Welcome to L319: Genetics Laboratory

Yeast: S. cerevisiae Fruit Fly: D. melanogaster Bacteria: E. coli Welcome to L319: Genetics Laboratory Instructor: Dr. Amy Berndtson Assistant Instructors: Adrienne Evans (Tuesday), Ke Xu (Thursday)

Yeast: S. cerevisiae Fruit Fly: D. melanogaster Bacteria: E. coli Course Specifics: Purpose of the course: Reinforce genetic principles through genetic research Independent of L311: Topics covered in L319 are not coordinated with L311 Parts of the Course: Lecture: Theoretical basis for experiments Laboratory: Experiments will overlap – need to keep track of syllabus Experiments: Designed for you to solve genetic problems - puzzles

Summary of Grading

Flowsheets: Read the Introduction handout for specific details (pp 4-5) • Cover Introduction and Material and Methods for a typical lab report • 2. Help you understand the “what and why” of an experiment • 3. Introductions are only due once for each experiment • Procedure and Rational are due each week of the lab except final data collections (see syllabus for detail) • 5. Flow sheets are due at the beginning of each class • Make copies of your flowsheets to take notes on during class • Concise and effective writing is rewarded! • Plagiarism is not tolerated

Introduction Procedure/ Rationale

Lab Reports: Read the Introduction handout for specific details (pp 5-7) • Cover the Results, Discussion and Appendix of a typical lab report • Detailed instructions for data analysis and report components will be posted on our class web site for each lab exercise • Reports take effort – you may need to ask us questions • Don’t procrastinate until the last minute! • Concise and effective writing is rewarded! • Plagiarism is not tolerated

Exams: Read the Introduction handout for specific details (pp 7-8) • Based on lab report analysis – problem solving • 2. Will post copies of old exams • Lab Participation: Read the Introduction handout for specific details (p 8) • Based on class participation, effort and attitude

To do well in this course: Attendance: Attend lecture and lab (require) Flowsheets:Follow the weekly guidelines and turn flowsheets in on time Reports: Start them early and seek help if you have questions Exams: Make sure you understand the data analysis in each report Lab Performance: Have a good attitude and participate

To do poorly in this class: Attendance: Don’t attend lecture or lab (2 non-excused absence = F for course) Flowsheets:Don’t follow the guidelines or fail to turn flowsheets in on time Reports: Procrastinate so that you don’t have time to ask questions Exams:Don’t understand the data analysis in each lab Lab Performance: Have a bad attitude and don’t participate

Experiment 1A: Mutant Search in Yeast • I. Genetics: Variation • A. Wild-type alleles • B. Mutant alleles • Source of Genetic Mutation • A. Point mutations • B. Frameshift mutations • C. Deletions • D. Insertions • Spontaneously: occur naturally at a relatively low frequency • A. Error in DNA replication or repair • B. Deamination of the 5’-methylcytosine • Induced: cause mutation to occur at a higher frequency • A. Chemical carcinogens • B. UV • C. X-rays • In this experiment we will use a chemical mutagen to induce mutation in S. cerevisiae

Ethylmethane Sulfonate: alkylating agent What kind of mutation is this? Point mutation – transition G:A

Position of mutation within a codon: Neutral Acidic Basic How could a missense mutation affect a protein? Could alter protein folding – protein function

Position of mutation within a codon: How could a nonsense mutation affect a protein? Truncates the protein – usually becomes inactive

Experiment 1A: Use EMS to induce point mutations • Silent (will not detect phenotypically) • Missense • Nonsense mutations • Experiment 2A: Use a strain of E. coli that has a high rate of spontaneous mutations • 1. Point mutations • a. Silent (will not detect phenotypically) • b. Missense • c. Nonsense • 2. Framshift • Major effect on protein function if occurs near the 5’ end of the coding sequence • Less effect on protein function if occurs near the 3’ end of a coding sequence or within an intron • 3. Deletion • a. Major effect on protein function if it occurs within the 5’ end of the coding sequence • b. Less of an effect on protein function if it occurs within the 3’ end of the coding sequence • 4. Insertion • a. Usually inactivates a protein when it inserts into both exons and introns • Non-revertible mutations

Life Cycle of Yeast: exist as both haploids and diploids Mating types We will start here Why is it easier to find mutants? Haploid: no other copy of an allele to mask a recessive trait

Facts about S. cerevisiae: • Studied for over 50 years • Eukaryotic Model System • a. Nucleus • b. Organelles • c. Exists as both haploids and diploids • d. Mitosis and Meiosis • e. 1N = 16 chromosomes • f. 1st genome sequenced (12.5 mega bases) • g. Many genes are mapped • 3. Isolation of mutants helped map many of the genes

Genetic Research Needs Mutants 1. Interested in a biological process Histidine biosynthesis in yeast 2. Need mutants that have phenotypes related to the process Histidine auxotroph 3. Need to isolate a large number of mutants because the biological process is most likely controlled by more than one gene 7 genes are involved with histidine biosynthesis in yeast

Histidine Biosynthesis in Yeast 4B 4A 1 6 7 4C 2 5 3

Genetic Research Needs Mutants 1. Interested in a biological process Histidine biosynthesis in yeast 2. Need mutants that have phenotypes related to the process Histidine auxotroph 3. Need to isolate a large number of mutants because the biological process is most likely controlled by more than one gene 7 genes are involved with histidine biosynthesis in yeast 4. Need an observable phenotype to isolate the mutants Histidine auxotrophs will not grow in media without histidine 5. Methods of isolating the mutant cells from wild-type cells Isolate histidine auotrophs from histidine prototrophs

What is a histidine auxotroph? Organism (yeast cell) that cannot synthesize histidine – to survive histidine must be provided exogenously What is a histidine prototroph? Organism (yeast cell) that can synthesize histidine – does not need exogenous histidine to survive

What amino acid were they unable to synthesize? How did they obtain this amino acid? In the book Jurassic Park, the dinosaurs were auxotrophs by design Lysine Through plants

Can’t grow Can’t grow Identifying Histidine Auxotrophs with a Genetic Screen Isolate his- mutants

Strategy to isolate histidine auxotrophs from the S. cerevisiae stain TD28 Spontaneous Frequency of mutation (10-7) Frequency of mutation (10-5 100 fold increase)

Spontaneous EMS - Induced Frequency of mutation (10-7) Frequency of mutation (10-5)) When isolating mutants through a genetic screen you want: 100 cell/plate How many plates would be required to isolate 1 mutant from the Unmutagenized culture? 105 plates How many plates would be required to isolate 1 mutant from the Mutagenized culture? 103 plates This is way too many plates to work with – impossible – too expensive

Key Steps TD28 is auxotrophic for Urea and Inositol Kill 99% Cells Kill 90% Cells Why? Decrease the number of histidine prototroph (His+) cells

Welcome to L319: Genetics Laboratory