Biology 141 - Week 9 slides - Fall 2011 - GM

Biology 141 - Week 9 slides - Fall 2011 - GM - Yeast:...

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Unformatted text preview: Yeast: Replica-plating and complementation L. Mclane and A. Escobar Review of definitions Chromosomes - Inherited genetic material - Prokaryotes vs. Eukaryotes Genes - Smallest unit of heritable material - Blueprint of a protein or functional RNA molecule Alleles - Alternative forms of genes L. Mclane and A. Escobar Complementation: the basics Cell X Haploid (n) Normal gene A X Cell Y Haploid (n) Mutant gene a Can Cell X survive after it has been mated with Cell Y? Why or why not? L. Mclane and A. Escobar Complementation in yeast Gene Z Gene Q Enzyme Z Enzyme Q A B a Z+Q- x C Metabolic Intermediates α Z-Q+ Z+QZ-Q+ L. Mclane and A. Escobar Gene Z a or α A Z+Q+ Gene Q Enzyme Z Enzyme Q B Gene Z a Z+Q­ mutant Gene q X Enzyme Z A Enzyme Q B C mutant Gene z Z­Q+ A Enzyme Z B Gene Z Z+Q­ Z­Q+ A Enzyme Z Metabolic Intermediates Gene Q X α C Metabolic Intermediates Enzyme Q C Metabolic Intermediates Gene Q B Enzyme Q C Metabolic Intermediates L. Mclane and A. Escobar Useful Auxotrophic Markers Some strains are prototrophic (wild type) Many laboratory strains are auxotrophic for one nutrient or another dependent on another source for a nutrient URA3 LEU2 HIS3 TRP1 ADE2 ADE3 LYS2 others L. Mclane and A. Escobar Genetic complementation of auxotrophic markers a x TRP1 Will grow without TRP supplemented, so it is Trp+ Will not grow without TRP supplemented, so it is Trp- α trp1 TRP1 trp1 L. Mclane and A. Escobar L. Mclane and A. Escobar L. Mclane and A. Escobar Canavanine: A special case Canavanine is a non-proteinogenic amino acid of certain leguminous plants. Canavanine is accumulated primarily in the seeds. Organisms that consume it can mistakenly incorporate it into their own proteins in the place of arginine, thereby producing structurally aberrant proteins that may not function properly. Canavanine was first isolated from Canavalia Ensiformis (jack bean). L. Mclane and A. Escobar Canavanine: A special case CAN1 encodes for arginine permease (transporter protein) Transports ARG and a toxic analog, canavanine (CAN) CAN competes with ARG during translation to be incorporated into proteins and makes them non- functional CAN1 cells die on plates containing CAN (canavanine), because functional CAN1 (arginine permease) brings the toxin into the cell, termed Can-Sensitive (CanS) can1 cells can grow on plates containing CAN (canavanine), because the transporter (arginine permease) does not work – no toxin enters the cell, termed Can-Resistant (CanR) IT’S GOOD TO BE A can1 MUTANT! L. Mclane and A. Escobar Wild-type yeast SD-complete arginine permease arginine Functional proteins that incorporated arginine Wild-type yeast SD-can arginine permease arginine canavanine Non-functional proteins that incorporated canavanine can1 yeast SD-can arginine permease arginine canavanine Functional proteins that incorporated arginine Plates SD: synthetic dextrose plate SD-Complete: synthetic complete plate YPD (to make a new master) unmarked (brownish in color) SD-complete = black/black YPD: Yeast Peptone Dextrose plates SD-ade = black/red SD-ade/his/leu: SD-complete plates minus the said amino acid SD-leu = green/blue SD-his = purple/green CAN = orange/green/green Two SD = red – PUT THESE ASIDE UNTIL THE END OF THE LAB YPD containing a lawn of a tester of genotype MATa trp1 YPD and SD-complete are equivalent – the SD-complete is just made from synthetic as opposed to natural materials YPD containing a lawn of a tester of genotype MATα trp1 L. Mclane and A. Escobar NEW YPD with 14 Unknowns MASTER PLATE MASTER PLATE SD Complete Velvet #2 6. SD-CAN abel the bottom of each late with full names, oom #, lab day, and late type late orientation mark on the OTTOM of the master, nd then on the new lates. REPLICA PLATE using he mark as a guide. n the BOTTOM of the master, umber the colonies 1-14. 4. SD-HIS SD-LEU YPD with MATa/trp1 2. 3. SD-ADE Velvet #1 1. 5. Remember, SD-CAN is an SD complete plate WITH CAN! 7. SD YPD with MATα/trp1 8. Tomorrow SD Plate #7 and #8 on SD after growth overnight Replica-Plating Purpose: To determine the auxotrophic and mating types of 14 unknown strains Labeling of plates: - What type of plate is it? - Orientation marker - Initials/Lab Day/Lab Room Orientation mark over Patch #2 should be at the TOP L. Mclane and A. Escobar The day after your lab, 2 people from each group need to come into lab to do the following: 1. Find your YPD with MATa that you mated to the MASTER. 2. Find your YPD with MATα that you mated to the MASTER. 3. Replica plate these plates each onto an SD plate. 4. Use a different velvet for each plating. Make sure your SD plates are LABELED as MATa or MATα, and with all other appropriate information (room, day, initials) 5. VELVET #1 YPD with MATa/trp1 SD VELVET #2 YPD with MATα/trp1 SD L. Mclane and A. Escobar Hypothesis • Prediction • Independent variable • • Dependent variable • Controls L. Mclane and A. Escobar YOU MUST COME TO LAB TOMORROW!!! Read the protocol instructions – you need to: Find your mated plates Replica plate them onto SD plates Make sure you LABEL so you can differentiate between your plates! Come to the lab between 9 am-1 pm. Two group members from each group should come so that you can double check your work! L. Mclane and A. Escobar Due Next Week • • Study for Quiz #5 Prelab of Week #10 protocol ...
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This note was uploaded on 10/25/2011 for the course BIO 141 taught by Professor Dr.cafferty during the Fall '11 term at Emory.

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