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Genetics 200A Monday, September 28, 2009 Day 5: Yeast Lecture #1 (Hiten Madhani) [email protected] Peter Walter’s Yeast Genetic Mystery mmm1 -> sick mmm1 smm1 -> healthy goal: find smm1 sequence genome: identical genomes cross to yeast KO collection to map: Not linked to any gene What’s going on? Fungi Fungi are eukaryotes There are many identified species (>70,000) Many are saprophytes (eat decaying plant matter) Some are human pathogens (e.g. Candida albicans, Aspergillus fumigatus) Are interesting for many biotechnology applications Sacchromyces cerevisiae a.k.a. “budding yeast” (but wt can form hyphae also!!!) Used as brewing yeast Can be grown on minimal media (carbon source, nitrogen source, some vitamins, salt, trace elements) Useful resource: www.yeastgenome.org S. cerevisiae genome 12.5 MB, ~6000 genes Linear chromosomes (range in size from 240 kB to 1 MB) Can run out the whole genome on a single gel (“pulse field gel”) Useful for identifying differences in chromosome copy number (aneuploidy) S. cerevisiae mitochrondial genome Is separate from the nuclear genome Mostly encodes hydrophobic components of the mitochondria (Some mitochondrial components are also encoded in the nuclear genome) Genome can be defective/absent (lacks genes for respiration) “petite” yeast BUT MUST STILL HAVE MITOCHONDRIA! S. cerevisiae L-A virus genome L-A virus is a cytoplasmic parasite of yeast Has a small dsRNA genome There are also some viruses that can parasitize L-A as well S. cerevisiae prions Most famous is [Psi+] Most are amyloid (-sheet aggregation) S. cerevisiae cell cycle Mitosis (asymmetric division: budding) G1 (growth phase, no division) S (DNA synthesis) Start (Checkpoint: enough nutrients --> proceed) G2 (Yeast don’t really do this, unless they get caught at Start) S. cerevisiae budding (cell division) Budding location is nonrandom (i.e., regulated) Two patterns: Axial budding: next bud near the last site Bipolar budding: next bud at opposite side of bud Pattern is cell-type dependent S. cerevisiae DNA replication (S) REQUIRES 1x CEN: Centromere 2x ARS: Origins of replication (1x ARS for circular plasmid) ***CEN-ARS plasmids are self-replicated in yeast*** Replicated DNA (“sister chromatids”) held together by cohesin S. cerevisiae Mitosis (M) Metaphase Each chromosome captures a single microtuble (from each centrosome) in yeast Centrosome is the microtuble organizing center Also called spindle pole body in yeast Are attached the nuclear envelope in yeast Must have 2 centrosomes at opposite ends of cell Anaphase Loss of sister chromatid cohesion Sister chromatids separate to opposite cell poles S. cerevisiae sex Two cell types: a and a secretes a factor, secretes factor a has factor receptor, has a factor receptor When cells detect pheromone (a, factor), they “shmoo” (or conjugate) Cells arrest in G1 (stop dividing) Grow toward mating partner (chemotropism) Cell-cell attachment (agglutination) Cell fusion Karyogamy (nuclei approaching) Nuclear fusion After conjugation, cells are diploid a/ N-starved cells form pseudohyphae C- and N-starved cells undergo sporulation Includes meiosis Produces 4 haploid spores (2x a, 2x ) Cell types are specified at MAT (mating type) locus MAT is on chromosome III MATa has two genes: a1, a2 MAT has two genes: 1, 2 In a cell: a1, a2 don’t do anything a is default state without MAT locus In cell: 1 up-regulates -specific genes (sgs) 2 down-regulates a-specific genes (asgs) In diploids: a1/2 down-regulates haploid-specific genes (hsgs) a1/2 down-regulates 1 too a1/2 down-regulates RME1, a repressor of meiosis S. cerevisiae meiosis Requires specific mechanisms to solve problems not encountered during mitosis One round of pre-meiotic S including sister cohesion Homolog pairing (e.g. both copies of chromosome 3 come together) Attachment of homologs via homologous recombination* *This mechanism is not universal and not required in all organsims Ex. Male flies don’t allow any recombination during meiosis Sister chromatid cohesion holds together homologs after recombination and during meiosis I Meiosis I involves monopolar spindle attachment, i.e., microtubules attach to one spindle pole body and not both S. cerevisiae meiosis (cont.) First meiotic division (Metaphase I) Loss of arm cohesion Maintenance of centromeric/peri-centromeric cohesion Leads to anaphase I (separation of homologs) Second meiotic division (No S phase) Metaphase II (as normal) Anaphase II (separation of sister chromatids) Four gametes (spores) == TETRAD!! S. cerevisiae nomenclature and markers Every gene is three letters and a number Gene names are italicized Wild type and dominant alleles are capitalized (e.g., HIS3) Recessive alleles are lowercase and the allele is designated by a second number (e.g., his3-1) Yeast markers are generally auxotrophies (nutrient requirements) “Drop-out” media lacks a nutrient Media is –His (histidine drop-out media) if it lacks histidine Phenotype is His- if it fails to grow (as opposed to His+ if it can) Replica plating allows preservation of colonies grown under permissive conditions (rich media), assayed under non-permissive conditions (drop-out media) Ben Schiller Email questions/corrections to [email protected] or any TAs/instructors