Alternating transcription of two flagellin genes in a Salmonella bacterium is caused by a conservative site-specific recombination event that inverts a small DNA segment containing a promoter.
In one orientation, the promoter activates transcription of the H2 flagellin gene as well as that of a repressor protein that blocks the expression of the H1 flagellin gene. Promoters and repressors are described in detail in Chapter 7; here we note simply that a promoter is needed to express a gene into protein and that a repressor blocks this from happening.
When the promoter is inverted, it no longer turns on H2 or the repressor, and the H1 gene, which is thereby released from repression, is expressed instead. The inversion reaction requires specific DNA sequences (red) and a recombinase enzyme that is encoded in the invertible DNA segment. This site-specific recombination mechanism is activated only rarely (about once in every 105 cell divisions). Therefore, the production of one or the other flagellin tends to be faithfully inherited in each clone of cells.
IN SPECIFIC TISSUE (e.g., LIVER)
Cre recombinase gene gene of interest gene of interest deleted from chromosome and lost as liver cells divide
IN OTHER TISSUES, THE GENE OF INTEREST IS EXPRESSED NORMALLY
Cre recombinase gene gene of interest mRNA(e.g., promoter active only in liver) protein of interest
Figure 5–66 How a conservative site-specific recombination enzyme from bacteria is used to delete specific genes from particular mouse tissues. This approach requires the insertion of two specially engineered DNA molecules into the animal’s germ line. The first contains the gene for a recombinase (in this case, the Cre recombinase from the bacteriophage P1) under the control of a tissue-specific promoter, which ensures that the recombinase is expressed only in that tissue. The second DNA molecule contains the gene of interest flanked by recognition sites (in this case, LoxP sites) for the recombinase. The mouse is engineered so that this is the only copy of this gene. Therefore, if the recombinase is expressed only in the liver, the gene of interest will be deleted there, and only there. The reaction that excises the gene is the same as that shown in Figure 5–64A. As described in Chapter 7, many tissue-specific promoters are known; moreover, many of these promoters are active only at specific times in development. Thus, it is possible to study the effect of deleting specific genes at different times during the development of each tissue.
• How does DNA replication contend with all the other processes that occur simultaneously on chromosomes, very early in development. The same strategy can also be used to inappropriately including DNA repair and gene express any specific gene in a tissue of interest; here, the triggered deletion joins transcription? a strong transcriptional promoter to the gene of interest. With this tool one can in principle determine the influence of any protein in any desired tissue of an intact • What is the basis for the low animal. frequency of errors in DNA replication observed in all cells? Is this the best that cells can do given the speed of replication and the limits of molecular The genomes of nearly all organisms contain mobile genetic elements that can move diffusion? Was this mutation rate from one position in the genome to another by either transpositional or conserva-selected in evolution to provide genetic variation?
tive site-specific recombination processes. In most cases, this movement is random and happens at a very low frequency. Mobile genetic elements include transposons, which move within a single cell (and its descendants), plus those viruses whose genomes can integrate into the genomes of their host cells.
ways of repairing it. Are there still
There are three classes of transposons: the DNA-only transposons, the retrovi other, undiscovered ways that cells ral-like retrotransposons, and the nonretroviral retrotransposons. All but the last have for repairing DNA?