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Two transcription factors, CRP and CytR, mediate positive and negative control of nine cistrons involved in nucleoside catabolism and recycling in Escherichia coli. The ability of multiple transcription factors to combine in different ways to confer differential gene regulation is of significant interest in both prokaryotic and eukaryotic gene regulation. Analysis of cooperative interactions between CytR and CRP at the deoP2 and udpP promoters has implicated the importance of promoter architecture in controlling repression and induction. These studies have also identified competition between CytR and CRP as an additional contributor to differential regulation. The pattern and energetics of CytR and CRP interactions at the cdd promoter, the most strongly activated of the CytR-regulated promoters, have been delineated using DNase I footprinting. Surprisingly, CRP has greater affinity for the promoter proximal site at cddP, CRP1, than for the distal site, CRP2, in contrast to promoters studied previously. This difference is a major contributor to unusually high CRP-mediated activation of cddP. Additionally, while cytidine binding to CytR nearly eliminates the pairwise interactions between CytR and CRP bound at CRP1, it has little effect on pairwise cooperativity between CytR and CRP bound at CRP2 or as a consequence on the overall cooperativity of the three-protein complex in which CRP is bound to both sites. The effect of cytidine binding on cooperativity differs between the three promoters studied thus far. We propose that the different patterns of interaction reflect the spacing between CytR half-sites and the location of the CytR operator in relation to the two CRP sites.

The AutoMate Express™ Forensic DNA Extraction System was developed for automatic isolation of DNA from a variety of forensic biological samples. The performance of the system was investigated using a wide range of biological samples. Depending on the sample type, either PrepFiler™ lysis buffer or PrepFiler BTA™ lysis buffer was used to lyse the samples. After lysis and removal of the substrate using LySep™ column, the lysate in the sample tubes were loaded onto AutoMate Express™ instrument and DNA was extracted using one of the two instrument extraction protocols. Our study showed that DNA was recovered from as little as 0.025 μL of blood. DNA extracted from casework-type samples was free of detectable PCR inhibitors and the short tandem repeat profiles were complete, conclusive, and devoid of any PCR artifacts. The system also showed consistent performance from day-to-day operation.

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