Tropomyosin, at 93 kd, sediments at 2.6S, whereas the 65-kd protein, hemoglobin

Because a monoclonal antibody recognizes a specific antigenic site (epitope), it binds only to the specific protein against which it was made.

Given the inexorable march of technology, it seems inevitable that the sensitivity of detection of molecules will ultimately be pushed beyond the yoctomole level (1024 mole).

If each cycle of PCR doubles the amount of DNA synthesized in the previous cycle, then 10 cycles will give a 103-fold amplification, 20 cycles will give a 106-fold amplification, and 30 cycles will give a 109-fold amplification.

To judge the biological importance of an interaction between protein A and protein B, we need to know quantitative details about their concentrations, affinities, and kinetic behaviors.

The rate of change in the concentration of any molecular species X is given by the balance between its rate of appearance and its rate of disappearance.

After a sudden increase in transcription, a protein with a slow rate of degradation will reach a new steady state level more quickly than a protein with a rapid rate of degradation.

A common step in the isolation of cells from a sample of animal tissue is to treat the tissue with trypsin, collagenase, and EDTA. Why is such a treatment necessary, and what does each component accomplish? And why does this treatment not kill the cells?

Tropomyosin, at 93 kd, sediments at 2.6S, whereas the 65-kd protein, hemoglobin, sediments at 4.3S. (The sedimentation coefficient S is a linear measure of the rate of sedimentation.) These two proteins are drawn to scale in Figure Q81. How is it that the bigger protein sediments more slowly than the smaller one? Can you think of an analogy from everyday experience that might help you with this problem?

Hybridoma technology allows one to generate monoclonal antibodies to virtually any protein. Why is it, then, that genetically tagging proteins with epitopes is such a commonly used technique, especially since an epitope tag has the potential to interfere with the function of the protein?

How many copies of a protein need to be present in a cell in order for it to be visible as a band on an SDS gel? Assume that you can load 100 g of cell extract onto a gel and that you can detect 10 ng in a single band by silver staining the gel. The concentration of protein in cells is about 200 mg/mL, and a typical mammalian cell has a volume of about 1000 m3 and a typical bacterium a volume of about 1 m3. Given these parameters, calculate the number of copies of a 120-kd protein that would need to be present in a mammalian cell and in a bacterium in order to give a detectable band on a gel. You might try an order-of-magnitude guess before you make the calculations.

You have isolated the proteins from two adjacent spots after two-dimensional polyacrylamide-gel electrophoresis and digested them with trypsin. When the masses of the peptides were measured by MALDI-TOF mass spectrometry, the peptides from the two proteins were found to be identical except for one (Figure Q82). For this peptide, the mass-to-charge (m/z) values differed by 80, a value that does not correspond to a difference in amino acid sequence. (For example, glutamic acid instead of valine at one position would give an m/z difference of around 30.) Can you suggest a possible difference between the two peptides that might account for the observed m/z difference?

You want to amplify the DNA between the two stretches of sequence shown in Figure Q83. Of the listed primers, choose the pair that will allow you to amplify the DNA by PCR.

In the very first round of PCR using genomic DNA, the DNA primers prime synthesis that terminates only when the cycle ends (or when a random end of DNA is encountered). Yet, by the end of 20 to 30 cyclesa typical amplificationthe only visible product is defined precisely by the ends of the DNA primers. In what cycle is a double-stranded fragment of the correct size first generated?

Explain the difference between a gain-of-function mutation and a dominant-negative mutation. Why are both these types of mutation usually dominant?

Discuss the following statement: We would have no idea today of the importance of insulin as a regulatory hormone if its absence were not associated with the human disease diabetes. It is the dramatic consequences of its absence that focused early efforts on the identification of insulin and the study of its normal role in physiology.

You have just gotten back the results from an RNAseq analysis of mRNAs from liver. You had anticipated counting the number of reads of each mRNA to determine the relative abundance of different mRNAs. But you are puzzled because many of the mRNAs have given you results like those shown in Figure Q84. How is it that different parts of an mRNA can be represented at different levels?

Examine the network motifs in Figure Q85. Decide which ones are negative feedback loops and which are positive. Explain your reasoning.

Imagine that a random perturbation positions a bistable system precisely at the boundary between two stable states (at the orange dot in Figure Q86). How would the system respond?

Detailed analysis of the regulatory region of the Lac operon has revealed surprising complexity. Instead of a single binding site for the Lac repressor, as might be expected, there are three sites termed operators: O1, O2, and O3, arrayed along the DNA as shown in Figure Q87. To probe the functions of these three sites, you make a series of constructs in which various combinations of operator sites are present. You examine their ability to repress expression of -galactosidase, using either tetrameric (wild type) or dimeric (mutant) forms of the Lac repressor. The dimeric form of the repressor can bind to a single operator (with the same affinity as the tetramer) with each monomer binding to half the site. The tetramer, the form normally expressed in cells, can bind to two sites simultaneously. When you measure repression of -galactosidase expression, you find the results shown in Figure Q87, with higher numbers indicating more effective repression. A. Which single operator site is the most important for repression? How can you tell? B. Do combinations of operator sites (Figure Q87, constructs 1, 2, 3, and 5) substantially increase repression by the dimeric repressor? Do combinations of operator sites substantially increase repression by the tetrameric repressor? If the two repressors behave differently, offer an explanation for the difference. C. The wild-type repressor binds O3 very weakly when it is by itself on a segment of DNA. However, if O1 is included on the same segment of DNA, the repressor binds O3 quite well. How can that be?