Prof. Fahd M. Nasr. Lebanese university Faculty of sciences I Department of Natural Sciences.

Transcription

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

2 B3206 Microbial Genetics

3 Eukaryotic M. G. The yeast Saccharomyces cerevisiae as a genetic model system Lecture X

4 Schizosaccharomyces pombe Schizosaccharomyces malidevorans A unicellular rod shaped euk. 2 to 3 mm in diameter and 7 to 14 mm in length Found in sugar-containing fermentations of alcohol Discovered in 1893 beer imported from East Africa P. Lindner Swahili word for beer pombe Identified as yeast fission yeast reproduces by fission Schizo- means different

5 Genome structure S. pombe genome was the sixth to be sequenced Small and compact genome 13.8Mb 3 linear chromosomes and 4,824 genes 4,730 introns in 43% of genes 15 is the largest number of introns 614 genes have two introns, 324 have three, 148 have four, 70 have five and 40 have six Similarities to proteins coded by 289 genes that are mutated, amplified, or deleted in human disease

6 Schizosaccharomyces pombe 50 genes human diseases: cystic fibrosis, hereditary deafness, and diabetes Largest gps of human disease-related genes cancer 23 genes processes in maintaining genomic stability DNA damage/repair, checkpoint controls, and the cell cycle, etc. Good model for cell cycle and checkpoint background Evolutionary aspects Evolution of one-celled and multi-celled euk. Which genes define eukaryotic cells and the transition from onecelled to multi-celled organisms

7 Life cycle of yeasts Fission yeast

8 Fission yeast S. pombe P. Nurse

9 The cell cycle in S. pombe

10 Schizosaccharomyces pombe - Fission yeast S. cerevisiae is as divergent to S. pombe as to humans H.s S.c S.p.

11 Fungi as model organisms Small genome relative to other eukaryotes Many fungal genes are homologous to those in other eukaryotes Easy to grow + short life cycles Haploid genomes amenable to mutation Sexual stage haploid spores products of meiosis Asexual (clonal) reproduction

12 Yeast genetics and molecular biology Saccharomyces cerevisiae a euk. cell with a sexual cycle Yeast genetics making mutants crossing yeast strains cloning yeast genes deleting genes Model systems studied in yeast

13 S. cerevisiae is an ascomycete A yeast cell 4-7mm

14 S. cerevisiae is a eukaryote Belongs to fungi, ascomycetes Unicellular organism with ability to produce pseudohyphae S. cerevisiae divides by budding two cells of unequal size: mother and daughter Yeast cells age and mothers die after about divisions

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17 S. cerevisiae is a eukaryote "bakers yeast" or "brewers yeast" ferments sugars CO2 + ethanol The CO2 is trapped as tiny bubbles dough rises Bread made with yeast was wonderful, light, tasty stuff Most intensively studied eukaryotic model Escherichia coli as the model prokaryote The microorganism behind the most common type of fermentation

18 The yeast Saccharomyces cerevisiae Yeast copes with a wide range of environmental conditions T from freezing to about 55 C are tolerated Yeasts proliferate from 12 C to 40 C Growth is possible from ph Almost complete drying is tolerated (dry yeast) Yeast can still grow and ferment at sugar concentrations of 3M Yeast can tolerate up to 20% alcohol

19 Yeast as an expression system Many recombinant proteins are successfully produced in S. cerevisiae Vaccines Hepatitis B virus surface antigen Diagnostics HIV-1 antigens Human therapeutic agents Insulin Human growth factor

20 Yeast as an expression system Single-celled, model for genetics, etc. Strong promoters, naturally occurring plasmid (2mm) vectors Post-translational modifications Engineered to secrete recombinant pro (Generally Recognized As Safe) organism (GRAS)

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22 Saccharomyces cerevisiae

23 Saccharomyces cerevisiae

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25 From 'open' to 'closed' mitosis

26 Yeast has a sexual life! Yeast cells can proliferate as 1n and as 2n 2n cells are 1.2-fold bigger Haploid cells a or alpha (a) Two haploid cells can mate zygote Yeast cannot move cells must grow towards each other (shmoos) The diploid zygote starts dividing from the junction

27 Yeast Life Cycle

28 Sequential steps during mating in Schizosaccharomyces pombe and Saccharomyces cerevisiae

29 Cytoduction Study of cytoplasmic heredity in yeast Cross two genetically marked yeast strains normal diploid zygotes and haploid nuclear cytoplasmic hybrids Nuclear markers of one parent and the cytoplasmic marker (rho+) of the other parent The autonomous cytoplasmic factor transfer cytoduction

30 Cytoduction Abortive form of yeast cell mating about 1% of cytoductants Test on cytoplasmic determination of some characters Constructing strains identical nucleus genotype with mt and other cytoplasmic factors of different origin

31 Cytoduction scheme Ustilago maydis, JOURNAL OF BACTERIOLOGY, Dec. 1992, p

32 KAR1 gene Mating between MATa and MATα diploids (n) cells adhere orderly removal of cell walls and plasma membrane cell fusion Nuclear envelope intact, (2n) nucleus direct fusion of the two parental nuclei A number of mutations block the karyogamy kar1-1 prevents nuclear fusion in 90-95% of matings KAR1 is essential for mitotic growth 3 functional domains

33 KAR1 gene KAR1 is essential for mitotic growth 3 functional domains An Nt domain important for the nuclear fusion Deletion kar1d15 allele viable and defective only in nuclear fusion kar1d15 mutation is unilateral unproductive matings if either parent is kar1d15

34 The end