General requirement for RNA polymerase II holoenzymes in vivo.

Yeast RNA polymerase II holoenzymes have been described that consist of RNA polymerase II, a subset of general transcription factors, and nine SRB regulatory proteins. The feature that distinguishes the RNA polymerase II holoenzymes from other forms of RNA polymerase II in the cell is their tight association with SRB proteins. We investigated the fraction of genes that require SRB proteins in vivo by examining the effect of temperature-sensitive mutations in SRB genes on transcription by RNA polymerase II. Upon transfer to the restrictive temperature, there is a rapid and general shutdown of mRNA synthesis in srb mutant cells. These data, combined with the observation that essentially all of the SRB protein in cells is tightly associated with RNA polymerase II molecules, argue that SRB-containing holoenzymes are the form of RNA polymerase II recruited to most promoters in the cell.

Intrinsic transcript cleavage activity of RNA polymerase.

The GreA and GreB transcript cleavage factors of Escherichia coli suppress elongation arrest and may have a proofreading role in transcription. With the use of E. coli greA-greB- mutant, RNA polymerase is demonstrated to possess substantial intrinsic transcript cleavage activity. Mildly alkaline pH mimics the effect of the Gre proteins by inducing transcript cleavage in ternary complexes and antagonizing elongation arrest through a cleavage-and-restart reaction. Thus, transcript cleavage constitutes the second enzymological activity of RNA polymerase along with polymerization/pyrophosphorolysis of RNA, whereas the Gre proteins merely enhance this intrinsic property.

Promoter architecture in the flagellar regulon of Bacillus subtilis: high-level expression of flagellin by the sigma D RNA polymerase requires an upstream promoter element.

Flagellin is one of the most abundant proteins in motile bacteria, yet its expression requires a low abundance sigma factor (sigma 28). We show that transcription from the Bacillus subtilis flagellin promoter is stimulated 20-fold by an upstream A+T-rich region [upstream promoter (UP) element] both in vivo and in vitro. This UP element is contacted by sigma 28 holoenzyme bound at the flagellin promoter and binds the isolated alpha 2 subassembly of RNA polymerase. The UP element increases the affinity of RNA polymerase for the flagellin promoter and stimulates transcription when initiation is limited by the rate of RNA polymerase binding. Comparison with other promoters in the flagellar regulon reveals a bipartite architecture: the -35 and -10 elements confer specificity for sigma 28, while promoter strength is determined largely by upstream DNA sequences.

Quantitative assessment of sense and antisense transcripts from genes involved in antigenic variation (vsp genes) and encystation (cwp 1 gene) of Giardia lamblia clone GS/M-83-H7.

Antigenic variation of the intestinal protozoan parasite Giardia lamblia is caused by an exchange of the parasite's variant surface protein (VSP) coat. Many investigations on antigenic variation were performed with G. lamblia clone GS/M-83-H7 which produces surface antigen VSP H7. To generate novel information on giardial vsp gene transcription, vsp RNA levels were assessed by quantitative reverse transcription-(RT)-PCR in both axenic VSP H7-type trophozoites and subvariants obtained after negative selection of GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. Our investigation was not restricted to the assessment of the sense vsp transcript levels but also included an approach aimed at the detection of complementary antisense vsp transcripts within the two trophozoite populations. We found that sense vsp H7 RNA predominated in VSP H7-type trophozoites while sense RNA from only one (vsp IVg) of 8 subvariant vsp genes totally analysed predominated in subvariant-type trophozoites. Interestingly, the two trophozoite populations exhibited a similar relative distribution regarding the vsp H7 and vsp IVg antisense RNA molecules. An analogous sense versus antisense RNA pattern was also observed when the transcripts of gene cwp 1 (encoding cyst wall protein 1) were investigated. Here, both types of RNA molecules only appeared after cwp 1 had been induced through in vitro encystation of the parasite. These findings for the first time demonstrated that giardial antisense RNA production did not occur in a constitutive manner but was directly linked to complementary sense RNA production after activation of the respective gene systems.

Q192R polymorphism of the paraoxonase-1 gene as a risk factor for obesity in Portuguese women

Introduction - Obesity became a major public health problem as a result of its increasing prevalence worldwide. Paraoxonase-1 (PON1) is an esterase able to protect membranes and lipoproteins from oxidative modifications. At the PON1 gene, several polymorphisms in the promoter and coding regions have been identified. The aims of this study were i) to assess PON1 L55M and Q192R polymorphisms as a risk factor for obesity in women; ii) to compare PON1 activity according to the expression of each allele in L55M and Q192R polymorphisms; iii) to compare PON1 activity between obese and normal-weight women. Materials and methods - We studied 75 healthy (35.9±8.2 years) and 81 obese women (34.3±8.2 years). Inclusion criteria for obese subjects were body mass index ≥30 kg/m2 and absence of inflammatory/neoplasic conditions or kidney/hepatic dysfunction. The two PON1 polymorphisms were assessed by real-time PCR with TaqMan probes. PON1 enzymatic activity was assessed by spectrophotometric methods, using paraoxon as a substrate. Results - No significant differences were found for PON1 activity between normal and obese women. Nevertheless, PON1 activity was greater (P<0.01) for the RR genotype (in Q192R polymorphism) and for the LL genotype (in L55M polymorphism). The frequency of allele R of Q192R polymorphism was significantly higher in obese women (P<0.05) and was associated with an increased risk of obesity (odds ratio=2.0 – 95% confidence interval (1.04; 3.87)). Conclusion - 55M and Q192R polymorphisms influence PON1 activity. The allele R of the Q192R polymorphism is associated with an increased risk for development of obesity among Portuguese Caucasian premenopausal women.

Frequency of the toxic shock syndrome toxin-1 gene in methicillin-susceptible and -resistant Staphylococcus aureus isolates from teaching hospitals in Shiraz, Iran

INTRODUCTION: Staphylococcus aureus produces a range of virulence factors such as toxic shock syndrome toxin-1. METHODS: In this cross-sectional study of 345 clinical S. aureus isolates, the presence of the tst gene was assessed by polymerase chain reaction (PCR). RESULTS: The study revealed 53/345 (15.4%) isolates were positive for the tst gene. The tst gene was present in 18.1% of methicillin-susceptible S. aureus (MSSA) isolates and 11.6% of methicillin-resistant S. aureus (MRSA) isolates (p = 0.136). CONCLUSIONS: These results reveal the remarkable risk of S. aureus infections in hospitals, regardless of methicillin-resistance status.

Molecular characterization of Echinococcus granulosusfrom Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene

Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10), have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44), sheep (41) and humans (14) from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4). The G1 genotype is considered the most widespread and infectious form of E. granulosusworldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

Estrogen receptor 1 gene polymorphisms and coronary artery disease in the Brazilian population

We examined the association of three established single nucleotide polymorphisms, IVS1-397T>C, IVS1-351A>G, and +261G>C, in the ESR1 gene with the prevalence and severity of coronary atherosclerosis in a southern Brazilian population of European ancestry. Three hundred and forty-one subjects (127 women and 214 men) with coronary artery disease (CAD) were classified as having significant disease (CAD+ patient group) when they showed 60% or more luminal stenosis in at least one coronary artery or major branch segment at angiography; patients with 10% or less luminal stenosis were considered to have minimal CAD (CAD- patient group). The control sample consisted of 142 subjects (79 women and 63 men) without significant disease, in whom coronary angiography to rule out the presence of asymptomatic CAD was not performed. The polymorphisms were investigated by polymerase chain reaction followed by restriction analyses. In the male sample, the +261G>C*C allele was more frequent in CAD+ than CAD- subjects (8 versus 1%, P = 0.024). Homozygosity for the C allele of the IVS1-397T>C polymorphism was also significantly associated with increased CAD severity (OR: 2.99; 95% CI = 1.35-6.63; P = 0.007). In agreement with previous findings, these results suggest that the IVS1-397T>C*C allele was associated with CAD severity independent of gender, whereas the association of the +261G>C variant with CAD was observed in males only. The relation between ESR1 variation and CAD may influence clinical decisions such as the use of hormone therapy, and additionally will be helpful to identify the genetic susceptibility determinants of cardiovascular disease development.

EBNA-1 gene sequences in Brazilian and American patients with Hodgkin's disease

We examined the types of Epstein-Barr virus-associated nuclear antigen-1 (EBNA-1) gene carboxy (C)-terminal mutations occurring in Hodgkin's disease (HD) and reactive tissues from two different geographic regions. Previously reported EBNA-1 C-terminal region amino acid sequence variants, based on the amino acid at codon 487, include Prototype (P)-ala, which is found in the B95.8-derived prototype virus, P-thr, Variant (V)-leu, V-val, and V-pro. Using polymerase chain reaction (PCR) to amplify portions of the EBNA-1 gene, followed by DNA sequencing, we found a single EBNA-1 gene sequence variant in each tissue, whether reactive or neoplastic and whether from Brazil or the United States. Variant EBNA-1 gene sequences were more common in both neoplastic and non-neoplastic tissues from different geographic areas than the so-called prototype sequence. In the 17 Brazilian HD cases, 4 cases had P-thr variants and 13 had V-leu variants. In the six reactive tissues from Brazil, one had a P-ala variant, two had P-thr variants, and three had V-leu variants. In the 12 American HD cases, 2 had P-ala variants, 6 had P-thr variants, and 4 had V-leu variants. The 11 American reactive tissues included 2 P ala variants, 5 P-thr variants, and 4 V-leu variants. In both countries, there were similar variant EBNA-1 sequences present in normal tissues and HD cases. Compared with the P ala and P-thr cases, the V-leu cases were more likely to have the 30-bp latent membrane protein 1 (LMP1) gene deletion (P = 0.0075). In addition, cases of HD with the V-leu were statistically associated with a substitution of asparagine for glutamine at codon 322 of the C-terminal portion of the LMP1 gene. Our results suggest that any variation in EBNA-1 gene sequence is caused by a polymorphism present in pre-existing viral strains in the underlying population, and not a mutation occurring during oncogenesis. (C) 1999 by the American Society of Hematology.

Deletion of Epstein-Barr virus latent membrane protein 1 gene in United States and Brazilian Hodgkin's disease and reactive lymphoid tissue: High frequency of a 30-bp deletion

A 30-basepair (bp) deletion in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene has been reported in nasopharyngeal carcinoma and EBV-associated malignant lymphomas. Prior studies have found the deletion in about 10% to 28% of cases of Hodgkin's disease (HD), particularly in cases with aggressive histology. We studied the prevalence of 30-bp LMP1 gene deletion in EBV-positive HD in the United States (US) (12 cases) and Brazil (26 cases) with comparison to reactive lymphoid tissues (21 cases) and HD without EBV-positive Reed-Sternberg cells (15 cases). We studied the status of the LMP1 gene by Southern blot hybridization of polymerase chain reaction (PCR) products obtained after amplification with primers spanning the site of the deletion. We also performed EBV typing, EBER1 in situ hybridization, and LMP1 protein immunohistochemistry. EBV was detected in 12/26 (46%) cases of HD from the US and 26/27 (96%) cases of Brazilian HD. The 30-bp LMP1 gene deletion was observed in 4/12 (33%) cases of EBV-positive HD from US, and 12/26 (46%) cases of Brazilian EBV-positive HD, including 3 cases of type B EBV, as compared with 12/21 (57%) reactive lymphoid tissues and 9/15 (60%) cases of EBV-negative HD. US and Brazilian HD showed a higher prevalence of the 30-bp LMP1 gene deletion, compared with studies of others. The unexpected finding of high incidence of 30-bp deletion in LMP1 gene in reactive lymphoid tissue and HD without EBV-positive Reed-Sternberg cells suggests that this deletion may not be relevant to HD pathogenesis in most cases. Copyright (C) 1997 by W.B. Saunders Company.

Critical protective role for annexin 1 gene expression in the endotoxemic murine microcirculation

The inflammatory response is a protective process of the body to counteract xenobiotic penetration and injury, although in disease this response can become deregulated. There are endogenous biochemical pathways that operate in the host to keep inflammation under control. Here we demonstrate that the counter-regulator annexin 1 (AnxA1) is critical for controlling experimental endotoxemia. Lipopolysaccharide (LPS) markedly activated the AnxA1 gene in epithelial cells, neutrophils, and peritoneal, mesenteric, and alveolar macrophages-cell types known to function in experimental endotoxemia. Administration of LPS to AnxA1-deficient mice produced a toxic response characterized by organ injury and lethality within 48 hours, a phenotype rescued by exogenous application of low doses of the protein. In the absence of AnxA1, LPS generated a deregulated cellular and cytokine response with a marked degree of leukocyte adhesion in the microcirculation. Analysis of LPS receptor expression in AnxA1-null macrophages indicated an aberrant expression of Toll-like receptor 4. In conclusion, this study has detailed cellular and biochemical alterations associated with AnxA1 gene deletion and highlighted the impact of this protective circuit for the correct functioning of the homeostatic response to sublethal doses of LPS. Copyright © American Society for Investigative Pathology.

Age-of-onset-dependent influence of NOD2 gene variants on disease behaviour and treatment in Crohn's disease

BACKGROUND: Influence of genetic variants in the NOD2 gene may play a more important role in disease activity, behaviour and treatment of pediatric- than adult-onset Crohn's disease (CD). METHODS: 85 pediatric- and 117 adult-onset CD patients were tested for the three main NOD2 CD-associated variants (p.R702W, p.G908R and p.10007fs) and clinical data of at least two years of follow-up were compared regarding disease behaviour and activity, response to therapy and bone mineral density (BMD). RESULTS: Chronic active and moderate to severe course of CD is associated in patients with pediatric-onset (p=0.0001) and NOD2 variant alleles (p=0.0001). In pediatric-onset CD the average PCDAI-Score was significantly higher in patients carrying NOD2 variants (p=0.0008). In addition, underweight during course of the disease (p=0.012) was associated with NOD2 variants. Interestingly, osteoporosis was found more frequently in patients carrying NOD2 variant alleles (p=0.033), especially in pediatric-onset CD patients with homozygous NOD2 variants (p=0.037). Accordingly, low BMD in pediatric-onset CD is associated with a higher PCDAI (p=0.0092), chronic active disease (p=0.0148), underweight at diagnosis (p=0.0271) and during follow-up (p=0.0109). Furthermore, pediatric-onset CD patients with NOD2 variants are more frequently steroid-dependent or refractory (p=0.048) and need long-term immunosuppressive therapy (p=0.0213). CONCLUSIONS: These data suggests that the presence of any of the main NOD2 variants in CD is associated with osteoporosis and an age of onset dependent influence towards underweight, higher disease activity and a more intensive immunosuppressive therapy. This observation supports the idea for an early intensive treatment strategy in children and adolescent CD patients with NOD2 gene variants.

Chlorophyll breakdown: Pheophorbide a oxygenase is a Rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 gene

Chlorophyll (chl) breakdown during senescence is an integral part of plant development and leads to the accumulation of colorless catabolites. The loss of green pigment is due to an oxygenolytic opening of the porphyrin macrocycle of pheophorbide (pheide) a followed by a reduction to yield a fluorescent chl catabolite. This step is comprised of the interaction of two enzymes, pheide a oxygenase (PaO) and red chl catabolite reductase. PaO activity is found only during senescence, hence PaO seems to be a key regulator of chl catabolism. Whereas red chl catabolite reductase has been cloned, the nature of PaO has remained elusive. Here we report on the identification of the PaO gene of Arabidopsis thaliana (AtPaO). AtPaO is a Rieske-type iron–sulfur cluster-containing enzyme that is identical to Arabidopsis accelerated cell death 1 and homologous to lethal leaf spot 1 (LLS1) of maize. Biochemical properties of recombinant AtPaO were identical to PaO isolated from a natural source. Production of fluorescent chl catabolite-1 required ferredoxin as an electron source and both substrates, pheide a and molecular oxygen. By using a maize lls1 mutant, the in vivo function of PaO, i.e., degradation of pheide a during senescence, could be confirmed. Thus, lls1 leaves stayed green during dark incubation and accumulated pheide a that caused a light-dependent lesion mimic phenotype. Whereas proteins were degraded similarly in wild type and lls1, a chl-binding protein was selectively retained in the mutant. PaO expression correlated positively with senescence, but the enzyme appeared to be post-translationally regulated as well.

Guideline to reference gene selection for quantitative real-time PCR

Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and P-actin have been used for that purpose. However, it has been reported that these genes as well as alternatives, like rRNA genes, are unsuitable references, because their transcription is significantly regulated in various experimental settings and variable in different tissues. Therefore, quantitative real-time PCR was used to determine the mRNA transcription profiles of 13 putative reference genes, comparing their transcription in 16 different tissues and in CCRF-HSB-2 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and ionomycin. Our results show that Classical reference genes are indeed unsuitable, whereas the RNA polymerase II gene was the gene with the most constant expression in different tissues and following stimulation in CCRF-HSB-2 cells. (C) 2003 Elsevier Inc. All rights reserved.

von Hippel Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin-proteasome pathway. Here, we identify von Hippel-Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel-Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin-VHL-proteasome pathway in the integration-transcription transition of the viral replication cycle.