Rapid detection of urinary tract infections using isotachophoresis and molecular beacons.

We present a novel assay for rapid detection and identification of bacterial urinary tract infections using isotachophoresis (ITP) and molecular beacons. We applied on-chip ITP to extract and focus 16S rRNA directly from bacterial lysate and used molecular beacons to achieve detection of bacteria specific sequences. We demonstrated detection of E. coli in bacteria cultures as well as in patient urine samples in the clinically relevant range 1E6-1E8 cfu/mL. For bacterial cultures we further demonstrate quantification in this range. The assay requires minimal sample preparation (a single centrifugation and dilution), and can be completed, from beginning of lysing to detection, in under 15 min. We believe that the principles presented here can be used for design of other rapid diagnostics or detection methods for pathogenic diseases.

A novel suppressor of ras1 in fission yeast, byr4, is a dosage-dependent inhibitor of cytokinesis.

A novel gene, designated byr4, was identified in Schizosaccharomyces pombe that affects the mitotic cell cycle and shows genetic interactions with the ras1 signaling pathways. Null alleles of byr4 cause cell cycle arrest in late mitosis and permit multiple rounds of septation. The multiple septa typically divide two nuclei, but the nuclei frequently do not stain equally with 4',6-diamidino-2-phenylindole (DAPI), suggesting that byr4 is required for proper karyokinesis. Overexpression of byr4 inhibits cytokinesis, but cell cycle progression continues leading to multinucleate cells. When byr4 is overexpressed, the early steps in the cytokinesis pathway, including formation of the medial F-actin ring, occur normally; however, the later steps in the pathway, including contraction of the F-actin ring, septation, and rearrangement of the medial F-actin following mitosis, rarely occur, byr4 shows two genetic interactions with ras1. The inhibition of cytokinesis by byr4 overexpression was exacerbated by null alleles of ras1 and scd1, suggesting a link between pathways needed for cell polarity and cytokinesis. Overexpression of byr4 also partially bypasses the need for ras1 for sporulation. The electrophoretic mobility of the byr4 protein varied in response to mutants that perturb cytokinesis and karyokinesis, suggesting interactions between byr4 and these gene products. A more rapidly migrating byr4 protein was found in cells with mutations in cdc16, which undergo repeated septation, and in cdc15, which fail to form a medial F-actin ring in mitosis. A slower migrating byr4 protein was found in cells with a mutation in the beta-tubulin gene, which arrests cells at the metaphase-anaphase transition.

Loss of Rhb1, a Rheb-related GTPase in fission yeast, causes growth arrest with a terminal phenotype similar to that caused by nitrogen starvation.

The Rheb GTPase is most similar in primary sequence to the Ras, Rap, R-Ras, and Ral GTPases, which regulate cell growth and differentiation in many cell types. A likely fission yeast homologue of mammalian Rheb, which we designated Rhb1, was identified by genome sequencing. Our investigation of rhb1 showed that rhb1(-) cells arrested cell growth and division with a terminal phenotype similar to that of nitrogen-starved cells. In particular, cells depleted of Rhb1 arrested as small, round cells with 1N DNA content, arrested more quickly in low-nitrogen medium, and induced expression of fnx1 and mei2 mRNA, two mRNAs that were normally induced by nitrogen starvation. Since mammalian Rheb binds and may regulate Raf-1, a Ras effector, we tested for functional overlap between Ras1 and Rhb1 in fission yeast. This analysis showed that Ras1 overexpression did not suppress rhb1(-) mutant phenotypes, Rhb1 overexpression did not suppress ras1(-) mutant phenotypes, and ras1(-) rhb1(-) double mutants had phenotypes equal to the sum of the corresponding single-mutant phenotypes. Hence, there is no evidence for overlapping functions between Ras1 and Rhb1. On the basis of this study, we hypothesize that Rhb1 negatively regulates entry into stationary phase when extracellular nitrogen levels are adequate for growth. If this hypothesis is correct, then Rhb1 and Ras1 regulate alternative responses to limiting nutrients.

Post-transcriptional action of ACTH in the control of P450c17 expression in rabbit adrenal glands.

Stimulation of transcription of the CYP17 gene by ACTH has been demonstrated previously by others using cultures of adrenal tissue from several species. In the present investigations we have demonstrated from measurements in pooled rabbit adrenal glands that after 4 or 6 days of ACTH injections no difference in amounts of CYP17 mRNA per microgram of total adrenal RNA was observed between ACTH and control animals. While the total amount of CYP17 mRNA per adrenal increased 1.4- to 1.7-fold over the injected and non-injected controls due to an increase in total RNA in the ACTH-stimulated adrenals, Western analysis of adrenal microsomal protein demonstrated that the amount of adrenal P450c17 protein, the product of the CYP17 gene, increased over 50-fold. The data show that ACTH is acting at both transcriptional and post-transcriptional loci to increase the amount of rabbit adrenal P450c17 and that the greater effect is post-transcriptional.

Genetic transformation of the Lyme disease agent Borrelia burgdorferi with coumarin-resistant gyrB.

No useful method to genetically manipulate Borrelia burgdorferi, the causative agent of Lyme disease, has been developed previously. We have used resistance to the coumarin antibiotic coumermycin A1, an inhibitor of DNA gyrase, as a genetic marker to monitor the transformation of B. burgdorferi by electroporation. Introduction of site-directed mutations into the gyrB gene demonstrated that transformation was successful, provided evidence that homologous recombination occurs on the chromosome, and established that mutations at Arg-133 of DNA gyrase B confer coumermycin A1 resistance in B. burgdorferi. The coumermycin A1-resistant gyrB marker and genetic transformation can now be applied toward dissecting the physiology and pathogenesis of the Lyme disease agent on a molecular genetic level.

ras1 and pat1 alleles interact to quantitatively and qualitatively alter conjugation in fission yeast.

To identify novel components of ras1+ signalling in Schizosaccharomyces pombe, extragenic suppressors of the mating defect of ras1 effector mutants were isolated. A novel allele of pat1, pat1-e1, was isolated that increases the mating of ras1-D43E mutants to near wild-type levels but does not suppress the mating defect of ras1-I41M, ras1-Y37F, or ras1-Y45I mutants. This allele-specific suppression is not a characteristic of all pat1 alleles since pat1-3 and pat1-114 partially and equally suppress ras1-D43E and ras1-I41M mutants. Analysis of mating cultures showed that ras1-D43E and pat1-e1 interact to qualitatively alter the mating response. While pat1-e1 ras1-D43E cells were delayed in agglutination, cell-cycle delay, and mat1-Pm transcription, they induce mat1-Mc at the same time and mate more rapidly than other mating cultures. These results suggest that pheromone signalling, but not nutritional signalling, is delayed in pat1-e1 ras1-D43E cells. We hypothesize that this delay causes an elevated pheromone response and thus suppression of the mating defect of the ras1-D43E mutant by pat1-e1.