Prof. Fahd M. Nasr Lebanese university Faculty of sciences [email protected] https://yeastwonderfulworld.wordpress.com/
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Prof. Fahd M. Nasr
Lebanese universityFaculty of sciences
https://yeastwonderfulworld.wordpress.com/
Mighty Yeasts
The yeast Saccharomyces cerevisiaeas a genetic model system
Yeast genetics• In 1996 Yeast genes
– 1/3 characterised by genetic analysis– 1/3 shows homology to known genes– 1/3 orphans
• 5% yeast genes with introns very few have more than one
• The intergenic space between genes is only between 200 and 1,000bp
Yeast genome analysis• Major goal function of every gene• Large projects and numerous approaches• Micro array analysis
– Gene expression profiles– Binding sites in the genome for all transcription factors
• A complete set of more than 6,000 deletion mutants is available for research
• Various approaches to analyse the properties of these mutants
• Tag yeast genes to GFP protein detection and microscopic localisation
• Different global protein interaction projects are ongoing
Yeast genes: nomenclature
Yeast genes: nomenclature• Many genes systematic sequencing : YDR518C,
YML016W..., where– Y stands for ”yeast”– The second letter represents the chromosome (D=IV,
M=XIII....)– L or R stand for left or right chromosome arm– The three-digit number stands for the ORF counted
from the centromere on that chromosome arm– C or W stand for ”Crick” or ”Watson” indicate the
strand or direction of the ORF• Some genes do not follow this nomenclature HO, MATa,
MATa
General pathway for mutational dissection of a biological process
“Forward Genetics”
Gene deletion in yeast
HIS3
CBK1X X
Chromosome XIV
DNA cassette~50nc
Chromosome XIV
HIS3
CBK1
DcbK1::HIS3
mmmm + histidine
+
+ -
+Transform the strain CBK1, his3 with DNA cassette
Select for transformants on mm
wild type(CBK1)
∆cbk1
DIC Calcofluor white
Yeast genetics: markers and strains
• Genetic markers for selection
• Commonly genetic markers HIS3, URA3, TRP1, LEU2, LYS2, ADE2
• The ade2 mutation cells turn red
• The first markers fermentation markers: SUC, MAL, GAL– GAL genes encode the enzymes needed to take up
galactose and convert it to glucose-6-phosphate
Purine nucleotide synthesis pathway in yeast
The art and design of genetic screens: yeast
Galactose metabolizing pathway of yeast
Galactose metabolizing pathway of yeast
• GAL genes are near each other but do not constitute an operon
• GAL4 unlinked gene repressor protein– Binds a promoter element UASG– UAS is located between GAL1 and GAL10– Transcription occurs in both directions from UASG
• Galactose absent GAL4p +GAL80p bind the UASGtranscription does not occur
• Galactose present galactose binds GAL80p and GAL4p amino acids are phosphorylated
• Galactose acts as an inducer by causing a conformation change in GAL4p/GAL80p
Transcriptional control of galactose-utilizing genes in yeast
Activation model of GAL genes in yeast
Regulation of galactose utilization in yeast
Repression of the GAL1 gene in yeast
How genes respond to environmental stimuli
Scer TTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCATATACASpar CTATGTTGATCTTTTCAGAATTTTT-CACTATATTAAGATGGGTGCAAAGAAGTGTGATTATTATATTACATCGCTTTCCTATCATACACASmik GTATATTGAATTTTTCAGTTTTTTTTCACTATCTTCAAGGTTATGTAAAAAA-TGTCAAGATAATATTACATTTCGTTACTATCATACACASbay TTTTTTTGATTTCTTTAGTTTTCTTTCTTTAACTTCAAAATTATAAAAGAAAGTGTAGTCACATCATGCTATCT-GTCACTATCACATATA
* * **** * * * ** ** * * ** ** ** * * * ** ** * * * ** * * *
Scer TATCCATATCTAATCTTACTTATATGTTGT-GGAAAT-GTAAAGAGCCCCATTATCTTAGCCTAAAAAAACC--TTCTCTTTGGAACTTTCAGTAATACGSpar TATCCATATCTAGTCTTACTTATATGTTGT-GAGAGT-GTTGATAACCCCAGTATCTTAACCCAAGAAAGCC--TT-TCTATGAAACTTGAACTG-TACGSmik TACCGATGTCTAGTCTTACTTATATGTTAC-GGGAATTGTTGGTAATCCCAGTCTCCCAGATCAAAAAAGGT--CTTTCTATGGAGCTTTG-CTA-TATGSbay TAGATATTTCTGATCTTTCTTATATATTATAGAGAGATGCCAATAAACGTGCTACCTCGAACAAAAGAAGGGGATTTTCTGTAGGGCTTTCCCTATTTTG
** ** *** **** ******* ** * * * * * * * ** ** * *** * *** * * *
Scer CTTAACTGCTCATTGC-----TATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTCGTCTSpar CTAAACTGCTCATTGC-----AATATTGAAGTACGGATCAGAAGCCGCCGAGCGGACGACAGCCCTCCGACGGAATATTCCCCTCCGTGCGTCGCCGTCTSmik TTTAGCTGTTCAAG--------ATATTGAAATACGGATGAGAAGCCGCCGAACGGACGACAATTCCCCGACGGAACATTCTCCTCCGCGCGGCGTCCTCTSbay TCTTATTGTCCATTACTTCGCAATGTTGAAATACGGATCAGAAGCTGCCGACCGGATGACAGTACTCCGGCGGAAAACTGTCCTCCGTGCGAAGTCGTCT
** ** ** ***** ******* ****** ***** *** **** * *** ***** * * ****** *** * ***
Scer TCACCGG-TCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAA-----TACTAGCTTTT--ATGGTTATGAASpar TCGTCGGGTTGTGTCCCTTAA-CATCGATGTACCTCGCGCCGCCCTGCTCCGAACAATAAGGATTCTACAAGAAA-TACTTGTTTTTTTATGGTTATGACSmik ACGTTGG-TCGCGTCCCTGAA-CATAGGTACGGCTCGCACCACCGTGGTCCGAACTATAATACTGGCATAAAGAGGTACTAATTTCT--ACGGTGATGCCSbay GTG-CGGATCACGTCCCTGAT-TACTGAAGCGTCTCGCCCCGCCATACCCCGAACAATGCAAATGCAAGAACAAA-TGCCTGTAGTG--GCAGTTATGGT
** * ** *** * * ***** ** * * ****** ** * * ** * * ** ***
Scer GAGGA-AAAATTGGCAGTAA----CCTGGCCCCACAAACCTT-CAAATTAACGAATCAAATTAACAACCATA-GGATGATAATGCGA------TTAG--TSpar AGGAACAAAATAAGCAGCCC----ACTGACCCCATATACCTTTCAAACTATTGAATCAAATTGGCCAGCATA-TGGTAATAGTACAG------TTAG--GSmik CAACGCAAAATAAACAGTCC----CCCGGCCCCACATACCTT-CAAATCGATGCGTAAAACTGGCTAGCATA-GAATTTTGGTAGCAA-AATATTAG--GSbay GAACGTGAAATGACAATTCCTTGCCCCT-CCCCAATATACTTTGTTCCGTGTACAGCACACTGGATAGAACAATGATGGGGTTGCGGTCAAGCCTACTCG
**** * * ***** *** * * * * * * * * **
Scer TTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCG--ATGATTTTT-GATCTATTAACAGATATATAAATGGAAAAGCTGCATAACCAC-----TTSpar GTTTT--TCTTATTCCTGAGACAATTCATCCGCAAAAAATAATGGTTTTT-GGTCTATTAGCAAACATATAAATGCAAAAGTTGCATAGCCAC-----TTSmik TTCTCA--CCTTTCTCTGTGATAATTCATCACCGAAATG--ATGGTTTA--GGACTATTAGCAAACATATAAATGCAAAAGTCGCAGAGATCA-----ATSbay TTTTCCGTTTTACTTCTGTAGTGGCTCAT--GCAGAAAGTAATGGTTTTCTGTTCCTTTTGCAAACATATAAATATGAAAGTAAGATCGCCTCAATTGTA
* * * *** * ** * * *** *** * * ** ** * ******** **** *
Scer TAACTAATACTTTCAACATTTTCAGT--TTGTATTACTT-CTTATTCAAAT----GTCATAAAAGTATCAACA-AAAAATTGTTAATATACCTCTATACTSpar TAAATAC-ATTTGCTCCTCCAAGATT--TTTAATTTCGT-TTTGTTTTATT----GTCATGGAAATATTAACA-ACAAGTAGTTAATATACATCTATACTSmik TCATTCC-ATTCGAACCTTTGAGACTAATTATATTTAGTACTAGTTTTCTTTGGAGTTATAGAAATACCAAAA-AAAAATAGTCAGTATCTATACATACASbay TAGTTTTTCTTTATTCCGTTTGTACTTCTTAGATTTGTTATTTCCGGTTTTACTTTGTCTCCAATTATCAAAACATCAATAACAAGTATTCAACATTTGT
* * * * * * ** *** * * * * ** ** ** * * * * * *** *
Scer TTAA-CGTCAAGGA---GAAAAAACTATASpar TTAT-CGTCAAGGAAA-GAACAAACTATASmik TCGTTCATCAAGAA----AAAAAACTA..Sbay TTATCCCAAAAAAACAACAACAACATATA
* * ** * ** ** **
GAL10
GAL1
TBP
GAL4 GAL4 GAL4
GAL4
MIG1
TBPMIG1
Factor footprint
Conservation island
Conservation of Motifs
Yeast genetics: markers and strains
• Certain antibiotic resistance markers used in transformation kanamycin resistance = kanR
• There are many yeast strains in use in the laboratories:W303-1A, S288C, S1278b, SK1, BY4741....
Yeast genetics: markers and strains
• Specific properties can be quite different and are different to wild or industrial strains
• The full genotype of W303-1A strain:MATa leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1 can1-100 GAL SUC2mal0
Auxotroph• Mutant organism requiring a specific growth
substance– Auxotrophy inability of an organism to synthesize a
particular organic compound required for its growth– An auxotroph is an organism that displays this
characteristic– Auxotrophic is the corresponding adjective
• Auxotrophy is the opposite of prototrophy– wild-type strain– Auxotrophic genetic markers are often used in
molecular genetics
Chemical structure of arginine compared to canavanine
Promoters on Vectors
Tetrad analysis for genetic analysis
Synthetic Lethal Screen
Tetrad Spore MAT leu ura his SUC NaCl
1 A a + + - - -
1 B alpha + - + - -
1 C a - - - + -
1 D alpha - + + + +
2 A a - - - - -
2 B a + + + + +
2 C alpha + - + - -
2 D alpha - + - + -
Yeast genetics: crossing strains
Replicate plating to isolate auxotrophic mutants: grow with His but not without His
Yeast genetics: meiosis• Yeast tetrad analysis
outcome of meiosis• 2n has two chromosomes• DNA replication two
chromosomes with two identical chromatids each
• Aligned chromosomes undergo recombination
• First meiotic division separate the chromosomes
• Second meiotic division separate the chromatids
• Each spore represents essentially one chromatid
X
Three kinds of patternsMATa TRP1URA3 x MATa trp1 ura3
Recombination Between Markers
RF = 1/2T +NPD If RF = 50% then genes are unlinked
When genes are linked, PDs exceed NPDs
Sulfate assimilation requires 3 different enzymes, encoded by MET3, MET14, and MET16
Met3p
SO42-
Met14p
APS PAPS
Met16p
SO32-
A mutation in any of the 3 genes will prevent sulfate assimilation required for de novo*
methionine synthesis
*de novo – no organic sulfur source is used
Ascus 1
MATa/α MET3/met3 MET14/met14
What happens when the Met+ diploid sporulates?
(We’ll ignore the MET16 and MATa loci)
Ascus 2
Each meiosis produces an ascus with 4 haploid spores
OR
1/4 spores will give rise to a Met+
haploid
Linkage analysis
# of tetradsGene pair PD NPD Tgal1/gal7 313 0 0gal1/gal10 59 0 0gal7/gal10 72 0 0gal1/gal4 21 23 56gal3/gal4 20 13 48
What does what?Enzymatic activities
Genotype Kinase Transferase Epimerasewild type 13-24 9-14 8-34gal1 0 14.9 87.2gal7 6 0 33.8gal10 2.7 2.2 0gal10/i- 13 15.5 0gal1/gal7 0 0 20.3gal1/gal10 0 6.1 0gal4 0 0 0gal4/i- 0 0 0
The end
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