Yeast Flashcards
What are they?
Baker’s yeast is Saccharomyces cerevisiae- how does it grow?
why is fermentation better than respiration?
why is respiration better?
what does fermentation form?
Single celled eukaryotic fungi- lower eukaryotes
fermentation and respiration
doesn’t require oxygen, uses glucose quicker (hence energy quicker), ethanol poisons competitors & can use ethanol for more energy
more energy efficient- 34-36ATP vs 2, but it is much slower
2 ATP, 2 CO2, 2 Ethanol
What is different between industrial yeast & wild yeast diversity?
Why is yeast advantageous for making recombinant drugs/insulin?
Wild yeast diversity = much larger as it hasn’t been domesticated/optimised
as they are eukaryotes- more similar to humans
History——
What did Leeuwenhoek show?
Schwann?
Liebig?
Pasteur?
Buchner?
Simple microscopes to observe yeast fermenting in beer but didn’t know if it was alive
yeast essential for fermentation as when removed it there was none
fermentation is a chemical reaction & that yeast was a catalyst but not alive
if heat unfermented mixture- kills yeast & prevents fermentation & also it doesn’t require oxygen
yeast cells produce enzyme promote fermentation even without the cells present- so the catalysts are the enzymes produced by living cells = fermentation
What is lager yeast?
Winge was the director of Carlberg- what did he discover?
why is yeast a good model?
hybrid of 2 species S cerevisiae (bakers yeast) & S. bayanus
yeast have sex- allows system for genetics research & model
well characterised, small genome, rapid growth, easy to manipulate, homologous to human genes (closely related more than e.coli)- so can knock out genes & insert human equivalent which can save them
What mating types breed?
What happens after the diploid forms?
What happens to the diploid cells under stress?
what are the spores?
what happens when they receive sufficient nutrients/are no longer stressed?
what is shmooing?
how is mating type controlled?
what do these genes express?
can mating types switch?
Alpha haploid (n) & a haploid (n) to form diploid a/alpha proliferate (2n)
proliferative vegetation- mother grows bud which increases in size to form daughter cell- separates in cell division (diploid)
undergo meiotic differentiation- and 2 meiotic divisions to form 4 haploid meiotic products = spores
tetrad of 2 alpha haploids & 2 a haploids
germinate & form haploid yeast cells of a & alpha type & often mate with eachother
the 2 mating types grow towards eachother- cells & nuclei fuse to make 1 diploid nucleus & cell
MAT locus- haploids have either MATa or MATalpha allele but diploids are heteroxygous & have both
haploid a makes pheromones & receptors to recognise alpha pheromones
haploid alpha makes alpha pheromones & receptors to recognise a pheromones
yes- not fixed
What’s the difference between auxotrophic & prototrophic mutants?
how can you identify auxotrophic mutants?
how can you find out what nutrients the auxotrophic mutants need?
auxotrophic = in this case need specific nutrients to grow for AA/NA/vitamins prototrophic = produce all 20AA by themselves (wild types)
mutagenize liquid culture of wild types with UV & dilute & plate cells onto rich extract & replica plate onto minimal medium (which wild type survive)
- cells that survive on rich but not minimal = auxotrophic
add defined nutrients to minimal medium- e.g add lysine so if auxotroph grows = lys- (can’t grow without lysine) whereas lys+ is wild type (can grow without)
what happens if you cross lys- a haploid x lys+ alpha haploid?
why?
what about crossing: a lys- met+ x alpha lys+ met-?
what about: a lys1- x alpha lys1-
a lys1- lys2+ x alpha lys1+ lys2-?
what is the complimentation test?
form a/alpha lys+/lys- mutant- which complement eachother so can grow without lysine due to lys+
wild type lys+ is dominant- so can grow without lysine present
complementation occurs- wildtypes of met in a & lys in alpha means they dominate in haploid so can grow on minimal media
both defective in same gene-can’t grow on minimal media so there is no complimentation. but more than 1 gene is often required- e.g lys2
wild type for lys1 & lys2 present in diploid so complementation so grow on minimal media
test to find out which diff genes defective in diff mutants
What happens if you cross 2 different strains so lys2+ and lys2- for tetrad analysis? in diploid
what is necessary for recombination?
what about lys2+ and ura7- on 1 chromosome & lys2- and ura7+ on other?
what happens if the mutations are close together?
what can you use tetrad analysis for?
they recombine & undergo meiosis & form 4 haploids 2 which are lys2+ and 2 which are lys2-
mutations to be far apart on chromosome- mutation segregation is random/independent so no genetic linkage
if far apart- recombine & get all possible combinations in haploids
crossover more rare so no new combinations- if you end up with new combinations it’s due to genetic linkage
calculate frequency of recombination & build up genetic map
what is a shuttle vector?
why would you want to clone a specific gene?
how would you do this?
what do you need to do first? how?
how would you undergo genomic library screening?
how can you test the candidate genes?
since we end up with lys2- mutant phenotype, how can we use the complementation test to check?
fuse e.coli plasmid & wild yeast plasmid & add genetic markers to allow selection for either yeast or e.coli = 2 different host species
find the defective gene in auxotrophic lys2- for example
cut up genomic DNA from wild type yeast & put pieces into plasmid & test each plasmid for ability to complement auxotrophic lys2- mutant to find plasmid with wild type lys2+ gene
- make gene library- restriction endonucleases to cut DNA & make sticky ends & ligate with shuttle vector
- transform into e.coli for amplification
transform lys2- with genomic library by finding wild type lys2+ gene by growing it on minimal media- then plasmid with wild type can complement the mutant lys2-
- cut gene with restriction enzyme
- ligate into know gene marker which has resistance to a specific drug
- transform plasmid into wild yeast cells
- transform with the corresponding drug
- if it survives- means the lys2+ gene has been knocked out by the anti drug gene & ended up with lys2- mutant
cross our alpha unknown lys diploid with a lys2-: if it doesn’t grow on minimal media no lys2+ is present so is auxotrophic- the gene can then be sequenced & analysed
How do yeasts become pathogenic?
Why is yeast used to recombinantly produce Artemisinin?
Unicellular yeast cells develop into invasive filaments & invade tissues
drug to combat malaria- cheaper to be made recombinantly & higher yield
Schizosaccharomyces pombe = model yeast, what kind of yeast is it? why is it a good model?
what is its haploid life cycle? why does it prefer this?
what happens under stress?
what happens to the spores under better conditions?
what happens if nutrients are scare without haploids of the other mating type?
what can you do in a lab but not in nature?
fission yeast- lots of genes have been characterised (like e.coli) but not all
- haploid cells elongate & divide by symmetrical division (mitosis) into 2 daughter cells (clones). prefered in vegetative cell cycle as long as conditions/nutrients are good
undergo sex role differentiation- where mating type H+ and H- fused to form diploids & undergo 2 levels meiosis to form 4 haploid spores in ascus (zygotic)
germinate & start haploid cycle again
diploid cells enter dormant state without sex role differentiation
take diploids before meiosis & grow in medium
what led to genetic diversity in yeast if it wasn’t mutations?
how can you see which genes determine phenotype?
horizontal gene transfer from bacteria to eukaryotes/fission yeast & also association with plants
GWAS genome wide association study- many mutants contributing to specific traits
why can we use fission yeast as a model for cellular ageing of non dividing cells?
how can you find out which phenotypes/genes cause longer lifespan?
what is a factor of age?
how can you find different strains of S. pombe to help malic acid deacidification in colder countries?
many cells in human body are non dividing but have to survive- and yeast cells stop dividing under stress
combine short living strains with long living strains with intercrossing for recombination & grow to see which variants live over time
allele frequency
test different strains- find ones which are most effective for fermentation, malic acid deacidification, reduce lvls of toxic products ^ generate appropriate aroma
