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Research examines the viable but nonculturable state of the bacteria at the molecular level for insights to develop a method for their detection. Data reveal that a gene is expressed during that state and the gene encoded mRNA activity is consistent with the resuscitation capability of the cells.

Results demonstrate that reverse transcriptase-polymerase chain reaction technique can be used for simultaneous transcriptional analysis. Data also indicate that the technique does not involve hybridization and is sensitive.

High loads of Norwalk-like viruses (NLVs) may be in sewage. That might justify more studies addressing the efficacy of NLV removal by sewage water treatment methods. NLV RNA was found by reverse transcriptase PCR (RT-PCR) in sewage water concentrates. Analysis of sequences of the RT-PCR products showed identical sequences in stools of patients and related sewage samples. In 6 of 11 samples not related to outbreaks, multiple NLV genotypes were seen.

Results describe the organization and order of the genes in the mercury resistance determinant and identification of two new transcription open reading frames between merA and merB genes. The differences in the functional activity and inducibility of the determinant in Bacillus cereus RC607, in relation to other bacteria, are presented.

A nested reverse transcriptase (RT) polymerase chain reaction (PCR) assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between one and 10 cells. Total RNA extracted from cultured bacteria, Atlantic salmon kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. After DNase treatment, extracted RNA was amplified by both RT PCR and PCR by employing primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 249-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain.

A study was conducted to analyze whether increased sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) expression in the soleus muscle by unloading is influenced by transcriptional activation. Findings showed that unloading transcriptionally activates the SERCA1 gene. Results also indicated that cis-acting elements sufficient for regulating unloading-induced transcriptional activation are contained in the promoter construct.

Reverse transcriptase polymerase chain reaction (RT-PCR) was used in assessing four methods of extraction and three methods of concentration of three enteric viruses from mussels. The mussels were contaminated by immersion in seawater with astrovirus, hepatitis A virus and poliovirus. Results showed that more RT-PCR-positive samples were obtained with the glycine solution and borate buffer extraction methods than with the saline beef extraction method. Detoxification of the samples by Sephadex LH20 filtration significantly diminished the efficiency of RT-PCR virus detection.

Reverse transcription-polymerase chain reaction techniques were used to clone the genes encoding the variable regions of a monoclonal anti-Bacillus cereus spore IgG. A fusion protein gene was constructed and joined to a strep tag sequence to create a bifunctional single-chain antibody gene. Although similar spore binding behaviors were displayed by the two antibodies, cross-reactions with the spores of B. subtilis and Clostridium perfringens were observed from the single-chain antibody absent in the native monoclonal antibody.

Research was conducted to investigate the structure of msDNA-Ec48, a multicopy single-stranded DNA in Escherichia coli produced by reverse transcriptase. Southern hybridization was utilized to map the location of the msDNA-containing region in the original clone. Results suggested that the msDNA-Ec48 is characterized by 1991 ribonucleotides. They also indicated that retron-Ec48 is related to a prophage associated with phages P2 and P4.

The occurrence of Na+/H+ exchanger 4 (NHE4) isoform in distal tubules and proximal tubules in whole rat kidney cortex was determined using reverse transcriptase-polymerase chain reaction. The recovered NHE4 exhibited an insensitivity to amiloride and amiloride analogs which was a distinct characteristic not previously shown by other NHE isoforms. Other properties that differentiate NHE4 to other isoforms include its reactions when transfected to mutant or acid loaded cells and lack of sensitivity to the anion transporter inhibitor SITS.

The cytochrome P450 2C24 gene is characterized by the capability to generate, in rat kidney, a transcript containing exons 2 and 4 spliced at correct sites but having the donor site of exon 4 directly joined to the acceptor site of exon 2 (exon scrambling). By reverse transcriptase-PCR analysis, it is now shown that the only exons present in the scrambled transcript are exons 2, 3, and 4 and that this molecule lacks a [poly(A).sup.+] tail. Furthermore, the use of PCR primers in both orientations of either exon 2 or exon 4 revealed that the orders of the exons in the scrambled transcript are 2-3-4-2 and 4-2-3-4, respectively. These results, combined with the observation that P450 2C24 is a single-copy gene, with no duplication of the exon 2 to exon 4 segment, suggest that the scrambled transcript has properties consistent with that of a circular molecule. In line with this is the observation of an increased resistance of the transcript to phosphodiesterase I, a 3[prime]-exonuclease. Moreover, an alternatively processed cytochrome P450 2C24 mRNA, lacking the three scrambled exons and having exon 1 directly joined to exon 5, has been identified in kidney and liver, tissues that express the scrambled transcript. This complete identity of the exons that are absent in the alternatively processed mRNA but present in the scrambled transcript is interpreted as indicative of the possibility that exon scrambling and exon skipping might be interrelated phenomena. It is therefore proposed that alternative pre-mRNA processing has the potential to generate not only mRNAs lacking one or more exons but also circular RNA molecules.

Low-salt diets elevate the neuronal form of the constitutive nitric oxide synthase (ncNOS) in renal cortical tissues whereas an opposite effect is observed with high-salt diets. Similar changes are also observed in the expression of renin and angiotensinogen mRNA. However, dietary salt intake has no effect on the mRNA levels for ncNOS from the inner medula. Reverse transcription-polymerase chain reaction and mutant deletion template analysis of Sprague-Dawley rats shows the inverse regulatory effect of salt intake on ncNOS expression in macula densa cells.

Cytokine-induced expression of both inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase by human islets seems to proceed only through the activation of tyrosine kinases. The combined effect of three human recombinant cytokines, namely, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, and interferon-gamma (IFN-gamma) induces the expression of iNOS mRNA. Reverse transcriptase-polymerase chain reaction and Northern blot analysis reveal IL-1beta and IFN-gamma to be the optimum cytokine combination.

The utilization of reverse transcription and polymerase chain reaction for the detection in activated sludge and surface waters of enteroviruses is discussed. RT-PCR was compared against direct hybridization and cell culture methods. The sensitivity of RT-PCR was overwhelmingly better compared to the other two. However, contaminants in activated sludge and concentrated surface waters must be monitored to avoid the inhibition of its enzymatic amplification.

Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription-PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome.

A study conducted on anoxic sediment samples from a productive freshwater lake using a technique for the rapid extraction of RNA from indigenous bacterial communities resulted in the identification of bacteria lineages related to Pirellula staleyi, a member of Planctomycetales and the OP5 bacterial division. The use of reverse transcriptase PCR revealed evidence of Pirellula-like bacteria that are active in an anoxic environment indicating the widespread occurrence of OP5 members in diverse environments.