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Modern Genetic Analysis Chapter 11. Applications of Recombinant DNA Technology
Problems

1. | Transgenic tobacco plants were obtained in which the vector Ti plasmid was designed to insert the gene of interest plus an adjacent kanamycinresistance gene. The inheritance of chromosomal insertion was followed by testing progeny for kanamycin resistance. Two plants typified the results obtained generally. When plant 1 was backcrossed with wild-type tobacco, 50 percent of the progeny were kanamycin resistant and 50 percent were sensitive. When plant 2 was backcrossed with the wild type, 75 percent of the progeny were kanamycin resistant, and 25 percent were sensitive. What must have been the difference between the two transgenic plants? What would you predict about the situation regarding the gene of interest?

2.

In Neurospora, which has seven chromosomes, the following chromosomal rearrangements were obtained in different strains:
a. A paracentric inversion of chromosome 1 (the largest chromosome)
b. A pericentric inversion of chromosome 1
c. A reciprocal translocation in which about half of chromosome 1 was exchanged with about half of chromosome 7 (the smallest chromosome)
d. A unidirectional insertional translocation, in which a part of one chromosome was inserted into another
e. A disomic (n + 1)
f. A monosomic (2 n- 1)
g. A tandem duplication of a large part of chromosome 1

From all these strains and a normal wild type, DNA was isolated carefully to prevent mechanical breakage, and the samples were subjected to pulsed field gel electrophoresis. Predict the bands that you would expect to see in each case.

See answer

3. | In a bacterial vector, you have cloned a plant gene that codes for a photosynthesis protein. Now you wish to find out if this gene is active in roots and other nonphotosynthetic tissue. How would you go about doing this? Describe the experimental details as well as you can.

4. | In Neurospora, you have two chromosome 5 probes that detect RFLP loci approximately 20 m.u. apart. In Southern blots of PstI digest of strain 1, probe A picks up two fragments of 1 and 2 kb, and probe B picks up two fragments of 4 and 1.5 kb. In Southern blots of strain 2, probe A detects one band of 3 kb, and probe B detects one band of 5.5 kb. Draw the Southern banding patterns that you would see in PstI digests of individual ascospore cultures from asci, using both probes simultaneously. Be sure to state how many different ascus types you expect, and be sure to account for the occurrence of crossovers.

5. | A cystic fibrosis mutation in a certain pedigree is due to a single nucleotide-pair change. This change destroys an EcoRI restriction site normally found in this position. How would you use this information in counseling members of this family about their likelihood of being carriers? State the precise experiments needed. Assume that you detect that a woman in this family is a carrier, and it transpires that she is married to an unrelated man who is also a heterozygote for cystic fibrosis, but, in his case, it is a different mutation in the same gene. How would you counsel this couple about the risks of a child's having cystic fibrosis?

6. | In yeast, you have sequenced a piece of wild-type DNA and it clearly contains a gene, but you do not know what gene it is. Therefore, to investigate further, you would like to find out its mutant phenotype. How would you use the cloned wild-type gene to do this? Show you experimental steps clearly. See answer

7. | How would you use pulsed field gel electrophoresis to find out what chromosome a cloned gene is on? See answer

8. | Bacterial glucuronidase converts a colorless substance called X-Gluc into a bright-blue indigo pigment. The gene for glucuronidase also works in plants if given a plant promoter region. How would you use this gene as a reporter gene to find out in which tissues a plant gene that you have just cloned is normally active? (Assume X-Gluc is easily taken up by the plant tissues.)

9.

In mouse HindIII restriction digests, a certain probe picks a simple RFLP consisting of two alternative alleles of 1.7 kb and 3.8 kb. A mouse heterozygous for a dominant allele for bent tail and heterozygous for the just described RFLP is mated with a wild-type mouse that shows only the 3.8-kb fragment. Forty percent of the bent-tail progeny are homozygous for the 3.8-kb allele, and 60 percent are heterozygous for the 3.8- and the 1.7-kb forms.
a. Is the bent-tail locus linked to the RFLP locus? Draw the parental and progeny chromosomes to illustrate your answer.
b. What RFLP types do you predict among the wild-type offspring, and in what proportions?

See answer

10.

Probes A and B are used to detect RFLPs in a study of two different haploid populations of yeast. In population 1, gene A gives morph A1 and gene B gives morph B1; in population 2, gene A gives A2 and gene B gives B2. These morphs are distinguished by the HindIII fragments bound by the probe (size is in kilobases).

Meiotic products are examined, and the DNA fragments hybridizing to probes A and B are given for each type:

a. How were the different spore DNAs produced?
b. Draw the appropriate chromosomal region(s).

(Problem 10 courtesy of Joan McPherson.)

See answer

11.

The plant Arabidopsis thaliana was transformed by using the Ti plasmid into which a kanamycinresistance gene had been inserted in the T-DNA region. Two kanamycin-resistant colonies (A and B) were selected, and plants were regenerated from them. The plants were allowed to self, and the results were as follows:

a. Draw the relevant plant chromosomes in both plants.
b. Explain the two different ratios.

12.

Two different circular yeast plasmid vectors (YP1 and YP2) were used to transform leu cells into leu+. The resulting leu+ cultures from both experiments were crossed with the same leu cell of opposite mating type. Typical results were as follows:

a. Explain the different action of these two plasmids during transformation.
b. If total DNA is extracted from transformants from YP1 and YP2 and digested with an enzyme that cuts once within the vector (and not the insert), predict the results of electrophoresis and Southern analyses of the DNA; use the specific plasmid as a probe in each case.
See answer

13.

A linear 9-kb Neurospora plasmid mar1 seems to be a neutral passenger, causing no apparent harm to its host. It has a restriction map as follows:

However, it was suspected that this plasmid sometimes integrates into genomic DNA. To test this idea, the central large BglII fragment was cloned into a pUC vector, and this was used as a probe in a Southern analysis of XbaI-digested genomic DNA from a mar1-containing strain. Predict what the autoradiogram would look like if
a. the plasmid never integrates.
b. the plasmid occasionally integrates into genomic DNA.

14.

The pedigree below shows a man affected with dominantly inherited Huntington disease. Both of his at-risk children have elected prenatal diagnosis, although they do not wish their status to be evaluated. The Southern blot has been typed for a two-allele system (with allelic fragments at 4.9 kb or 3.7 and 1.2 kb) that shows 4 percent recombination with Huntington disease. For each of the two fetal samples tested, calculate the chance that the fetus will inherit the allele for Huntington.

(Problem 14 from Steve Wood.)

15.

A Neurospora geneticist is interested in the genes that control hyphal extension. He decides to clone a sample of these genes. It is known from previous mutational analysis of Neurospora that a common type of mutant has a small-colony ("colonial") phenotype on plates, caused by abnormal hyphal extension. Therefore, he decides to do a tagging experiment by using transforming DNA to produce colonial mutants by insertional mutagenesis. He transforms Neurospora cells by using a bacterial plasmid carrying a gene for benomyl resistance (ben-R) and recovers resistant colonies on benomyl-containing medium. Some colonies show the colonial phenotype being sought, so he isolates and tests a sample of these colonies. The colonial isolates prove to be of two types as follows:

a. Explain the difference between these two types of results.
b. Which type should he use to try to clone the genes affecting hyphal extension?
c. How should he proceed with the tagging protocol?
d. If a probe specific for the bacterial plasmid is available, which progeny should be hybridized by this probe?

Unpacking the Problem

a. What is hyphal extension, and why do you think anyone would find it interesting?
b. How does the general approach of this experiment fit in with the general genetic approach of mutational dissection?
c. Is Neurospora haploid or diploid, and is it relevant to the problem?
d. Is it appropriate to transform a fungus (a eukaryote) with a bacterial plasmid? Does it matter?
e. What is transformation, and in what way is it useful in molecular genetics?
f. How are cells prepared for transformation?
g. What is the fate of transforming DNA if successful transformation occurs?
h. Draw a successful plasmid entry into the host cell, and draw a representation of a successful stable transformation.
i. Does it make any difference to the protocol to know what benomyl is? What role is the benomyl-resistance gene playing in this experiment? Would the experiment work with another resistance marker?
j. What does the word colonial mean in the present context? Why did the experimenter think that finding and characterizing colonial mutations would help in understanding hyphal extension?
k. What kind of "previous mutational studies" do you think are being referred to?
l. Draw the appearance of a typical petri plate after transformation and selection. Pay attention to the appearance of the colonies.
m. What is tagging? How does it relate to insertional mutagenesis? How does insertion cause mutation?
n. How are crosses made and progeny isolated in Neurospora?
o. Is recombination relevant to this question? Can an RF value be calculated? What does it mean?
p. Why are there only two colonial types? Is it possible to predict which type will be more common?
q. What is a probe? How are probes useful in molecular genetics? How would the probing experiment be done?
r. How would you obtain a probe specific to the bacterial plasmid?

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