+) = mutants complement

each other ; (-) = mutants do not complement each other

Complementation groups: 1. A,D, 1, 3, 4 2. B, 5, 6 3. C,E, 2
Mutants in the same complementation groups have mutations in the same gene

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Epistatic Analysis
Epistasis - the interaction between two or more genes to

control a single phenotype

Epistatic Analysis: determine the order and/or relation ship of genes in a pathway

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Example of Epistatic analysis
Example: Adenine biosynthesis mutants ade2 and ade3 (unlinked

genes):
ade2 mutants are Ade-, make red colonies
ade3 mutants are Ade-, make white colonies
Double mutant will reveal position of genes/gene products in the adenine biosynthesis pathway relative to each other

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Scenario I
Scenario II
Two possibilities of order of action of Ade2p and

Ade3p in this pathway:

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ade2 mutant, α mating type
Ade -, RED
ade3 mutant, a mating type
Ade-,

creamy white

Diploid is white, Ade+

sporulate

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Possible distribution of chromosomes during meiosis
Parental Ditype - uninformative
All Ade-

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Nonparental Ditype – informative (two spores carry both mutations)
Two Scenarios

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2 x Ade+, white
Double mutant, Ade-, RED
A -------> B (red pigment)

------> C ------> D …

The ADE2 gene product catalyzes a reaction upstream of the ADE3 gene product. A mutation of ade2 blocks adenine synthesis at a point where the intermediate is a red pigment

Scenario 1

Ade3p

Ade2p

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2 x Ade+, white
Double mutant 2x Ade-, WHITE
The ADE3 gene product

catalyzes a reaction upstream of the ADE2 gene product. A mutation of ade3 blocks adenine synthesis at a point upstream of the formation of the red pigment. The cells are white.

Scenario 2

A -----> B ------> C ------> D (red pigment)---->…

Ade3p

Ade2p

Слайд 53

or
ade 3
ade3
ADE3
ADE 3
ADE2
ade 2
ade2
ADE 2
Tetratype

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Tetratype
Ade-, white
Ade+, white
Ade-, red
Ade-, white
Ade-, white
Scenario1
Scenario2
A -----> B ------> C ------>

D (red pigment)---->…

Ade3p

Ade2p

A -------> B (red pigment) ------> C ------> D …

Ade3p

Ade2p

Yeast Genetics and Molecular Biology. Lecture I. Yeast basics and classical yeast genetics

Содержание

What is Yeast Genetics?Definition of Genetics in Wikipedia: “Genetics (from Ancient

This slide was nicked from internet lecture notes of a course

Pioneers of yeast geneticsØjvind Winge (1886-1964), Carlsberg laboratory, Kopenhagen: http://www.genetics.org/cgi/content/full/158/1/1 Discovery

Baker’s YeastSaccharomyces cerevisiae:- Also “Budding yeast”- Ascomycete (ascus as fruiting body)-

Requirements for Model Organisms:

Yeast similarity to human cells

Yeast as a Model Organism David Botstein, Steven A. Chervitz, and J.

“Bacterial” aspects of yeast:Single cell organismHaploid growth phase -> phenotype of

Processes that can be studied in yeastCell cycle (mitosis, meiosis)(Principles of)

Growth requirements of Baker’s YeastWild type S. cerevisiae: prototrophic as long

Crabtree effect and oxygen requirements of S. cerevisiaePreferred carbon source: glucose,

Examples of Fermentable and Non-Fermentable Carbon Sources

Diauxic shiftYeast prefers alcoholic fermentation if the carbon source allows for

Growth Media“Favorite” Media (RICH media):YP (Yeast extract and Peptone=peptic digest of

Synthetic complete mediaContain all the amino acids, some nucleic acid precursors

Minimal mediaCarbon source and Nitrogen source (YNB)Only wild type yeast can

Yeast Gene and Gene Product NomenclatureDominant alleles are written in italicised

In most cases the wild type allele is denoted in upper

Classical yeast geneticsPre-molecular biology

The Life Cycle of Saccharomyces cerevisiaeWild type strainsMost laboratory strains (ho-)2n1n2n1nStarvation(no

Yeast has a haploid growth phase Phenotype of mutation apparent immediatelyEvery

Genetic ManipulationAbility to mate yeast cells allows combining of mutations Meiotic

Genetic analysis of a simple mutation“Wild type” strain (Leu+)“mate”

Segregation of two alleles involved in Leucine biosynthesis Cells are Leu+,

Meiosis 1: separation of the homologous chromosomesMeiosis 2: separation of the

Digest off cell wallTetrad with 3 spores visible in one focal

Line up on gridNon-selective media (e.g. YPD)Selective media (SC- Leu)Leu-Leu-

Original Dissection on Non-selective plateReplica on selective plate (e.g. Leu- strain

Segregation of two unlinked genesExample: TRP1, LEU2Haploid, Leu+, Trp-Haploid, Leu-, Trp+Diploid,

Possible distribution of chromosomes during meiosisResulting tetrads after sporulationparental ditype (Trp+,

ortrp1trp1TRP1TRP1Tetratype

Ratios of different types of tetrads! (NOT spores)

Distances between linked genes can be calculated by counting the different

Dissecting Metabolic Pathways in YeastQuestion: What enzymes are involved in the

Replica plating:

YPDSC - UraMost colonies still wild type – can grow on

Sorting of mutationsIn our hypothetical screen, we have identified several haploid

Complementation analysis Scenario 1: mutations are in the same geneUra-Ura-Ura-Diploid cannot

Complementation analysis Scenario 2: mutations are in different genesUra-Ura-Ura+Mutant A, α

Complementation of mutants in the uracil biosynthesis pathway(+) = mutants complement

Epistatic AnalysisEpistasis - the interaction between two or more genes to

Example of Epistatic analysisExample: Adenine biosynthesis mutants ade2 and ade3 (unlinked

Scenario IScenario IITwo possibilities of order of action of Ade2p and

ade2 mutant, α mating typeAde -, REDade3 mutant, a mating typeAde-,

Possible distribution of chromosomes during meiosisParental Ditype - uninformativeAll Ade-

Nonparental Ditype – informative (two spores carry both mutations)Two Scenarios

2 x Ade+, whiteDouble mutant, Ade-, REDA -------> B (red pigment)

2 x Ade+, whiteDouble mutant 2x Ade-, WHITEThe ADE3 gene product

orade 3ade3ADE3ADE 3ADE2ade 2ade2ADE 2Tetratype

TetratypeAde-, whiteAde+, whiteAde-, redAde-, whiteAde-, whiteScenario1Scenario2A -----> B ------> C ------>

Похожие презентации

What is Yeast Genetics?
Definition of Genetics in Wikipedia: “Genetics (from Ancient

Pioneers of yeast genetics
Øjvind Winge (1886-1964), Carlsberg laboratory, Kopenhagen: http://www.genetics.org/cgi/content/full/158/1/1
Discovery

Baker’s Yeast
Saccharomyces cerevisiae:
- Also “Budding yeast”
- Ascomycete (ascus as fruiting body)
-

“Bacterial” aspects of yeast:
Single cell organism
Haploid growth phase -> phenotype of

Processes that can be studied in yeast
Cell cycle (mitosis, meiosis)
(Principles of)

Growth requirements of Baker’s Yeast
Wild type S. cerevisiae: prototrophic as long

Crabtree effect and oxygen requirements of S. cerevisiae
Preferred carbon source: glucose,

Diauxic shift
Yeast prefers alcoholic fermentation if the carbon source allows for

Growth Media
“Favorite” Media (RICH media):
YP (Yeast extract and Peptone=peptic digest of

Synthetic complete media
Contain all the amino acids, some nucleic acid precursors

Minimal media
Carbon source and Nitrogen source (YNB)
Only wild type yeast can

Yeast Gene and Gene Product Nomenclature
Dominant alleles are written in italicised

Classical yeast genetics
Pre-molecular biology

The Life Cycle of Saccharomyces cerevisiae
Wild type strains
Most laboratory strains (ho-)
2n
1n
2n
1n
Starvation
(no

Yeast has a haploid growth phase
Phenotype of mutation apparent immediately
Every

Genetic Manipulation
Ability to mate yeast cells allows combining of mutations
Meiotic

Genetic analysis of a simple mutation
“Wild type” strain (Leu+)
“mate”

Segregation of two alleles involved in Leucine biosynthesis
Cells are Leu+,

Meiosis 1: separation of the homologous chromosomes
Meiosis 2: separation of the

Digest off cell wall
Tetrad with 3 spores visible in one focal

Line up on grid
Non-selective media (e.g. YPD)
Selective media (SC- Leu)
Leu-
Leu-

Original Dissection on Non-selective plate
Replica on selective plate (e.g. Leu- strain

Segregation of two unlinked genes
Example: TRP1, LEU2
Haploid, Leu+, Trp-
Haploid, Leu-, Trp+
Diploid,

Possible distribution of chromosomes during meiosis
Resulting tetrads after sporulation
parental ditype
(Trp+,

or
trp1
trp1
TRP1
TRP1
Tetratype

Dissecting Metabolic Pathways in Yeast
Question: What enzymes are involved in the

YPD
SC - Ura
Most colonies still wild type – can grow on

Sorting of mutations
In our hypothetical screen, we have identified several haploid

Complementation analysis Scenario 1: mutations are in the same gene
Ura-
Ura-
Ura-
Diploid cannot

Complementation analysis Scenario 2: mutations are in different genes
Ura-
Ura-
Ura+
Mutant A, α

Complementation of mutants in the uracil biosynthesis pathway
(+) = mutants complement

Epistatic Analysis
Epistasis - the interaction between two or more genes to

Example of Epistatic analysis
Example: Adenine biosynthesis mutants ade2 and ade3 (unlinked

Scenario I
Scenario II
Two possibilities of order of action of Ade2p and

ade2 mutant, α mating type
Ade -, RED
ade3 mutant, a mating type
Ade-,

Possible distribution of chromosomes during meiosis
Parental Ditype - uninformative
All Ade-

Nonparental Ditype – informative (two spores carry both mutations)
Two Scenarios

2 x Ade+, white
Double mutant, Ade-, RED
A -------> B (red pigment)

2 x Ade+, white
Double mutant 2x Ade-, WHITE
The ADE3 gene product

or
ade 3
ade3
ADE3
ADE 3
ADE2
ade 2
ade2
ADE 2
Tetratype

Tetratype
Ade-, white
Ade+, white
Ade-, red
Ade-, white
Ade-, white
Scenario1
Scenario2
A -----> B ------> C ------>