Exam 8: Analyzing Cells, Molecules, and Systems

You would like to clone a gene by PCR. The sequence of the two ends of the coding strand for the gene is provided below. Which following pair of primers would direct the amplification of this gene? All sequences are written from 5? to 3?. ATGAAATCTACGTTTCAC……CCCCCAGTACCCCCCTTA

Loss-of-function mutations in either imp or yfgL genes in Escherichia coli result in an increased permeability of the outer membrane to small molecules and therefore confer antibiotic sensitivity, as the antibiotics can enter the cell more readily. Interestingly, double mutants, in which both genes are defective, are resistant to antibiotics. Similar to the wild-type strain, but unlike the single mutants, they can grow on media containing antibiotics. This is an example of …

The AgeI restriction enzyme recognizes the sequence ACCGGT and cuts each strand between the first (A) and the second (C) nucleotide. This can be represented as A|CCGGT, with the vertical bar indicating the cleavage position. If a DNA fragment is cut with AgeI at both ends, cutting a plasmid with which of the following enzymes would then allow insertion of the fragment into the plasmid by simple ligation of sticky ends?

Consider a protein composed of only 21 amino acids encoded by a short ORF that is located between two known genes in a mammalian genome. Due to its length, the ORF had not been annotated as a gene. Which of the following methods can demonstrate that it is indeed a gene that is actively translated to protein?

You performed a co-immunoprecipitation experiment to identify cellular proteins associated with protein X. For the experiment, you used antibodies against protein X that were immobilized on beads. You incubated these beads with cell lysates from either wild-type cells or cells lacking the gene encoding protein Y. After washing and eluting the bound protein off the beads, you separated each bound and unbound fraction by SDS-PAGE. Finally, you performed immunoblotting using antibodies against either protein Y or protein Z. The results of the Western blots are presented in the following schematic drawings. According to these results, do you think protein Y enhances (E) or inhibits (I) binding of protein X to protein Z? Write down E or I as your answer.

Where do individual peptides get fragmented in a tandem mass spectrometry (MS/MS) set-up?

The wild-type nematode Caenorhabditis elegans has a bent body with the shape of a wave. In the presence of increasing concentrations of ethanol, however, the body bend is progressively diminished. Loss-of-function mutations in SLO-1, a gene that codes for an ion channel, confer ethanol resistance and allow the worm to maintain its body bend up to higher ethanol concentrations. The channel function is defined based on the ability of the channel to conduct ions across the membrane. According to these observations, would you expect the channel to open or close in the presence of ethanol in wild-type worms? Would a gain-of-function mutation in SLO-1 tend to increase or decrease body bend?

Consider the binding of a transcription regulatory protein (A) to a promoter DNA (p) according to the following scheme. A + p?A:p At steady state, which of the following formulae correctly yields the bound promoter fraction, i.e. [A:p]/([A:p] + [p])? The association constant is designated as K.

Indicate which of the following network motifs represent negative feedback, positive feedback, incoherent feed-forward, and coherent feed-forward, respectively. Your answer would be a four-letter string composed of letters A to D only, e.g. BACD.

You have set up a system of differential equations to describe the concentration of a protein as a function of time based on transcription, translation, and degradation rates. You simulate two conditions (1 and 2) that are identical except that the transcription rate for the protein is set tenfold higher in one of them (1) compared to the other (2). Starting from a concentration of zero, the time required to reach 50% of the final steady-state concentration is …

A portion of RNA-seq data obtained from two tissue samples is plotted in the following schematic diagram. Which region (A to E) corresponds to an alternative exon?

For the two following graphs, the same purified protein has been subjected to velocity sedimentation and gel filtration, as indicated. Two absorption peaks are visible in each graph, corresponding to the monomeric and dimeric forms of the protein. Which peaks correspond to the protein dimer?

Indicate whether each of the following descriptions applies to transformed cell lines (T), nontransformed cell lines (N), or both (B). Your answer would be a four-letter string composed of letters T, N, and B only, e.g. BBNN. ( ) They can be thawed and recultured after long-term storage in liquid nitrogen. ( ) They often grow only when attached to a surface, such as that of a culture dish. ( ) They proliferate to very high densities. ( ) They usually cause tumors when injected into immunocompromised animals.

You have created transgenic mice that carry the gene encoding green fluorescent protein (GFP) under the control of a ubiquitous promoter. The GFP-encoding gene, however, is inactive unless recombination by Cre removes a blocking sequence between the promoter and the gene. These mice have been mated with those harboring the Cre recombinase under the control of the insulin promoter. The Cre recombinase is fused to the estrogen receptor, which keeps the fusion in the cytosol unless it binds to tamoxifen, in which case it allows nuclear entry. After mating, you select adult mice harboring both of these transgenes and inject them with either tamoxifen or water. Which of the following would you expect to observe later as a result when you examine brain and pancreas tissue samples under a fluorescence microscope?

Consider a biochemical pathway in petal cells of a plant, in which a white precursor is turned into a final red pigment through a yellow and an orange intermediate by the activity of three enzymes encoded by genes A, B, and C. The petal color in plants harboring functional (capital letter) or nonfunctional (lowercase letter) alleles of these genes is presented in the following table. What do you think is the order in which the enzymes act in the pathway? Your answer would be a three-letter string composed of letters A to C, e.g. BCA.

Which mass spectrometry instrument is most suitable for determining all the proteins present in an organelle?

The p53 gene is an important tumor suppressor gene in our genome. It codes for a transcription regulatory protein that can assemble into tetramers. A number of mutations in p53 render the protein inactive. These mutant protein molecules form mixed tetramers with wild-type p53 proteins and, in this case, the tetramer is inactive. The mutations can therefore be categorized as …

Indicate whether each of the following descriptions better applies to forward (F) or reverse (R) genetics. Your answer would be a four-letter string composed of letters F and R only, e.g. RFFF. ( ) It involves new gene discovery by genetic screens. ( ) It may also be called genome editing. ( ) In this approach, mutations are introduced deterministically in a known gene. ( ) It is involved in creating knockout mice lacking a particular gene.

Susceptibility to drug and alcohol addiction is partially genetic. For example, the human dopamine receptor DRD2 is associated with cocaine and alcohol abuse; individuals carrying an A nucleotide, instead of the more common C nucleotide, at a certain position within the sixth intron of the DRD2 gene are at a significantly increased risk of abuse. This variation in sequence is an example of …

Sort the following steps in the common procedure to create transgenic plants. Your answer would be a four-letter string composed of letters A to D, e.g. DABC. (A) Callus growth (B) Agrobacterium infection (C) Shoot/root induction (D) Removal of leaf tissue from a plant