Worked Problems

1. DNA from a strain of bacteria with genotype a+ b+ c+ d+ e+ was isolated and used to transform a strain of bacteria that was a- b- c- d- e-. The transformed cells were tested for the presence of donated genes. The following genes were cotransformed:

What is the order of genes a, b, c, d, and e on the bacterial chromosome?

The rate at which genes are cotransformed is inversely proportional to the distance between them: genes that are close together are frequently cotransformed, whereas genes that are far apart are rarely cotransformed. In this transformation experiment, gene c+ is cotransformed with both genes e+ and d+, but genes e+ and d+

are not cotransformed; therefore the c locus must be between the d and e loci:

Gene e+ is also cotransformed with gene b+; so the e and b loci must be located close together. Locus b could be on either side of locus e. To determine whether locus b is on the same side of e as locus c, we look to see whether genes b+ and c+ are cotransformed. They are not; so locus b must be on the opposite side of e from c:

Gene a+ is cotransformed with gene d+; so they must be located close together. If locus a were located on the same side of d as locus c, then genes a+ and c+ would be cotransformed. Because these genes display no cotransformation, locus a must be on the opposite side of locus d:

a d c e b which are ara+ and which are leu+. Results from these experiments are as follows:

Selected marker Cells with cotransduced genes (3%) leu+ 3 thr+

76 ara+

How are the loci arranged on the chromosome?

Notice that, when we select for leu+ (the top half of the table), most of the selected cells also are ara+. This finding indicates that the leu and ara genes are located close together, because they are usually cotransduced. In contrast, thr+ is only rarely cotransduced with leu+, indicating that leu and thr are much farther apart. On the basis of these observations, we know that leu and ara are closer together than are leu and thr, but we don't yet know the order of three genes — whether thr is on the same side of ara as leu or on the opposite side, as shown here:

2. Consider three genes in E. coli: thr+ (the ability to synthesize threonine), ara+ (the ability to metabolize arabinose), and leu+ (the ability to synthesize leucine). All three of these genes are close together on the E. coli chromosome. Phages are grown in a thr+ ara+ leu+ strain of bacteria (the donor strain). The phage lysate is collected and used to infect a strain of bacteria that is thr~ ara~ leu~. The recipient bacteria are then tested on medium lacking leucine. Bacteria that grow and form colonies on this medium (leu+ transductants) are then replica plated onto medium lacking threonine and medium lacking arabinose to see which are thr+ and which are ara+.

Another group of recipient bacteria are tested on medium lacking threonine. Bacteria that grew and formed colonies on this medium (thr+ transductants) were then replica plated onto medium lacking leucine and medium lacking arabinose to see leu ara

We can determine the position of thr with respect to the other two genes by looking at the cotransduction frequencies when thr+ is selected (the bottom half of the table). Notice that, although the cotransduction frequency for thr and leu also is 3%, no thr+ ara+ cotransductants are observed. This finding indicates that thr is closer to leu than to ara, and therefore thr must be to the left of leu, as shown here:

thr leu ara

COMPREHENSION QUESTIONS]_

* 1. List some of the characteristics that make bacteria and viruses ideal organisms for many types of genetic studies.

2. Explain how auxotrophic bacteria are isolated.

3. Briefly explain the differences between F+, F", Hfr, and F' cells.

* 4. What types of matings are possible between F+, F", Hfr, and F' cells? What outcomes do these matings produce? What is the role of F factor in conjugation?

* 5. Explain how interrupted conjugation, transformation, and transduction can be used to map bacterial genes. How are these methods similar and how are they different?

6. What types of genomes do viruses have?

7. Briefly describe the differences between the lytic cycle of virulent phages and the lysogenic cycle of temperate phages.

8. Briefly explain how genes in phages are mapped.

* 9. How does specialized transduction differ from generalized transduction?

*10. Briefly explain the method used by Benzer to determine whether two different mutations occurred at the same locus.

11. What is the difference between a positive-strand RNA virus and a negative-strand RNA virus?

*12. Explain how a retrovirus, which has an RNA genome, is able to integrate its genetic material into that of a host having a DNA genome.

13. Briefly describe the genetic structure of a typical retrovirus.

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