Genetic architecture of thermal adaptation in Escherichia coli.

Elucidating the genetic basis of adaptation on a genomewide scale has evaded biologists, but complete genome sequences and DNA high-density array technology make genomewide surveys more tractable. Six lines of Escherichia coli adapted for 2,000 generations to a stressful high temperature of 41.5 degrees C were examined on a genomewide scale for duplication/deletion events by using DNA high-density arrays. A total of five duplication and deletion events were detected. These five events occurred in three of the six lines, whereas the remaining three lines contained no detectable events. Three of the duplications were at 2.85 Mb of the E. coli chromosome, providing evidence for the replicability of the adaptation to high temperature. Four candidate genes previously shown to play roles in stress and starvation survival were identified in the region of common duplication. Expression of the two candidate genes examined is elevated over expression levels in the ancestral lines or the lines without the duplication. In the two cases where the duplication at 2.85 Mb has been further characterized, the timing of the genome reorganization is coincident with significant increases in relative fitness. In both of these cases, the model for the origin of the duplication is a complex recombination event involving insertion sequences and repeat sequences. These results provide additional evidence for the idea that gene duplication plays an integral role in adaptation, specifically as a means for gene amplification.

Global gene expression profiling in Escherichia coli K12. The effects of integration host factor.

We have used nylon membranes spotted in duplicate with full-length polymerase chain reaction-generated products of each of the 4,290 predicted Escherichia coli K12 open reading frames (ORFs) to measure the gene expression profiles in otherwise isogenic integration host factor IHF(+) and IHF(-) strains. Our results demonstrate that random hexamer rather than 3' ORF-specific priming of cDNA probe synthesis is required for accurate measurement of gene expression levels in bacteria. This is explained by the fact that the currently available set of 4,290 unique 3' ORF-specific primers do not hybridize to each ORF with equal efficiency and by the fact that widely differing degradation rates (steady-state levels) are observed for the 25-base pair region of each message complementary to each ORF-specific primer. To evaluate the DNA microarray data reported here, we used a linear analysis of variance (ANOVA) model appropriate for our experimental design. These statistical methods allowed us to identify and appropriately correct for experimental variables that affect the reproducibility and accuracy of DNA microarray measurements and allowed us to determine the statistical significance of gene expression differences between our IHF(+) and IHF(-) strains. Our results demonstrate that small differences in gene expression levels can be accurately measured and that the significance of differential gene expression measurements cannot be assessed simply by the magnitude of the fold difference. Our statistical criteria, supported by excellent agreement between previously determined effects of IHF on gene expression and the results reported here, have allowed us to identify new genes regulated by IHF with a high degree of confidence.

Cell-cycle regulated expression of Rap1 in regenerating liver.

Rap1 proteins are capable of competing with Ras p21 for binding to effectors, and of antagonizing some Ras-induced effects, but their participation in normal growth regulation has not been established. The level of Rap1 protein and the expression of the rap1A gene were examined by immunoblotting and Northern analysis during the regenerative growth response in rat liver following partial hepatectomy. Protein and mRNA were significantly down-regulated prior to and during the onset of DNA synthesis. The timing of this effect is consistent with a model in which expression of Rap1 is turned off or down to allow the initiation of proliferation.