Topic C & F: Genetics and Molecular Evolution Flashcards
What is paused RNA Polymerase II and what is it’s function?
RNA polymerase machinery prepares for gene transcription but sits and waits until genes are needed to be transcribed. A cell might not need a particular gene expressed all of the time, but the paused RNA polymerase allows it to be more rapidly expressed when it is needed.
What is the role of enhancers in transcription and describe a mechanism by which they act?
typically upstream of a gene, may be greater than 1 kb away, they enhancer or promote txn of a gene by interacting with a promoter by bringing sites into direct contact by forming a loop like structure, can act in cis or trans
How does DNA methylation repress gene transcription?
DNA methylation typically occurs on Cs in CpG island and block TFs and other proteins from accessing DNA
Describe two mechanism that can lead to short telomeres
Telomeres aren’t capped so they are degraded, ageing causes telomeres to shorten
How does the enzyme telomerase extend the ends?
iono add
Describe an experiment that would allow you to determine whether a long non-coding RNA is critical for cell differentiation. How would you distinguish that RNA itself is or the act of txn is responsible for differentation?
design an shRNA to knockdown the ncRNA in cells. You would need cells where you induce a shRNA that doesn’t target your ncRNA of interest as control and you then determine if the knockdown of ncRNA affects differentation. To determine if it is the RNA itself or the act of txn you could use homologous recombination in mice to insert a seq generating a mutant copy of the lncRNA. If you see the same effect of the differentation, it is most likely an effect of the txn. If not, it is most likely an effect of the RNA itself.
When an E.coli is growing in medium containing glucose as its only carbon source, the lac operon txn rate is low but not zero. Why is it likely to be advantageous for E.coli that the lac operon txn rate is not high?
- The proteins encoded by the lac operon do not provide any function when glucose is the sole carbon source.
* *There is more to this question
List three genetic experiments that you would perform in a model organism to determine how a missense allele affects a gene’s function.
- You can do CRISPR-Cas9.
- RNA knockdown
- Transgenic rescue
idk
What two types of alleles are the most dangerous and why?
antimorphic or dominant negative where abnormal gene product interferes with the function of the gene and causes a more severe phenotype and neomorphic.
amorphic or null alleles knock out the gene and can be lethal. check answers to problem set.
Briefly discuss why null alleles of a gene are useful to model organism geneticists but also have limitations in terms of understanding gene function.
null alleles provide a complete knock down of a gene allowing you to determine the gene function however a complete knockdown maybe lethal so you have to use hypermorphs and hypomorphs to look at a spectrum of functions
What are ultra conserved elements and why are they of interest?
Ultraconserved elements are extremely conserved genomic regions across evolutionary distant species. Since they are highly conserved , they must have very important function.
**What is the most common form of selection in the genome?
Purifying
What is the neutral theory of evolution?
The vast majority of evolutionary changes at the molecular level are caused by random drift of selectively of neutral mutants (not affecting fitness).
What effect will a 1.selective sweep have on the time to most recent common ancestry and shape of a genealogy?
2.balancing selection
- It will increase the time and looking like a star shaped genoalogy (population bottleneck, growth)
- decrease the time and it will looking chicken legs. (population subdivision)
What is the effect of purifying selection on rates of nucleotide divergence between species? What about positive selection?
decrease (you dont want change)
Increase (favoring change)
Do coalescence events take longer in a larger or smaller population?
Larger
Assume that an X-linked recessive trait observed in 2.5% of all of the males. What are the genotype frequencies among the females of this population if the population if the population is in HWE?
For male, q = 0.025 p = 0.975. The allele frequency should be the same for male and female. So genotype frequency is q^2, 2pq, q^2.
What effect will a selective sweep have on linkage disequilibrium?
Increase levels of linkage disequilbrium
Which demography events mimic positive directional selection? balancing selection? How can you discriminate between demographic and selective effects?
Population bottleneck and growth mimic positive directional selection. Population subdivision mimics balancing selection. HKA test avoids this problem by comparing patterns of variability at two regions in the same individuals. The McDonald-Kreitman test compares the ratio of ns and synonmous SNPs within species to the ratio of ns to synonmous fixed difference between species, which should not be affected by the shape of the tree
Define effective population size.
The number of different chromsomes segregating in a population aka the number of breeding organisms of a population.
Studies of global patterns of genetic diversity suggest that (choose one):
a) Genetic variation is highest between populations rather than within population
b) Genetic variation is highest within populations rather than between populations
c) African populations have lowest levels of genetic variation
d) Asian populations have highest levels of genetic variation
b
In general an average of 85% of genetic variation exists within local populations, 7% is between local populations within the same continent, and 8% of variation occurs between large groups living on different continents.
Diseases may differ in prevalence among ethnic groups due to (choose one):
a) genetic drift
b) natural selection c) new mutation
d) all of the above
d
Briefly describe Wrights Fst measure of genetic differentiation
Fst is equal to Ht-Hs/Ht
where H represents the average heterozygosity across loci within populations (S) and in the total sample(t)
If Fst is close to zero that means they is little or no genetic differentiation
If Fst is close to one, differentiation is very high
In a natural population Fst is low in humans 7%
This means that 93% of all variability in humans is present in every population.
Briefly describe the Transmission Disequilibrium Test (TDT) for family-based association test.
The TDT measures the over-transmission of an allele from heterozygous parents to affected offsprings. Looking at n families, count the number of informative (heterozygous) parents that pass on a marker allele to an affected child. If this is significantly different from n/2 by chi-square test, then that marker is associated with the disease.
A specificity of the TDT is that it will detect genetic linkage only in the presence of genetic association. While genetic association can be caused by population structure, genetic linkage will not be affected, which makes the TDT robust to the presence of population structure.
Look at heterozygote parents and see which allele was transmitted and which not to the affected and unaffected children. Looking for things linked to your phenotype. Ends up being chi-square dis- tributed – T test
Briefly describe what is measured in codon bias test statistics.
frequency of occurrence of synonymous codons in coding DNA.
You study two closely linked autosomal SNP loci, A (alleles A1, A2) and B (alleles B1, B2). Assume that the allele frequency are A1 (0.8), A2 (0.2), B1 (0.3), and B2 (0.7). Knowing nothing else, what do you predict for the frequency of two-locus genotypes in the pop- ulation? State the assumption you make.
Suppose the frequency of the combination A2 B1 on a chromosome was found to be 0.18. Discuss the implication of this observation. You might wish to include: recombination fraction between A and B, linkage disequilibrium, association, haplotypes, or changes in frequency over generations.
assume hardy weinberg populations
just multiply the frequencies
and assume they are independent
We are observing 3X more than we would expect if these alleles were independent so this implies that there is linkage between the loci. The changes in allele frequency for these are not indepen- dent. loci in LD. A2B1 is a haplotype. Recombination fraction between A and B is low.
Among 500 men genotyped by your classmate, she found the following numbers of individuals with each allelic combination: 45 A1B1, 55 A1B2, 255 A2B1, and 145 A2B2.Are the alleles at the A and B loci associated? Explain.
Allele frequencies based on counts from the 500 men A1=.2 A2=.8 B1=.6 B2=.4
they are associated otherwise you would expect equal proportions of genotypes
To quantify how associated they are use a Chi-square test to come up with a P-value that they are not associated using equal expectations for each genotype with (2-1)(2-1)=1 degree of freedom
Briefly describe ‘homeobox genes’.
genes involved in the regulation of patterns of development (morphogenesis) have homeodomain – when translated becomes homeo-domain – DNA binding
What is meant by ‘long-range regulatory determinants’? How have they been discovered? What and how do they work? Do they acts in ‘cis’ or ‘trans’?
Locus Control Regions – can regulate multiple genes in the region They were discovered by enhancer traps, 3C, etc.
They act in cis
Models for how they work inlude looping, tracking, mix (looping and tracking), linking
What is a ‘complementary group’?
A group of mutations that fail to complement because they affect the same locus
Mobile DNA systems are keys players in genome evolution. Name a mobile DNA element, describe its mechanisms of replication and mobilization, and specify an effect on phenotype or genomic change.
Retrotransposons – ‘copy and paste’, transcribed into RNA and reverse transcribed to integrate into the genome in a new location. Can move far away from original location. If it integrates into a gene it could cause a loss of function for that gene. Over evolutionary time these elements are responsible for adding to the length of the genome
The sole active autonomous mobile element in human beings is a member which class: DNA transposons, SINE, non-LTR retrotransposon, or LTR retrotransposon.
Non-LTR retrotransposons
The human genome is composed to a considerable degree of sequences derived from genomic parasites. Name two such parasites and describe their replication mechanisms.
DNA transposons: use a cut-and-paste mechanism to move from one place to another.
Non-LTR retrotransposons: LINEs, SINEs, the only active ones in human, use a copy-and-paste mechanism. First transcribe DNA into RNA with retrotransposase to make a copy of the RNA into DNA, then insert in a new location.
Briefly explain the terms ‘suppressor screen’, ‘enhancer trap’, and ‘synthetic lethal’.
Suppressor screen: Start with pool of mutants with some phenotype and mutate them again and see which are rescued from the phenotype
Enhancer traps - screens to find enhancers for the expression of a given gene – usually done in cell type specific. Randomly fragments from genome and create reporter vectors to test gene expression activity.
Synthetic lethal: When you are null for two genes and the phenotype is lethal but each individual knock out is not
The genomes of almost every organism are infested with genetic parasites, i.e. selfish DNA sequences, that covalently integrate their own sequences into the host cell genome. Name 3 specific examples and briefly describe their replication strategy.
DNA transposons: Cut-and-paste
LTR Retrotransposons: Copy-and-paste, RNA intermediate, makes its own polymerase
Non-LTR Retrotransposons (autonomous): Copy-and-paste, RNA intermediate, makes its own poly- merase
Non-LTR Retrotransposons (non-autonomous): Copy-and-paste, RNA intermediate, requires anotherretrotransposon to replicate
You are getting ready to perform a genetic screen for fly mutants that lack wings. Thus you will want to obtain mutants that are homozygous viable and can be screened for such phenotype. Explain how a balancer chromosome can be used to identify homozygous mutants.
cross balancer/mut X balancer/mut and the mut/mut genotypes will not have the balancer phenotypes
What are off-target effects of RNAi and how might one minimize such effects?
A siRNA could end up targeting other genes, instead of the intended target gene. We can have multiple siRNAs targeting the same genes, or do cDNA rescue.
Redundancy: design multiple siRNAs for the same target and see if the phenotype is the same Rescue: rescue with an RNAi resistant transcript of the target mRNA
In characterizing an allele of gene X, you find that homozygous mutant embryos obtained from crossing two heterozygous parents appear to develop normally but fail to hatch. Interestingly, an injection of RNAi molecule specific for gene X results in embryos lacking abdomen. Assuming that con- trols are done and the RNAi result is specific to knocking down gene X, how can you explain the result?
Maternal effect – the protein of gene X (or mRNA) was oringally in the placenta of the develop- ing embryo but the embryo couldn’t produce it on its own so with the RNAi the embryo can not develop in the begining as it can when the mother’s gene is available
Of the following types of sequences, which makes up the smallest fraction of the human genome: L1 element, exons, introns, or Alu element.
exons
Why are null alleles so important?
Complete removal of gene product is the most rigorous way to assess what processes require that gene (necessity).
However, for genes that play multiple roles in the organism, the null mutant phenotype may reveal only he earliest essential role, and may not allow assessment of later roles. Hypomorphs, conditional alleles or tissue-specific KOs are useful in this case.
Also, for genes that function redundantly with other genes, the null mutant phenotype may not reveal its function. Dominant negative or hypermorphic alleles can be useful in this case.
Finally, null mutations do not reveal sufficiency. For example, does a gene product actually regulate a process vs. being passively required for it?
In a genetically tractable organism like C. elegans or Drosophila, what criteria are typically used to determine if a given allele is likely to be null?
*Recessive phenotype? (lf alleles often recessive, if alleles usually dominant) *Severe molecular lesion? (large deletion, early nonsense or frameshift) *Reduced gene product? (undetectable mRNA and/or protein)
*RNAi mimics the allele
*High frequency of isolating similar alleles (lf alleles more common, or alleles rarer) *Phenotype of allele is most severe (penetrant, expressive) in 1allelic series’
Look with qPCR for mRNA levels and also look at protein levels with a western
As dosages of the mutant allele increase the phenotype doesn’t change and as doses of the wildtype allele increases the phenotype becomes more wild-type.
What are the limitation of null alleles and what other types of alleles might also be useful?.
May be a lethal phenotype so difficult to study. Hypomophs and hypermorphs would be useful in order to get an ‘allelic series’ and a spectrum of phenotypes of different degrees.
What is the underlying principle of genetic analysis? In other words, how does this approach differ from the approach a biochemist might use to study a biological process?
Biochemical approach: in vitro recreate things in a test tube/ reconstruct desired result/ put things together. (ex. knock-out genes)
Genetic analysis:
• take things apart & see if organism cares
• “Don’t know what you have until it is gone”
• relies on mutations
• start with defect & find the basis of the defect
Advantages of genetic analysis:
• general, can be used for any process, in any organism • predictable, mutations are stable & inheritable
• reveals gene functions that are necessary in vivo.
• Technically simple, conceptually complex
Genetic analysis can be used in 2 ways:
(1) gene identification, function: start w/ process of interest & search for mutations that affect this process.
• gene identification: saturation screen - all genes that can mutate to a particular phenotype modification screen (suppressor/enhancer) - mutations that modify another mutations
• gene function: phenotype, interaction w/ another mutant (2) As tools to study other genes
• markers
change morphology or physiology in a useful way
What is a mosaic?
An organism that has two different populations of cells with multiple genotypes but both arise from the same fertilized egg (eg. result of trisomy rescue, x-inactivation)
What method can be used to generate mosaics?
Transplantation experiments: cells from a blastula stage embryo from one genetic background are aspired out and injected into a blastula stage embryo of different genetic background. Induce recombination (delete out a gene) Flip recombinase in a tissue (or Cre-Lox system on subset of cells)
What does it mean to say that a gene functions “cell non-autonomously” for a given function? What result in a mosaic analysis would lead to this conclusion?
cell non-automous function: genotypically mutant cells cause other cells, regardless of genotype to exhibit a mutant phenotype
If in a mosaic analysis you see WT genotype cells next to mutant genotype cells but they are all exhibiting mutant phenotype
Besides mosaic analysis, what other experimental approaches can be used to address the question of where a gene functions?
Reporter constructs of Lacz to see where that gene is being expressed
In a hypothetical genetic pathway, gene 1 knockouts have a fat phenotype while gene 2 knockouts have a thin phenotype. gene 1;gene 2 double knockouts have a fat phenotype:Which gene mutation is epistatic?
Imagine that gene 1 encodes a microRNA. Given the microRNAs often inhibit target gene ex- pression, propose a type of genetic screen that might identify gene 1 targets.
Gene 1 is epistatic to gene 2
G2 − − | G1 −− > thin
G1 −−> G2 −−> thin
Random mutagenesis of gene 1 mutant to rescue mutant phenotype. Loss of function mutation in a gene that rescues mutant phenotype is likely to be target of gene 1 miRNA.
