Chapter 17 Practice Problems Flashcards
bind to promotors
basal factors
bind to enhancers
repressors
site of DNA methylation
CpG
pattern of expression depends on which parent transmitted the allele
imprinting
prevents or reduces gene expression post transcriptionally
miRNA
bind to activators
coactivators
heritable change in gene expression not caused by DNA sequence mutation
epigenetic effect
organizes enhancer/promoter interactions
insulator
activates gene transcription temporal and tissue specifically
enhancer
identifies DNA binding sites of transcription factors
ChIP-Seq
You have synthesized an enhancer less GFP reporter gene in which the jellyfish GFP cDNA is placed downstream of a basal promoter that functions in mice. You will now fuse this enhancerless reporter to the three types of sequences listed below (x-z)
a. which of the three types of sequences would you use for which of the three listed purposes (I-iii)? in each case, explain how the particular fusion would address the particular use
types of sequences fused to the reporter:
x. random mouse genome sequences
y. known mouse kidney specific enhancer
z. fragments of genomic DNA surrounding the transcribed part of a mouse gene
uses:
i. to identify a gene’s enhancer
ii. to express GFP tissue specifically
iii. to identify genes expressed in neurons
b. which fo the sequences x-z would you fuse to a particular mouse gene of interest in order to express the protein product of the gene ectopically, that is, in a tissue in which the gene is not usually expressed? why might you want to do this experiment in the first place?
a. (i) To identify a gene’s enhancer(s), fuse the reporter to (z), fragments of genomic DNA around the gene. A genomic fragment that includes an enhancer will cause GFP expression in at least some of the tissues in which the endogenous gene is normally expressed in mice. (The particular pattern of reporter expression observed will depend on how many enhancers the endogenous gene contains and how many of these enhancers were fused to the reporter gene.)
(ii) To express GFP tissue- specifically, fuse the reporter to (y), a known kidney-specific enhancer. GFP will be expressed in the kidney. An alternative answer is (z), fragments of genomic DNA surrounding a gene. This approach will work if the gene is known to be expressed tissue-specifically; one or more fragments of the DNA in the vicinity of the gene are likely to contain an enhancer that determines this tissue-specific expression.
(iii) To identify genes expressed in neurons, fuse the reporter to (x), random mouse genome sequences. Those that contain enhancers active in neurons will cause GFP expression in neurons; these enhancers can be used to identify the genes that they control normally.
b. As one example of ectopic expression, assume that the gene of interest is not expressed in the kidneys normally. If you fused the mouse gene to (y), a known kidney-specific enhancer, the gene should then be ectopically expressed in the kidneys. This approach should work with any characterized enhancer because you could then predict where the fusion gene will be expressed.
You might want to express a gene ectopically to determine whether a mutant phenotype will result; the nature of that phenotype can sometimes reveal information about the gene’s normal function.
Yeast genes have cis-acting elements upstream of her promoters, similar to enhancers, called upstream activating sequences or UASs. Several target genes involved in galactose utilization are regulated by one type of UAS called UASg which has four binding sites for an activator called GALA. Two target genes regulated by UASG are GAL7 and GAL10. The GAL80 protein is an indirect repressor of GAl7 and GAL10 transcription: at UASS, GAL80 binds to GAL 4 protein and blocks GAL4’s activation domain. In the presence of galactose, GAL80 no longer binds GAL4
In which gene(s) (GAL4 and GAL80) should you be able to isolate mutations that allow the constitutive expression of the target genes GAL7 and GAL10 in the absence of galactose? In each case, what characteristics of the protein would the mutation disrupt?
Constitutive expression of the galactose genes in yeast will be observed if GAL4 binding to UASG cannot be prevented. Therefore, a GAL80 mutation in which the protein is either not made or is made but cannot bind GAL4 protein will prevent indirect repression of the galactose genes and lead to constitutive synthesis. A GAL4 mutation that prevents GAL4 protein from binding GAL80 protein would also lead to constitutive expression of the galactose genes.
a. assume that two transcription factors are required for expression of the blue pigmentation genes in pansies. (Without the pigment, the flowers are white) what phenotypic ratios would you expect from crossing strains heterozygous for wild type and recessive amorphsc alleles for each of the genes encoding these transcription factors?
b. Now assume hat either transcription factor is sufficient to get blue color. What phenotypic ratios would you expect from crossing the same two heterozygous strains?
a. If both transcription factors are required for expression (complementary gene action), only the A– B– genotype will produce blue flowers. Diagram the cross:
Aa Bb × Aa Bb →
9/16 A– B– (blue) : 3/16 A– bb (white) : 3/16 aa B– (white) : 1/16 aa bb (white)
= 9/16 blue : 7/16 white flowered plants.
b. If either transcriptional factor is sufficient for blue color (redundant gene action), then three of the four genotypic classes from a dihybrid cross will produce blue flowers:
9/16 A– B– (blue) : 3/16 A– bb (blue) : 3/16 aa B– (blue) : 1/16 aa bb (white)
= 15/16 blue : 1/16 white flowers.
Genes in prokaryotes and eukaryotes are regulated by activators and repressors
a. compare and contrast the mechanism of function of prokaryotic repressor with a typical eukaryotic repressor protein.
b. compare and contrast the mechanism of function of a prokaryotic activator with a typical eukaryotic activator protein
a. Prokaryotic repressors, like the Lac repressor, directly block RNA polymerase from binding the promoter and/or block the movement of RNA polymerase bound at the promoter to the transcription start site (see Fig. 16.5(b)). Eukaryotic repressors recruit corepressor proteins that either close chromatin or prevent pol II from binding the promoter (see Fig. 17.10).
b. Prokaryotic activators, like CAP, attract RNA polymerase to the promoter by touching the polymerase directly. Eukaryotic activators bind coactivators that displace nucleosomes to open chromatin, or recruit RNA pol II to the promoter through interactions with other proteins such as those in the Mediator complex (see Fig. 17.6).
Lysine 4 of histone H3 (H3K4) is methylated in the nucleosomes of many transcriptionally active genes. Suppose you want to determine all the places in the human genome where nucleosomes contain methylated H3K4
a. starting with an antibody that specifically binds only to the tails of histone H3s that have K4 methylation, what kind of expreiment would you perform? Outline the major steps of this experiment
b. do you think that ou would get the same results if your starting material was skin cells in one experiment and blood precursor cells in a second experiment? explain
c. Describe a follow up experiment that could determine if your data from part a are consistent with the idea that H3K4 methylation marks appear only at transcriptionally active agents
a. You would perform a ChIP-Seq experiment similar to that in Fig. 17.18. (i) Isolate chromatin from cell nuclei; (ii) crosslink DNA and proteins; (iii) fragment DNA by sonication; (iv) add antibody to methylated H3K4; (v) co-immunoprecipitate methylated H3K4 protein and DNA; (vi) purify DNA and sequence.
b. No; blood precursor cell and skin cell chromatin would be H3K4-methylated in different patterns because different genes are transcribed in these cells. Some overlap would exist in the genes identified, because they transcribe some common genes.
c. The gene sequences identified in part (a) could be compared to the genes identified in a RNA-Seq experiment. If the hypothesis is correct, you would see that many RNA-Seq reads would correspond with the DNA that co-immunoprecipitates with methylated H3K4, and you would perform a statistical test to make sure that this correlation is statistically significant, using DNA that fails to co-immunoprecipitate as a control.
