Basic Exercises

1. Arrange the following events in chronological order, beginning

with the earliest: (a) splicing of an RNA molecule,
(b) migration of an mRNA molecule into the cytoplasm,
(c) transcription of a gene, (d) degradation of an mRNA
molecule, (e) polypeptide synthesis.
Answer: c-a-b-e-d.
2. What factor induces the expression of the hsp70 gene in
Drosophila?
Answer: The hsp70 gene is induced by heat stress.
3. Indicate whether each of the following phenomena related
to the regulation of gene expression occurs in the nucleus
or the cytoplasm of a eukaryotic cell.
(a) Stimulation of gene expression by a transcription factor.
(b) Alternate splicing of the primary transcript of a gene.
(c) Polyadenylation of a genes primary transcript.
(d) Translation of a messenger RNA.
(e) Inhibition of translation by a microRNA binding to a
messenger RNA.
(f) Degradation of a messenger RNA induced by a short
interfering RNA.
(g) Binding of a peptide hormone to its receptor.
(h) Binding of a steroid hormone to its receptor.
(i) Silencing of gene expression by heterochromatin.
(j) Whole chromosome inactivation.

Answer: Item (h) may take place in the cytoplasm or the nucleus,
depending on the particular steroid hormone. Items
(a), (b), (c), (i), and (j) take place in the nucleus. All other
items take place in the cytoplasm.
4. What are some differences between euchromatin and
heterochromatin?
Answer: Heterochromatin stains darkly throughout the cell
cycle; euchromatin does not stain darkly during interphase.
Heterochromatin is rich in repeated DNA sequences and in
transposable elements; euchromatin may contain repeated
sequences and transposons, but usually not to the extent that
heterochromatin does. Heterochromatin has few proteincoding
genes; euchromatin has many protein-coding genes.
5. Indicate whether the following are associated with gene
activity or inactivity: (a) DNA methylation, (b) histone acetylation,
(c) histone methylation, (d) heterochromatin, (e) locus
control region, (f ) GAL4 protein, (g) DNase I sensitivity.
Answer: (a) inactivity, (b) activity, (c) inactivity, (d) inactivity,
(e) activity, (f ) activity, (g) activity.
6. How is the level of X-linked gene expression equalized in
the two sexes of (a) humans, (b) fl ies, (c) worms?
Answer: (a) In humans, one of the two X chromosomes in
females is randomly inactivated. (b) In fl ies, the single X
chromosome in males is hyperactivated. (c) In worms, the
two X chromosomes in hermaphrodites are hypoactivated.

Testing Your Knowledge

1. The bacterial lacZ gene for _-galactosidase was inserted into
a transposable P element from Drosophila (Chapter 17) so
that it could be transcribed from the P element promoter.
This fusion gene was then injected into the germ line of
a Drosophila embryo along with an enzyme that catalyzes
the transposition of P elements. During development, the
modifi ed P element became inserted into the chromosomes
of some of the germ-line cells. Progeny from this
injected animal were then individually mated to fl ies from
a standard laboratory stock to establish strains that carried
the P/lacZ fusion gene in their genomes. Three of these
strains were analyzed for lacZ expression by staining dissected
tissues from adult fl ies with X-gal, a chromogenic
substrate that turns blue in the presence of _-galactosidase.
In the fi rst strain, only the eyes stained blue, in the second,
only the intestines stained blue, and in the third, all the tissues
stained blue. How do you explain these results?
Answer: The three strains evidently carried different insertions
of the P/lacZ fusion gene (see accompanying diagram).
In each strain, the expression of the P/lacZ fusion gene
must have come under the infl uence of a different regulatory
sequence, or enhancer, capable of interacting with
the P promoter and initiating transcription into the lacZ
gene. In the fi rst strain, the modifi ed P element must have
inserted near an eye-specifi c enhancer, which would drive

transcription only in eye tissue. In the second strain, it must

have inserted near an enhancer that drives transcription
in the intestinal cells, and in the third strain, it must have
inserted near an enhancer that drives transcription in all,
or nearly all, cells, regardless of tissue affi liation. Presumably
each of these different enhancers lies near a gene that
would normally be expressed under its control. For example,
the eye-specifi c enhancer would be near a gene needed
for some aspect of eye function or development. These
results show that random insertions of the P/lacZ fusion
gene can be used to identify different types of enhancers
and, through them, the genes they control. These fusion
gene insertions are therefore often called enhancer traps.
2. In their seminal paper on RNA interference, Andrew Fire
and coworkers (1998 Nature 391: 806811) describe the
results of experiments in which RNA derived from the
mex-3 gene was injected into C. elegans hermaphrodites.
Embryos obtained from these injected hermaphrodites
were analyzed by in situ hybridization using probes
for mex-3 RNA. The probes were designed to bind to
mex-3 messenger RNA, which normally accumulates in
the gonads of hermaphrodites and in their embryos. Binding
of the probe molecules to mRNA in the embryos is
easily detected if the probe molecules have been labeled.
When Fire and his colleagues performed these in situ

hybridization experiments, they found that embryos from

worms that had been injected with double-stranded mex-3
RNA were not labeled by the probe molecules, whereas
embryos from worms that had been injected with singlestranded
RNA complementary to mex-3 mRNAthat is, with
antisense mex-3 RNAwere labeled, though not quite
as intensively as embryos from worms that had not been
injected at all. What do these results indicate about the
effi cacy of double-stranded versus single-stranded antisense
RNA to silence gene expression?
Answer: The results of these in situ hybridization experiments
indicate that double-stranded RNA is a strong silencer
of mex-3 gene expression in C. elegans embryos. By contrast,
single-stranded antisense RNA barely has an effect
on mex-3 gene expression. The embryos from worms injected
with double-stranded mex-3 RNA did not carry any
detectable mex-3 messenger RNA. The absence of mex-3
messenger RNA in these embryos is the result of RNA
interference induced by the injected double-stranded
RNA. The embryos from worms injected with singlestranded
antisense mex-3 RNA did carry some mex-3
messenger RNA. Thus, single-stranded antisense mex-3
RNA is not as effective as double-stranded mex-3 RNA in
the induction of RNAi.
3. The patchy phenotype of tortoiseshell cats (Chapter 5)
results from random inactivation of X chromosomes in
females that are heterozygous for different alleles of an
X-linked gene for fur color; one allele leads to light-colored
fur, the other to dark-colored fur. The patchy phenotype
of gynandromorphs in Drosophila (Chapter 6)

results from nondisjunction of the X chromosomes

during one of the early cleavage divisions. If an XX
zygote is heterozygous for wild-type and mutant alleles
of the X-linked white gene, nondisjunction can produce a
lineage of XO cells that carry only the mutant allele, and
if these cells form an eye, or part of an eye, that eye tissue
will be white. By contrast, tissue derived from XX cells
will be red because those cells carry the wild-type allele
of the white gene. Is either of these patchy phenotypes an
example of epigenetic regulation of gene expression?
Explain your answer.
Answer: The patchy phenotype of tortoiseshell cats results from
an epigenetic phenomenonrandom inactivation of an X
chromosome in each of the cells destined to form pigmentproducing
cells in the adult. All the pigment-producing
cells are genetically equivalentthat is, they have the same
DNA content. The tortoiseshell phenotype is not due to
a change in the underlying genotype during the animals
embryological development. Rather, it is due to a change
in the state of one of the X chromosomes, the X that is
inactivated, and this state is inherited clonally through cell
division. Thus, the light and dark patches of fur in the cat
differ epigenetically, not genetically. In contrast, the patchy
phenotype of Drosophila gynandromorphs is due to a genetic
change that occurs during development. One of the
X chromosomes is lost. The red and white patches of tissue
in a gynandromorphs eye are not genetically equivalent.
The difference between them is therefore genetic rather
than epigenetic.
Questions and Problems
19.1 Operons are common in bacteria but not in eukaryotes.
Suggest a reason why.
19.2 In bacteria, translation of an mRNA begins before
the synthesis of that mRNA is completed. Why is this
coupling of transcription and translation not possible
in eukaryotes?
19.3 Muscular dystrophy in humans is caused by mutations
in an X-linked gene that encodes a protein called dystrophin.
What techniques could you use to determine if
this gene is active in different types of cells, say skin cells,
nerve cells, and muscle cells?
19.4 Why do steroid hormones interact with receptors inside
the cell, whereas peptide hormones interact with receptors
on the cell surface?
19.5 In the polytene chromosomes of Drosophila larvae
(Chapter 6), some bands form large puffs when the
larvae are subjected to high temperatures. How could you

show that these puffs contain genes that are vigorously

transcribed in response to this heat-shock treatment?
19.6 How would you distinguish between an enhancer and a
promoter?
19.7 Tropomyosins are proteins that mediate the interaction of
actin and troponin, two proteins involved in muscle contractions.
In higher animals, tropomyosins exist as a family
of closely related proteins that share some amino acid
sequences but differ in others. Explain how these proteins
could be created from the transcript of a single gene.
19.8 A polypeptide consists of three separate segments of
amino acids, ABC. Another polypeptide contains
segments A and C, but not segment B. How might you
determine if these two polypeptides are produced by
translating alternately spliced versions of RNA from a
single gene or by translating mRNA from two different
genes?

19.9 What techniques could be used to show that a plant gene

is transcribed when the plant is illuminated with light?
19.10 When introns were fi rst discovered, they were thought
to be genetic junkthat is, sequences without any useful
function. In fact, they appeared to be worse than junk
because they actually interrupted the coding sequences
of genes. However, among eukaryotes, introns are pervasive
and anything that is pervasive in biology usually
has a function. What function might introns have? What
benefi t might they confer on an organism?
19.11 The GAL4 transcription factor in yeast regulates two
adjacent genes, GAL1 and GAL10, by binding to DNA
sequences between them. These two genes are transcribed
in opposite directions on the chromosome, one to the left
of the GAL4 proteins binding site and the other to the
right of this site. What property of enhancers does this
situation illustrate?
19.12 Using the techniques of genetic engineering, a researcher
has constructed a fusion gene containing the
heat-shock response elements from a Drosophila hsp70
gene and the coding region of a jellyfi sh gene (gfp) for
green fl uorescent protein. This fusion gene has been
inserted into the chromosomes of living Drosophila by
the technique of transposon-mediated transformation
(Chapter 17). Under what conditions will the green fl uorescent
protein be synthesized in these genetically transformed
fl ies? Explain.
19.13 Suppose that the segment of the hsp70 gene that was used
to make the hsp70/g fp fusion in the preceding problem
had mutations in each of its heat-shock response elements.
Would the green fl uorescent protein encoded by this fusion
gene be synthesized in genetically transformed fl ies?
19.14 The polypeptide products of two different genes, A and B,
each function as transcription factors. These polypeptides
interact to form dimers: AA homodimers, BB homodimers,
and AB heterodimers. If the A and B polypeptides are
equally abundant in cells, and if dimer formation is
random, what is the expected ratio of homodimers to
heterodimers in these cells?
19.15 A particular transcription factor binds to enhancers in
40 different genes. Predict the phenotype of individuals
homozygous for a frameshift mutation in the coding sequence
of the gene that specifi es this transcription factor.
19.16 The alternately spliced forms of the RNA from the
Drosophila doublesex gene encode proteins that are needed
to block the development of one or the other set of sexual
characteristics. The protein that is made in female animals
blocks the development of male characteristics, and
the protein that is made in male animals blocks the development
of female characteristics. Predict the phenotype
of XX and XY animals homozygous for a null mutation in
the doublesex gene.
19.17 The RNA from the Drosophila Sex-lethal (Sxl) gene is
alternately spliced. In males, the sequence of the mRNA

derived from the primary transcript contains all eight

exons of the Sxl gene. In females, the mRNA contains
only seven of the exons because during splicing exon 3 is
removed from the primary transcript along with its fl anking
introns. The coding region in the females mRNA is
therefore shorter than it is in the males mRNA. However,
the protein encoded by the females mRNA is longer
than the one encoded by the males mRNA. How might
you explain this paradox?
19.18 In Drosophila, expression of the yellow gene is needed
for the formation of dark pigment in many different
tissues; without this expression, a tissue appears yellow
in color. In the wings, the expression of the yellow gene
is controlled by an enhancer located upstream of the
genes transcription initiation site. In the tarsal claws,
expression is controlled by an enhancer located within
the genes only intron. Suppose that by genetic engineering,
the wing enhancer is placed within the intron and
the claw enhancer is placed upstream of the transcription
initiation site. Would a fl y that carried this modifi ed
yellow gene in place of its natural yellow gene have darkly
pigmented wings and claws? Explain.
19.19 A researcher suspects that a 550-bp-long intron contains
an enhancer that drives expression of an Arabidopsis gene
specifi cally in root-tip tissue. Outline an experiment to
test this hypothesis.
19.20 What is the nature of each of the following classes of
enzymes? What does each type of enzyme do to
chromatin? (a) HATs, (b) HDACs, (c) HMTs.
19.21 In Drosophila larvae, the single X chromosome in males
appears diffuse and bloated in the polytene cells of the
salivary gland. Is this observation compatible with the
idea that X-linked genes are hyperactivated in Drosophila
males?
19.22 Suppose that the LCR of the _-globin gene cluster was
deleted from one of the two chromosomes 11 in a man.
What disease might this deletion cause?
19.23 Would double-stranded RNA derived from an intron be
able to induce RNA interference?
19.24 RNA interference has been implicated in the regulation
of transposable elements. In Drosophila, two of the key
proteins involved in RNA interference are encoded by the
genes aubergine and piwi. Flies that are homozygous for
mutant alleles of these genes are lethal or sterile, but fl ies
that are heterozygous for them are viable and fertile. Suppose
that you have strains of Drosophila that are heterozygous
for aubergine or piwi mutant alleles. Why might the
genomic mutation rate in these mutant strains be greater
than the genomic mutation rate in a wild-type strain?
19.25 Suppose female mice homozygous for the a allele of the
Igf2 gene are crossed to male mice homozygous for the
b allele of this gene. Which of these two alleles will be
expressed in the F1 progeny?

19.26 Epigenetic states are transmitted clonally through cell

division. What kinds of observations indicate that these
states can be reversed or reset?
19.27 A researcher hypothesizes that in mice gene A is actively
transcribed in liver cells, whereas gene B is actively transcribed
in brain cells. Describe procedures that would
allow the researcher to test this hypothesis.
19.28 Suppose that the hypothesis mentioned in the previous
question is correct and that gene A is actively transcribed
in liver cells whereas gene B is actively transcribed
in brain cells. The researcher now extracts equivalent
amounts of chromatin from liver and brain tissues and
treats these extracts separately with DNase I for a limited
period of time. If the DNA that remains after the
treatment is then fractionated by gel electrophoresis, transferred
to a membrane by Southern blotting, and hybridized
with a radioactively labeled probe specifi c for gene A,
which sample (liver or brain) will be expected to show the
greater signal on the autoradiogram? Explain your answer.
19.29 Why do null mutations in the msl gene in Drosophila have
no effect in females?
19.30 Suppose that a woman carries an X chromosome in which
the XIST locus has been deleted. The womans other X
chromosome has an intact XIST locus. What pattern

of X-inactivation would be observed throughout the

womans body?
19.31 In Drosophila, the variegated phenotype of the white
mottled allele is suppressed by a dominant autosomal
mutation that knocks out the function of the gene for
heterochromatin protein 1 (HP1), an important factor
in heterochromatin formation. Flies with the white mottled
allele and the suppressor mutation have an almost
uniform red color in their eyes; without the suppressor
mutation, the eyes are mosaics of red and white tissue.
Can you suggest an explanation for the effect of the
suppressor mutation?
19.32 The sheep Dolly (Chapter 2) was the fi rst cloned
mammal. Dolly was created by implanting a nucleus
from a cell taken from the udder of a female sheep into an
enucleated egg. This nucleus had two X chromosomes,
and because it came from a differentiated cell, one of
them must have been inactivated. If the udder cell was
heterozygous for at least one X-linked gene whose
expression you could assay, how could you determine
if all of Dollys cells had the same X chromosome
inactivated? If, upon testing, Dollys cells prove to
be mosaic for X chromosome activitythat is, different
Xs are active in different clones of cellswhat must have
happened during her embryological development

Genomics on the Web at

http://www.ncbi.nlm.nih.gov
The human _-globin genes are located in a cluster on the short
arm of chromosome 11.
1. Search for the namesake gene of the cluster, the adult
_-globin gene, in the human genome database. What is the
offi cial symbol of this gene? How many exons does it contain?
2. Use the Map Viewer function to locate the _-globin gene
cluster on the ideogram of chromosome 11. In what cytological
band does it reside? Is it closer to the telomere of the short
arm or to the centromere?
3. Use the Sequence Viewer to inspect the adult _-globin gene
in detail. Is the gene transcribed toward the centromere or
toward the telomere? How long is the transcript of the gene?
How long is the mature mRNA? How many amino acids does
the mRNA specify? What are the fi rst three amino acids, and
what codons specify them?

4. Bring up the text sequence of the adult _-globin gene by

clicking the ATGC button on the Sequence Viewer page.
Locate the initiation codon for methionine in the fi rst exon.
Because the sequence in the window is that of the template
strand of the DNA, this codon reads 5_-CAT-3_ from left to
right on the screen.
5. GATA1 and MyoD are two transcription factors that
recognize short sequences in mammalian genomes. The
sequence recognized by GATA1 is 5_-TGATAG-3_, and
the sequence recognized by MyoD is 5_-CAAATG-3_.
Copy the sequence of the transcribed portion of the adult
_-globin gene into a text fi le and scan it for each of these
recognition sequences. Where are they located? Which
of these two transcription factors might be involved in
regulating the expression of the adult _-globin gene?