Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 95
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In some instances, the F plasmid and similar types of plasmidscan become incorporated into the bacterial chromosome so that the plasmid genes allowing DNA transfer arethemselves part of the bacterial chromosome. In this case, the plasmid is said to have been integrated into thechromosome. Because it can exist either separate from the chromosome or incorporated into it, the F factor is anexample of an episome, a term that refers to any genetic element that can exist free in the cell or as a segment ofDNA integrated into the chromosome.Conjugation begins with physical contact between a donor cell and a recipient cell.
A tubular projection from thedonor cell forms a passageway between the donor and recipient cells. Through this passageway, a copy of thedonor DNA moves from the donor to the recipient. In the final stage, which requires recombination if the donorcontains an integrated plasmid, a segment of the transferred donor DNA becomes part of the genetic complementof the recipient. If the donor contains a free plasmid, then only the plasmid DNA is transferred and takes upresidence in the recipient.Let us begin with a description of the genetic properties of plasmids and their transfer and then examine plasmidmediated chromosomal transfer.PlasmidsPlasmids are circular DNA molecules that are capable of replicating independently of the chromosome and rangein size from a few kilobases to a few hundred kilobases (Figure 8.6).
The F factor is approximately 100 kb inlength and contains many genes for its maintenance in the cell and its transmission between cells. Plasmids havebeen observed in many bacterial species and are usually not essential for growth of the cells. For studying plasmidsin the laboratory, a culture of cells derived from a single plasmid-containing cell is used; because plasmidsreplicate and are inherited, all cells of the culture contain the plasmid of interest. Plasmids not only contain adiversity of genes for their own maintenance, which are not present in the bacterial chromosome, but plasmids canalso acquire chromosomal genes from their host by several mechanisms. The presence of certain plasmids inbacterial cells is made evident by phenotypic characteristics of the host cell conferred by genes in the plasmid. Forexample, a plasmid containing the tet-rPage 315gene will make the recipient bacterial cell resistant to tetracycline.Plasmids rely on the DNA-replication enzymes of the host cell for their reproduction, but initiation of replication iscontrolled by plasmid genes.
The number of copies of a particular plasmid in a cell varies from one plasmid to thenext, depending on its particular mode of regulation of initiation. High-copy-number plasmids are found in asmany as 50 copies per host cell, whereas low-copy-number plasmids are present to the extent of 1 or 2 copies percell. Plasmid DNA can be taken up by cells and become permanently established in the bacteria. The ability ofplasmid DNA to transform cells genetically has made plasmids important in genetic engineering (Chapter 9).From the point of view of bacterial genetics, the F plasmid is of greatest interest because of its role in mediatingconjugation between cells of E.
coli. Cells that contain F are donors and are designated F+ (''F plus"); those lackingF are recipients and are designated F- ("F minus"). The F plasmid is a low-copy-number plasmid. A typical F+ cellcontains one or two copies of F. These F plasmids replicate once per cell cycle and segregate to both daughter cellsin cell division.The F plasmid contains a set of genes for establishing conjugation between cells and for transferring DNA fromdonor to recipient. A copy of the F plasmid can be transferred in conjugation from an F+ cell to an F- cell.
Transferis always accompanied by replication of the plasmid. Contact between an F+ and an F- cell initiates rolling-circlereplication of F (Section 5.4), which results in the transfer of a single-stranded linear branch of the rolling circle tothe recipient cell. During transfer, DNA is synthesized in both donor and recipient (Figure 8.7).
Synthesis in thedonor replaces the transferred single strand, and synthesis in the recipient converts the transferred single strand intodouble-stranded DNA. When transfer is complete, the linear F strand becomes circular again in the recipient cell.Note that because one replica remains in the donor while the other is transferred to the recipient, after transfer bothcells contain F and can function as donors. The transfer of FFigure 8.6Electron micrograph of a ruptured E.
coli cell showing released chromosomal DNA andseveral plasmid molecules.[Courtesy of David Dressler and Huntington Potter.]Page 316Figure 8.7Transfer of F from an F+ to an F- cell. Pairing of the cells triggers rolling-circle replication. Pink representsDNA synthesized during pairing. For clarity, the bacterial chromosome is not shown, and the plasmidisdrawn overly large; the plasmid is in fact much smaller than a bacterial chromosome.Page 317Figure 8.8Integration of F (blue circle) by recombination between a nucleotide sequence in F and a homologoussequence in the bacterial chromosome.
The F plasmid DNA is shown greatly enlarged relative to the sizeof thebacterial chromosome. In reality, the length of the DNA molecule in F is about 2 percent of the lengthof the bacterial chromosome.requires only a few minutes. In laboratory cultures, if a small number of donor cells is mixed with an excess ofrecipient cells, F spreads throughout the population in a few hours, and all cells ultimately become F+. Transfer isnot so efficient under natural conditions, and only about 10 percent of naturally occurring E.
coli cells contain the Ffactor.Hfr CellsThe F plasmid occasionally becomes integrated into the E. coli chromosome by an exchange between a sequence inF and a sequence in the chromosome (Figure 8.8). The bacterial chromosome remains circular, though enlargedabout 2 percent by the F DNA.
Integration of F is an infrequent event, but single cells containing integrated F canbe isolated and cultured. The cells in such a strain are called Hfr cells. Hfr stands for high frequency ofrecombination, which refers to the relatively high frequency with which donor genes are transferred to therecipient. Integrated F mediates the transfer of DNA from the bacterial chromosome in an Hfr cell; thus a replica ofpart of the bacterial chromosome, as well as part of the plasmid, is transferred to the F- cell.The Hfr × F- conjugation process is illustrated in Figure 8.9. The stages of transfer are much like those by which Fis transferred to F- cells: coming together of donor and recipient cells, rolling-circle replication in the donor cell,and conversion of the transferred single-stranded DNA into double-stranded DNA by lagging-strand synthesis inthe recipient.
However, in the case of Hfr matings, the transferred DNA does not become circular and is notcapable of further replication in the recipient because the transferred F factor is not complete. The replication andassociated transfer of the chromosomal DNA are controlled by the integrated F and are initiated in the Hfrchromosome at the same point in F at which replication and transfer begin within an unintegrated F plasmid. A partof F is the first DNA transferred, chromosomal genes are transferred next, and the remaining part of F is the lastDNA to enter the recipient.
Because the conjugating cells usually break apart long before the entire bacterialchromosome is transferred, the final segment of F is almost never transferred into the recipient.Page 318Figure 8.9Stages in the transfer and production of recombinants in an Hfr × F- mating. Pairing initiates rolling-circlereplication within the F sequence in the Hfr cell, resulting in the transfer of a single strand of DNA. The single strandis converted into double-stranded DNA in the recipient. The mating cells usually break apart before the entirechromosome is transferred. Recombination takes place between the Hfr fragment and the F- chromosomeand leads to recombinants containing genes from the Hfr chromosome. Note that only a part of F istransferred. This part of F is not incorporated into the recipient chromosome.
The recipient remains F-.Page 319Several differences between F transfer and Hfr transfer and notable.• It takes 100 minutes under the usual conditions for an entire bacterial chromosome to be transferred, in contrastwith about 2 minutes for the transfer of F. The difference in time is a result of the relative sizes of F and thechromosome (100 kb versus 4700 kb).• During transfer of Hfr DNA into a recipient cell, the mating pair usually breaks apart before the entirechromosome is transferred. Under usual conditions, several hundred genes are transferred before the cells separate.• In a mating between Hfr and F- cells, the F- recipient remains F- because cell separation usually takes place beforethe final segment of F is transferred.• In Hfr transfer, some regions in the transferred DNA fragment become incorporated into the recipientchromosome.
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