Fate of the Incoming DNA after Uptake

Irrespective of its mode of entry, DNA that enters a bacterial cell has one of three possible fates. It may survive as an independent DNA molecule, it may be completely degraded, or part may survive by integration or recombination with the host chromosome before the rest is degraded.

For incoming DNA to survive inside a bacterial cell as a self-replicating DNA molecule, it must be a replicon. In other words it must have its own origin of replication and lack exposed ends. For survival in the vast majority of bacteria, this means that it must be circular. In those few bacteria, such as Borrelia and Streptomyces (see Ch. 5) with linear replicons, the ends must be properly protected. In eukaryotes, long-term survival of a linear DNA molecule requires a replication origin, a centromere sequence, and telomeres to protect the ends (see Ch. 5).

A linear fragment of double-stranded DNA that enters a bacterial cell will normally be broken down by exonucleases that attack the exposed ends. For any of its asexual or vegetative reproduction Form of reproduction in which there is no reshuffling of the genes between two individuals binary fission Simple form of cell division in which the cell elongates and divides down the middle after replication of the DNA conjugation Process in which genes are transferred by cell to cell contact donor cell Cell that donates DNA to another cell recipient cell Cell that receives DNA from another cell sexual reproduction Form of reproduction that involves reshuffling of the genes between two individuals transduction Process in which genes are transferred inside virus particles transformation Process in which genes are transferred into a cell as free molecules of DNA

FIGURE 18.01 Recombination allows survival of transformed DNA

In most cases, incoming linear DNA molecules are degraded by the host cell exonucleases. If there are homologous regions between incoming DNA and the host chromosome, crossing over may replace regions of the host chromosome with part of the incoming DNA.

genes to survive, they must be incorporated into the chromosome of the recipient cell by the process of recombination (see Ch. 14). For recombination to occur, crossovers must form between regions of DNA of similar sequence—i.e. homologous sequences. DNA between two crossover points will be swapped by the two DNA molecules (Fig. 18.01). Consequently, if genes from incoming DNA are incorporated, the corresponding original genes of the recipient cell are lost.

Such homologous recombination normally only occurs between closely related molecules of DNA—e.g. DNA from two strains of the same bacterial species. Unrelated DNA may be incorporated by recombination provided it is surrounded by sequences that are related (Fig. 18.02). Another possibility is that the incoming DNA contains a transposon that can function in the recipient cell. If so, then the transposon may survive by abandoning the incoming DNA molecule and jumping into the chromosome of the new host cell.

If the incoming DNA is a plasmid that can replicate on its own, recombination into the chromosome is not necessary for survival. For genetic engineering purposes, it is usually more convenient to avoid recombination. Consequently, molecular biologists often put the genes they are working with onto plasmids (see Ch. 22).

In addition to exonuclease attack, incoming DNA is often susceptible to restriction. This is a protective mechanism designed to destroy incoming foreign DNA. Most bacteria assume that foreign DNA is more likely to come from an enemy, such as a virus, than from a harmless relative and they cut it into small fragments with restriction enzymes. This applies to both linear and circular DNA, as the degradative enzymes are endonucleases that cut DNA molecules in the middle (see Ch. 22 for details). Only DNA that has been modified by methylating the correct recognition sequences is accepted as friendly. In genetic engineering, restriction negative host strains are used to surmount this obstacle.

Homologous recombination

(CROSSING OVER)

Incoming genes may be preserved from destruction by recombination onto the host chromosome.

Incoming circular DNA with its own origin of replication can survive without recombination.

Restriction enzymes degrade foreign DNA, whether linear or circular.

Transformation is Gene Transfer by Naked DNA 487

FIGURE 18.02 Incorporation of Unrelated DNA

Incoming DNA does not have to be entirely related to the host in order for recombination to occur. In some instances, the incoming DNA has regions that are related (purple) and regions that are totally unrelated (green). The regions of homology may be large enough to allow recombination, thus integrating an unrelated piece of DNA into the host chromosome. Receiving new genetic material may provide the host cell with a new trait that is desirable to changing environments. In organisms that make identical clones during reproduction, this strategy is critical to evolutionary survival.

Transformation is Gene Transfer by Naked DNA 487

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