STUDY HINTS
Most of the material we have discussed so far in this primer has been directed at diploid animals and plants. Although the genetic code and role of DNA are essentially the same in eukaryotic and prokaryotic cells, there are important differences. A key difference is that eukaryotic cells have a nucleus (eu- true, kary- nucleus) and genetic transmission is based on the behavior of chromosomes during meiosis and mitosis. Prokaryotic cells, on the other hand, have no nucleus and are haploid. Since they do not have meiosis, they also lack the genetic transmission associated with sexual reproduction. Mendelian rules do not apply to them. In addition, there are also DNA elements, such as transposons, in eukaryotic cells that provide a special, but very important, exception to traditional genetic transfer.
There are, however, several mechanisms of genetic exchange. Although the method of DNA transfer differs in each case, it can result in a partial diploid for any genes that are carried by the DNA fragment entering the prokaryotic cell. These not only help generate genetic diversity, they also offer geneticists powerful tools to manipulate and study the prokaryotic genome.
· Transformation – small “naked” DNA fragments are transported into the cell directly from the cell’s environment.
· Transduction – DNA picked up by a virus in one cell can be transferred to another cell when that virus infects it.
· Conjugation – exchange of DNA between a donor and a recipient cell connected by means of hairlike structures, the pili. The donor carries a plasmid (such as the F plasmid in Escherichia coli) that carries genes coding for pili formation; the recipient cell lacks the plasmid. A plasmid is a circular piece of DNA that replicates independently of the bacterial genome and does not carry genes required for normal bacterial development. There are two common situations in which this plasmid transfer can also involve bacterial genes.
o High-Frequency Recombination (Hfr) – occurs when a plasmid like F becomes incorporated into the bacterial chromosome (making it an Hfr chromosome) and then carries this DNA to the recipient cell it replicates. The F factor may integrate at different places in the bacterial chromosome and drives replication from its own origin replication; the genes near the integration site are always replicated first. Since the conjugation bridge may not remain intact long enough for complete bacterial chromosomal transfer, the genes replicated first will have the highest probability of being transferred to the recipient cell:
o Sexduction – the F plasmid incorporated in an Hfr chromosome can become excised again but carry with it a segment of bacterial DNA (called an F′ plasmid). When this modified plasmid replicates and transfers to a recipient cell, it also replicates and transfers the inserted bacterial DNA segment.
The proper genetic markers must be used for the recipient chromosome as well as for the transforming, transducing, and Hfr DNA. By making the partial diploid (merozygote) heterozygous at a number of loci, recombination can be studied. In these experiments, variable lengths of donor DNA enter the recipient cell, and then pairing and recombination take place between the entering linear DNA segment and the circular recipient chromosome. This circularity of the host chromosome requires an even number of multiple crossovers, with only one of the products, the intact chromosome, being recovered.
Mapping in bacteria can be accomplished, but it is usually not as straightforward as we find it in Drosophila, corn, Neurospora, or certain other organisms. On the other hand, the ability to work with very large numbers of organisms and with small pieces of chromosome makes it possible to map very closely linked genes (fine-structure mapping). Furthermore, the ingenuity of the investigators in this field is a pleasure to behold.
IMPORTANT TERMS
Auxotroph
Conditional mutation
Conjugation
Episome
F factor
Hfr
Lysis
Plasmid
Prophage
Prototroph
Sexduction
Temperate phage
Time-of-entry mapping
Transduction
Transformation
Virulent phage
PROBLEM SET 13
1. In bacterial matings ’twas found
Results that did simply astound.
Gene B followed C
But then C followed B!
The reason? The gene string is
(a) ground,
(b) bound,
(c) wound,
(d) sound,
(e) round.
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2. In a transformation study, E. coli is incubated with transforming DNA carrying the linked genes B+D+T+. The bacterium is B−D−T−. Single transformations recovered include B+, D+, T+; double transformants are B+T+ and B+D+but not D+T+. Triple transformants (B+D+T+) are also obtained. What are the relative positions of the three linked genes?
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3. If three closely linked genes are cotransduced, two at a time (two-factor transduction), and if gene W can be cotransduced with gene P, and if gene P can be cotransduced with gene L, but if gene W is never cotransduced with gene L, what can you conclude as to the order of these three genes? Alternatively, if A and B, as well as B and C, can be cotransduced easily, but if A and C are only infrequently cotransduced, does this tell you anything about the relative position of the three genes?
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4. Assume a region of the E. coli chromosome is paired with a length of DNA brought
in by an Hfr strain. The positions of the genes involved with the production of five different amino acids are indicated in the above diagram (chromosomal DNA above, plasmid below). Numbers represent six intervals between genes where crossovers can occur. If phenylalanine is the unselected marker here, then you would expect that the cells able to grow in minimal medium supplemented with phenylalanine would be
(a) Phe−;
(b) Phe+;
(c) predominantly Phe−; with perhaps a few Phe+;
(d) either Phe− or Phe+, with equal probability.
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5. Explain, using both words and diagram(s), how the F factor can become integrated on the E. coli chromosome to produce different Hfr strains, whose transmission of the male chromosome shows these patterns:
Please construct the physical map of the E. coli chromosome, using these data. The first gene donated is the first gene listed.
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6. At time zero, an Hfr strain with the genetic makeup b−h−k−m− (markers listed here alphabetically, not necessarily by their real gene order) is mixed with an F-strain that is auxotrophic for all markers (that is, b−h−k−m−). Furthermore, the Hfr strain is streptomycin sensitive (strs) and the F− strain is streptomycin resistant (strr). The samples were then plated onto selective medium, and the frequencies of the b−strrrecombinants that had received the h, k, and/or m genes from the Hfr cell were estimated. The following graph shows the number of recombinants against time. What was the order of transfer from Hfr to F−?
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7. Referring again to the experiment in question 6, which two genes are the closest together?
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8. Four different E. coli Hfr strains donate the genetic markers in the order shown in the following table. All of these Hfr strains are derived from the same F+ strain. What is the order of markers on this chromosome?
|
Strain 1: |
R |
S |
J |
C |
|
Strain 2: |
C |
A |
T |
D |
|
Strain 3: |
L |
D |
T |
A |
|
Strain 4: |
D |
L |
W |
R |
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ANSWERS TO PROBLEM SET 13
1. The answer is (e), but this “strange” idea was met with skepticism when proposed by Wollman and Jacob in 1957. See the answer to question 5 for more information on this.
2. To illustrate the principle here, first let us consider a different three-factor transformation experiment in which the paired region has three heterozygous loci and four regions in which an exchange can occur. In the following figure, single transformations result from an exchange on either side of the gene.
If A+ is incorporated into the E. coli chromosome, it requires exchanges in regions 1 and 2. For gene incorporation of gene B+, exchanges are needed in regions 2 and 3. For C+, exchanges are needed in regions 3 and 4. For the triple transformation, crossovers in regions 1 and 4 are required. The double transformants are interesting here and can tell us the relative positions of the three loci. A and B, as well as B and C, can be doubly transformed by crossovers in regions 1 and 3 for A and B, and in regions 2 and 4 for B and C. For the outside loci, A and C, to be doubly transformed (without B+) we need four exchanges, one in each region:
This is much less likely to occur and, in the study described in the problem, was not recovered, though all other possible transformants were reported. Turning now to the problem, the double transformant not seen was D+T+, which tells us that these are on the outside (and B+ is in the middle).
3. In transduction, the phage vector can carry only a small piece of the donor chromosome to the cell about to be transduced. It has been estimated that only about 2 percent of the E. coli genome can fit into a transducing phage, so that only genes that are closely linked can be cotransduced. Genes that are separated by a length of DNA that comprises more than about 2 percent of the total chromosome will not be cotransduced. In the first part of the question, Wand P, and P and L, can be cotransduced, so we conclude that they are close enough to be located on a piece of E. coli chromosome that is less than 2 percent of the total length; but the same cannot be said for W and L, which are not cotransduced, so the segment of chromosome that carries both W and L must exceed the 2 percent limit. This indicates that the gene sequence is WPL. In the second part of the question, the sequence is A B C, and the A—C distance is less than that of the W—L region and is close to the 2 percent limit. In order to have a transducing piece that includes both A and C, the breaks in the chromosome would have to be immediately outside the two loci, and this can only be expected to occur rarely, leading to the observed infrequent cotransduction of A and C.
4.
(c) If we do not require the incorporation of Phe+ onto the E. coli chromosome, then cells capable of growing in minimal medium supplemented with phenylalanine would need only the Lys+, Trp+, Glu+, and Val+ genes. This could be produced by a double exchange in regions 4 and 6. An infrequent quadruple crossover in regions 2 and 3, in addition to 4 and 6, would give us an occasional surviving cell with the Phe+ allele also incorporated.
5. There are a number of sites on the E. coli chromosome that are recognized by the F factor and that permit the insertion of the F factor into the E. coli chromosome. During conjugation, a break occurs in one strand of the integrated F factor, and this strand moves across the conjugation bridge, taking with it the attached chromosome strand. When the F factor becomes integrated, it does so with one of two alternate polarities, which determine whether host material to the left of the F factor is transferred or whether material to the right of the F factor is transferred. It is obvious from the transmission pattern that Hfr 1 and Hfr 2 have opposite polarities. The determination of which gene goes first is based on both the site and the polarity of F-factor integration into the chromosome. The fact that the F factors become integrated at different positions and that we have overlapping in the transmission patterns permits us to draw the map, which makes sense of the data only if the chromosome is a circle. The map and the positions of the F factors in the different Hfr strains are indicated in the accompanying figure. The order in which the genes enter the cell is reversed.
6. The genes closest to b+ (one of the selected markers) will enter most often, whereas those that are least closely linked will run the greatest risk that transfer will be interrupted before transfer of the DNA is complete. The gene order is b m h k.
7. Genes h and k are transferred with the shortest time interval between them and are, therefore, the most closely linked.
8. The order of markers and the orientation of each strain are indicated by arrowheads.
CROSSWORD PUZZLE 13
Mapping in Bacteria and Viruses
Across
1. Transduction of two or more bacterial genetic markers by the same virus
5. Self-reproducing circular DNA strand that is often infectious, when present in bacteria they often confer antibiotic resistance to the bacteria
6. Transfer of bacterial genes from one bacterium to another by an F′ plasmid, also called F-duction
9. Man who did the mutation fine-structure mapping of the virus rII locus
12. Tubular structures that form on a bacterial cell when infected with the F′ plasmid, involved in conjugation
15. Fertility plasmid that produces a donor bacterium, thus allowing conjugation with a recipient
17. Movement of genetic material in microorganisms from one organism to another when connected by pili
20. Strain of E. coli that has a high frequency of recombination because the F plasmid becomes a part of the bacterial chromosome
21. Term for the rupturing of the membrane of a cell, e.g., as by a virulent virus after replication in a cell
23. Phage that lyses its host
Down
2. Use of a virus to transfer DNA from one bacterial cell to another
3. Genetic unit that is synonymous with the word gene, but is defined by the cis–trans test
4. Two mutants that when present together produce a wild-type
5. Bacterial virus
7. ___-acting, term that describes a genetic element that is on a separate chromosome and affects the function of another gene
8. Term for the alteration in a cell’s makeup because of introduced DNA from another organism
10. Term used for a plasmid that is able to incorporate into the genome of an organism
11. ___-acting, term for a genetic element that must be on the same chromosome as another gene in order to influence that gene
13. Mutation involving the loss of base pairs from a chromosome; also called deficiency
14. Phage genome that is integrated into a host’s genome
16. Form of transduction in which a prophage becomes virulent and carries bacterial genes from one cell to another cell
18. Term for a phage that can produce a prophage
19. Form of transduction that involves the transmission of bacterial DNA by the accidental packing of a phage head with a bacterial DNA fragment
22. Hole that a virus makes on a layer of host cells, by either killing or slowing the host cell growth