Exam 8: Analyzing Cells, Molecules, and Systems

You have used a variation of fluorescence in situ hybridization (FISH) called flow-FISH. In this technique, DNA probes are labeled with fluorescence and hybridized to their targets inside the cells in a tissue. The cells are then sorted using a fluorescence-activated cell sorter based on their fluorescent signal intensity. The probes in your experiment are designed to hybridize to human telomeric sequences at the ends of the chromosomes. You perform flow-FISH using these probes on two human cell types, an early primary fibroblast culture and a late secondary culture that is showing the signs of replicative cell senescence. The sorting results are presented in the following histograms. Which histogram (1 or 2) would you expect to correspond to the primary culture?

In SDS-PAGE of proteins, …

Which of the following can limit the use of RNA interference in studying gene expression and function?

In a bacterium that is normally cylindrical in shape, loss of either of two genes A and B results in round cells. Additionally, gain-of-function mutations in gene A can rescue the phenotype of gene B loss-of-function mutants. What phenomenon best describes this observation? The product of which gene is more likely to function upstream of the other one in determining cell shape in this bacterium?

To create cellular factories for monoclonal antibody production, cells derived from an immortalized B lymphocyte cell line (M) are cultured together with antibody-secreting B lymphocytes (B) on a so-called HAT medium. The medium contains an inhibitor of DHFR, an enzyme essential for de novo nucleotide biosynthesis. Additionally, it contains substrates for the only alternative nucleotide biosynthetic pathway called the salvage pathway. The enzyme HGPRT is essential for the salvage pathway. One of the cells mentioned above lacks the gene encoding one of the enzymes mentioned. Considering the role of the HAT medium in selecting for hybrid cells that are both immortalized and secrete antibodies, which cell (B or M) do you think is missing an enzyme? Which enzyme is missing in this cell?

The binding of protein A to two other proteins, B and C, has been studied with fluorescence anisotropy, and the results of the measurements have been plotted in the following schematic diagram. Given equal kₒn rate constants, which complex do you think has a longer half-life? What is the value of K?ₐ (the association constant) for this complex?

Two loci on a human chromosome are about 1,500,000 nucleotide pairs apart and are found to recombine in about 2% of gametes during gametogenesis. Consistently, each genetic map unit (which corresponds to 1% recombination) in humans is equivalent to a physical distance of about … nucleotide pairs.

Fill in the blank in the following paragraph regarding plant cell cultures. Do not use abbreviations. "Plant cells can be easily rendered totipotent to give rise to all plant tissues on their own. This can be done by culturing a plant tissue in sterile nutrient media to produce an amorphous cell mass called a …"

Indicate whether each of the following descriptions better applies to Illumina sequencing (I), Ion Torrent™ sequencing (T), or both sequencing technologies (B). Your answer would be a four-letter string composed of letters B, I, and T only, e.g. IBBB. ( ) It uses fluorescently labeled nucleotides. ( ) It uses PCR-generated copies of DNA. ( ) It is a second-generation sequencing technology and employs a cyclic wash-and-measure paradigm. ( ) It relies on the fidelity of DNA polymerase for its accuracy.

Indicate whether each of the following manipulations or procedures used in current recombinant DNA technology commonly rely on an enzyme (E) or are done nonenzymatically (N). Your answer would be a six-letter string composed of letters E and N only, e.g. EEEEEE. ( ) Cleavage of DNA at specific sites ( ) DNA ligation ( ) DNA cloning ( ) Nucleic acid hybridization ( ) Synthesis of DNA of any desired specific sequence ( ) DNA sequencing

Consider two proteins A and B with equal transcription and translation rates. The mean lifetime of A, however, is twice as long as that of B. Starting with no protein, the time it takes for a protein to reach 99% of its steady-state concentration is …

Bistability in a biological system can be accompanied by hysteresis. Consider switchlike cellular responses to a signal molecule, as shown in the following graphs. In each graph, the solid curve corresponds to the change in cellular response when the signal concentration is being increased over time, whereas the dashed curve corresponds to the change when the signal concentration is being decreased. Which graph do you think corresponds to a system with a greater degree of hysteresis? What mechanism is responsible for this behavior?

You have carried out a genetic screen in a species of fish, and have identified a new gene in this organism. You have cloned and sequenced the gene, but do not know anything about the biochemical function of the gene product. Which of the following is the most sensible next step in order to get clues about the function?

Everything else being equal, if the mean lifetime of a protein is doubled, its concentration at steady state would …

Indicate whether each of the following descriptions better applies to equilibrium sedimentation (E) or velocity sedimentation (V). Your answer would be a four-letter string composed of letters E and V only, e.g. EEEE. ( ) It separates subcellular components based on their buoyant density. ( ) It can conveniently separate ¹⁵N-containing and ¹⁴N-containing DNA. ( ) It uses a shallow sucrose gradient to stabilize the sedimentation bands against convection. ( ) In this method, subcellular components may either move up or down to form discrete bands.

Your friend has cut a 3000-nucleotide-pair circular plasmid with either or both restriction enzymes A and B, and has separated the digestion products by electrophoresis. The results are shown in the schematic drawing below, with the approximate size of the fragments indicated above each band. Based on these results, what is the closest distance (in nucleotide pairs) between an A restriction site and a B restriction site on this plasmid?

A piece of DNA has been sequenced by Ion Torrent™ sequencing. The result corresponding to a six-nucleotide segment of this DNA is presented in the graph below, in which pH change is recorded after the addition of each of the four nucleotide triphosphates in cycles. What is the sequence of this segment? All sequences are written from 5′ to 3′.

You have devised genetic screens to identify gain- and loss-of-function mutations in the Escherichia coli DNA ligase gene. One of the screens (1) seeks mutant cells that are infected more readily with a ligase-deficient strain of T4 phage. The other screen (2) seeks mutant cells that have increased radiation sensitivity at high temperatures. Indicate whether each of the following descriptions better applies to screen 1 (1) or screen 2 (2). Your answer would be a three-digit number composed of digits 1 and 2 only, e.g. 122. ( ) It would identify gain-of-function DNA ligase mutants. ( ) It would identify loss-of-function DNA ligase mutants. ( ) It would identify conditional mutants.

When a promoter p is not saturated with bound regulatory protein A, doubling the total concentration of A would …

A pool of five barcoded yeast mutants, each deleted for a different gene (genes A to E), has been grown under three different conditions (1 to 3). After the growth phase was completed, genomic DNA was isolated from the entire pool and the relative amount of each mutant was determined by quantifying the amount of DNA containing each barcode. The results are presented in the following graph. Which gene (A to E) is essential under condition 1 but not under conditions 2 or 3?