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Welcome to L319: Genetics Laboratory PowerPoint PPT Presentation


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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.

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Welcome to L319: Genetics Laboratory

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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)


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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


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Summary of Grading


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  • 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


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Introduction

Procedure/

Rationale


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  • 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


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  • 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


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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


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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


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  • 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


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Ethylmethane Sulfonate: alkylating agent

What kind of mutation is this?

Point mutation – transition G:A


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Position of mutation within a codon:

Neutral

Acidic

Basic

How could a missense mutation affect a protein?

Could alter protein folding – protein function


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Position of mutation within a codon:

How could a nonsense mutation affect a protein?

Truncates the protein – usually becomes inactive


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  • 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


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    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


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    • 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


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    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


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    Histidine Biosynthesis in Yeast

    4B

    4A

    1

    6

    7

    4C

    2

    5

    3


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    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


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    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


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    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


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    Can’t grow

    Can’t grow

    Identifying Histidine Auxotrophs with a Genetic Screen

    Isolate his- mutants


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    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)


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    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


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    Key Steps

    TD28 is auxotrophic

    for Urea and Inositol

    Kill 99%

    Cells

    Kill 90%

    Cells

    Why?

    Decrease the number of histidine prototroph

    (His+) cells


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