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Modern Genetic Analysis Chapter 10. Recombinant DNA Technology
Problems

1.

A circular bacterial plasmid (pBP1) has a single HindIII restriction-enzyme site in the middle of a tetracycline-resistance gene (tetR). Fruit fly genomic DNA is digested with HindIII, and a library is made in pBP1. Probing reveals that clone 15 contains a specific Drosophila gene of interest. Clone 15 is studied by restriction analysis with HindIII and another restriction enzyme, EcoRV. The ethidium bromidestained electrophoretic gel shows bands as in the accompanying diagram (the control was an uninserted plasmid pBP1). The sizes of the bands (in kilobases) are shown alongside. (note: Circular molecules do not give intense bands on this type of gel, so you can assume that all bands represent linear molecules.)

a. Draw restriction maps for plasmid pBP1 with and without the insert, showing the sites of the target sequences and the approximate position of the tetR gene.
b. If the same tetR gene cloned in a completely nonhomologous vector is made radioactive and used as a probe in a Southern blot of this gel, which bands do you expect to appear radioactive on an autoradiogram?
c. If the same gene of interest from a fly closely related to Drosophila has been cloned in a nonhomologous vector and this clone is used as a probe for the same gel, what bands do you expect to see on the autoradiogram?
Unpacking the Problem
a. Which plasmid described in this chapter seems closest in type to pBP1?
b. What is the importance of the single HindIII restriction site?
c. Why is it important that the single site is in a resistance gene? Would it be useful if not?
d. What is the effect of insertion of donor DNA into the resistance gene? Is this effect important for this problem?
e. What is a library? What type was used in this experiment and does it matter for this problem?
f. What kind of probing would have shown that clone 15 contains the gene of interest? And is this relevant to the present problem?
g. What is an electrophoretic gel?
h. What function does ethidium bromide serve in this experiment?
i. Does the gel shown represent a Southern or a Northern blot or neither?
j. Generically, what types of molecules are visible on the gel?
k. How many fragments are produced if a circular molecule is cut once?
l. How many fragments are produced if a circular molecule is cut twice?
m. Can you write a simple formula relating the number of restriction-enzyme sites in a circular molecule to the number of fragments produced?
n. If one enzyme produces n fragments and another produces m fragments, how many fragments are produced if both enzymes are used?
o. In the diagram, at what positions were the DNA samples loaded into the gel?
p. Why are the smaller molecular weight fragments at the bottom of the gel?
q. What is the total molecular weight of the fragments in all the lanes? What patterns do you see?
r. Is it a coincidence that the 3- and 2-kb fragments together equal the 5-kb fragment in size?
s. Is it a coincidence that the 1.5- and 1-kb fragments together equal the 2.5-kb fragment in size?
t. If a fragment produced by one enzyme disappears when the DNA is treated with that same enzyme plus another enzyme, what does this signify?
u. What determines whether a probe will hybridize to a DNA blot (denatured)?
v. In part c, why is it stressed that a nonhomologous vector is used?

Now attempt to solve the problem.

2. | The restriction enzyme EcoRI cuts DNA at the sequence GTTAAC, and the enzyme HaeIII cuts DNA at the sequence GGCC. On average, how frequently will each enzyme cut double-stranded DNA? (In other words, what is the average spacing between restriction sites?) See answer

3. | You have a purified DNA molecule, and you wish to map restriction-enzyme sites along its length. After digestion with EcoRI, you obtain four fragments: 1, 2, 3, and 4. After digestion of each of these fragments with HindII, you find that fragment 3 yields two subfragments (31 and 32) and that fragment 2 yields three (21, 22, and 23). After digestion of the entire DNA molecule with HindII, you recover four pieces: A, B, C, and D. When these pieces are treated with EcoRI, piece D yields fragments 1 and 31, A yields 32 and 21, and B yields 23 and 4. The C piece is identical with 22. Draw a restriction map of this DNA. See answer

4.

After Drosophila DNA has been treated with a restriction enzyme, the fragments are attached to plasmids and selected as clones in E. coli. By using this "shotgun" technique, David Hogness has recovered every DNA sequence of Drosophila in a library.
a. How would you identify the clone that contains DNA coding for the protein actin, whose amino acid sequence is known?
b. How would you identify a clone coding for a specific tRNA?

5.

You have isolated and cloned a segment of DNA that is known to be a unique sequence in the genome. It maps near the tip of the X chromosome and is about 10 kb in length. You label the 5 ends with 32P and cleave the molecule with EcoRI. You obtain two fragments: one is 8.5 kb long; the other is 1.5 kb. You separate the 8.5-kb fragment into two fractions, partly digesting one with HaeIII and the other with HindII. You then separate each sample on an agarose gel. By autoradiography, you obtain the following results:

Draw a restriction-enzyme map of the complete 10-kb molecule.

See answer

6. | Calculate the average distances (in nucleotide pairs) between the restriction sites in organism X for the following restriction enzymes:

7.

A linear fragment of DNA is cleaved with the individual restriction enzymes HindIII and SmaI and then with a combination of the two enzymes. The fragments obtained are

a. Draw the restriction map.
b. The mixture of fragments produced by the combined enzymes is cleaved with the enzyme EcoRI, resulting in the loss of the 3-kb fragment (band stained with ethidium bromide on an agarose gel) and the appearance of a band stained with ethidium bromide representing a 1.5-kb fragment. Mark the EcoRI cleavage site on the restriction map.

(Problem 7 courtesy of Joan McPherson. From A. J. F. Griffiths and J. McPherson, 100+ Principles of Genetics. W. H. Freeman and Company, 1989.)

See answer

8.

A viral DNA fragment carrying a specific gene V is introduced into a muscle cell culture by transformation. After incubation with 32P-labeled ribonucleotides, the virus-encoded RNA product is isolated at two timed intervals. The radiolabeled viral RNA is treated as follows. First, it is hybridized to a specific cDNA previously constructed from viral-gene-V mature mRNA. Second, the hybrid is treated with RNase. Finally, the hybrid is denatured and electrophoresed on a gel, which is then subjected to autoradiography. The following results suggest that the pathologic nature of the virus is time-related (the number of nucleotides is indicated on the bands observed):

a. What is the size of the mature mRNA for gene V?
b. Draw a diagram of each hybrid and indicate what the illustrated bands represent.
c. Why is protein V not produced until after 2 hours?

(Problem 8 courtesy of Joan McPherson. From A. J. F. Griffiths and J. McPherson, 100+ Principles of Genetics. W. H. Freeman and Company, 1989.)

9.

The gene for b -tubulin has been cloned from Neurospora and is available. List a step-by-step procedure for cloning the same gene from the related fungus Podospora, using as the cloning vector the pBR E. coli plasmid shown here, where kan = kanamycin and tet = tetracycline:

See answer

10.

A circular bacterial plasmid containing a gene for tetracycline resistance was cut with restriction enzyme BglII. Electrophoresis showed one band of 14 kb.
a. What can be deduced from this result?

The plasmid was cut with EcoRV and electrophoresis produced two bands, of 2.5 and 11.5 kb.
b. What can be deduced from this result?

Digestion with both enzymes together resulted in three bands of 2.5, 5.5, and 6 kb.
c. What can be deduced from this result?

Plasmid DNA cut with BglII was mixed and ligated with donor DNA fragments also cut with BglII, to make recombinant DNA molecules. All recombinant clones proved to be tetracycline sensitive.
d. What can be deduced from this result?

One recombinant clone was cut with BglII, and fragments of 4 and 14 kb were observed.
e. Explain this result.

The same clone was treated with EcoRV and fragments of 2.5, 7, and 8.5 were observed.
f. Explain these results by showing a restriction map of the recombinant DNA.

See answer

11.

a.A fragment of mouse DNA with EcoRI sticky ends carries the gene M. This DNA fragment, which is 8 kb long, is inserted into the bacterial plasmid pBR322 at the EcoRI site. The recombinant plasmid is cut with three different restrictionenzyme treatments. The patterns of ethidium bromide fragments, after electrophoresis on agarose gels, are shown in this diagram:

A Southern blot is prepared from gel iii. Which fragments will hybridize to a probe (32P) of pBR plasmid DNA?
b. Gene X is carried on a plasmid consisting of 5300 nucleotide pairs (5300 bp). Cleavage of the plasmid with the restriction enzyme BamHI gives fragments 1, 2, and 3, as indicated in the following diagram. (B = BamHI restriction site). Tandem copies of gene X are contained within a single BamHI fragment. If gene X encodes a protein X of 400 amino acids, indicate the approximate positions and orientations of the gene X copies.

(Problem 11 courtesy of Joan McPherson. From A. J. F. Griffiths and J. McPherson, 100+ Principles of Genetics. W. H. Freeman and Company, 1989.)

12.

In functional complementation in both prokaryotes and eukaryotes, prototrophy is often the phenotype selected to detect transformants. Prototrophic cells are used for donor DNA extraction; then this DNA is cloned and the clones are added to an auxotrophic recipient culture. Successful transformants are identified by plating the recipient culture on minimal medium and looking for colonies. What experimental design would you use to make sure that a colony that you hope is a transformant is not, in fact,
a. a prototrophic cell that has entered the recipient culture as a contaminant?
b. a revertant of the auxotrophic mutation?

13.

In any particular transformed eukaryotic cell (say, of Neurospora), how could you tell if the transforming DNA (carried on a circular bacterial vector)
a. replaced the resident gene of the recipient by double crossing-over or single crossing-over?
b. was inserted ectopically?

14. | In an electrophoretic gel across which is applied a powerful electric alternating pulsed field, the DNA of the haploid fungus Neurospora crassa (n = 7) moves slowly but eventually forms seven bands, which represent DNA fractions that are of different sizes and hence have moved at different speeds. These bands are presumed to be the seven chromosomes. How would you show which band corresponds to which chromosome?

15.

A linear phage chromosome is labeled at both ends with 32P and digested with restriction enzymes. EcoRI produces fragments of sizes 2.9, 4.5, 6.2, 7.4, and 8.0 kb. An autoradiogram developed from a Southern blot of this digest shows radioactivity associated with the 6.2 and 8.0 fragments. BamHI cleaves the same molecule into fragments of sizes 6.0, 10.1, and 12.9, and the label is found associated with the 6.0 and 10.1 fragments. When EcoRI and BamHI are used together, fragments of sizes 1.0, 2.0, 2.9, 3.5, 6.0, 6.2, and 7.4 kb are produced.
a. Draw a restriction-enzyme target-site map of this molecule, showing relative positions and distances apart.
b. A radioactive probe made from a cloned phage gene X is added to Southern blots of singleenzyme digests of phage DNA. The autoradiograms show hybridization associated with the 4.5-, 10.1-, and 12.9-kb fragments. Draw in the approximate location of gene X on the restriction map.

(Problem 15 courtesy of Joan McPherson. From A. J. F. Griffiths and J. McPherson, 100+ Principles of Genetics. W. H. Freeman and Company, 1989.)

See answer

16. | The cDNA clone and genomic clone for a phosphatase enzyme have been isolated. From the following data, the structural characteristics of the gene and its transcript can be determined:

cDNA Map

The fragment of cDNA was excised from a plasmid and end labeled with 32P.

Combined digest with HaeIII and TaqI enzymes

Electrophoretic fragments stained with ethidium bromide:

Gel from the combined digestion exposed to X-ray film; that is, the autoradiogram pattern:

Digestion with TaqI enzyme alone

a. Determine the cDNA map.
Genomic DNA Map

The fragment of genomic DNA was cleaved from a l -phage clone with EcoRI enzyme, and its restriction characteristics were determined. The fragment was end labeled and digested.

Complete BamHI digest

b. Draw the genomic map, marking the restriction sites.
c. A labeled cDNA probe hybridized to the 3.4- and 2.2-kb genomic fragments. The 1.2-kb TaqI fragment hybridized to the 3.4-kb genomic fragment. If the phosphatase gene is a single-copy gene, to which genomic fragment might the 1.1-kb TaqI fragment hybridize?
d. Which part of the gene does the 3.0-kb genomic fragment represent?

(Problem 16 courtesy of Joan McPherson. From A. J. F. Griffiths and J. McPherson, 100+ Principles of Genetics. W. H. Freeman and Company, 1989.)

17.

a -Interferon is encoded by a gene that does not contain introns. The BamHI restriction fragment containing the complete gene can be identified on a Southern blot by hybridization to a specific interferon cDNA probe (32P), which, under the conditions of hybridization, will detect only interferon sequences.

To determine the cause of unknown immune deficiencies, blood samples from patients and unaffected people are screened for the a -interferon gene and its expression. The following drawing represents autoradiograms for Southern and Northern blots and a Western blot probed by antibody that recognizes a -interferon sequences. Persons 1 and 2 are normal for immune capacity; persons 3, 4, and 5 have immune deficiencies.

a. Which persons are homozygous for the a -interferon gene?
b. What do you think are the causes of the immune deficiencies of persons 3, 4, and 5? Describe the type of gene mutation for each person.

(Problem 17 courtesy of Joan McPherson. From A. J. F. Griffiths and J. McPherson, 100+ Principles of Genetics. W. H. Freeman and Company, 1989.)

18. | A fragment of human genomic DNA for gene P is excised from a l vector by using HindIII, and the ends of the gene fragment (GP) are labeled with 32P. The fragment is initially digested with Bam, giving two fragments, of 1.0 kb and 10 kb. The 10-kb fragment is partly digested with HpaII and the products are electrophoresed, giving the following results:

Autoradiogram of

HpaII Digest of GP Fragment

a. Map the genomic fragment, indicating the Bam site and the HpaII sites.

The cDNA for gene P can be cut by HpaII into only two fragments. Both are labeled, and each is used as a 32P probe to investigate fragments from a complete digest of GP by using HpaII and Bam enzymes, on Southern blots.

Southern Blots of Complete Digest

b. Draw a diagram comparing genomic DNA with cDNA. Indicate the restriction sites. Explain the differences.
c. Mark the HpaII site on the cDNA and the orientation of the two Hpa fragments.

(Problem 18 courtesy of Joan McPherson. From A. J. F. Griffiths and J. McPherson, 100+ Principles of Genetics. W. H. Freeman and Company, 1989.)

19. | Two children are investigated for the expression of a gene (D) that encodes an important enzyme for muscle development. The results of the studies of the gene and its product follow.

Child 1

For child 2, the enzyme activity of each stage was very low and could be estimated only at approximately 0.1 unit at ages 1, 2, 3, and 4.

Child 2

a. For both children, draw graphs representing the developmental expression of the gene. (Fully label both axes.)
b. How can you explain the very low levels of active enzyme for child 2? (Protein degradation is only one possibility.)
c. How might you explain the change in the Southern blot for child 2 compared with that for child 1?
d. If only one mutant gene has been detected in family studies of the two children, define the individual children as either homozygous or heterozygous for gene D.

(Problem 19 courtesy of Joan McPherson. From A. J. F. Griffiths and J. McPherson, 100+ Principles of Genetics. W. H. Freeman and Company, 1989.)

20.

A cloned fragment of DNA was sequenced by using the dideoxy method. A part of the autoradiogram of the sequencing gel is represented here.

a. Deduce the nucleotide sequence of the DNA nucleotide chain synthesized from the primer. Label the 5 and 3 ends.
b. Deduce the nucleotide sequence of the DNA nucleotide chain used as the template strand. Label the 5 and 3 ends.
c. Write out the nucleotide sequence of the DNA double helix (label the 5 and 3 ends).
d. How many of the six reading frames are "open" as far as you can tell?

21. | The cDNA clone for the human gene encoding tyrosinase was used in a Southern analysis of EcoR1-digested genomic DNA of wild-type mice. Three mouse fragments were found to be radioactive (were bound by the probe). Albino mice were used in a Southern analysis, but, in this case, no genomic fragments bound to the probe. Explain these results in relation to the nature of the wild-type and mutant mouse alleles.

22. | The protein encoded by the alkaptonuria gene is 445 amino acids long, yet the gene spans 60 kb (see Genetics in Process 10-1). How is this possible?

© 1999 by W. H. Freeman and Company. All rights reserved.