Genetic Recombination Help (page 2)
There are three basic mechanisms by which DNA can be transferred from one bacterial cell to another: transformation, conjugation or transduction. These processes are known generally as horizontal gene transfer. If the donor DNA is incorporated, or recombined, into the genome of the recipient cell, a recombinant organism with one or more new phenotypes may result.
Bacterial transformation is the transfer of naked DNA from one bacterial cell to another. When a bacterial cell ruptures (in a process called lysis), its circular DNA is released into the environment and can be taken in by competent cells. Competence is the ability of a cell to take up naked DNA from its environment. Not all bacteria are capable of competence and those that are only become competent during certain parts of their life cycle. To become competent, the cell produces competence proteins that modify the cell wall to allow binding of naked DNA and aid in the absorption and/or incorporation of the foreign DNA into the cell's chromosome. DNA that has been transferred from a donor cell to a recipient cell is called donor DNA, or an exogenote; the recipient DNA is the native DNA of the recipient cell, also called the endogenote. A bacterial cell that has received donor DNA is initially diploid for part of its genome, and is said to be a merozygote. Once taken in by a competent cell, the donor DNA becomes coated with a protein (such as the RecA-protein of E. coli) that aids in recombination of the donor DNA with the native chromosome. Once the donor DNA is integrated into the recipient DNA, the cell is no longer a merozygote.
If the donor DNA contains a different allele than the recipient chromosome, the resulting recombinant DNA will contain one or more mismatched base pairs, and is referred to as a heteroduplex. If progeny cells are to inherit the new allele, a process called mismatch repair must occur by excising a segment of the recipient DNA strand and using the donor DNA strand as a template for its replacement. Also, more than one gene may be contributed by the donor DNA, and if this occurs, the recipient cell is cotransformed. The frequency of cotransformation is a function of the linkage distance between the two genes.
EXAMPLE 10.5 In 1928 Fred Griffith discovered the first example of bacterial transformation. Streptococcus pneumoniae is a bacterium that causes human pneumonia and can also kill mice. The virulent strain of this bacterium contains a polysaccharide capsule that tends to resist destruction by immune cells of the host species. A nonvirulent strain of the pneumococcus does not have a capsule. The virulent strain forms colonies with smooth borders on nutrient agar plates and is thus designated the S (smooth) strain; the nonvirulent strain forms colonies with rough borders and is designated the R (rough) strain. When mice were injected with both heat-killed S strain and live R strain, they died, and live S-strain bacteria were recovered from their bodies. Griffith did not know how to explain these results, but he called the process ''transformation'' and named the responsible substance ''transforming principle.'' Later studies by Avery and others demonstrated that the transforming principle was naked DNA. In the Griffith experiment, the exogenote from the S strain contained the gene responsible for capsule formation. When this exogenote was incorporated into the endogenote of the R strain the transformant cells had the ability to make the capsule and thus became virulent S-type cells.
Bacterial conjugation involves the temporary union of two cells of opposite mating type, followed by unidirectional transfer of some genetic material through a cytoplasmic bridge from the donor cell to the recipient cell, and then disunion of the cells (exconjugants). An episome is an extra-chromosomal genetic element that may exist either as an autonomously replicating circular DNA molecule or as an integrated DNA sequence within the host chromosome (e.g., phage lambda).A plasmidwas originally defined as a small, circular DNA molecule that replicates autonomously of the bacterial chromosome and is incapable of integration into the bacterial chromosome. Plasmids often carry genes for antibiotic resistance that confer a selective advantage upon their host cells when antibiotics are in their environment. Currently, however, the word "plasmid" is often used for both episome and plasmid. Most plasmids are between 1=10 and 1=100 the size of the bacterial chromosome.
In some strains of E. coli, there is an episomal fertility factor called the F plasmid. Strains that carry an F plasmid are called males, designated F+, and can manufacture a protein called pilin from which a conjugation connection, or pilus, is constructed. Contraction of the pilus connecting two cells brings the conjugating cells into close contact for DNA exchange. Cells that do not have an F plasmid are called females and are designated F_. When an F+ cell conjugates with an F_ cell, replication of the F plasmid is initiated. One strand of the F plasmid is broken, and replication by the rolling-circle mechanism (Fig. 10-4) causes the 5' end of the broken strand to enter the recipient cell through the pilus (Fig. 10-8), where it is copied into a double-stranded DNA molecule. The other strand of the F plasmid in the donor cell also replicates simultaneously so the donor cell does not lose its F plasmid (it remains F+). The recipient cell thus becomes F+.
An F plasmid has little homology with the bacterial chromosome, so homologous recombination between these two DNA circles rarely occurs. In approximately 1 in 105 cells, however, a nonhomologous recombination event causes F to become integrated into a site on the bacterial chromosome. Thus, F is a plasmid that can exist chromosomally or extra-chromosomally. A cell with F integrated into its chromosome is called an Hfr (high frequency of recombination) cell. It is so designated because many chromosomal genes can now be transferred from donor to recipient with high frequency. In E. coli, integration of the F factor is known to occur at about 10 specific sites on the chromosome. The integration site and the orientation of the integrated F determines the order with which chromosomal genes will be transferred during conjugation (Fig. 10-9).
DNA replication begins in the Hfr cell at the F locus in such a way that a small part of F is at the beginning of the donated segment; normal bacterial genes then following sequence, and finally the remaining portion of the F is replicated last. About 90 min is required for E. coli to transfer its entire genome at 37_C under laboratory conditions. Thermal agitation of molecules (Brownian movement) usually causes the pilus to rupture before DNA transfer is complete, so a complete F particle is rarely recovered in the recipient cell. For this reason, the F_ recipient cell usually remains F_ after conjugation with an Hfr cell. The presence of the donor DNA in the recipient cell activates a recombination system that causes genetic exchange to occur. In order to recover these recombinant bacteria, it is convenient to use an Hfr cell that is sensitive to some antibiotic and a recipient cell that is resistant to that same antibiotic. The locus of the antibiotic-resistance gene in the recipient cell ideally should be so far from the origin of the donor DNA that the pilus will almost always rupture before that locus can be transferred.
EXAMPLE 10.6 Suppose that an Hfr strain is able to synthesize leucine and is sensitive to the antibiotic streptomycin (leu+, str-s ) and that a recipient F_ strain is unable to synthesize leucine, but is resistant to streptomycin (leu-, str-r ). Mixing of these two strains allows conjugation to occur. Recombinants are selected by plating the mixture on minimal medium (without leucine) containing streptomycin. The Hfr strain cannot grow in the presence of streptomycin. The recipient strain cannot grow unless it has received the leu+ gene by conjugation. Only the recombinants of genotype leu+, str-r will form colonies on the plate. In this case, leu+ is the selected marker; str-r is the counterselective marker that prevents growth of any cell other than a recombinant on this type of medium.
The integration of F factor with the host chromosome is a reversible process. Normally, the excision event that releases the F factor from the host chromosome involves a crossover at the same position at which it was integrated. Occasionally, however, the excision event is aberrant, and the released F factor contains one or a few bacterial genes that were close to the integrated F (Fig. 10-10).
Such a cell is symbolized F'. This cell thus has a deletion in part of its chromosome; the missing material is present in the F plasmid. If the chromosomal genes in the plasmid are essential genes, the F0 cell becomes dependent upon both the host chromosome and the F plasmid for its survival. Normal binary fission of an F0 cell produces progeny that are also F'. However, when an F' cell conjugates with an F_ recipient cell, the recipient cell becomes partially diploid for the small piece of chromosomal material carried in the F' particle. Because the F' plasmid in this new F' cell contains genetic material that is homologous with a segment of the host chromosome, there is a higher probability that this DNA may become integrated at the homologous region. Furthermore, the new F' cell can conjugate with F_ recipients and transfer the F' plasmid information to all its exconjugants with high frequency. This process is also referred to as sex-duction.
Plasmids carry genetic information for their own replication, but usually none that is essential for the life of the cell in normal environments. However, plasmids may carry one or more genes that confer selective advantage to the cell in certain environments, such as in the presence of antibiotics. Plasmids that carry genes for resistance to one or more substances normally toxic to the host are designated R plasmids or R factors. Some strains of E. coli carry a colicinogenic plasmid (Col) that can synthesize colicin, a protein that kills closely related bacterial strains that lack the Col plasmid. The best known of the bacterial plasmids are designated F, R, and Col.
The transfer of genetic material from one bacterium to another, using a bacterial virus (bacteriophage, phage) as a vector, is called transduction. This aspect of bacterial gene transfer is discussed in Chapter 11. However, the basic principle of a donor strain that provides DNA to a recipient strain is the same as in the previously discussed mechanisms. The primary difference is that the DNA is transferred through a bacteriophage intermediate.
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