Exam 4

Question 1

Complementation test

Answer 1

If the genes are on the same gene, then it fails to compliment (negative).

If the genes are on different genes, then it will compliment (positive).

Question 2

Define Amorph

Answer 2

Complete loss of function mutants

Recessive

Question 3

To study genetic interactions, you need to make sure that…

Answer 3

You are looking at two different genes. Via complementation tests

Question 4

Define Hypomorph

Answer 4

Gene product still works, but not at full capacity. 10% working. A decrease in gene dose with less phenotype.

Recessive

Question 5

Define Hypermorph

Answer 5

An increase in gene dose that causes a greater effect in phenotype

gain of function- dominant

Question 6

Define Antimorph

Answer 6

Type of mutant that opposes the normal function of a gene. Could be a dominant negative mutation

Dominant

Question 7

Define Neomorph

Answer 7

A mutant gene that has a different function than a wildtype gene. New product with different functions

gain of new function- dominant

Question 8

Define haploinsufficiency

Answer 8

Wildtype allele is recessive, and the genes normal function is impaired so that the remainder of the gene is unable to produce enough gene product.

-dominant

Question 9

Define epistasis

Answer 9

is a circumstance where the expression of one gene is modified (e.g., masked, inhibited or suppressed by the expression of one or more other genes.

EX: White eyes are epistatic (masks) to other pigment mutants.

Question 10

What is an epistatic gene?

Answer 10

The gene who’s phenotype is displayed

Question 11

What is a hypostatic gene?

Answer 11

A gene that is masked/ covered by the epistatic gene

Question 12

What is the main question linear epistasis is trying to answer?

Answer 12

Do two genes act in the same or different pathways?

Question 13

What is the main question ordering epistasis is trying to answer?

Answer 13

If the genes are in the same pathway, then which gene is upstream vs. downstream in the pathway?

Question 14

What are the assumptions needed for a linear epistasis test?

Answer 14

1. Complete loss of function mutants (null/ amorph)
2. Mutants have the same/ similar phenotypes (some exceptions)

Question 15

How do you interpret results of a linear epistasis test?

Answer 15

If the resulting phenotype is a combination effect= genes are in DIFEERENT pathways

If the resulting phenotype shows a REGULAR phenotype with not combination effect= genes are in the SAME pathways

Question 16

How do you conduct a linear and ordering epistasis test?

Answer 16

Generate double mutants (null) and observe phenotype, compare this to single mutant phenotypes.

-If you cant identify an epistatic gene than one of the assumptions is not true

Question 17

What are the requirements of an ordering epistasis test?

Answer 17

1. Genes must be in a linear pathway (one gene either positively or negatively regulates the other)
2. Genes in the pathway are on/off (no intermediate states)
3. Mutants must have distinguishable (different) phenotypes
4. What is the regulation? Positive or negative?

Question 18

What are the interpretations of ordering epistasis tests?

1. Negative regulation
2. Positive regulation
3. Constitutively active

Answer 18

1. If it is negatively regulated then the epistatic gene will be downstream!
2. If it is positively regulated then the epistatic gene will be upstream !- substrate dependent pathways
3. If constitutively active mutant involved, epistatic gene is downstream

Question 19

What should you keep in mind when drawing a pathway?

Answer 19

The pathway should represent the wildtype functions of the genes.

Keep in mind the positive/ negative relationships. Will it inhibit or activate?

Question 20

How would you approach a substrate dependent pathway epistasis?

Answer 20

Treat as a positive regulator.
The epistatic enzyme will be upstream of the pathway.

Question 21

Define synthetic enhancement

Answer 21

The double mutant phenotype is more sever than a single mutant

Question 22

Define synthetic suppression

Answer 22

The double mutant phenotype is less severe than a single mutant - more like wild type

Question 23

What do synthetic enhancement and suppression phenotypes indicate?

Why is this?

Answer 23

That a genetic interaction is happening but not what the interaction is

Mutations in genes involve hypomorphs

Question 24

What is the goal of GWAS (Genome wide association studies)?

Answer 24

To identify genetic loci that contribute to disease phenotypes in humans

Relies on differences in SNPs, where an associated SNP marks
a region of genome that is associated with disease

Question 25

What is the methodology of GWAS?

Answer 25

recruit large population, including people w/ and w/o the condition of interest and utilize SNP array

Question 26

What are the challenges in GWAS studies? How do you get over them?

Answer 26

1. Sample size (particularly if disease being studied is rare) 2. Statistical tests - high false positive rate 3. Many SNPs to sort through (so can be hard to sort through what’s signal and what’s noise) 4. By definition, what you’re looking at is many modifiers that can be acting at once, so this is in general just hard Helps to have good phenotyping to distinguish different allelic combinations, modifiers, associated with disease - SNP’s for specific, narrow phenotypes (e.g., loss of lung function vs. every possible symptom assoc. w/ disease) - quantitative measurements (e.g., blood pressure)

Question 27

How does GWAS work?

Answer 27

compare SNP occurrence in affected vs. unaffected population SNPs that are significantly associated with diseased population (above dotted line) are promising candidates for further analysis this narrows down where the gene is, not the mutation

Question 28

What is the difference between forward and reverse genetics?

Answer 28

Forward: Start with a phenotype and then identify genes/mutants Reverse: Start with a gene, and then look for a phenotype

Question 29

Define a knockout mutant

Answer 29

the gene is no longer expressed in an organism

Question 30

Define a knockdown mutant

Answer 30

Expression of the gene is reduced

Question 31

Define a knock-in mutant

Answer 31

a mutant (usually point mutant or expressed truncation) is targeted to the endogenous locus, replacing the wild-type gene -replacing function

Question 32

Define a transgenic mutant.

Answer 32

a gene (often a mutant) is introduced to the genome at an exogenous location (the endogenous genes are usually still present). -adding more function, random location

Question 33

How would you create a knockout mutant by HR?

Answer 33

An integration cassette with homology to genomic sequences on either side of the gene is inserted into cells. Also contains a selective factor (antibiotic resistance) In some cells, crossing over between the integration cassette and the genomic sequence will replace the gene with the cassette sequence (2 cross over events needed)

Question 34

How do you create a knock-DOWN mutant with RNAi?

Answer 34

Create dsRNA for the given mRNA. 1. Dicer dices the dsRNA into small interfering RNA siRNA 2. siRNA is incorporated into RISC 3. Recognized target mRNA- induced cleavage used to downregulate translation. this results in knockdown not knock out

Question 35

What is a morpholino?

Answer 35

A method to knockdown a gene Morpholine rings instead of ribose that block translation

Question 36

How would you create a mutant gene with site directed mutagenesis?

Answer 36

1. Start with wild-type gene on plasmid 2. PCR with mutagenic primers, making copies of whole (mutant) plasmid 3. Degrade parental wild-type plasmid with enzyme 4. Amplify in bacteria and purify results in plasmid with mutant gene

Question 37

How could using a mutant plasmid result in a knock in mutant?

Answer 37

1. Can introduce into WT cells as is, or cells where the gene is deleted 2. Can replace the endogenous gene in the genome (same mechanism as integration cassettes)

Question 38

What is CRISPR? What does it require?

Answer 38

RNA guided site specific DNA cleavage Requires PAM sequences

Question 39

What does CRISPR result in?

Answer 39

1. Non homologous end joining that introduces random mutations that can then be screened. Knockout method 2. Homology directed repair that can add DNA, knock in

Question 40

What are some challenges with CRISPR?

Answer 40

Off target effects are unknown on target effects where it effects the region around the cut site

Question 41

What is promoter/enhancer mapping?

Answer 41

If the region you stuck (random i guess) in front of a reporter gene (GFP) is an enhancer (for gene in a plasmid), then the reporter expression will mimic expression patterns of the gene itself. This doesn't mean that the entire sequence stuck in is the promoter, so you need to slowly chip away the sequence and see if the expression patterns remain the same or not.

Question 42

What is a transcriptional/promoter fusion? What is it used for?

Answer 42

If you want to determine where, when, or conditions that a gene is being transcribed Does not tell you where the protein localizes

Question 43

What should be included in a transcriptional fusion construct?

Answer 43

1. Genes regulatory sequences (promoter, enhancers, poly a signal) 2. Reporter gene 3. GOI is NOT included

Question 44

What is a translational fusion? What is it used for?

Answer 44

If you want to examine where in the cell (subcellular localization) a protein is found. can see under microscope used for protein purification MIGHT tell where in the organism it is expressed

Question 45

What do you need in a translational fuson construct?

Answer 45

1. Regulatory elements (constitutively active promoter from another organism, poly a signal) 2. reporter integrated with the GOI

Question 46

How would you identify protein protein interactions via affinity tagging/pull down?

Answer 46

1. Make translational fusion of GOI with an AFFINITY TAG - the tag is made to bind to a specific substrate in a column 2. Incubate cell extract with specific substrate 3. Target protein is pulled down out of extract (and proteins that is also associates with come with it) 4. Identify associated proteins with SDS page, western blot, mass spectrometry -You can adjust conditions to get tight associations or loose ones

Question 47

What are the biotechnology design principals?

Answer 47

1. Where (what organism) is it being expressed? 2. Where (what organism) is the sequence coming from? 3. Does the coding region require modifications?(Tags) (only needed if you want to purify a protein) 4. What regulatory elements are needed? 5. How is the gene construct being delivered?