B3206 Microbial Genetics

Transkriptio

1 Prof. Fahd M. Nasr Lebanese university Faculty of sciences I Department of Natural Sciences fnasr@ul.edu.lb B3206 Microbial Genetics 1

2 Eukaryotic M. G. The yeast Saccharomyces cerevisiae as a genetic model system Lectures XIII and XIV Why yeast as genetics model Basic cellular mechanisms conserved Unicellular Grow on readily controlled, defined media Ideal life cycle Very compact genome Quick to map a phenotype producing gene Single gene deletion mutants One third of the genes have counterparts in human 2

3 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 3

4 Yeast genes: nomenclature 3 letters and up to 3 numbers: CDC28 Wild type capital letters in italics: MEC1, HOG1, CDC28... Recessive mutant small letters in italics: tps1, rho1, cdc28 Mutant alleles are designated with a dash and a number tps1-1, rho1-23, cdc28-2 Gene deletion mutation write genetic marker used for deletion tps1d::his3 A protein a capital letter at the beginning and not in italics often a "p" is added at the end: Tps1p, Rho1p, Cdc28p 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 4

5 Two possible approaches A reverse genetic approach A gene of interest is disrupted The resulting phenotype is studied A forward genetic approach A phenotype of interest is chosen Screen a pool of randomly generated mutants Both have been equally effective The yeast genome Excellent model 5

6 Gene deletion in yeast ~50nc X WT CBK1 HIS3 CBK1 DNA cassette X Chromosome XIV Dcbk1::HIS3 HIS3 Chromosome XIV 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 6

7 Schematic representation of purine metabolism in S. cerevisiae 7

8 The art and design of genetic screens: yeast Transcriptional control of galactose-utilizing genes in yeast 3 genes (GAL1, GAL7 and GAL10) enzymes that function in the galactose metabolic pathway GAL1 galactokinase GAL7 galactose transferase GAL10 galactose epimerase 8

9 Galactose metabolizing pathway of yeast Transcriptional control of galactose-utilizing genes in yeast Pathway produces D-glucose 6-phosphate glycolytic pathway and is metabolized by genes that are continuously transcribed In absence of galactose, GAL genes are not transcribed GAL genes rapidly induced by galactose and absence of glucose Analogous to E. coli lac operon repression by glucose 9

10 Transcriptional control of galactose-utilizing genes in yeast GAL genes are near each other but do not constitute an operon GAL4 unlinked gene repressor protein Binds a promoter element UAS G UAS is located between GAL1 and GAL10 Transcription occurs in both directions from UAS G Galactose absent GAL4p +GAL80p bind the UAS G transcription 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 Absence of galactose 10

11 Presence of galactose Repression of the GAL1 gene in yeast 11

12 How genes respond to environmental stimuli 12

13 Scer TTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCATTACCACCATATACA Spar CTATGTTGATCTTTTCAGAATTTTT-CACTATATTAAGATGGGTGCAAAGAAGTGTGATTATTATATTACATCGCTTTCCTATCATACACA GAL10 Smik GTATATTGAATTTTTCAGTTTTTTTTCACTATCTTCAAGGTTATGTAAAAAA-TGTCAAGATAATATTACATTTCGTTACTATCATACACA Sbay TTTTTTTGATTTCTTTAGTTTTCTTTCTTTAACTTCAAAATTATAAAAGAAAGTGTAGTCACATCATGCTATCT-GTCACTATCACATATA * * **** * * * ** ** * * ** ** ** * * * ** ** * * * ** * * * TBP Scer TATCCATATCTAATCTTACTTATATGTTGT-GGAAAT-GTAAAGAGCCCCATTATCTTAGCCTAAAAAAACC--TTCTCTTTGGAACTTTCAGTAATACG Spar TATCCATATCTAGTCTTACTTATATGTTGT-GAGAGT-GTTGATAACCCCAGTATCTTAACCCAAGAAAGCC--TT-TCTATGAAACTTGAACTG-TACG Smik TACCGATGTCTAGTCTTACTTATATGTTAC-GGGAATTGTTGGTAATCCCAGTCTCCCAGATCAAAAAAGGT--CTTTCTATGGAGCTTTG-CTA-TATG Sbay TAGATATTTCTGATCTTTCTTATATATTATAGAGAGATGCCAATAAACGTGCTACCTCGAACAAAAGAAGGGGATTTTCTGTAGGGCTTTCCCTATTTTG ** ** *** **** ******* ** * * * * * * * ** ** * *** * *** * * * GAL4 GAL4 GAL4 Scer CTTAACTGCTCATTGC-----TATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTCGTCT Spar CTAAACTGCTCATTGC-----AATATTGAAGTACGGATCAGAAGCCGCCGAGCGGACGACAGCCCTCCGACGGAATATTCCCCTCCGTGCGTCGCCGTCT Smik TTTAGCTGTTCAAG ATATTGAAATACGGATGAGAAGCCGCCGAACGGACGACAATTCCCCGACGGAACATTCTCCTCCGCGCGGCGTCCTCT Sbay TCTTATTGTCCATTACTTCGCAATGTTGAAATACGGATCAGAAGCTGCCGACCGGATGACAGTACTCCGGCGGAAAACTGTCCTCCGTGCGAAGTCGTCT ** ** ** ***** ******* ****** ***** *** **** * *** ***** * * ****** *** * *** GAL4 Scer TCACCGG-TCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAA-----TACTAGCTTTT--ATGGTTATGAA Spar TCGTCGGGTTGTGTCCCTTAA-CATCGATGTACCTCGCGCCGCCCTGCTCCGAACAATAAGGATTCTACAAGAAA-TACTTGTTTTTTTATGGTTATGAC Smik ACGTTGG-TCGCGTCCCTGAA-CATAGGTACGGCTCGCACCACCGTGGTCCGAACTATAATACTGGCATAAAGAGGTACTAATTTCT--ACGGTGATGCC Sbay GTG-CGGATCACGTCCCTGAT-TACTGAAGCGTCTCGCCCCGCCATACCCCGAACAATGCAAATGCAAGAACAAA-TGCCTGTAGTG--GCAGTTATGGT ** * ** *** * * ***** ** * * ****** ** * * ** * * ** *** MIG1 Scer GAGGA-AAAATTGGCAGTAA----CCTGGCCCCACAAACCTT-CAAATTAACGAATCAAATTAACAACCATA-GGATGATAATGCGA------TTAG--T Spar AGGAACAAAATAAGCAGCCC----ACTGACCCCATATACCTTTCAAACTATTGAATCAAATTGGCCAGCATA-TGGTAATAGTACAG------TTAG--G Smik CAACGCAAAATAAACAGTCC----CCCGGCCCCACATACCTT-CAAATCGATGCGTAAAACTGGCTAGCATA-GAATTTTGGTAGCAA-AATATTAG--G Sbay GAACGTGAAATGACAATTCCTTGCCCCT-CCCCAATATACTTTGTTCCGTGTACAGCACACTGGATAGAACAATGATGGGGTTGCGGTCAAGCCTACTCG **** * * ***** *** * * * * * * * * ** MIG1 TBP Scer TTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCG--ATGATTTTT-GATCTATTAACAGATATATAAATGGAAAAGCTGCATAACCAC-----TT Spar GTTTT--TCTTATTCCTGAGACAATTCATCCGCAAAAAATAATGGTTTTT-GGTCTATTAGCAAACATATAAATGCAAAAGTTGCATAGCCAC-----TT Smik TTCTCA--CCTTTCTCTGTGATAATTCATCACCGAAATG--ATGGTTTA--GGACTATTAGCAAACATATAAATGCAAAAGTCGCAGAGATCA-----AT Sbay TTTTCCGTTTTACTTCTGTAGTGGCTCAT--GCAGAAAGTAATGGTTTTCTGTTCCTTTTGCAAACATATAAATATGAAAGTAAGATCGCCTCAATTGTA * * * *** * ** * * *** *** * * ** ** * ******** **** * Scer Spar Smik Sbay Scer Spar Smik Sbay Conservation of Motifs TAACTAATACTTTCAACATTTTCAGT--TTGTATTACTT-CTTATTCAAAT----GTCATAAAAGTATCAACA-AAAAATTGTTAATATACCTCTATACT TAAATAC-ATTTGCTCCTCCAAGATT--TTTAATTTCGT-TTTGTTTTATT----GTCATGGAAATATTAACA-ACAAGTAGTTAATATACATCTATACT TCATTCC-ATTCGAACCTTTGAGACTAATTATATTTAGTACTAGTTTTCTTTGGAGTTATAGAAATACCAAAA-AAAAATAGTCAGTATCTATACATACA TAGTTTTTCTTTATTCCGTTTGTACTTCTTAGATTTGTTATTTCCGGTTTTACTTTGTCTCCAATTATCAAAACATCAATAACAAGTATTCAACATTTGT * * * * * * ** *** * * * * ** ** ** * * * * * *** * TTAA-CGTCAAGGA---GAAAAAACTATA TTAT-CGTCAAGGAAA-GAACAAACTATA TCGTTCATCAAGAA----AAAAAACTA.. TTATCCCAAAAAAACAACAACAACATATA * * ** * ** ** ** GAL1 Factor footprint Conservation island Yeast genetics: markers and strains Certain antibiotic resistance markers used in transformation kanamycin resistance = kan R There are many yeast strains in use in the laboratories: W303-1A, S288C, S1278b, SK1, BY

14 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 SUC2 mal0 Yeast genetics: crossing strains Cross two haploid strains with different mutations diploid strain Analysis mutations in the same or different genes Sporulation tetrads dissection spores individual colonies 14

15 Yeast genetics: crossing strains Genetic crosses map genes on chromosomes The last genetic map more than 1,000 genes and turned out to be very accurate Today genetic crosses yeast strains with new combination of mutations The genome fully sequenced genetic screens for new mutations Auxotrophy/markers An auxotroph needs a specific substance to grow Auxotrophic genetic markers are often used in molecular genetics HIS3, LEU2, TRP1, URA3, ADE2, and CAN1 Positive and/or negative selection LacZ: transgenic reporter, colony blue colored in X-Gal containing medium upon expression 15

16 Yeast genetics: crossing strains To cross two strains mix on agar plates and allow to mate MATa leu2 URA3 x MATa LEU2 ura3 2n cells will be heterozygous grow on medium lacking both leucine and uracil 2n will be grown and plated on sporulation medium asci/tetrads The ascus wall is digested with a specific enzyme mix spores are separated with a micromanipulator on agar plates Yeast genetics: crossing strains Spores will germinate colonies can be studied individually Properties of the meiotic progeny can be studied directly Pattern of growth how two mutations separate The spore colonies are replicated to different media in order to characterize the properties of the spores and to follow the genetic markers 16

17 Yeast genetics: crossing strains Tetrad Spore MAT leu ura his SUC NaCl 1 A a B alpha C a D alpha A a B a C alpha D alpha

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