Lecture 26- yeast cloning

genetic markers

-are used to follow chromosomes in genetic crosses, to select diploids in genetic crosses, to select transformants in transformation with plasmids or integration of genes into the genome

-commonly genetic markers cause auxotrophies (traditional markers on plasmids/yeast found by complementation)

-HIS3, URA3, TRP1, LEU2, LYS2, ADE2

-human genetic markers won’t work in yeast b/c we have introns and yeast does not splice

ade2 mutation

-cells turn red

first markers in yeast genetics

-fermentation markers, genes that confer the ability to catabolize certain substrates

-SUC, MAL, GAL

SUC gene (SUC1-7)

-encodes invertase (periplasmic enzyme) and can be located on different chromosomes in different yeast strains (telomere location)

MAL loci (MAL1-6)

-encode each 3 genes: maltase, maltose transporter and a transcriptional activator; also a telomer location

GAL genes

-encode the enzymes needed to take up galactose and convert it to glucose-6-phosphate

Kanamycin resistance (kanR)

-antibiotic resistance marker used in transformation

yeast genetics and molecular biology

-yeast are classic eukaryotes

cloning in E.coli

-construct a plasmid library of wt E. coli DNA

-transform a his- strain with our library of plasmids; if one of the library plasmids has the wt hisG gene inserted in it and it gets into a cell by transformation; this cell will be able to synthesize histidine and grow in the absence of histidine in the media

-therefore we know we have a clone of the hisG gene

identification of E. coli genes

-lots of E. coli gene clones were identified using analogous gene complementation

-complementation of E. coli mutants with eukaryotic gene libraries did not work with a few exceptions, some yeast clones did complement E. coli mutations

-yeast promoters do not look like bacterial promoters, signals for translation are different also

cloning my complementation

-frequently, when one has isolated a number of mutants and classified them into complementation groups the nature of the gene is not known

-to identify the gene it is cloned from a gene library by complementation of the mutation

a gene library

-is a large population of plasmids containing different fragments of genomic yeast DNA, cumulatively representing the entire yeast genome

-such libraries are constructed by digesting the entire yeast DNA partially with a nuclease such as Sau3A (cutting site GATC), which cuts frequently; this strategy generates many overlapping fragments, and it ensures that all genes are functionally represented; Sau3A fragments can be cloned into BamHI (GGATCC) cut plasmid

-if the fragments cloned are 5-9kb on average, 2,000 plasmids represent the genome once and 10,000 plasmids give a more than 90% probability that all genes are functionally represented

-the library is transformed into the yeast mutant of interest

-transformants are screened or selected for restoration of the wt phenotype

-plasmids are prepared from positive clones, transformed into E.coli and further analyzed; some sequence information reveals the identity of the clone

-retransformation into the yeast mutant verifies that the plasmid contains a truly complementing gene; this is necessary because yeast cells can take up more than one kind of plasmid

cloning by complementation

-can only be done with recessive mutants

-cloning of genes with dominant mutants requires a gene library from each mutant and transformed into the wt strain; transformants showing the mutant phenotype are then selected

-complementation of mutations does not mean that the cloned gene is the one that is defective in the mutant, it could be a multi-copy suppressor; this can happen with centromeric vectors because selective pressure can drive up the copy number of plasmids

a multi-copy suppressor

-a gene that overcomes the primary defect in the mutant when expressed at high levels; this is a common phenomenon

-so common a useful approach to clone new genes is starting from a certain mutant

to demonstrate that the cloned gene is the one that is mutated in the mutant

-a deletion mutant has to be constructed by homologous recombination using the cloned gene as template

-if the original and the deletion mutant have the same phenotype, this is good evidence that the 2 genes are the same

-final proof is by crossing 2 mutants; if the diploid has the mutant phenotype too and all spores isolated from the diploid aswell, this is proof that the 2 genes are the same

yeast genes complemented E. coli mutations

-HIS3, TRP1, LEU2, LYS2, ADE2: first eukaryotic clones that could be identified; identified by function

-if have a plasmid clone that really encodes the yeast HIS3 gene, it should complement a his3- yeast strain to his+

-yeast transformation with plasmids was worked out using this strategy to recognize rare transformants

cloning in yeast

-yeast can maintain replicating plasmids but the copy number is much smaller than in E. coli, usually between 1 and 50 per cell

-yeast can maintain more than one type of plasmid at the same time

-cloning and plasmid prep from yeast is very ineffective

-therefore, cloning in yeast uses E. coli as a plasmid production system:

• -plasmids are constructed in vitro

• -plasmids are transformed into E. coli and the constructions are confirmed

• -plasmids are produced in bacteria and then transformed into yeast

-yeast-E. coli shuttle vectors

-yeast has a very efficient and reliable system for homologous recombination, which can be used for cloning

yeast-E. coli shuttle vectors

-integrative plasmids (YIP) consist of the backbone of a E. coli vector such as pBR322, pUC19, pBLUESCRIPT, and consist of a yeast selection marker such as URA3, HIS3, TRP1, LEU2

-lack replication origin for yeast: hence, they are propagated only through the integration into the genome

-not selecting from the plasmid and it never disappears

-has to integrate into the genome to survive

integration of plasmids into the yeast genome

-integration occurs by homologous recombination, this means a plasmid like YIp5 will integrate into the URA3 locus

-integration results in the duplication of the target sequence

-duplicated DNA flanks the vector

-f there is more than one yeast gene on the plasmid, integration can be targeted by linearization within one of the sequences: cut DNA is highly recombinogenic

-integrated plasmids are stably propagated but occasionally pop-out by recombination between the duplicated sequences

deleting a yeast gene

-using the cloned gene the ORF is deleted in vitro and replaced by a marker gene; the marker gene flanked by sequencing originating from the gene that has to be deleted