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Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; 2000.

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An Introduction to Genetic Analysis. 7th edition.

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Problems

1. The two strands of λ phage differ from each other in their GC content. Owing to this property, they can be separated in an alkaline cesium chloride gradient (the alkalinity denatures the double helix). When RNA synthesized by λ phage is isolated from infected cells, it is found to form DNA-RNA hybrids with both strands of λ DNA. What does this tell you? Formulate some testable predictions.

Because RNA can hybridize to both strands, the RNA must be transcribed from both strands. This does not mean, however, that both strands are used as a template within each gene. The expectation is that only one strand is used within a gene but that different genes are transcribed in different directions along the DNA. The most direct test would be to purify a specific RNA coding for a specific protein and then hybridize it to the lambda genome. Only one strand should hybridize to the purified RNA.

2. The data in the following table represent the base compositions of two double-stranded DNA sources and their RNA products in experiments conducted in vitro.

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a.

From these data, can you determine whether the RNA of these species is copied from a single strand or from both strands of the DNA? How? Drawing a diagram will make it easier to solve this problem.

b.

Explain how you can tell whether the RNA itself is single stranded or double stranded.

(Problem 2 is reprinted with the permission of Macmillan Publishing Co., Inc., from M. Strickberger, Genetics. Copyright ©1968, Monroe W. Strickberger.)

3. Before the true nature of the genetic coding process was fully understood, it was proposed that the message might be read in overlapping triplets. For example, the sequence GCAUC might be read as GCA CAU AUC:

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Devise an experimental test of this idea.

A single nucleotide change should result in three adjacent amino acid changes in a protein. One amino acid change and two adjacent amino acid changes would be expected to be much rarer than the three changes. This is directly the opposite of what is observed in proteins.

4. In protein-synthesizing systems in vitro, the addition of a specific human mRNA to the E. coli translational apparatus (ribosomes, tRNA, and so forth) stimulates the synthesis of a protein very much like that specified by the mRNA. What does this result show?

5. Which anticodon would you predict for a tRNA species carrying isoleucine? Is there more than one possible answer? If so, state any alternative answers.

6. a.  In how many cases in the genetic code would you fail to know the amino acid specified by a codon if you knew only the first two nucleotides of the codon?

b.

In how many cases would you fail to know the first two nucleotides of the codon if you knew which amino acid is specified by it?

7. Deduce what the six wild-type codons may have been in the mutants that led Brenner to infer the nature of the amber codon UAG.

The codon for amber is UAG. The following list includes the amino acids that would need to have been inserted to continue the wild-type chain and their codons:

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In each case, the codon with an asterisk would require a single base change to become UAG.

8. If a polyribonucleotide contains equal amounts of randomly positioned adenine and uracil bases, what proportion of its triplets will code for (a) phenylalanine, (b) isoleucine, (c) leucine, (d) tyrosine?

(a)

1/8;

(b)

1/4;

(c)

1/8;

(d)

1/8.

9. You have synthesized three different messenger RNAs with bases incorporated in random sequence in the following ratios: (a) 1U:5C, (b) 1A:1C:4U, (c) 1A:1C:1G:1U. In a protein-synthesizing system in vitro, indicate the identities and proportions of amino acids that will be incorporated into proteins when each of these mRNAs is tested. (Refer to Figure10-27.)

10. One of the techniques used to decipher the genetic code was to synthesize polypeptides in vitro, with the use of synthetic mRNA with various repeating base sequences—for example, (AGA) n , which can be written out as AGAAGAAGAAGAAGA. . . . Sometimes the synthesized polypeptide contained just one amino acid (a homopolymer), and sometimes it contained more than one (a heteropolymer), depending on the repeating sequence used. Furthermore, sometimes different polypeptides were made from the same synthetic mRNA, suggesting that the initiation of protein synthesis in the system in vitro does not always start on the end nucleotide of the messenger. For example, from (AGA) n , three polypeptides may have been made: aa1 homopolymer (abbreviated aa1-aa1), aa2 homopolymer (aa2-aa2), and aa3 homopolymer (aa3-aa3). These probably correspond to the following readings derived by starting at different places in the sequence:

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The table below shows the actual results obtained from the experiment done by Khorana.

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[Note: The order in which the polypeptides or amino acids are listed in the table is not significant except for (UAUC) n and (UUAC) n .]

a.

Why do (GUA) n and (GAU) n each encode only two homopolypeptides?

b.

Why do (GAUA) n and (GUAA) n fail to stimulate synthesis?

c.

Assign an amino acid to each triplet in the following list. Bear in mind that there often are several codons for a single amino acid and that the first two letters in a codon usually are the important ones (but that the third letter is occasionally significant). Also remember that some very different looking codons sometimes encode the same amino acid. Try to carry out this task without consulting Figure 10-27.

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To solve this problem requires both logic and trial and error. Don’t be disheartened: Khorana received a Nobel Prize for doing it. Good luck!

(Problem 10 is from J. Kuspira and G. W. Walker, Genetics: Questions and Problems. McGraw-Hill, 1973.)

a.

(GAU) n encodes Asp (GAU), Met (AUG), and stop (UGA). (GUA) n encodes Val (GUA), Ser (AGU), and stop (UAG). One reading frame contains a stop codon.

b.

Each of the three reading frames contains a stop codon.

11. You are studying a gene in E. coli that specifies a protein. A part of its sequence is

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You recover a series of mutants for this gene that show no enzymatic activity. By isolating the mutant enzyme products, you find the following sequences:

Image ch10fb13.jpg

What is the molecular basis for each mutation? What is the DNA sequence that specifies this part of the protein?

Mutant 1: A simple substitution of Arg for Ser exists, suggesting a nucleotide change. Two codons for Arg are AGA and AGG, and one codon for Ser is AGU. The final U for Ser could have been replaced by either an A or a G.

Mutant 2: the Trp codon (UGG) changed to a stop codon (UGA or UAG).

Mutant 3: Two frameshift mutations occurred:

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Mutant 4: An inversion occurred after Trp and before Cys. The DNA original sequence was:

Image app3fb59.jpg

Therefore, the complementary RNA sequence was:

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The DNA inverted sequence became:

Image app3fb61.jpg

Therefore, the complementary RNA sequence was

Image app3fb62.jpg

12. A single nucleotide addition and a single nucleotide deletion approximately 15 sites apart in the DNA cause a protein change in sequence from

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a.

What are the old and the new mRNA nucleotide sequences? (Use Figure 10-26).

b.

Which nucleotide has been added and which has been deleted?

(Problem 12 is from W. D. Stansfield, Theory and Problems of Genetics. McGraw-Hill, 1969.)

a.

Image app3fb63.jpg

b.

Image app3fb64.jpg

13. Suppressors of frameshift mutations are now known. Propose a mechanism for their action.

14. Use Figure 10-26 to complete the following table. Assume that reading is from left to right and that the columns represent transcriptional and translational alignments.

Image ch10fb15.jpg

3′-CGT ACC ACT GCA-5′

5′-GCA TGG TGA CGT-3′

5′-GCA UGG UGA CGU-3′

3′-CGU ACC ACU GCA-5′

NH3-Ala-Trp stop Arg

15. A mutational event inserts an extra nucleotide pair into DNA. Which of the following outcomes do you expect? (1) No protein at all; (2) a protein in which one amino acid is changed; (3) a protein in which three amino acids are changed; (4) a protein in which two amino acids are changed; (5) a protein in which most amino acids after the site of the insertion are changed.

16. Consider the gene that specifies the structure of hemoglobin. Arrange the following events in the most likely sequence in which they would occur.

a.

Anemia is observed.

b.

The shape of the oxygen-binding site is altered.

c.

An incorrect codon is transcribed into hemoglobin mRNA.

d.

The ovum (female gamete) receives a high radiation dose.

e.

An incorrect codon is generated in the DNA of the hemoglobin gene.

f.

A mother (an X-ray technician) accidentally steps in front of an operating X-ray generator.

g.

A child dies.

h.

The oxygen-transport capacity of the body is severely impaired.

i.

The tRNA anticodon that lines up is one of a type that brings an unsuitable amino acid.

j.

Nucleotide-pair substitution occurs in the DNA of the gene for hemoglobin.

f, d, j, e, c, i, b, h, a, g

17. An induced cell mutant is isolated from a hamster tissue culture because of its resistance to α-amanitin (a poison derived from a fungus). Electrophoresis shows that the mutant has an altered RNA polymerase; just one electrophoretic band is in a position different from that of the wild-type polymerase. The cells are presumed to be diploid. What does this experiment tell you about ways in which to detect recessive mutants in such cells?

Cells in long-established culture lines usually are not fully diploid. For reasons that are currently unknown, adaptation to culture frequently results in both karyotypic and gene-dosage changes. Such changes can result in hemizygosity for some genes, which allows for the expression of previously hidden recessive alleles.

18. A double-stranded DNA molecule with the sequence shown here produces, in vivo, a polypeptide that is five amino acids long.

Image ch10fb16.jpg

a.

Which strand of DNA is transcribed and in which direction?

b.

Label the 5′ and the 3′ ends of each strand.

c.

If an inversion occurs between the second and third triplets from the left and right ends, respectively, and the same strand of DNA is transcribed, how long will the resultant polypeptide be?

d.

Assume that the original molecule is intact and that transcription is of the bottom strand from left to right. Give the base sequence, and label the 5′ and 3′ ends of the anticodon that inserts the fourth amino acid into the nascent polypeptide. What is this amino acid?

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By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21874

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