Employing a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques, the complete coding sequence of cDNA for the equine SRY gene was determined. We also attempted to make clear whether the equine SRY gene transcript is expressed in the adult testis, and whether the type of transcript is expressed as linear or circular RNA. As a result, in total a 1420 bp cDNA sequence was determined. Accomplishment of 3' RACE infers that equine SRY gene was expressed as a linear RNA transcript in testicular tissue just after puberty, in contrast to the situation in mice. (+info)
Molecular differentiation of Renibacterium salmoninarum isolates from worldwide locations.
Renibacterium salmoninarum is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of R. salmoninarum. We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of R. salmoninarum is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of R. salmoninarum obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences. (+info)
Randomly amplified polymorphic DNA analysis of clinical and environmental isolates of Vibrio vulnificus and other vibrio species.
Vibrio vulnificus is an estuarine bacterium that is capable of causing a rapidly fatal infection in humans. A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed for use in detecting V. vulnificus, as well as other members of the genus Vibrio. The resulting RAPD profiles were analyzed by using RFLPScan software. This RAPD method clearly differentiated between members of the genus Vibrio and between isolates of V. vulnificus. Each V. vulnificus strain produced a unique band pattern, indicating that the members of this species are genetically quite heterogeneous. All of the vibrios were found to have amplification products whose sizes were within four common molecular weight ranges, while the V. vulnificus strains had an additional two molecular weight range bands in common. All of the V. vulnificus strains isolated from clinical specimens produced an additional band that was only occasionally found in environmental strains; this suggests that, as is the case with the Kanagawa hemolysin of Vibrio parahaemolyticus, the presence of this band may be correlated with the ability of a strain to produce an infection in humans. In addition, band pattern differences were observed between encapsulated and nonencapsulated isogenic morphotypes of the same strain of V. vulnificus. (+info)
A randomly amplified polymorphic DNA marker specific for the Bacillus cereus group is diagnostic for Bacillus anthracis.
Aiming to develop a DNA marker specific for Bacillus anthracis and able to discriminate this species from Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides, we applied the randomly amplified polymorphic DNA (RAPD) fingerprinting technique to a collection of 101 strains of the genus Bacillus, including 61 strains of the B. cereus group. An 838-bp RAPD marker (SG-850) specific for B. cereus, B. thuringiensis, B. anthracis, and B. mycoides was identified. This fragment included a putative (366-nucleotide) open reading frame highly homologous to the ypuA gene of Bacillus subtilis. The restriction analysis of the SG-850 fragment with AluI distinguished B. anthracis from the other species of the B. cereus group. (+info)
Class I integrons in Gram-negative isolates from different European hospitals and association with decreased susceptibility to multiple antibiotic compounds.
Class I integrons are associated with carriage of genes encoding resistance to antibiotics. Expression of inserted resistance genes within these structures can be poor and, as such, the clinical relevance in terms of the effect of integron carriage on susceptibility has not been investigated. Of 163 unrelated Gram-negative isolates randomly selected from the intensive care and surgical units of 14 different hospitals in nine European countries, 43.0% (70/163) of isolates were shown to be integron-positive, with inserted gene cassettes of various sizes. Integrons were detected in isolates from all hospitals with no particular geographical variations. Integron-positive isolates were statistically more likely to be resistant to aminoglycoside, quinolone and beta8-lactam compounds, including third-generation cephalosporins and monobactams, than integron-negative isolates. Integron-positive isolates were also more likely to be multi-resistant than integron-negative isolates. This association implicates integrons in multi-drug resistance either directly through carriage of specific resistance genes, or indirectly by virtue of linkage to other resistance determinants such as extended-spectrum beta-lactamase genes. As such their widespread presence is a cause for concern. There was no association between the presence of integrons and susceptibility to cefepime, amikacin and the carbapenems, to which at least 97% of isolates were fully susceptible. (+info)
Use of molecular subtyping to document long-term persistence of Corynebacterium diphtheriae in South Dakota.
Enhanced surveillance of patients with upper respiratory symptoms in a Northern Plains community revealed that approximately 4% of them were infected by toxigenic Corynebacterium diphtheriae of both mitis and gravis biotypes, showing that the organism is still circulating in the United States. Toxigenic C. diphtheriae was isolated from five members of four households. Four molecular subtyping methods-ribotyping, multilocus enzyme electrophoresis (MEE), random amplified polymorphic DNA (RAPD), and single-strand conformation polymorphism-were used to molecularly characterize these strains and compare them to 17 archival South Dakota strains dating back to 1973 through 1983 and to 5 isolates collected from residents of diverse regions of the United States. Ribotyping and RAPD clearly demonstrated the household transmission of isolates and provided precise information on the circulation of several distinct strains within three households. By MEE, most recent and archival South Dakota strains were identified as closely related and clustered within the newly identified ET (electrophoretic type) 215 complex. Furthermore, three recent South Dakota isolates and eight archival South Dakota isolates were indistinguishable by both ribotyping and RAPD. All of these molecular methods showed that recent South Dakota isolates and archival South Dakota isolates were more closely related to each other than to the C. diphtheriae strains isolated in other parts of the United States or worldwide. The data also supported the improbability of importation of C. diphtheriae into this area and rather strongly suggest the long-term persistence of the organism in this region. (+info)
Isolation and characterization of a novel promoter for the bovine growth hormone receptor gene.
The use of alternative promoters represents an important mechanism for the regulation of growth hormone receptor (GHR) gene expression. Two promoters have been isolated previously for the GHR gene: the P1 promoter that drives liver-specific expression, and the P2 promoter that drives ubiquitous expression. In the present study, we isolated a third GHR promoter termed P3. The P3 promoter was GC-rich and TATA-less. The P3 promoter was able to drive the expression of a luciferase reporter gene in cell lines Hep G2, PLC/PRF/5, and BHK-21. In vivo, the P3 promoter initiated transcription from two major sites in exon 1C of the GHR gene in many tissues. In the adult bovine liver, the P3-transcribed GHR mRNA represented only 10% of the total GHR mRNA pool. In non-hepatic tissues such as kidney, skeletal muscle, mammary gland, and uterus, P3-transcribed GHR mRNA represented 30-40% of the total GHR mRNA pool. Within the bovine GHR gene, the P3 promoter was located immediately downstream from the P2 promoter. In transfected cells, the P2 promoter served as an enhancer for the P3 promoter. Existence and co-regulation of two ubiquitous promoters may be a mechanism for achieving a high level of expression of the GHR gene in multiple tissues. (+info)
Identification of a glucose response element in the promoter of the rat glucagon receptor gene.
We cloned the 5' upstream region of the rat glucagon receptor gene, demonstrating that the 5' noncoding domain of the glucagon receptor mRNA contained two untranslated exons of 131 and 166 nucleotides (nt), respectively, separated by two introns of 0.6 and 3.2 kilobase pairs. We also observed an alternative splicing involving the 166-base pair exon. Cloning of up to 2 kilobase pairs of the newly identified genomic domain and transfection of various constructs driving a reporter gene, in pancreatic islet cell line INS-1, uncovered a strong glucose regulation of the promoter activity of plasmids containing up to nucleotide -868, or more, upstream from the transcriptional start point. This promoter activity displayed threshold-like behavior, with low activity of the promoter below 5 mM glucose, and maximal activation as of 10 mM glucose. This glucose regulation was mapped to a highly palindromic 19-nucleotide region between nt -545 and -527. Indeed, deletion or mutation of this sequence abolished the glucose regulation. This domain contained two palindromic "E-boxes" CACGTG and CAGCTG separated by 3 nt, a feature similar to the "L4 box" found in the pyruvate kinase L gene promoter. This is the first description of a G protein-coupled receptor gene promoter regulated by glucose. (+info)