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CDB > Yeast Genetics > Flashcards

Yeast Genetics Flashcards

1
Q

What makes Yeast an ideal model organism?

A
  • Strong genetic and coding gene homology to humans and eukaryotes
  • Useful for studying cell cycle
  • Unicellular, few cell types, cheap, smaller genome and fast to grow
  • Haploid and diploid lifecycle allows cross over, recombination and haploid experimentation
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2
Q

What are the core steps in forward genetics?

A
  • Recessive or dominant
  • How many mutations per cell for phenotype
  • How many unique genes linked to phenotype
  • Metabolic/genetic heirarchy
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3
Q

What are the core steps in reverse genetics?

A
  • Fluoresce protein
  • Knock out or down
  • Overexpress
  • Chip-seq
  • Protein-protein interactions
  • Crossing mutants
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4
Q

How do you study essential genes?

A
  • Conditional mutants: temperature sensitive
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5
Q

What are the two species of yeast used for?

A
  • S. cerevisiae (budding): tetrad analysis, forward genetics,
  • S. pombe(fission): similar to higher eukaryotes, RNAi, centromeric structure, mitosis regulation
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6
Q

What are complementation tests?

A
  • Test whether two mutants are in the same gene
  • cross two recessive mutants
  • If cross still KOs same gene in all offspring then they are same gene
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7
Q

What does a cdc2 complementation screen show?

A
  • Recessive mutants show cdc phenotype, failure to divide leads to elongation
  • Dominant mutants show wee phenotype, divide at smaller size
  • When inactivated cannot enter mitosis but dominant mutant disrupts regulation instead
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8
Q

What are the stages of S. c meiosis called?

A
  • Haploid spores: Ascus

- Haploid cells: Tetrads

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9
Q

What is a tetrad dissection?

A
  • As each division creates a spore they are distinguishable form other divisions
  • Seperated by micromanipulator, needle and microscope, and digestive enzyme
  • Moved to defined place to investigate phenotype ratios
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10
Q

What ratios will arise from tetrad analysis of unlinked genes?

A
  • 1:1 parental ditype and non-parental ditype

- Each combination equally likely in random combination

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11
Q

What ratios arise from tetrad analysis of linked genes?

A
  • Linked means they are on same chromosome
  • If very tightly linked only PDs will be present
  • If recombination is allowed the ratio will be 1:1:4 and the distance is derived from the equation
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12
Q

How can double mutants be utilized?

A
  • Tell you how and in which order mutants interact with eachother
  • Generated via tetrad analysis, take the NPDs
  • Two genes in same pathway double mutant phenotype similar to upstream gene
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13
Q

How are nutrient markers used?

A
  • Some mutants prevent yeast from producing necessary compounds, auxotrophic
  • Can screen via selection(survive) and counterselection(cannot grow) media
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14
Q

How are genetic nutrient markers to used to isolate diploid cells?

A
  • Complementation using recessive auxotrophic mutations which together grow in SD
  • Using dominant markers to select ones that can grow in two drugs
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15
Q

How do yeast vectors work?

A
  • Yeast plasmids can propagate and have markers in both yeast and E. coli
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16
Q

How many yeast plasmids are lost per divide?

A

1-10%

17
Q

How are ts yeast vectors made for cloning?

A
  • Create library with thousands of plasmids with different fragments of genomes and markers
  • Plate ts mutant with library
  • At low temperature they should all grow
  • At high temperature only ones with WT gene will grow
18
Q

How does targeted deletion of yeast genes work?

A
  • Only works with dominant genes as wt copy remains
  • Yeast recombination machinery makes use of flanking homology
  • Use tetrad analysis to test success 2:2 ratio
19
Q

How do epitope markers work?

A
  • Specific antibodies hard to make
  • So attach epitope with known antibody to gene end to mark protein
  • GFP is a common example
20
Q

What is a regulateable promoter(for experiments)?

A
  • Normal promoter replaced by regulateable which can be regulated by addition of transcription factor
21
Q

What is a heat inducible degron?

A
  • Tagged with degron which targets protein for degredation

- Only acts as degron at high temperature

22
Q

What are suppressors?

A
  • A further change in genome (plasmid, mutantion) that counteracts phenotype of mutant
23
Q

What are intragenic suppressors?

A
  • Mutations in same gene that counteract mutant phenotype

- Means that supressor and mutation are highly linked

24
Q

What are some types of information maps that can been collected in yeast functional genomics?

A
  • genome wide seq such as ChIP, RNA-seq, ATAC
  • Deletion mutants for phenotypes and genetics interactions
  • Epitope tagging for protein-protein interactions
  • GFP for protein localization
25
Q

What can deletion libraries be used for?

A
  • Either individual analysis or competitive growth
26
Q

What are the possible differences in phenotype between single and double mutants?

A
  • Positive interactions: double mutant weaker
    >Mutation to both regulator and transcription factor stops toxin production
  • Negative interactions: phenotype gets worse
    >Redundancy: single mutant no effect due to redundancy
    >Hypomorphic mutation essential pathway: Single mutant weak phenotype, double essential
  • Null: no difference
27
Q

What is a synthetic gene array?

A
  • Make double mutant between gene of interest and all other mutants in library
  • Slow and labour intensive
28
Q

What are HIP, HOP and HIPHOP?

A
  • HIP: Assumes single copy of gene makes hypersensitive to drug
  • HOP: Assumes genes that regulate target will be sensitive to drug
  • HIPHOP: Looks at drugs and finds both direct and indirect targets of drugs
29
Q

How can yeast be used to study human mutants?

A
  • In cases lacking homology you can just insert human diseased gene in yeast to see what happens
30
Q

What is chronological life span (CLS) concept in yeast?

A
  • How long cell survives in non replicative environment

- Assessed by colony formation capacity over time

31
Q

Why are daughter cells younger than mother cells?

A
  • Ageing factors segregate to mother
  • ERCs: extra chromosomal rRNA circles
  • Defective mitochondria
  • Aggregated proteins
  • Enlarged highly acidic vacuoles
32
Q

What are some drugs produced synthetically in yeast?

A
  • Artemisinin antimalaria drug has a complex pathway that had to be reproduced
  • Morphine
33
Q

What are some examples of long ncRNAs in yeast?

A
  • Stable Unannortated Transcripts (SUTs): structurally similar to mRNAs
  • Cryptic Unstable Transcripts: (CUTs): expressed at low levels, overlap with coding sequences

CDB (13 decks)

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