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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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cAMP receptor protein (CRP) and CytR mediate positive and negative control of nine genes in Escherichia coli, most of which are involved in nucleoside catabolism and recycling. Five promoters share a common architecture in which tandem CRP sites flank an intervening CytR operator (CytO). CytR and CRP bind cooperatively to these promoters to form a three-protein, DNA-bound complex that controls activation and repression, the levels of which vary markedly among the promoters. To understand the specific combinatorial control mechanisms that are responsible for this outcome, we have used quantitative DNase I footprinting to generate individual site isotherms for each site of protein-DNA interaction. The intrinsic affinities of each transcription factor for its respective site and the specific patterns of cooperativity and competition underlying the molecular interactions at each promoter were determined by a global analysis of these titration data. Here we present results obtained for nupGP and tsxP2, adding to results published previously for deoP2, udpP, and cddP. These data allowed us to correlate the reported levels of activation, repression, and induction with the ligation states of these five promoters under physiologically relevant conditions. A general pattern of transcriptional regulation emerges that allows for complex patterns of regulation in this seemingly simple system.

Sexual assault samples are among the most difficult sample types encountered by forensic laboratories. Typically, a sexual assault sample poses multiple challenges including small quantity of male DNA and a relatively high quantity of female DNA. Differential extraction procedures are time-consuming and labor-intensive, particularly with microscopic sperm identification. A rapid upfront screening or triage assay prior to use of differential extraction procedures could ensure that male DNA is present prior to the use of full DNA workflows. Previously reported rapid male screening assays do not provide a confirmation of whether sperm are present and therefore still require the use of microscopic sperm identification. This work was focused on an improved rapid male DNA screening assay with an upfront sperm identification using mRNA profiling.The rapid male screening assay consists of a brief lysis using only a small tip portion of a swab, to obtain both RNA (eluted) and DNA (extraction 'waste') followed by a one-step reverse transcription-high resolution melt assay for PRM2, a sperm-specific mRNA. Without additional purification, the DNA 'waste' fraction can be quantitated and used to obtain an upfront Y-STR profile of the sperm donor. The assay takes 30 min for lysis followed by ∼2 h for quantitation and sperm identification.Here, we demonstrate the specificity of the assay to detect male DNA in semen samples with no cross reactivity with other body fluids and no PRM2 detection in vasectomized males. RNA and DNA profiling results were obtained with as little as ∼0.15 μl of semen in vaginal-semen admixtures. In all samples tested, Y-STR profiling results were obtained when male DNA was indicated based on quantitation results.