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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. Describe the state of the F factor in an Hfr, F+, and F strain.

An Hfr strain has the fertility factor, F, integrated into the chromosome. An F+ strain has the fertility factor free in the cytoplasm. An F strain lacks the fertility factor.

2. How does a culture of F+ cells transfer markers from the host chromosome to a recipient?

3. Draw an analogy between gene transfer and integration of the transferred gene into the recipient genome in

a.

Hfr crosses by conjugation and generalized transduction.

b.

F′ derivatives such as F′lac and specialized transduction.

a.

Hfr cells undergoing conjugation transfer host genes in a linear fashion. The genes transferred depend on both the Hfr strain and the length of time during which the transfer occurred. Therefore, a population containing several different Hfr strains will appear to have an almost random transfer of host genes. This event is similar to generalized transduction, in which the viral protein coat forms around a specific amount of DNA rather than specific genes. In generalized transduction, any gene can be transferred.

b.

F′ factors arise from improper excision of an Hfr from the bacterial chromosome. They can have only specific bacterial genes on them because the integration site is fixed for each strain. Specialized transduction resembles this event in that the viral particle integrates into a specific region of the bacterial chromosome and then, on improper excision, can take with it only specific bacterial genes. In both cases, the transferred gene exists as a second copy.

4. Why can generalized transduction transfer any gene, but specialized transduction is restricted to only a small set?

5. A microbial geneticist isolates a new mutation in E. coli and wishes to map its chromosomal location. She uses interrupted-mating experiments with Hfr strains and generalized-transduction experiments with phage P1. Explain why each technique, by itself, is insufficient for accurate mapping.

6. In E. coli, four Hfr strains donate the following markers, shown in the order donated:

Image ch7fb6.jpg

All these Hfr strains are derived from the same F+ strain. What is the order of these markers on the circular chromosome of the original F+?

Image app3fb49.jpg

The regions with the bars above or below are identical in sequence (and “close” the circular chromosome). The correct order of markers on this circular map is

Image app3fb50.jpg

7. Four E. coli strains of genotype a+b are labeled 1, 2, 3, and 4. Four strains of genotype ab+ are labeled 5, 6, 7, and 8. The two genotypes are mixed in all possible combinations and (after incubation) are plated to determine the frequency of a+b+ recombinants. The following results are obtained, where M = many recombinants, L = low numbers of recombinants, and 0 = no recombinants.

Image ch7fb7.jpg

On the basis of these results, assign a sex type (either Hfr, F+, or F) to each strain.

An F strain will respond differently to an F+ (L) or an Hfr (M) strain, whereas Hfr × Hfr, Hfr × F+, F+ × F+, and F × F will give 0. Thus strains 2, 3, and 7 are F. Strains 1 and 8 are F+, and strains 4, 5, and 6 are Hfr.

8. An Hfr strain of genotype a+b+c+dstrs is mated with a female strain of genotype abcd+strr. At various times, the culture is shaken vigorously to separate mating pairs. The cells are then plated on agar of the following three types, where nutrient A allows the growth of a cells; nutrient B, of b cells; nutrient C, of c cells; and nutrient D, of d cells (a plus indicates the presence of streptomycin and a nutrient, and a minus indicates its absence):

Image ch7fb8.jpg

a.

What donor genes are being selected on each type of agar?

b.

The table below shows the number of colonies on each type of agar for samples taken at various times after the strains are mixed. Use this information to determine the order of the genes a, b, and c.

Image ch7fb9.jpg

c.

From each of the 25-minute plates, 100 colonies are picked and transferred to a dish containing agar with all of the nutrients except D. The numbers of colonies that grow on this medium are 89 for the sample from agar type 1, 51 for the sample from agar type 2, and 8 for the sample from agar type 3. Using these data, fit gene d into the sequence of a, b, and c.

d.

At what sampling time would you expect colonies to first appear on agar containing C and streptomycin but no A or B?

(Problem 8 is from D. Freifelder, Molecular Biology and Bio-chemistry. Copyright © 1978 by W. H. Freeman and Company.)

9. You are given two strains of E. coli. The Hfr strain is arg+ala+glu+pro+leu+Ts; the F strain is argalagluproleuTr. The markers are all nutritional except T, which determines sensitivity or resist-ance to phage T1. The order of entry is as given, with arg+ entering the recipient first and Ts last. You find that the F strain dies when exposed to penicillin (pens) but the Hfr strain does not (penr). How would you locate the locus for pen on the bacterial chromosome with respect to arg, ala, glu, pro, and leu? Formulate your answer in logical, well-explained steps and draw explicit diagrams where possible.

10. A cross is made between two E. coli strains: Hfr arg+bio+leu+ × Fargbioleu. Interrupted-mating studies show that arg+ enters the recipient last, so arg+ recombinants are selected on a medium containing bio and leu only. These recombinants are tested for the presence of bio+ and leu+. The following numbers of individuals are found for each genotype:

Image ch7fb10.jpg

a.

What is the gene order?

b.

What are the map distances in recombination percentages?

a.

The gene order is arg bio leu.

b.

arg–bio: RF = 12.76 m.u.

bio–leu: RF = 2.12 m.u.

11. Linkage maps in an Hfr bacterial strain are calculated in units of minutes (the number of minutes between genes indicates the length of time it takes for the second gene to follow the first in conjugation). In making such maps, microbial geneticists assume that the bacterial chromosome is transferred from Hfr to F at a constant rate. Thus, two genes separated by 10 minutes near the origin end are assumed to be the same physical distance apart as two genes separated by 10 minutes near the F-attachment end. Suggest a critical experiment to test the validity of this assumption.

The most straightforward way would be to put an Hfr at both ends of the same sequence and measure the time of transfer between two specific genes. For example,

Image app3fb51.jpg

12. In the cross Hfr aro+arg+eryrstrs × Faroargerysstrr, the markers are transferred in the order given (with aro+ entering first), but the first three genes are very close together. Exconjugants are plated on a medium containing Str (streptomycin, to counterselect Hfr cells), Ery (erythromycin), Arg (arginine), and Aro (aromatic amino acids). The following results are obtained for 300 colonies from these plates isolated and tested for growth on various media: on Ery only, 263 strains grow; on Ery + Arg, 264 strains grow; on Ery + Aro, 290 strains grow; on Ery +  Arg + Aro, 300 strains grow.

a.

Draw up a list of genotypes, and indicate the number of individuals in each genotype.

b.

Calculate the recombination frequencies.

c.

Calculate the ratio of the size of the arg-to-aro region to the size of the ery-to-arg region.

13. A particular Hfr strain normally transmits the pro+ marker as the last one in conjugation. In a cross of this strain with an F strain, some pro+ recombinants are recovered early in the mating process. When these pro+ cells are mixed with F cells, the majority of the F cells are converted into pro+ cells that also carry the F factor. Explain these results.

The best explanation is that the integrated pro+ was incorporated onto an F′ factor that was transferred into recipients early in the mating process. These cells now carry the F factor and are able to transmit F+ in the second cross as part of the F′ factor, which still carries pro+.

14. F′ strains in E. coli are derived from Hfr strains. In some cases, these F′ strains show a high rate of integration back into the bacterial chromosome of a second strain. Furthermore, the site of integration is often the same site that the sex factor occupied in the original Hfr strain (before production of the F′ strains). Explain these results.

The high rate of integration and the preference for the same site originally occupied by the sex factor suggest that the F′ contains some homology with the original site. The source of homology could be a fragment of the sex factor or it could be the chromosomal copy of the bacterial gene (most likely).

15. You have two E. coli strains, F strr ala and Hfr strs ala+, in which the F factor is inserted close to ala+. Devise a screening test to detect strains carrying F′ ala+.

16. Five Hfr strains A through E are derived from a single F+ strain of E. coli. The following chart shows the entry times of the first five markers into an F strain when each is used in an interrupted-conjugation experiment:

Image ch7fb11.jpg

a.

Draw a map of the F+ strain, indicating the positions of all genes and their distances apart in minutes.

b.

Show the insertion point and orientation of the F plasmid in each Hfr strain.

c.

In the use of each of these Hfr strains, state which gene you would select to obtain the highest proportion of Hfr exconjugants.

17. Streptococcus pneumoniae cells of genotype strsmtl are transformed by donor DNA of genotype strrmtl+ and (in a separate experiment) by a mixture of two DNA’s with genotypes strrmtl and strsmtl+. The adjoining table shows the results.

Image ch7fb12.jpg

b.

What does the second line of the table tell you? Why?

18. A transformation experiment is performed with a donor strain that is resistant to four drugs: A, B, C, and D. The recipient is sensitive to all four drugs. The treated recipient cell population is divided up and plated on media containing various combinations of the drugs. The table below shows the results.

Image ch7fb13.jpg

a.

One of the genes is obviously quite distant from the other three, which appear to be tightly (closely) linked. Which is the distant gene?

b.

What is the probable order of the three tightly linked genes?

(Problem 18 is from Franklin Stahl, The Mechanics of Inheritance, 2d ed. Copyright © 1969, Prentice Hall, Englewood Cliffs, New Jersey. Reprinted by permission.)

19. Recall that in Chapter 5 we considered the possibility that a crossover event may affect the likelihood of another crossover. In the bacteriophage T4, gene a is 1.0 m.u. from gene b, which is 0.2 m.u. from gene c. The gene order is a, b, c. In a recombination experiment, you recover five double crossovers between a and c from 100,000 progeny viruses. Is it correct to conclude that interference is negative? Explain your answer.

The expected number of double recombinants is 2. Interference = −1.5. By definition, the interference is negative.

20. You have infected E. coli cells with two strains of T4 virus. One strain is minute (m), rapid-lysis (r), and turbid (tu); the other is wild type for all three markers. The lytic products of this infection are plated and classified. Of 10,342 plaques, the following numbers are classified as each genotype:

Image ch7fb14.jpg

a.

Determine the linkage distances between m and r, between r and tu, and between m and tu.

b.

What linkage order would you suggest for the three genes?

c.

What is the coefficient of coincidence (see Chapter 6) in this cross, and what does it signify?

(Problem 20 is reprinted with the permission of Macmillan Publishing Co., Inc., from Monroe W. Strickberger, Genetics. Copyright © 1968 by Monroe W. Strickberger.)

21. With the use of P22 as a generalized transducing phage grown on a pur+pro+his+ bacterial donor, a recipient strain of genotype purprohis is infected and incubated. Afterward, transductants for pur+, pro+, and his+ are selected individually in experiments I, II, and III, respectively.

a.

What media are used for these selection experiments?

b.

The transductants are examined for the presence of unselected donor markers, with the following results:

Image ch7fb15.jpg
What is the order of the bacterial genes?

c.

Which two genes are closest together?

d.

On the basis of the order that you proposed in part c, explain the relative proportions of genotypes observed in experiment II.

(Problem 21 is from D. Freifelder, Molecular Biology and Biochemistry. Copyright © 1978 by W. H. Freeman and Company, New York.)

22. Although most λ-mediated gal+ transductants are inducible lysogens, a small percentage of these transductants in fact are not lysogens (that is, they contain no integrated λ). Control experiments show that these transductants are not produced by mutation. What is the likely origin of these types?

In a small percentage of the cases, gal+ transductants can arise by recombination between the gal+ DNA of the λdgal transducing phage and the gal gene on the chromosome. This will generate gal+ transductants without phage integration.

23. An ade+arg+cys+his+leu+pro+ bacterial strain is known to be lysogenic for a newly discovered phage, but the site of the prophage is not known. The bacte-rial map is:

Image ch7fb16.jpg

The lysogenic strain is used as a source of the phage, and the phages are added to a bacterial strain of genotype adeargcyshisleupro. After a short incubation, samples of these bacteria are plated on six different media, with the supplementations indicated in the table below. The table also shows whether colonies were observed on the various media.

Image ch7fb17.jpg

(In this table, a plus sign indicates the presence of a nutrient supplement, a minus sign indicates supplement not present, N indicates no colonies, and C indicates colonies present.)

a.

What genetic process is at work here?

b.

What is the approximate locus of the prophage?

a.

Specialized transduction is at work here. It is characterized by the transduction of one to a few markers.

b.

The prophage is located in the cys–leu region, which is the only region that gave rise to colonies when tested against the six nutrient markers.

24. You have two strains of λ that can lysogenize E. coli; the following figure shows their linkage maps:

Image ch7fb18.jpg

The segment shown at the bottom of the chromosome, designated 1–2–3, is the region responsible for pairing and crossing over with the E. coli chromosome. (Keep the markers on all your drawings.)

a.

Diagram the way in which λ strain X is inserted into the E. coli chromosome (so that the E. coli is lysogenized).

b.

It is possible to superinfect the bacteria that are lysogenic for strain X by using strain Y. A certain percentage of these superinfected bacteria become “doubly” lysogenic (that is, lysogenic for both strains). Diagram how this will occur. (Don’t worry about how double lysogens are detected.)

c.

Diagram how the two λ prophages can pair.

d.

It is possible to recover crossover products between the two prophages. Diagram a crossover event and the consequences.

25. You have three strains of E. coli. Strain A is Fcys+trp1/cys+trp1 (that is, both the F′ and the chromosome carry cys+ and trpI, an allele for tryptophan requirement). Strain B is F cystrp2 Z (this strain requires cysteine for growth and carries trp2, another allele causing a tryptophan requirement; strain B is lysogenic for the generalized transducing phage Z). Strain C is F cys+trp1 (it is an F derivative of strain A that has lost the F′). How would you determine whether trp1 and trp2 are alleles of the same locus? (Describe the crosses and the results expected.)

26. A generalized transducing phage is used to transduce an abcde recipient strain of E. coli with an a+b+c+d+e+ donor. The recipient culture is plated on various media with the results shown in the table below. (Note that a determines a requirement for A as a nutrient, and so forth.) What can you conclude about the linkage and order of the genes?

Image ch7fb19.jpg

Recognize that, if a compound is not added and growth occurs, the E. coli has received the genes for it by transduction. Thus, the BCE culture must have received a+ and d+. The BCD culture received a+ and e+. The ABD culture received c+ and e+. The order is thus d a e c. Notice that b is never cotransduced and is therefore distant from this group of genes.

27. In a generalized transduction system using P1 phage, the donor is pur+nad+pdx and the recipient is purnadpdx+. The donor allele pur+ is initially selected after transduction, and 50 pur+ transductants are then scored for the other alleles present. Here are the results:

Image ch7fb20.jpg

a.

What is the cotransduction frequency for pur and nad?

b.

What is the cotransduction frequency for pur and pdx?

c.

Which of the unselected loci is closest to pur?

d.

Are nad and pdx on the same side or on opposite sides of par? Explain. (Draw the exchanges needed to produce the various transformant classes under either order to see which requires the minimum number to produce the results obtained.)

28. In a generalized transduction experiment, phages are collected from an E. coli donor strain of genotype cys+leu+thr+ and used to transduce a recipient of genotype cysleuthr. Initially, the treated recipient population is plated on a minimal medium supplemented with leucine and threonine. Many colonies are obtained.

a.

What are the possible genotypes of these colonies?

b.

These colonies are then replica plated onto three different media: (1) minimal plus threonine only, (2) minimal plus leucine only, and (3) minimal. What geno- types could, in theory, grow on these three media?

c.

It is observed that 56 percent of the original colonies grow on medium 1, 5 percent grow on medium 2, and no colonies grow on medium 3. What are the actual genotypes of the colonies on media 1, 2, and 3?

d.

Draw a map showing the order of the three genes and which of the two outer genes is closer to the middle gene.

a.

The colonies are all cys+ and either + or − for the other two genes.

b.

(1) cys+ leu+ thr+ and cys+ leu+ thr

(2) cys+ leu+ thr+ and cys+ leu thr+

(3) cys+ leu+ thr+

c.

Because none grew on minimal medium, no colony was leu+ thr+. Therefore, medium 1 had cys+ leu+ thr, and medium 2 had cys+ leu thr+. The remaining cultures were cys+ leu thr, and this genotype was in 39 percent of the colonies.

d.

Image app3fb52.jpg

*29. In 1965, Jon Beckwith and Ethan Signer devised a method of obtaining specialized transducing phages carrying the lac region. In a two-step approach, the researchers first “transposed” the lac genes to a new region of the chromosome and then isolated the specialized transducing particles. They noted that the integration site, designated att80, for the temperate phage ø80 (a relative of phage λ) was located near one of the genes, termed tonB, that confer resistance to the virulent phage T1:

Image ch7fb21.jpg

Beckwith and Signer used an Flac episome that could not replicate at high temperatures in a strain carrying a deletion of the lac genes. By forcing the cell to remain lac+ at high temperatures, the researchers could select strains in which the episome had integrated into the chromosome, thereby allowing the F′ lac to be maintained at high temperatures. By combining this selection with a simultaneous selection for resistance to T1 phage infection, they found that the only survivors were cells in which the F′ lac had integrated into the tonB locus, as shown in the accompanying figure.

Image ch7fb22.jpg

This placed the lac region near the integration site for phage ø80. Describe the subsequent steps that the researchers must have followed to isolate the specialized transducing particles of phage ø80 that carried the lac region.

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: NBK22021

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