Principles of Genetics Flashcards
1
Q
In a mutant experiment, adding wild type copies rescues the phenotype, the underlying mutation is _____.
A
Loss of function
2
Q
In a mutant experiment, adding wild type copies makes the phenotype worse, the underlying mutation is _____.
A
Gain of function
3
Q
Recessive mutations are when the mutation is ______ with itself. Dominant mutations are when the mutation is present as a _______.
A
Homozygous
Heterozygote
4
Q
Recessive mutations yield which classes of mutations?
How about dominant?
A
Amorphs (null allele) and Hypomorphs (less gene function or less gene product than WT)
Hypermorphs, atimorphs, and neomorphs (more gene product or function that WT)
5
Q
Amorphs have \_\_\_\_\_ function Hypomorphs have \_\_\_\_ function Hypermorphs have \_\_\_\_\_ function Antimorphs have \_\_\_\_ function and act \_\_\_\_\_\_\_ Neomorphs have \_\_\_\_ function
A
No Partial Increased Dominant negative, against the WT allele Novel
6
Q
Under what conditions can recessive mutations act in a dominant fashion?
A
Through haploinsufficiency
7
Q
If the heterozygote and the WT have the same phenotype, the mutation must be _____.
A
Recessive
8
Q
Conditional alleles are usually _____ mutations. And are conditional on __________.
The normal condition is called _____ and the mutation condition is called _____. At an intermediate point between these 2 conditions, the allele may function as a _______.
A
Missence
Changes in environmental factors (i.e. temp)
Permissive. Restrictive.
Hypomorph.
9
Q
What is the difference between reverse and forward genetics?
A
Reverse = gene of interest to examine phenotype
Forward = phenotype to elucidate gene responsible by introducing random mutations throughout the genome.
10
Q
How would you perform a reverse genetic experiment? Forward?
A
Reverse = gene knockout or mutation
Forward = introduce random mutations
11
Q
Why does an increased concentration of mutagen lead to decreased cell survival?
A
Increased chances of mutating an essential gene
12
Q
Using comparisons, how could you tell that a mutation is recessive?
A
mut/del = mut/mut < mut/WT = WT/WT = mut/WT;WTdup
13
Q
Higher levels of mutagen risk _____ but low levels mean _____.
A
Mutating an essential gene.
More work, more organisms to screen
14
Q
During mutagenesis, what kinds of genes can you miss? (3)
A
- Essential genes
- Short genes
- Genes with redundant function
15
Q
It is always possible that you have more than one mutation in a single gene, therefore the first thing you must do is __________.
A
Backcross your mutant - mate your mutant to a WT, choose offspring that have phenotype of interest, and mating to another WT.
16
Q
What are signs that a screen is not saturated yet?
A
- No normal distribution
2. Many genes with 1 or 2 mutations
17
Q
What is the difference between a screen and selection?
A
Screen = identification of a mutant organism manually (only WT cells survive) - will have negatives and positives
Selection = impose growth conditions so that only mutant cells with phenotype of interest survive - will only have positives
18
Q
Selections are preferred to screens when ______.
A
The phenotype of interst is known
19
Q
What is the mating type of yeast haploids? Diploids?
A
Haploids: A or alpha
Diploids: A/alpha
20
Q
What is the process by which diploid yeast become haploid?
A
Sporulation - undergoes meiosis to produce 4 haploid cells
21
Q
What is the difference between an auxotrphic and antibiotic marker?
A
Auxotrphic marker = when an organism cannot synthesize all essential metabolites, the marker provides the gene it lacks
Antibiotic resistance marker = provides resistance to antibiotics
22
Q
The selectable marker is usually positioned on a construct so that it is _______ the gene of interest
A
Linked to
23
Q
Which yeast ploidy is used for transformation? Why?
A
Diploid.
Because off target recombination occurs more frequently in a haploid transformation
24
Q
What comonent facilitates transformation into yeast cells
A
Homology arms
25
If a cell has a URA selectable marker linked to the transformation construct, how would you know that the transformation was successful?
Plate on -URA plates and the colonies that survived have taken up the construct
26
After sporulation, the 4 products are contained within the ____ and are referred to as a _____.
Ascus membrane
Tetrad
27
How can you force sporulation?
Under nutrient poor conditions
28
In a tetrad that has been replica plated on a selective plate, how many colonies do you expect to see?
2 (there are 2 transformed haploids and 2 WT haploids)
29
Complementation tests require ______ mutants
Recessive
30
When mutations are on the same gene, they ____ in a complementation assay. When they are on different genes, they ____.
Do not complement.
Complement
31
When mutants are "in the same complementation group" this means that they are on same/different genes and do/do not complement?
Same
| Do not
32
What are the possible reasons that the same gene might complement each other? (3)
1. Different domains
2. Reduced dosage
3. Stabilization of products (Suppression)
33
What are possible reasons that mutations on different genes might not complement each other ? (2)
1. Haploinsufficiency
| 2. Dominant negative
34
What are the 2 classes of complementation assay exceptions?
1. Intragenic complementation
| 2. Non-allelic non complementation
35
What is "selective power?"
Immediately isolating mutant cells with the phenotype of interest/death
36
What are the pros and cons of selections and screens?
Selections:
- powerful, faster
- can only use with certain phenotypes
- need to be sure what your phenotype of interest is
Screens:
- unbiased (dont need to know the exact phenotype of interest)
- requires more time and resources
- allows recovery of more genes
37
What are the 3 subclasses of intragenic complementation? What are the 2 subclasses of non-allelic non-complementation?
1. Different domains
2. Reduced dosage
3. Stabilization of products (suppression)
1. Dosage (haploinsufficiency) = threshold
2. Poison = dominant negative
38
How can you trigger yeast meiosis and sporulation?
Starve them
39
What does it mean to be a prototroph?
Can synthesize everything you need in order to survive. Opposite of auxotroph.
40
What does minimal media for yeast contain? How about YPD?
Salts, minerals, glucose.
Yeast extract, peptone, dextrose
41
What percentage of yeast genes are essential?
30%
42
When performing point mutations in yeast, should you use haploid or diploid yeast?
Haploid.
43
Is there any way to recover an essential gene in a complementation test?
Yes, only if it is a conditional allele (a partial LoF)
44
Yeast spores will contain which mating types?
2 a
2 alpha
45
Complementation analysis is a test of _______ because the interpretation of the results is based on ______.
Function
Phenotype.
46
If complementation tests function, what tests position?
Analysis of recombination during meiosis using linkage analysis
47
During recombination, what 3 segregation patterns can be seen as a result?
1. 2 recombinants, 2 parental (1 recombination event, tetratype)
2. 4 recombinants (2 recombination events - non-parental ditype)
3. 4 parental (no recombination events, parental ditype)
48
What is the tetrad segregation pattern with 2 linked genes?
Same gene OR close genes on the same chromosome = recombo unlikely = linked
PD > T > NPD
49
What is the tetrad segregation pattern with 2 unlinked genes?
Different genes on the same OR different chromosomes = recombo is likely = unlinked
PD = NPD > T
50
What yields an intermediate tetrad segregation pattern?
Genes that are unlinked, and on the same chromosome
51
What 2 assumptions should you make when assessing tetrad unlinkage?
- Genes are on different chromosomes
| - No recombo between gene and centromere
52
If 2 genes are unlinked, why does PD = NPD?
Because of random segregation
53
Define synthetic lethality.
Two non-lethal muations that when combined in the same cell, result in cell inviability.
54
Is a synthetic genetic array a screen or selection?
Screen
55
How many yeast genes are non-essential?
5000
56
What types of genes can't be screened in SGA?
Essential ones
57
What is measured in SGA?
Mutant colony size (diameter) compared to WT
58
In SGA, if AB = 1, Ab = 0.75, and aB = 0.5, what is ab?
| What is this model called?
0.375
The multiplicative model
59
What are the assumptions of the multiplicative model?
What are the potential outcomes?
1. Genes are independent
2. If genes are in the same pathway, you will get a different result
Grows better = + interaction
Grows worse = negative interaction
Dead = synthetic lethal
60
In an SGA, if a and b negatively interact, and b and c negatively interact, what is the interaction between a and c? What does this say about a, b, and c?
Negative interaction.
They are all functionally related.
61
How do you perform the "Can't lose plasmid" screen in yeast?
1. Start with a plasmid that has WT gene of interest and URA3 marker
2. Transform plasmid into yeast strain that contains a mutation or deletion of the gene of interest
3. Mutagenize yeast with EMS
4. Plate on non-selective media
5. To find synthetic lethal genes, replica plate onto 5-FOA
6. Colonies that have the plasmid (and are synthetic lethal) will not grow on 5-FOA
62
What is 5-FOA?
A chemical that turns into a toxic metabolite when URA3 is around
63
How do you perform the "Can't lose plasmid" screen in bacteria?
1. Start with a highly unstable plasmid that has WT gene of interest, with ampR and LacZ
2. Transform plasmid into bacterial strain that contains a deletion or mutation of the gene of interest
3. Perform transposon mediated mutagenesis, inserting knar
4. Screen library on Kan, IPTG, and X-gal plates
5. Bacteria that have the transposon will grow, IPTG will express LacZ, and the colony will turn blue from the X-gal if it has the plasmid (synthetic lethal)
64
When performing the "Can't lose plasmid" screen in bacteria, why is amp not used on the plates?
So that the plasmid is not maintained if not needed for growth
65
In addition to the can't lose plasmid, what is another way to screen for synthetic lethality in bacteria?
Tn-seq
66
What does synthetic lethality tell us?
Parallel pathways
Physically interacting proteins
67
What is a suppressor?
A compensatory mutation that restores WT phenotype
68
What is the difference between intragenic and extragenic suppressors?
Intragenic = suppressor is in the same gene
Extragenic = suppressor is in a different gene
69
What are the different Intragenic suppressor subtypes? What don't they let you find?
1. True revertant
2. Second site mutation (affecting splice sites or amino acid sequence)
Genes in a pathway
70
What are the different extragenic suppressor subtypes? Which ones can rescue null mutants?
1. Interaction
2. Bypass
3. Epistatic
4. Mass Action
5. Dosage
6. Nonsense
Bypass and epistatic
71
What is the mechanism of function of a nonsense suppressor?
Allows read-through of a premature stop codon via a tRNA gene
72
Which extragenic suppressors don't enable the discovery of genes in a pathway?
Nonsense suppressors
73
What is the mechanism of function of an interaction suppressor?
An allele specific conformational mutation that re-enables the interaction of 2 proteins after the misfolding of one
74
What is the mechanism of function of a bypass suppressor?
Cell no longer needs the function of the gene of interest, as it confers a novel function to a protein
75
What is the mechanism of function of an epistatic suppressor?
A type of bypass suppressor where in the WT, one protein is repressed by the other...when the repressor protein is mutated, the other protein is not repressed...an epistatic suppressor will mutate the "repressed" protein so that is becomes repressed again.
76
What is the mechanism of function of a dosage suppressor?
Dosage compensation restores balance in a system
77
What is the mechanism of function of a mass action suppressor?
A type of dosage suppressor that stabilizes the mutant by overexpressing the interaction partner to increase the probability of binding.
78
What types of genes can be missed in suppressor screens
1. Duplicated genes
2. Essential genes
3. Small genes
4. Genes that have an incorrectly assumed phenotype
79
What is the purpose of epistasis?
Group genes by pathways and determine their order
80
What is required for epistatic analysis?
1. Null mutants
2. An intermediate or terminal phenotyp
3. Comparing mutants with opposite phenotypes
4. Comparing single mutants to double mutants
81
What is the phenotype observed that would allow you to claim that A is epistatic to B?
If the double mutant phenotype is the same as the single mutant A phenotype,
82
What are the 2 types of epistatic pathways?
Intermediate epistasis
| Terminal epistasis
83
What is the difference between intermediate and terminal epistasis in terms of epistatic classifications?
1. Intermediate - epistatic gene is upstream
| 2. Terminal - epistatic gene is downstream
84
What gene deletion/phenotype interaction is considered epistatic
Gene whose single mutant phenotype is similar to the double mutant phenotype.
85
Intermediate phenotypes in epistatic analysis often imply __________.
That the 2 genes are in parallel pathways, or the pathway is more complex than thought
86
C. elegans can be male and hermaphrodite, what are the odds that a hermaphrodite self fertilizes and has a male offspring? Why would this happen?
Non-disjunction occurs 1 in 1000 meioses
87
Hermaphrodites can be only mated to ________.
Males
88
During a C. elegans cross, which generation can dominant mutations be detected? Recessive?
F1
| F2
89
What is the unique feature of the hawiian strain of c elegans? This enables ______ and ______ to be done at the same time.
Has a SNP every Kb
Mapping and Cloning
90
What are the steps in using the hawaiian strain of c elegans to map/clone genes?
1. Mate hawaiian and Bristol worms
2. Self cross heterozygous F1 progeny
3. Select F2 for the mutant phenotype
4. Self cross the chosen F2, pool and sequence the offspring
5. Compare sequence to a population where the mutation wasn't selected
6. The mutation will map to a region that is homozygous for mutatnt strain and devoid of hawaiian
91
RNAi and morpholinos are used to study the function of ____________________.and leads to __________.
a specific gene by preventing the translation of mRNA into protein
leads to mRNA degradation
92
What types of RNAi are introduced synthetically?
long dsRNA, shRNA, and siRNA
93
Which type of RNAi is not processed by DICER?
siRNA
94
RNAi is incorporated into bacteria via ______, c elegans via _______ and d melano via ________.
Transfection.
Feeding.
Transgenic integration
95
Morpholinos are most commonly used in which model organisms? How are they administered?
Zebrafish and Xenopus
Microinjection
96
What is unique about the structure of morpholinos?
1. Contain a morpholine ring instead of a ribose sugar
2. Contains a non-charged phosphoro-diamidate backbone
3. Prevents electrostatic binding to proteins, which leads to less toxicity and more diffusion upon microinjection
4. Are resistant to nucleases, which leads to longer lasting effects
97
How are morpholinos different than siRNA in their mechanism of action?
Instead of leading to cleavage and degradation of mRNA, morpholinos sterically blocks ribosomes and blocks translation from occurring
98
Morpholinos can be used to _______ and _______.
Block translation
| Exon skipping
99
What are the caveats of RNAi and morpholinos?
Only leads to decrease in translation and not a knockdown of the protein
siRNA can bind non-specifically
In 15-20% of morpholinos, systemic effects are related to protein specific knockdown cause neural toxicity...this can be mitigated by introducing a p53 morpholino
100
How do bacteria store genetic information?
chromosomes and plasmids
101
How do bacteria transfer genetic information?
Binary fission, transformation, conjugation, transduction
102
Are bacterial chromosomes circular or linear?
Mostly circular but can be linear in a few species
103
Circular chromosomes have a single ______ which allows for ______.
ori
bidirectional replication
104
Define operon?
Group of genes controlled by a single promoter
105
What are some features of bacterial plasmids?
1. Found in 1-100 copies per cell
2. Usually don't contain essential genes
3. Much smaller than chromosomes
4. May exist as its own entity or integrate into the genome
106
What is the purpose of primer sites in a plasmid?
In order to confirm the presence of the gene in the plasmid via sanger sequencing
107
What is the most common method of bacterial DNA transfer?
Binary fission - genome is duplicated and identical copies given to each daughter cell
108
What are 2 ways to accomplish transformation?
1. Electroporation
| 2. Heat shock + CaCl2
109
Directed mutagenesis involves making changes to the gene's _______ or _______.
Sequence or structure
110
Suicide vectors involve ____________ events.
2 recombination
111
Suicide vectors contain which elements?
1. Non functional ori
2. Antibiotic resistance
3. SacB (or secondary counter selectable marker)
112
Allele exchange leads to ______ but not _____.
Gene deletion/total loss
Inactivation (partial/near total loss)
113
Name 2 reasons why you would want to inactivate a gene instead of deleting it.
1. The gene is essential
| 2. You want to assess high-low transition of expression
114
What are some considerations for gene inactivation/overactivation expression levels?
- Inducer concentration
| - Expression from chromosome vs plasmid (integrating vs replicating)
115
What is one strategy to perform inducible degradation?
CRISPRi (ssrA tag with SspB adapter)
116
What organism can RNAi not be used in?
Bacteria
117
Define transposon.
A type of transferable DNA element that often contains inverted repeats which help facilitate transfer
118
Insertion of a transposon can lead to ____ and ____ mutations
Deletion and truncation
119
What primers are needed for Tn-seq?
A forward primer complementary to the transposon, a library of random primers to account for Tn insertion anywhere in the genome
120
What does tn-seq show you? What does this allow you to determine?
Shows how frequently a transposon inserts itself into every gene in the genome. Determines the essential genome and conditional essentiality
121
A protospacer is a sequence of _________ base pairs found in ________ next to the ________.
30-40
Bacteriaphage DNA, PAM
122
What's the difference between CRISPR classes?
Class 1 = multiple proteins bind RNA
Class II = single protein binds RNA
123
```
CRISPR class I includes?
CRISPR class II includes?
```
```
1 = 1, 3, 4
2 = 2, 5, 6
```
124
Which CRISPR subtype destroys viral RNA instead of DNA?
Type 6
125
Which CRISPR type is normally used for gene editing? Why?
Type 2:
- simplistic, only require 1 cas protein to bind crRNA to become functional
- make precise ds cut in the DNA
-
126
Why isn't CRISPR type III or I used?
```
III = transcription dependent
1 = chews up DNA sequence unpredictably
```
127
In which 2 major ways are CRISPR systems modified?
1. crRNA and tracRNA fused to become sgRNA
| 2. NLS tagged to Cas9
128
What does Cas9 need to recognize a target?
1. Complementarity between target and sgRNA
| 2. A PAM
129
The PAM is on the ______ strand of the target and is _________ by the sgRNA
Non-target strand
Not recognized
130
What are 2 ways in which Cas9 can cut off target?
Target sequence similarity
PAM flexibility (NAGG or NAGC)
131
What is the most common method to insert CRISPR/Cas9 into mammalian cells? What does the plasmid need?
Transfection
spCas9, sgRNA with RE sties so you can clone in the sequence of the genome that you want to target, selectable markers (GFP or puromycin resistance)
132
What must be considered when choosing a cut site/sgRNA?
1. gRNA that cuts within an exon
2. That exon must be common to all splice variants of the gene
3. High on-target cutting/recognition
4. Low off-target cutting recognition
133
What's the most common way to assay a CRISPR knockout experiment?
Western blot (but must have antibody to protein of interest)
134
How can CRISPR propagate gene drive over normal inheritance?
Through use of selfish gene elements that act as transposons...WT copy is cut and mutant form is inserted through HDR
135
What is a yeast 2 hybrid used for?
To identify binding partners for proteins of interest from any organim who's genes can be expressed in yeast
136
How can you test to determine if a mutation is recessive or dominant in yeast?
Cross mutant haploid to WT haploid and give a diploid WT/mut, grow on selective media --> if phenotype is rescued, the phenotype is recessive
137
In a complementation test, - means _____ and + means _____.
same gene
different gene
138
Ts libraries are most useful for studying________.
Essential genes
139
Outcrosses to the hawaiian strain of worm is used in _____ genetics.
Forward when you don't know the gene you're looking at
140
Reverse genetics is a screen or selection? Howa about forward?
Reverse = screen
Forward = selection
141
Reverse genetics are restricted to ______.
only predicted genes
142
What is the use of an OriT? What about a f1 Ori?
Required for conjugation
Required for ssDNA transfer into a viral capsid
143
Ori determines _____ and _____.
Copy number and host range (broad or narrow)
144
What is a rop?
Repressor of primer, keeps copy number low in a plasmid
145
What is the major disadvantage to genetic screens?
They contain more bias
146
What does it mean that yeast have facile genetics?
They are easy to work with i.e. they can undergo homo recombo more easily
147
Complementation screens are done with haploid/diploid yeast.
Mate 2 haploids to get a diploid
148
What features of a genome can make mutagenesis difficult?
Large genoes with vast portions of non-coding regions
149
Cas9 makes ds breaks where?
3 nt upstream of PAM
150
Breaks formed by Cas9 are repaired by _____ or _____
NHEJ or HDR
151
CRISPR Cas systems can be designed to target all coding genes by targetting ______.
5' exons
152
Why are multiple copies of sgRNA per gene necessary for CRISPR screens?
Want to ensure that mutations aren't due to off target effects...if all 4 sgRNAs map to the same gene, we will know that it worked
153
How do you generate a library of sgRNAs? How do you deliver?
Synthesize oligos on an array
Lentivirus
154
What fraction of cells will be knocked out for a particular genes - assuming 100% cutting efficiency for the sgRNA
44% (2/3 chance of a frameshift x 2 copies of a gene)
0.66 X 0.66 = 0.4356
155
What is the sgRNA library workflow?
1. Oligo synthesis and detachment
2. Anneal and ligate sgRNA pool
3. Take a lentiviral vector with a cut site and sticky ends to allow insertion of sgRNA
4. Transduce cells with packaged and pooled lentiviral sgRNA library
5. sgRNA amplification and nextgen seq
156
What is the difference between arrayed vs pooled sgRNA library approaches?
Arrayed = each lentivirus has its own sgRNA, very costly, rarely performed
Pooled = All lentiviral particles and sgRNAs are in the same tube, cost effective, but you have to find the phenotype you want after the fact
157
How do you identify and rank sgRNA hits?
Sequence the mutant and control populations and see which sgRNAs increase in abundance (fold enrichment and redundancy in mapping to the same gene)
158
20, 000 genes with 4 sgRNAs per gene = _____ sgRNAs...if you want 100x coverage of each sgRNA = _____ transduction events....if you want an MOI of 0.1 = transduce ______ cells with _____ viral particles and then ________.
```
80,000
8x10^6
80x10^6
8x10^6
select the transduced cells
```
159
How would you distinguish a real hit in a sgRNA screen? How can you increase confidence in hits?
Identify multiple independent sgRNAs targetting the same gene
Repeat the screen using independently transduced population of cells so the signal is the same, but the noise is different
160
What is the "noise" in an sgRNA screen? (2)
Double transduction events that may be enriched
Spontaneous resistance (if you repeat the experiment, it will enrich a different set of sgRNAs)
161
When using CRISPR-Cas to increase gene expression, what sequences would you target in the genome and what would you design the system to recruit?
Promoter proximal regions
Use Cas9 as landing pad for TFs or transcriptional activators
162
What is the purpose of MOI?
To prevent multiple transudction events
163
Which suppressors are useful for deciphering pathways? (and why)
Which suppressors is allele specific? (and why)
Which suppressors can rescue a deletion mutation? (and why)
1. Epistatic suppressor
2. Interaction suppressor, true revertant
3. Bypass, epistatic
