Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

526 Chapter 8: Analyzing Cells, Molecules, and Systems
8–9 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?
8–10 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 μg 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 μm 3 and a typical bacterium a volume
of about 1 μm 3 . 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.
8–11 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 Q8–2). 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?
abundance abundance
3706
3786
m/z (mass-to-charge ratio)
Figure Q8–2
Masses of peptides
measured by
MALDI-TOF mass
spectrometry
(Problem 8–11).
Only the numbered
peaks differ between
the two protein
samples.
(1) 5′-GACCTGTGGAAGC-3′
(2) 5′-CTGGACACCTTCG-3′
(3) 5′-CGAAGGTGTCCAG-3′
(4) 5′-GCTTCCACAGGTC-3′
DNA to be amplified
5′-GACCTGTGGAAGC
CATACGGGATTGA-3′
3′ -CTGGACACCTTCG GTATGCCCTAACT-5′
primers
(5) 5′-CATACGGGATTGA-3′
(6) 5′-GTATGCCCTAACT-3′
(7) 5′-TGTTAGGGCATAC-3′
(8) 5′-TCAATCCCGTATG-3′
Figure Q8–3 DNA to be amplified and potential PCR primers
(Problem 8–12).
8–14 Explain Problems the difference p8.21/8.17 between a gain-of-function
mutation and a dominant-negative mutation. Why are
both these types of mutation usually dominant?
8–15 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.”
8–16 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 Q8–4. How is it that different
parts of an mRNA can be represented at different
levels?
8–17 Examine the network motifs in Figure Q8–5.
Decide which ones are negative feedback loops and which
are positive. Explain your reasoning.
8–18 Imagine that a random perturbation positions a
bistable system precisely at the boundary between two stable
states (at the orange dot in Figure Q8–6). How would
the system respond?
8–12 You want to amplify the DNA between the two
stretches of sequence shown in Figure Q8–3. Of the listed
primers, choose the pair that will allow you to amplify the
DNA by PCR.
Problems p8.09/8.08
8–13 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 cycles—a typical
amplification—the 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?
reads
mRNA
exons
1 2 3 4 5
Figure Q8–4 RNA-seq reads for a liver mRNA (Problem 8–16). The
exon structure of the mRNA is indicated, with protein-coding segments
indicated in light blue and untranslated regions in dark blue. The
numbers of sequencing reads are indicated by the heights of the
vertical lines above the mRNA.