3Screen vs. SelectionScreen: ALL colonies are looked at, we chose the ones with the right phenotype (e.g. expressing galactosidase in a screen designed to identify mutants in a certain regulatory pathway we pick the blue colonies)Selection: Only the colonies we are interested in will survive (e.g. selecting for transformants)
4Suppression vs. complementation Definition of complementation: The production of a wild-type phenotype by re-introduction of the wild type gene into the mutant (either by plasmid transformation or mating with a strain that carries the wild type copy of the gene).Complementation also by introduction of a functional homologueA suppressor is generally defined as a mutation that completely or partially restores the mutant phenotype of another mutationMulticopy suppression: overexpression of one gene can completely or partially restore the mutant phenotype of another mutation
5Yeast markers Marker definition: A) allele of a gene that allows identification of the strain itselfe.g. Mutations (“marker mutation”) in biosynthetic pathways (in a strain: mutations in ade2,ura3, leu2, trp1….)Example:Yeast strain W1536 5B:MATa; ade2Δ; ade3Δ; leu2-3; his3-11; his3-12; trp1-1; ura3-1 (Rothstein W303 derivative)
6Yeast markersB) Gene on a plasmid/piece of DNA that allows for the identification of a plasmid in the cella gene that confers a certain ability to the strain : ADE2, URA3, LEU2, TRP1, KanMX = geneticin resistance
7Plasmids/VectorsWhat is a Plasmid?What is a Vector?
8Genomic DNA Library Genomic library: Collection of plasmids/vectors carrying pieces of genomic DNA from your organism of choiceGenomic library:Cut into little pieces with restriction enzymesIsolate DNANucleus with DNALigate all different pieces into vectorCollection of plasmids representing the entire genome (or rather a large fraction thereof)cDNA library: containing DNA fragments reverse transcribed from mRNA
93070 fragments/library members required “complexity of the library” Number of fragments of genomic DNA required to be cloned into vector to cover entire genomeln (1-P)N=ln (1-F)N: number of library members requiredP: Probability that gene is in libraryF:Average size of FragmentsSize of genomeExample: yeast about 104 kb; choose P=99%, 15 kb fragment sizeln (1-0.99)~3070 fragments/library members required“complexity of the library”15104Ln ( )
11Finding a nutritional marker (e. g Finding a nutritional marker (e.g. LEU2 involved in leucine biosynthesis)
12Generation of genetic tools: How to find the ingredients to make a yeast vector
13Transform E. coli leuB- mutants with library containing yeast DNA Selectable marker: Cloning of a yeast gene involved in Leucine biosynthesisTransform E. coli leuB- mutants with library containing yeast DNAgene WoriblaLEU2oriblageneXoriblaCells are Leu- (cannot grow on media lacking Leucine)geneYoriblaE. coli cell, with leuB mutationHigh frequency transformation!Select for bacterial transformants on minimal media (lacking Leucine)Cloning by functional complementationIsolate plasmidLEU2oribla
14Transform Plasmid into Leu- strain oriblaLow frequency transformation!Recombination event required!Select for yeast transformants on synthetic media lacking LeucineoriSC-LeublaINEFFICIENT!Integrating Vectors)LEU2leu2-leu2-LEU2blaori
15+ Double Single Recombination event Reversible! (somewhat unstable) orioriblablaDouble SingleRecombination eventLEU2LEU2leu2-leu2-oriblaLEU2Leu2-+blaleu2-oriReversible! (somewhat unstable)“Popout” eventIrreversible!(very stable)LEU2leu2-LEU2Transformation possible but very low frequency/ inefficient
162. Finding an origin of replication (ARS = automomously replicating sequence)
17Cloning of ARS fragment LEU2oriblaYeast genomic DNAleu2-(make yeast genomic DNA library)Transform yeastLow efficiencyPick transformants (keep a stock)SC-LeuGrow non-selectively (e.g. on YPD) for several generations
18Plate cells from non-selective culture on Non-selective plate (e. g Plate cells from non-selective culture on Non-selective plate (e.g. YPD)Replica plate on selective culture (SCD– Leu)SC-LeuSC-LeuSC-LeuSC-LeuLeu+ - marker lost at high frequency -> plasmid- borne (not integrated in genome)! Isolate DNA
19High frequency of transformation! Re-transformLEU2oriblaARSleu2-Transform yeastSC-LeuHigh frequency of transformation!But: Unstable (Lost at high frequency Need centromeric function on plasmid
21Make new yeast library with marker gene and ARS + genomic DNA insert LEU2orileu2-ARSblaTransformYeast genomic DNASC-LeuHigh frequency of transformation(collect large number of colonies)
22 Isolate plasmids, retransform to confirm stability, sequence… Grow non-selectively (e.g. YPD) will enrich for stable transformants ( which have CEN fragment) other cells will lose plasmid if without centromeric sequenceTransformant with stable plasmid: 20% of cellsAfter 3 doublings282828288Transformant with stable plasmid: 50% of cells viable on SC - LeuEnrichment for cells containing stable plasmid isolate after large number of generations growing non-selectively! (Plate cells on SC- Leu) Isolate plasmids, retransform to confirm stability, sequence…
23Yeast PlasmidsCEN based vectors: containing yeast centromeric fragment and yeast ARS, relatively stable (lost~5-10% every generation)centromere is important forProper segregationControl of replication (only once during cell cycle)2 m minicircle – based (naturally occuring plasmid in yeast cir+ cells) multicopy (10-50 copies per cell), relatively stable (lost at about 5-10% per generation)Integrating Vectors (low trafo frequency, but very stable when integrated
26YACs (Yeast Artificial Chromosomes) TEL: The telomere which is located at each chromosome end, protects the linear DNA from degradation by nucleases.CEN: The centromere which is the attachment site for mitotic spindle fibers, "pulls" one copy of each duplicated chromosome into each new daughter cell.ORI: Replication origin sequences which are specific DNA sequences that allow the DNA replication machinery to assemble on the DNA and move at the replication forks.It also contains few other specific sequences like:A and B: selectable markers that allow the easy isolation of yeast cells that have taken up the artificial chromosome.Recognition site for the two restriction enzymes EcoRI and BamHI.
27- While DNA cloning into a plasmid allows the insertion of DNA fragment of about 10,000 nucleotide base pairs, DNA cloning into a YAC allows the insertion of DNA fragments up to 1,000,000 nucleotide base pairs.- Why is it so important to be able to clone such large sequences?To map the entire human genome (3x1,000,000,000 nucleotide base pairs) it would require more than 1,000,000 plasmid clones.In principle, the human genome could be represented in about 10,000 YAC clones.- YACs can be isolated in their full size by pulsed field gel electrophoresis (PFGE)- Techniques for cloning genomic DNA into yeast artificial chromosomes (YAC) make it possible to analyze very long DNA sequences like human genes
28Yeast transformation: Usually done in PEG/DTT/LiAcetate, heat shock (45oC; minutes)Simple one-step procedure (102 – 104 transformants per mg of DNA) (45 minutes)Or procedure involving growth steps and transformation at a specific growth stage ( transformants per mg of DNA) (half a day)
30YIp plasmids were used for gene knockouts/replacements in yeast (2-step gene replacement) FOA (5-fluoro-orotic acid) kills cells that carry a functonal URA3 -gene selection for plasmid “popout”
31+ Double Single Recombination event Irreversible Reversible! oriblaDouble SingleRecombination eventURA3IrreversibleYfg1-1yfg1-1YFG1YFG1yfg1-1YFG1URA3Yfg1-1+Reversible!oriblaURA3YFG1YFG1URA3yfg1-1Transformation possible but very low frequency/ inefficientYFG1
32One-step gene replacement: Uses a marker gene with flanking sequences homologous to the gene of interestCan be generated by PCR:YFG 5’YFG 3’KANMXYFG 5’YFG 3’KANMXoriblaYFGIntegration via homologous recombinationPCR reactionyfg::KANMXYFG 5’YFG 3’KANMXSelect for geneticin resistance
33Isolate yeast DNA from transformants - Verify integration by PCR yfg::KANMXYFG1.1.3 kbProduct 1.7kb2.Product (1.2 kb)No product3.Product (0.8 kb)No productAbout 2-3 weeks to create and confirm a yeast knockout! How much in Mouse?
34Plasmid shuffle: Done when working with essential gene: Phenotype of mutant versions of the gene can be investigated in a deletion background
35Introduction into the Diploid and tetrad dissection/selection of the haploid transformant RFT1CENRFT1URA3pGAL1tCYC1rft1::KANMXura3-sporulate
36Dissect! 4 spores of a tetrad rft1::KANMX ura3- rft1::KANMX ura3- RFT1 CENRFT1URA3pGAL1tCYC1ura3-RFT1CENrft1URA3pGAL1tCYC1Dissect!
37Dissect on YPGalactose!!!! ura3-rft1::KANMXura3-RFT1RFT1ura3-ura3-rft1::KANMXCENRFT1URA3pGAL1tCYC1CENRFT1URA3pGAL1tCYC1oreitherDEAD!CENRFT1URA3pGAL1tCYC1RFT1CENRFT1URA3pGAL1tCYC1rft1::KANMXura3-ura3-
38Select cells that carry the knockout allele plus plasmid A. Select on SC – ura (Galactose)CENRFT1URA3pGAL1tCYC1RFT1CENRFT1URA3pGAL1tCYC1rft1::KANMXura3-ura3-RFT1Can growCan growura3-DEAD!
39B. Select for growth on YPGalactose - geneticin CENRFT1URA3pGAL1tCYC1RFT1CENRFT1URA3pGAL1tCYC1rft1::KANMXura3-ura3-DEAD!Can grow
40DEAD! (cannot lose plasmid) C. Test Growth on SC-Galactose – FOA (5-fluoro-orotic acid)CENRFT1URA3pGAL1tCYC1RFT1CENRFT1URA3pGAL1tCYC1rft1::KANMXura3-ura3-DEAD! (cannot lose plasmid)Can grow (can lose plasmid)
41Perform experiments with isolate CENRFT1URA3pGAL1tCYC1rft1::KANMXura3-Grow on Raffinose – switch to glucose repression of RFT1 phenotype of Rft1p depletion can be studiedGrow on Raffinose – switch to galactose activation of RFT1 phenotype of Rft1p overexpression can be studied
42Reporter genesReporter genes are heterologous genes that confer and easily detectable phenotype to the object of studyReporter genes are often used in the analysis of gene expressionexamples:b- galactosidase (E. coli lacZ gene):can be assayed quantitatively (cleavage of ONPG)cleaves X-gal to produce blue colour (visual assay)>TranscriptionPromoter of YFGReporter Gene (e.g lacZ)Use: for example identify genes involved in repression by glucose; fuse promoter of glucose-repressed gene to b- galactosidase; mutagenize screen for blue colonies on glucose = yeast cells mutant for glucose repression
43Reporter genes (continued) examples:HIS3 (involved in Histidine biosythesis) can be competitively inhibited by 3-amino-triazole can be used in selection for viability on media lacking histidine; especially used to detect strong activating activity of transcription factors (e.g. in two-hybrid screens)CAT (chloramphenicol acetyl transferase) Transfers radioactive acetyl groups to chloramphenicol; detection by thin layer chromatography and autoradiography