Variable coordination of cotranscribed genes in Escherichia coli following antisense repression

BACKGROUND: A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target. RESULTS: To examine the effects of antisense inhibition on cotranscribed genes, we constructed a plasmid expressing the two reporter genes gfp and DsRed as one transcriptional unit. Incubation with antisense peptide nucleic acid (PNA) targeted to the mRNA start codon region of either the upstream gfp or the downstream DsRed gene resulted in a complete expression discoordination from this artificial construct. The same approach was applied to the three cotranscribed genes in the endogenously expressed lac-operon (lacZ, Y and A) and partial downstream expression coordination was seen when the lacZ start codon was targeted with antisense PNA. Targeting the lacY mRNA start codon region showed no effect on the upstream lacZ gene expression whereas expression from the downstream lacA gene was affected as strongly as the lacY gene. Determination of lacZ and lacY mRNA levels revealed a pattern of reduction that was similar to the Lac-proteins, indicating a relation between translation inhibition and mRNA degradation as a response to antisense PNA treatment. CONCLUSION: The results show that antisense mediated repression of genes within operons affect cotranscribed genes to a variable degree. Target transcript stability appears to be closely related to inhibition of translation and presumably depends on translating ribosomes protecting the mRNA from intrinsic decay mechanisms. Therefore, for genes within operons and clusters it is likely that the nature of the target transcript will determine the inhibitory effects on cotranscribed genes. Consequently, no simple and specific methods for expression control of a single gene within polycistronic operons are available, and a thorough understanding of mRNA regulation and stability is required to understand the results from both knock-down and knock-out methods used in bacteria

Antisense PNA accumulates in Escherichia coli and mediates a long post-antibiotic effect

Antisense agents that target growth-essential genes display surprisingly potent bactericidal properties. In particular, peptide nucleic acid (PNA) and phosphorodiamidate morpholino oligomers linked to cationic carrier peptides are effective in time kill assays and as inhibitors of bacterial peritonitis in mice. It is unclear how these relatively large antimicrobials overcome stringent bacterial barriers and mediate killing. Here we determined the transit kinetics of peptide-PNAs and observed an accumulation of cell-associated PNA in Escherichia coli and slow efflux. An inhibitor of drug efflux pumps did not alter peptide-PNA potency, indicating a lack of active efflux from cells. Consistent with cell retention, the post-antibiotic effect (PAE) of the anti-acyl carrier protein (acpP) peptide-PNA was greater than 11 hours. Bacterial cell accumulation and a long PAE are properties of significant interest for antimicrobial development.Peer reviewe

Competitive inhibition of natural antisense Sok-RNA interactions activates Hok-mediated cell killing in Escherichia coli

Short regulatory RNAs are widespread in bacteria, and many function through antisense recognition of mRNA. Among the best studied antisense transcripts are RNA antitoxins that repress toxin mRNA translation. The hok/sok locus of plasmid R1 from Escherichia coli is an established model for RNA antitoxin action. Base-pairing between hok mRNA and Sok-antisense-RNA increases plasmid maintenance through post-segregational-killing of plasmid-free progeny cells. To test the model and the idea that sequestration of Sok-RNA activity could provide a novel antimicrobial strategy, we designed anti Sok peptide nucleic acid (PNA) oligomers that, according to the model, would act as competitive inhibitors of hok mRNA::Sok-RNA interactions. In hok/sok-carrying cells, anti Sok PNAs were more bactericidal than rifampicin. Also, anti Sok PNAs induced ghost cell morphology and an accumulation of mature hok mRNA, consistent with cell killing through synthesis of Hok protein. The results support the sense/antisense model for hok mRNA repression by Sok-RNA and demonstrate that antisense agents can be used to out-compete RNA::RNA interactions in bacteria. Finally, BLAST analyses of ≈200 prokaryotic genomes revealed that many enteric bacteria have multiple hok/sok homologous and analogous RNA-regulated toxin–antitoxin loci. Therefore, it is possible to activate suicide in bacteria by targeting antitoxins

Targeting nucleic acids in bacteria with synthetic ligands

There is a need for new antibacterial agents, and one attractive strategy is to develop nucleic acid ligands that inhibit pathogen genes selectively. Also, such ligands can be used as molecular biology probes to study gene function and nucleic acid structures. In this thesis, bacterial genes were selectively inhibited with antisense peptide nucleic acid (PNA) and the higher order structures formed by (GAA)n repeats were probed with the intercalator benzoquinoquinoxaline (BQQ). A majority of bacterial genes belong to tight clusters and operons, and regulation within cotranscribed genes has been difficult to study. We examined the effects of antisense silencing of individual ORFs within a natural and synthetic operon in Escherichia coli. The results indicate that expression can be discoordinated within a synthetic operon but only partially discoordinated within a natural operon. Bacteria use natural antisense mechanisms to regulate gene expression and an established model for sense/antisense RNA pairing is the hok/sok toxin antitoxin (TA) locus. We aimed to test this model in cells and also the idea that sequestration of Sok-RNA could provide a novel antimicrobial strategy using antisense agents. The results support the hok/sok sense/antisense interaction model and the idea that PNA can out-compete this interaction and provide potent killing activity. Certain antisense agents are effective in bacteria, yet it is unclear how these relatively large molecules overcome stringent bacterial barriers. Here we determined the transit kinetics of peptide-PNAs and observed an accumulation of cell-associated PNA in E. coli and slow efflux. Consistent with cell accumulation and retention, the post-antibiotic effect (PAE) of a PNA that targets the growth essential fatty acid biosynthesis gene acpP was greater than seven hours. At the DNA level, polypyrimidine/polypurine rich sequences have the potential to form intramolecular triple helix structures (H-DNA). Triplet (GAA)n repeats are pathological in Friedreich s ataxia (FA) disease, and may form H-DNA. Here we probed for triplex structures in (GAA)n sequences using the triple-helix specific stabilizing compound BQQ. The results showed that E. coli plasmid carrying a (GAA)n repeat sequence forms H-DNA, suggesting that these structures may play a role in the pathology of FA

Evaluation of the effects of Salvia hypoleuca on the cAMP-responsive element modulator (CREM) gene expression and spermatogenesis in rat

Objective: Salvia species which are medicinal herbs have been used to improve male reproductive functions in traditional medicine. They also, have been used to treat several diseases. In this present study, effects of Salvia hypoleuca on male rat reproductive function were investigated by sperm analysis and assessment of CREM expression at mRNA and protein levels. Materials and methods: Two hundred and fifty mg/kg/day of S. hypoleuca as treatment was given to 10-week old male wistar rats for 56 consecutive days. Sperm analysis, RT-PCR and western blot were carried out to investigate rat reproductive function. Results: Results indicated significant increase in the weights of testis, epididymal sperm counts, and sperm motility compared to control group. RT-PCR and western blot showed an increase in the expression of both CREM mRNA and protein levels. Conclusion: The findings in this study showed S. hypoleuca induced spermatogenesis by CREM activation in testis and improved male fertility

An Immunohistochemical Study of Retinal Collagen IV Expression during Pre- and Postnatal Postnatal Periods in Balb/c Mice

Objective: Basement membranes are specialized extracellular matrices which playimportant roles such as cell regulation, proliferation and migration. Collagen fibers,especially type IV, are the most important basement membrane constituents. As retinais one of the target organs in diabetes mellitus, and nephropathy is a major cause ofend stage renal and retinal diseases resulting in increased morbidity and mortality,early diagnosis leads to better treatment. Hence, in this investigation, the appearanceand distribution of collagen IV during gestational days and early postnatal periodswere observed.Materials and Methods: 24 intact female Balb/c mice were kept under normal conditions.After mating, appearance of a vaginal plug was assumed as day zero of the pregnancy.From days 13-18 of gestation, the pregnant mice were euthanised and theirembryos as well as pups from days 1 to 5 were collected. For histochemichal studies,heads of the specimens were fixed, serially sectioned and immunohistochemicallstudies were performed by using monoclonal antibodies for tracing of collagentype IV.Results: Our findings revealed that the amount of collagen IV in the internal limiting membrane(ILM) and extra cellular matrix (ECM) of the retina, as well as vessels of the vitreusbody appear on embryonic day 16. Also, a patchy distribution was observed in the pigmentedepithelium which continued to further develop until the end stage of embryoniclife. Strong labeling was observed until postnatal day 3 but did not increase significantlythereafter.Conclusion: These findings establish the importance of collagen IV during the critical periodof retinal development. In addition, this study indicates that high levels of collagen IVare present in the basal membrane (BM) of the inner limiting membranes and pigmentedepithelium (3rd post natal on the 3rd postnatal day

The Role of Type IV Collagen in Developing Lens in Mouse Fetuses

Objective(s)Extracellular matrix (ECM) and basement membrane (BM) play important roles in many developmental processes during development and after birth. Among the components of the BM, collagen fibers specially type IV are the most important parts. The aim of this study was to determine the time when collagen type IV appears in the BM of lens structure during mouse embryonic development.Materials and MethodsIn this experimental study, 22 female Balb/C mice were randomly selected and were kept under normal condition, finding vaginal plug was assumed as day zero of pregnancy. From embryonic day 10 to 20, all specimens were sacrificed by cervical dislocation and their heads were fixed, serially sectioned and immunohistochemistry study for tracing collagen type IV in lens were carried out.ResultsOur data revealed that collagen type IV appeared at the early stage of gestation day 12 in BM of anterior epithelial lens cells and the amount of this protein gradually increased until days 15-17 in ECM and posterior capsule epithelium. After this period, severe reaction was not observed in any part of the lens.ConclusionThese findings establish the important role of collagen IV in developing optic cup and any changes during critical period of pregnancy may be result in severe visual system defec