Probing the sequence and structure of in vitro synthesized antisense and target RNAs from the replication control system of plasmid pMV158.

Antisense RNAII is a replication control element encoded by promiscuous plasmid pMV158. RNAII binds to its complementary sequence in the copG-repB mRNA, thus inhibiting translation of the replication initiator repB gene. In order to initiate the biochemical characterization of the pMV158 antisense RNA-mediated control system, conditions for in vitro transcription by T7RNA polymerase were set up that yielded large amounts of antisense and target run-off products able to bind to each other. The run-off antisense transcript was expected, and confirmed, to span the entire RNAII as synthesized by the bacterial RNA polymerase, including the intrinsic transcription terminator at its 3'-terminus. On the other hand, two different target transcripts, mRNA60 and mRNA80, were produced, characterized and tested for efficient binding to the antisense product. The mRNA60 and mRNA80 run-off transcripts supposedly spanned 60 and 80 nucleotides, respectively, on the copG-repB mRNA and lacked terminator-like structures at their 3'-termini. Probing of the sequence and conformation of the main products, along with modeling of their secondary structures, showed that both target transcripts were actually longer-than-expected, and contained a 3'-terminal hairpin wherein the extra nucleotides base-paired to the expected 3'-terminus of the corresponding run-off transcript. These longer products were proposed to arise from the RNA-dependent polymerizing activity of T7RNA polymerase on correct run-off transcripts primed by extremely short 3'-selfcomplementarity. Seizing of the target mRNA sequence complementary to the 5'-terminus of RNAII in a stable 3'-terminal hairpin generated by this activity seemed to cause a 3-fold decrease in the efficiency of binding to the antisense RNA.

Translation initiation of the replication initiator repB gene of promiscuous plasmid pMV158 is led by an extended non-SD sequence.

RepB is the pMV158-encoded protein that initiates rolling-circle replication of this promiscuous plasmid. Availability of RepB is rate-limiting for the plasmid replication process, and therefore the repB gene encoding the protein is subjected to strict control. Two trans-acting plasmid elements, CopG and the antisense RNAII, are involved in controlling the synthesis of the initiator at the transcriptional and translational level, respectively. In addition to this dual control of repB expression that senses and corrects fluctuations in plasmid copy number, proper availability of RepB also relies on the adequate functionality of the transcription and translation initiation regulatory signals. Translation of repB has been postulated to depend on an atypical ribosome binding site that precedes its start codon, although such a hypothesis has never been proved. To define sequences involved in translation of repB, several mutations in the translation initiation region of the repB mRNA have been characterized by using an Escherichia coli in vitro expression system wherein the synthesis of RepB was detected and quantified. We showed that translation of repB is not coupled to that of copG and depends only on its own initiation signals. The atypical ribosome binding site, as it was defined, is not involved in translation initiation. However, the sequence just upstream of the repB start codon, encompassing the proximal box of the atypical ribosome binding site and the four bases immediately downstream of it, is indeed important for efficient translation of repB. The high degree of conservation of this sequence among the rep genes of plasmids of the same pMV158 family supports its relevancy as a translation initiation signal in mRNAs without a recognizable Shine-Dalgarno sequence.

Construction of a plasmid vector based on the pMV158 replicon for cloning and inducible gene expression in Streptococcus pneumoniae.

We report the construction of a plasmid vector designed for regulated gene expression in Streptococcus pneumoniae. The new vector, pLS1ROM, is based on the replicon of the streptococcal promiscuous rolling circle replication (RCR) plasmid pMV158. We inserted the controllable promoter P(M) of the S. pneumoniaemalMP operon, followed by a multi-cloning site sequence aimed to facilitate the insertion of target genes. The expression from P(M) is negatively regulated by the transcriptional repressor MalR, which is released from the DNA operator sequence by growing the cells in maltose-containing media. To get a highly regulated expression of the target gene, MalR was provided in cis by inserting the malR gene under control of the constitutive P(tet) promoter, which in pMV158 directs expression of the tetL gene. To test the functionality of the system, we cloned the reporter gene gfp from Aequorea victoria, encoding the green fluorescent protein (GFP). Pneumococcal cells harboring the recombinant plasmid rendered GFP fluorescence in a maltose-dependent mode with undetectable background levels in the absence of the inducer. The new vector, pLS1ROM, exhibits full structural and segregational stability and constitutes a valuable tool for genetic manipulation and regulated gene expression in S. pneumoniae.

Influence of renal involvement on peripheral blood mononuclear cell expression behaviour of tumour necrosis factor-alpha and interleukin-6 in type 2 diabetic patients.

BACKGROUND: Type 2 diabetes is associated with a high cardiovascular risk, which is even increased if renal damage is superimposed. Peripheral blood mononuclear cells (PBMCs) and pro-inflammatory cytokines are key factors linking type 2 diabetes and atherosclerosis. We investigated the influence of renal damage on serum, urinary and PBMCs expression behavior of TNF-alpha and IL-6 in these patients. METHODS: PBMCs were isolated by density gradient centrifugation (Ficoll-Paque method) from fasting blood samples of 22 non-diabetic control subjects and 78 diabetic patients with normal renal function and different stages of diabetic nephropathy (18 with normoalbuminuria, 29 with microalbuminuria and 31 with macroalbuminuria). Expression levels of TNF-alpha and IL-6 were analyzed by real-time quantitative RT-PCR. Serum and urinary TNF-alpha and IL-6 concentrations were measured by a solid-phase, chemiluminescent immunometric assay. RESULTS: The mean percent increases in the serum and urinary levels of TNF-alpha and IL-6 in diabetic patients with respect to control subjects were 176% (P < 0.0001), 250% (P < 0.0001), 114% (P < 0.0001) and 39.6% (P = 0.01), respectively. The mRNA expression level of TNF-alpha was higher by 68.8% (P < 0.001) and IL-6 mRNA levels were higher by 64.1% (P < 0.001) with respect to non-diabetic controls. TNF-alpha mRNA expression in patients with macroalbuminuria was higher by 84.8% with respect to subjects with normalbuminuria (P < 0.001) and by 29% with respect to individuals with microalbuminuria (P < 0.05). Likewise, microalbuminuric patients showed a 44.5% increase in TNF-alpha mRNA expression compared to subjects with normoalbuminuria (P < 0.05). Concerning IL-6, the mRNA expression levels of this cytokine was higher by 63.1% with respect to normoalbuminuric subjects (P < 0.01), and by 23.1% with respect to patients with microalbuminuria (P < 0.05). However, with respect to controls, diabetic patients with normoalbuminuria had similar serum TNF-alpha and urinary excretion of IL-6, without any differences in the mRNA expression levels of these cytokines in PBMCs. Partial correlation and multiple regression analysis using TNF-alpha and IL-6 mRNA levels as the dependent variables showed that urinary albumin excretion (UAE) was direct and independently associated with the expression profile of these pro-inflammatory cytokines in PBMCs. CONCLUSIONS: These data show for the first time the relationship between inflammatory activation of PBMCs (reflected by enhanced mRNA expression of TNF-alpha and IL-6) and renal involvement (reflected by increased UAE) in type 2 diabetic patients. These results provide potential insights for the increased inflammation, accelerated atherosclerosis and cardiovascular risk associated with nephropathy in type 2 diabetes.

The 5'-tail of antisense RNAII of pMV158 plays a critical role in binding to the target mRNA and in translation inhibition of repB.

Rolling-circle replication of streptococcal plasmid pMV158 is controlled by the concerted action of two trans-acting elements, namely transcriptional repressor CopG and antisense RNAII, which inhibit expression of the repB gene encoding the replication initiator protein. The pMV158-encoded antisense RNAII exerts its activity of replication control by inhibiting translation of the essential repB gene. RNAII is the smallest and simplest among the characterized antisense RNAs involved in control of plasmid replication. Structure analysis of RNAII revealed that it folds into an 8-bp-long stem containing a 1-nt bulge and closed by a 6-nt apical loop. This hairpin is flanked by a 17-nt-long single-stranded 5'-tail and an 8-nt-long 3'-terminal U-rich stretch. Here, the 3' and 5' regions of the 5'-tail of RNAII are shown to play a critical role in the binding to the target mRNA and in the inhibition of repB translation, respectively. In contrast, the apical loop of the single hairpin of RNAII plays a rather secondary role and the upper stem region hardly contributes to the binding or inhibition processes. The entire 5'-tail is required for efficient inhibition of repB translation, though only the 8-nt-long region adjacent to the hairpin seems to be essential for rapid binding to the mRNA. These results show that a "kissing" interaction involving base-pairing between complementary hairpin loops in RNAII and mRNA is not critical for efficient RNA/RNA binding or repB translation inhibition. A singular binding mechanism is envisaged whereby initial pairing between complementary single-stranded regions in the antisense and sense RNAs progresses upwards into the corresponding hairpin stems to form the intermolecular duplex.

Association of IL-6 gene polymorphisms and COPD in a Spanish population.

Interleukin-6 (IL-6) is a potential mediator of systemic effects of Chronic Obstructive Pulmonary Disease (COPD). In the present case-control study we investigated the association of promoter polymorphisms of this gene and COPD in a cohort of 191 patients, smokers without COPD (n=75) and a healthy control population (n=296). Besides spirometry, exercise capacity (6MWD, 6 min walking distance) and body mass index (BMI) were measured in COPD patients. Genotyping of the IL-6 polymorphisms at positions -174, -572 and -597 was performed. The -597G/A and -174G/C polymorphisms were not associated with the disease. However, the -572G/C polymorphism was significantly associated with COPD susceptibility under a dominant model of inheritance. The frequency of the genotypes containing the C allele was significantly lower in the COPD cases (9.9%) compared with the healthy control group (16.9%) and smokers (23.1%), (OR=0.46, p=0.032 and OR=0.28, p=0.012, respectively). The GCG (-597/-572/-174) haplotype was significantly associated with the disease (OR=0.37, p=0.022, COPD cases vs. healthy subjects and OR=0.17, p=0.011, COPD cases vs. smokers). Moreover, a borderline association was also found for the -572G allele and hypoxemia (PaO(2)<60 mmHg) (p=0.05). Our data suggest that the IL-6 -572C allele may confer a diminished risk of developing COPD.

High-level mupirocin resistance within methicillin-resistant Staphylococcus aureus pandemic lineages.

The methicillin-resistant Staphylococcus aureus (MRSA) population in the Hospital Universitario Nuestra Señora de Candelaria over a 5-year period (1998 to 2002) was marked by shifts in the circulation of pandemic clones. Here, we investigated the emergence of high-level mupirocin resistance (Hi-Mup(r)). In addition to clonal spread, transfer of ileS2-carrying plasmids played a significant role in the dissemination of Hi-Mup(r) among pandemic MRSA lineages.