Global profile changes in transcripts induced with a phosphate analogue: implications for gene regulation.

Previous work has shown that thiophosphate, a phosphate analogue, leads to a global shift in the distribution of cellular proteins in a variety of organisms. Thiophosphate, when added to culture media, gets incorporated into the nucleic acids of cells resulting in nuclease-resistant phosphorothioate linkages. Using Escherichia coli, as a model organism, it was found that the global changes in protein expression induced with thiophosphate could be accounted for by significant changes in the absolute transcription levels of more than 1500 genes detected via RNA seq analysis. In fact, 58% of transcripts detected in RNA seq studies using total RNA were increased an average of 44 × fold while the remaining 42% were decreased an average of 20 × fold in thiophosphate-treated cells. Furthermore, microarray results showed no correlation between the transcriptional changes observed and the known stability of the corresponding mRNAs measured. Overall, the total amount of non-ribosomal RNA accumulated in TP-treated cells was increased relative to rRNA ~ 4 × fold (1.5-6 ×). The results further indicated that metabolic changes may play a role in inducing the transcriptional profiles observed with thiophosphate. Indeed, pathway analysis of transcripts showed an increase in routes for phosphoribosyl pyrophosphate (PRPP) synthesis and related derivatives, presumably due to a reduction in RNA turnover. These results raise the possibility that the energy savings with reduced RNA turnover could lead to an increased energy charge in the cell that induces transcriptional changes leading to an increase in biosynthetic processes.

Testing the efficacy of kitasamycin for use in the control and treatment of swine dysentery in experimentally infected pigs.

BACKGROUND: Swine dysentery (SD) caused by Brachyspira hyodysenteriae is an important disease in Australia. AIM: The aim of this study is to evaluate the macrolide antibiotic kitasamycin for use in SD control. METHODS: The minimum inhibitory concentrations (MICs) of kitasamycin, tylosin and lincomycin for 32 Australian isolates of B. hyodysenteriae were evaluated. Mutations in the 23S rRNA gene were examined. Isolate '13' with a low kitasamycin MIC was used to challenge weaner pigs. Sixty pigs were housed in 20 pens each containing three pigs: pigs in four pens received 2 kg/tonne of a product containing kitasamycin (3.1% active) prophylactically in their food starting 4 days before B. hyodysenteriae challenge (group 1); pigs in four pens were challenged and received the same dose therapeutically once one pig in a pen showed diarrhoea (group 2); four pens were challenged and received 4 kg/tonne of the product therapeutically (group 3); four pens were challenged but not medicated (group 4); two pens were unmedicated and unchallenged (group 5) and two pens received 2 kg/tonne and were unchallenged (group 6). Pigs were monitored for B. hyodysenteriae excretion and disease. RESULTS: Macrolide resistance was widespread, and mutations in the 23S rRNA gene were identified in 23 isolates. Four isolates with kitasamycin MICs < 5 µg/mL were considered susceptible. Following experimental challenge, 10 of 12 unmedicated pigs developed SD. No pigs receiving kitasamycin prophylactical or therapeutically developed SD. Medicated pigs shed low numbers of B. hyodysenteriae in their faeces. CONCLUSIONS: Kitasamycin can help control SD in pigs infected with susceptible isolates of B. hyodysenteriae.

Oligonucleotide microarray: a new rapid method for screening the 23S rRNA gene of Helicobacter pylori for single nucleotide polymorphisms associated with clarithromycin resistance.

BACKGROUND AND AIM: Resistance to antibiotics in Helicobacter pylori is increasing and becoming a serious problem in eradication treatment. Resistance of H. pylori to clarithromycin has been found to be associated with 2142 A-to-G, 2143 A-to-G and 2182 C-to-T point mutations in the 23S rRNA gene. Thus, the purpose of the present study was to develop a new method to analyze single nucleotide polymorphism (SNPs) of 23S rRNA gene using oligonucleotide microarray and to determine the prevalence of each mutation in H. pylori-positive patients. METHODS: Gastric tissue biopsy specimens were obtained from patients undergoing upper gastrointestinal endoscopy. After DNA extraction, asymmetric PCR was employed to prepare single-stranded target DNA labeled with a fluorescent dye. The PCR products that amplified a portion of 23S rRNA from H. pylori isolates were hybridized on DNA microarray, specific for SNP genotyping and mutation detection. The optimal signal intensity and efficiency of hybridization were observed for capture probes in the detection of DNA sequence variation. The relevant mutation was confirmed by DNA sequencing analysis. RESULTS: Fifty-four gastric biopsy specimens yielded H. pylori-positive results and were studied to detect mutations in the 23S rRNA gene. There were no samples with A-to-G transition at position 2142. The 2143 A-to-G and 2182 C-to-T mutations were present in 11.11% and 12.96% of H. pylori strains examined, respectively. The relevant mutation was confirmed by analysis of DNA sequencing to be the same as that described at position 2142, 2143 and 2182 using oligonucleotide microarray. CONCLUSIONS: Oligonucleotide microarray of the PCR product permits a rapid and accurate screening of SNPs of 23S rRNA gene from H. pylori. It is now possible to apply this hybridization technology in clinical diagnosis and treatment.

The phosphoprotein pp135 is an essential constituent of the fibrillar components of nucleoli and of coiled bodies.

We examined the distribution of the silver-stainable phosphoprotein, pp135, within Ehrlich tumor and HEp-2 cells by a postembedding Lowicryl immunogold labeling procedure. Identical labeling patterns were obtained in both cell types. During interphase, gold particles were found not only over the dense fibrillar component but were also evident over the fibrillar centers of nucleoli. By contrast, the granular component did not display any significant label. When rRNA synthesis was inhibited by actinomycin D, the same labeling was observed in segregated nucleoli; both fibrillar components were labeled. Aside from the nucleolar labeling, label was also consistently present in coiled bodies. During metaphase, label was visualized in silver-stainable material of the nucleolus organizing regions. It thus appears that, unlike the two major silver-stained proteins, nucleolin/C23 and B23, pp135 remains located in all major silver-stainable structures during the whole cell cycle. This finding strongly suggests that pp135 could be the component responsible for in situ silver staining. On the other hand, the maintenance of pp135 in the fibrillar centers throughout the cell cycle, like RNA polymerase I, upstream binding factor, and DNA topoisomerase I, suggests that pp135 could be a component involved in transcription of the rRNA genes.