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PubMed2005

Overexpression of the M2-2 protein of respiratory syncytial virus inhibits viral replication.

The M2-2 protein of respiratory syncytial virus (RSV) is involved in regulation of viral RNA transcription and replication. Encoded by the next-to-last gene of RSV, the M2-2 open reading frame (ORF) overlaps with the upstream M2-1 ORF, suggesting that the production of the M2-2 protein might be tightly regulated during virus replication. To evaluate the effect of M2-2 overexpression on RSV replication, the M2-2 gene was separated from M2-1 by leaving it at the position prior to the M2-1 or moving it to the promoter proximal position as an independent transcriptional unit in the RSV A2 genome. Although recombinant viruses bearing the shuffled M2-2 gene were recovered and expressed higher levels of M2-2, most of these viruses grew poorly in HEp-2 cells. Sequence analysis revealed that various mutations (substitution, insertion, and deletion) occurred in the M2-2 gene, resulting in reduced M2-2 activity as measured by the RSV minigenome system. Further examination of the M2-2 sequence and its function showed that either one of the first two AUG codons located at the 5' end of M2-2 could be used to produce a functional M2-2 protein and that deletion of the first six amino acids from its N terminus or four amino acids from its C terminus greatly reduced its function. The effect of M2-2 protein on RSV replication was also studied by examining RSV replication in cells transiently expressing M2-2. The M2-2 protein expressed at a high level completely inhibited RSV replication. These results strongly suggested that the level of the M2-2 protein produced in the infected cells is critical to RSV replication.

Animals↗
PubMed1992

Nucleotide sequence of the gene for an alkaline endoglucanase from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis.

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M(r) of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M(r) of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGCGGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.

Amino Acid Sequence↗
PubMed1989

Topography of early HPV 16 transcription in high-grade genital precancers.

The extent to which human papillomavirus (HPV) type 16 is transcribed and the nature of the transcripts produced in genital precancers has not been clearly defined. The authors analyzed 28 cases of cervical (CIN) or vulvar (VIN) intraepithelial neoplasia by RNA-RNA in situ hybridization, using probes generated from HPV 16 open reading frames (ORFs) either upstream (E6-E7) or downstream (E2-E5-L2) to the E1 ORF, where HPV 16 genomic integration most commonly occurs. Hybridization signals corresponding to one or both probes were detected in a high proportion of cells throughout the lesional epithelium of low- and high-grade CIN, including basal layers. In serial sections analyzed with the two probes, hybridization signals were obtained from both, and in similar proportion, irrespective of CIN grade. The distribution and character of hybridization signals suggests that the morphologic progression of precancers is not associated with either cessation of HPV 16 early transcription or a change in the general character of the transcripts produced.

DNA, Neoplasm↗
PubMed2001

Complex control of the developmental regulatory locus brlA in Aspergillus nidulans.

brlA is a primary regulator of asexual development in Aspergillus nidulans. Activation of brlA is necessary and sufficient for conidiophore development. It is known that brlA produces two overlapping transcripts, designated brlAalpha and brlAbeta. We found that expression of brlA is subject to complex regulation, in that activation of the two brlA transcripts is regulated at different levels. While brlAalpha is regulated at the transcriptional level, brlAbeta is regulated at both the transcriptional and translational levels. brlAalpha expression requires both abaA and brlA, but overexpression of brlAbeta can induce brlAalpha in an abaA mutant. brlAbetamuORF, a short ORF located upstream of the brlA initiator codon, regulates expression of brlA by damping translation of the brlAbeta ORF, and translational repression of brlA expression prevents premature development in A. nidulans. Transcriptional control of brlAbeta is apparently independent of BrlA. In order to understand better the transcriptional control of brlAalpha and brlAbeta, we have made 5' deletions in the essential approximately 2-kb upstream control sequences that regulate brlAbeta transcription and fused them to the E. coli lacZ reporter gene. Various deletions in this region resulted in only minor changes in the regulation of beta-galactosidase expression. The results of the deletion experiments indicate that there are probably several cis-acting control sequences involved in the regulation of brlAbeta. As a complementary approach, we fused various fragments of the 2034-bp brlAbeta and 754-bp brlAalpha control sequences to an otherwise inactive amdS::lacZ fusion, in order to search for regions that are sufficient to place the reporter under developmental control. We identified two approximately 600-bp brlAbeta fragments extending from -2901 to -2293 and -967 to -414, respectively, and a approximately 150-bp brlAalpha segment from -271 to -127, that confer activity on the inactive amdS promoter. brlA is overexpressed in an abaA null mutant and one site for abaA-dependent repression is apparently located in the -742 to -414 brlAbeta fragment. This indicates that abaA-mediated repression of brlA expression occurs through control of brlAbeta, but apparently involves a mechanism that does not require AbaA binding to brlA(p) sequences, because there are no AbaA binding sites in this region.

Aspergillus nidulans↗
PubMed2002

The Rhizobium etli gene iscN is highly expressed in bacteroids and required for nitrogen fixation.

Sequence analysis of the rpoN (2)- fixA intergenic region in the genome of Rhizobium etli CNPAF512 has uncovered three genes involved in nitrogen fixation, namely nifU, nifS and nifW. These genes are preceded by an ORF that is highly conserved among nitrogen-fixing bacteria. It encodes a putative gene product of 105 amino acids, belonging to the HesB-like protein family. A phylogenetic analysis of members of the HesB-like protein family showed that the R. etli HesB-like protein clusters with polypeptides encoded by ORFs situated upstream of the nifUS nitrogen fixation regions in the genomes of other diazotrophs. The R. etli ORF that encodes the HesB-like protein was designated iscN. iscN is co-transcribed with nifU and nifS, and is preferentially expressed under free-living microaerobic conditions and in bacteroids. Expression is regulated by the alternative sigma factor RpoN and the enchancer-binding protein NifA. A R. etli iscN mutant displays a reduction in nitrogen fixation capacity of 90% compared to the wild-type strain. This Nif(-) phenotype could be complemented by the introduction of intact copies of R. etli iscN.

Acetylene↗
PubMed2005

Transcripts encoding K12, v-FLIP, v-cyclin, and the microRNA cluster of Kaposi's sarcoma-associated herpesvirus originate from a common promoter.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three malignancies associated with AIDS and immunosuppression. Tumor cells harbor latent virus and express kaposin (open reading frame [ORF] K12), v-FLIP (ORF 71), v-Cyclin (ORF 72), and latency-associated nuclear antigen (LANA; ORF 73). ORFs 71 to 73 are transcribed as multicistronic RNAs initiating from adjacent constitutive and inducible promoters upstream of ORF 73. Here we characterize a third promoter embedded within the ORF 71-to-73 transcription unit specifying transcripts that encode ORF 71/72 or K12. These transcripts may also be the source of 11 microRNAs arranged as a cluster between K12 and ORF 71. Our studies reveal a complex arrangement of interlaced transcription units, incorporating four important protein-encoding genes required for latency and pathogenesis and the entire KSHV microRNA repertoire.

Base Sequence↗
PubMed2001

Prevalence and genotypic distribution of TT virus in Athens, Greece.

The prevalence of TT virus (TTV) infection in various population groups from Athens, Greece, was assessed by the polymerase chain reaction (PCR) using two primer sets from distinct regions of the genome: the conventional set derived from the open reading frame-1 (ORF-1) and the new, highly sensitive set targeting the region that includes the TATA signal localized upstream of ORF-2. Based on both primer sets, TTV DNA was detected in 42/50 (84.0%) healthy individuals, 42/50 (84.0%) chronic hepatitis C patients, 31/39 (79.5%) acute non-A-E hepatitis patients (group I), 14/16 (87.5%) renal failure patients with acute non-A-E hepatitis (group II), 47/50 (94.0%) intravenous drug users (IVDU), 36/50 (72.0%) hemophiliacs, and 21/31 (67.7%) hemodialysis patients. The presence of TTV was not associated with any particular risk group, and no differences were observed in relation to demographic, biochemical and virological characteristics between TTV DNA-positive and -negative patients. TTV did not seem to have a profound effect on the course of chronic C or acute non-A-E hepatitis either. Phylogenetic analysis revealed that TTV strains circulating in the greater metropolitan area of Athens belong not only to the G1 and G2 genotypes that are encountered worldwide, but also to G3 and to G5 that are found mainly in Europe and Asia, respectively. Further studies will shed light on the role of this highly prevalent virus.

Adolescent↗
PubMed1990

Characterization of three different nitrogen-regulated promoter regions for the expression of glnB and glnA in Azospirillum brasilense.

The complete nucleotide sequence of the open reading frame (ORF) located upstream of the glnA structural gene for glutamine synthetase (GS) in Azospirillum brasilense Sp7 was determined. This ORF, which codes for a 12 kDa protein, was identified as glnB, the structural gene for the PII protein, a component of the adenylylation cascade involved in the regulation of GS activity in some gram-negative bacteria. Transcription analysis and mRNA mapping of glnB and glnA of A. brasilense was performed with bacteria grown under different physiological conditions. The glnA gene can be transcribed either as a glnB-A mRNA of 2.4 kb or as a glnA mRNA of 1.5 kb. Differential expression of the two mRNAs was found to depend on the nitrogen source. The glnB-A mRNA was the major transcript under nitrogen fixation conditions, while the synthesis of the glnA mRNA was almost completely abolished. The glnA mRNA was predominantly produced in NH4(+)-containing medium. Transcription start site analysis revealed the presence of three different types of nitrogen-regulated promoters. GlnB-A mRNA was transcribed selectively from tandem promoters. One of them is similar to the NtrA-dependent promoter and the other to the Escherichia coli sigma 70 promoter. The synthesis of glnA mRNA was regulated by a promoter, which was repressed (or non-activated) only under conditions of nitrogen fixation, when moleuclar nitrogen was the sole nitrogen source. The transcriptional initiation site in front of glnA is not preceded by a canonical E. coli sigma 70 promoter. A sequence reminiscent of the NtrA-dependent promoter consensus, except for a fundamental mismatch, was found at positions -33 to -21. This sequence overlapped a putative "weak" NtrC-binding site, similar to those identified in enteric bacteria. From these results, it is postulated that glnA mRNA is controlled by a novel type of nitrogen-regulated promoter.

Amino Acid Sequence↗
PubMed1988

Accumulation of RNA homologous to human papillomavirus type 16 open reading frames in genital precancers.

The accumulation of human papillomavirus type 16 (HPV-16)-specific RNAs in tissue sections from biopsies of patients with genital precancers was studied by in situ hybridization with single-stranded 35S-labeled RNA. These analyses revealed that the most abundant early-region RNAs were derived from the E4 and E5 open reading frames (ORFs). RNAs homologous to the E6/E7 ORFs were also detected, whereas RNAs homologous to the intervening E1 ORF were not. This suggests that the E4 and E5 mRNAs are derived by splicing to the upstream E6/E7 ORFs, consistent with studies of HPV-11 in condylomata (L. T. Chow et al., Cancer Cells (Cold Spring Harbor) 5:55-72, 1987). Abundant RNAs homologous to the 5' portion of L1 were also detected. These RNAs were localized to the apical strata of the epithelium. HPV-16 RNAs accumulated in discrete regions of these lesions, and when present were most abundant in the upper cell layers of the precancerous epithelium. RNAs homologous to early ORFs were also detected in some germinal cells within the basal layer of the epithelium.

Epithelium↗
PubMed1995

A murC gene in Porphyromonas gingivalis 381.

The gene encoding a 51 kDa polypeptide of Porphyromonas gingivalis 381 was isolated by immunoblotting using an antiserum raised against P. gingivalis alkaline phosphatase. DNA sequence analysis of a 2.5 kb DNA fragment containing a gene encoding the 51 kDa protein revealed one complete and two incomplete ORFs. Database searches using the FASTA program revealed significant homology between the P. gingivalis 51 kDa protein and the MurC protein of Escherichia coli, which functions in peptidoglycan synthesis. The cloned 51 kDa protein encoded a functional product that complemented an E. coli murC mutant. Moreover, the ORF just upstream of murC coded for a protein that was 31% homologous with the E. coli MurG protein. The ORF just downstream of murC coded for a protein that was 17% homologous with the Streptococcus pneumoniae penicillin-binding protein 2B (PBP2B), which functions in peptidoglycan synthesis and is responsible for antibiotic resistance. These results suggest that P. gingivalis contains a homologue of the E. coli peptidoglycan synthesis gene murC and indicate the possibility of a cluster of genes responsible for cell division and cell growth, as in the E. coli mra region.

Alkaline Phosphatase↗
PubMed1988

Sequencing and expression of the 6'-N-acetyltransferase gene of transposon Tn1331 from Klebsiella pneumoniae.

Plasmid-mediated amikacin resistance in Klebsiella pneumoniae resides on a 1.5-kilobase BamHI fragment which is part of the Tn3-related multiresistance transposon Tn1331. In this work, we present the complete nucleotide sequence of the amikacin resistance gene and the neighboring sequences. Maxicell experiments detected only one polypeptide of 23 kilodaltons, the product of one of the open reading frames identified as ORF I. Comparison of the complete sequence with that of Tn3 indicated that 396 base pairs located just upstream from ORF I are identical to a region between the end of the tnpR gene and the first six amino acids of the beta-lactamase transcript. Sequences which may act as hot spots for recombination were identified. One was located just after amino acid 6 of beta-lactamase, and the other was located at the end of the amikacin resistance gene.

Acetyltransferases↗
PubMed1988

First committed step of lipid A biosynthesis in Escherichia coli: sequence of the lpxA gene.

The min 4 region of the Escherichia coli genome contains genes (lpxA and lpxB) that encode proteins involved in lipid A biosynthesis. We have determined the sequence of 1,350 base pairs of DNA upstream of the lpxB gene. This fragment of DNA contains the complete coding sequence for the 28.0-kilodalton lpxA gene product and an upstream open reading frame capable of encoding a 17-kilodalton protein (ORF17). In addition there appears to be an additional open reading frame (ORF?) immediately upstream of ORF17. The initiation codon for lpxA is a GUG codon, and the start codon for ORF17 is apparently a UUG codon. The start and stop codons overlap between ORF? and ORF17, ORF17 and lpxA, and lpxA and lpxB. This overlap is suggestive of translational coupling and argues that the genes are cotranscribed. Crowell et al. (D.N. Crowell, W.S. Reznikoff, and C.R.H. Raetz, J. Bacteriol. 169:5727-5734, 1987) and Tomasiewicz and McHenry (H.G. Tomasiewicz and C.S. McHenry, J. Bacteriol. 169:5735-5744, 1987) have demonstrated that there are three similarly overlapping coding regions downstream of lpxB including dnaE, suggesting the existence of a complex operon of at least seven genes: 5'-ORF?-ORF17-lpxA-lpxB-ORF23-dnaE-ORF37-3 '.

Acyltransferases↗
PubMed1991

The influence of antisense RNA on transcriptional mapping of the 5' terminus of a baculovirus RNA.

S1 nuclease protection and primer extension analyses were used to determine the 5' end of the Autographa californica nuclear polyhedrosis virus 603 open reading frame (ORF) transcript upstream of and on the opposite strand to the polyhedrin gene. These analyses suggested that the 5' end of the 603 ORF was located near the initiation site for polyhedrin gene transcription. Primer extension products of reverse transcription of 603 RNA were dependent on the presence of the TAAG sequence, an essential polyhedrin promoter element. The results could be interpreted as indicating bidirectional transcription from the TAAG element. However, the data could also be due to an artefact of antisense transcripts and RNA duplex formation in this region. Since the bidirectional transcriptional model is not consistent with other mapping data and Northern blot analysis, we conclude that the presence of antisense RNA can result in mapping artefacts and that caution must be taken in interpreting data where overlapping sense and antisense RNAs are present.

Animals↗
PubMed1994

Sequence of the essential early region of phi C31, a temperate phage of Streptomyces spp. with unusual features in its lytic development.

The temperate phage phi C31 is the most studied bacteriophage infecting Streptomyces spp., and has been used to develop an extensive and widely used series of cloning vectors. The sequence of 10 kb of phi C31 DNA containing most or all of the essential early genes was determined. Among the ORFs, 14 (perhaps 15) appear to be protein-coding, and these have been designated ORF1 to ORF14 and ORFX. Previously mapped transcripts appear to initiate upstream from ORFs 1, 8, 11 and 12, and within ORF3 and ORF12, in each case close to one example of the unusual ('21-mer') sequences that appear to serve as a recognition site for RNA polymerase early in the phi C31 lytic cycle [Ingham et al., Mol. Microbiol. 9 (1993) 1267-1274]. Further copies of the 21-mer are upstream from ORF2 and ORF13. There are four recognisable examples of a conserved inverted repeat sequence motif (CIR) thought to bind phi C31 repressor [Smith and Owen, Mol. Microbiol. 5 (1991) 2833-2844]. Only one CIR is closely associated with a 21-mer sequence, though three are located between known transcription units. Of all 14 ORFs, only one (ORF11) would encode a protein unmistakably resembling other known proteins; its product appears to be a DNA polymerase. Strikingly, two codons, TTA (Leu) and AGG (Arg), are absent from the 14 ORFs.

Amino Acid Sequence↗
PubMed1991

The nucleotide sequence and genome structure of the geminivirus miscanthus streak virus.

A tandem dimer of miscanthus streak virus (MiSV) DNA was inserted into the T-DNA of the binary plasmid vector pBIN19 and agroinoculated into several monocotyledonous plants (monocots) using Agrobacterium tumefaciens or A. rhizogenes. Disease symptoms and geminate particles were produced in maize and Panicum milaceum plants, and MiSV-specific double-stranded and single-stranded DNAs were found in these plants. The nucleotide sequence of the infectious MiSV clone, consisting of 2672 nucleotides, was determined. Four open reading frames (ORFs) for proteins of Mr greater than 10K were identified, two (V0 and V2) in the virus (+) sense and two (C1 and C2) in the complementary (-) sense, although C2 did not have an ATG start codon. Unlike other geminiviruses infecting monocots, complementary-sense ORFs did not overlap. Potential splicing donor and acceptor sites were identified in the sequence of the border region between the C terminus of ORF C1 and the N terminus of ORF C2. Amino acid sequences predicted from three (V2, C1 and C2) of these ORFs showed significant homology with the corresponding ORFs of other geminiviruses infecting monocots. A fifth ORF (V1), which showed some homology with ORF V1 of other monocot-infecting geminiviruses despite having a coding capacity for a product of Mr 8.8K, was found just upstream of ORF V2 as observed in those geminiviruses. ORF V0 showed no significant homology with ORFs present in any other geminiviruses. A mutation of V0 indicated that the C-terminal 30% of this ORF was not necessary for infection in maize, but that sequences around the mutated LspI site might have some regulatory role.

Amino Acid Sequence↗
PubMed1997

Flagellar flhA, flhB and flhE genes, organized in an operon, cluster upstream from the inv locus in Yersinia enterocolitica.

The inv gene of Yersinia enterocolitica codes for invasin, a member of the invasin/intimin-like protein family, which mediates the internalization of the bacterium into cultured epithelial cells. The putative inclusion of inv into a pathogenicity island was tested by investigating its flanking sequences. Indeed, the enteropathogenic Escherichia coli (EPEC) intimin, a member of the same family of proteins, is encoded by eaeA, a gene which belongs to a pathogenicity island. An ORF located upstream from inv was of particular interest since it appeared homologous both to the flagellar flhA gene and to sepA, an EPEC gene lying inside the same pathogenicity island as eaeA. A mutant in this ORF was non-motile and non-flagellated while its invasion phenotype remained unaffected. These data indicated that the ORF corresponded to the flhA gene of Y. enterocolitica. Subsequently, the flhB and flhE genes, located respectively upstream and downstream from flhA, were identified. The three flh genes appear to be transcribed from a single operon called flhB, according to the nomenclature used for Salmonella typhimurium. Intergenic sequence between flhE and inv includes a grey hole, with no recognizable function. Downstream from inv, we have detected the flagellar flgM operon as already reported. Finally, the incongruous localization of inv amidst the flagellar cluster is discussed; while transposition could explain this phenomenon, no trace of such an event was detected.

Adhesins, Bacterial↗
PubMed1998

Structure of catechol 2,3-dioxygenase gene from Alcaligenes eutrophus 335.

Catechol 2,3-dioxygenase (C23O), one of extradiol-type dioxygenases cleaving aromatic C-C bond at meta position of dihydroxylated aromatic substrates, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. As our ongoing study to characterize biochemical and genetic properties of the extradiol-type dioxygenases at molecular level, a C23O gene encoded in chromosomal DNA of Alcaligenes eutrophus 335, a strain degrading phenol and p-cresol, was cloned. The C23O gene was localized in an 1.4-kb PstI fragment from A. eutrophus 335, and was expressed in E. coli HB101. The C23O exhibited the highest aromatic ring-fission activity to catechol as a substrate, and its relative activity to other dihydroxylated aromatic substrates was in order of catechol >> 4-methylcatechol > 3-methylcatechol, protocatechuate, 4-chlorocatechol > 3,4-dihydroxy-phenylacetate > 2,3-dihydroxybiphenyl. Nucleotide sequence of the 1.4-kb fragment has revealed that an open reading frame (ORF) corresponding to the C23O gene was composed of 930 base pairs. A putative ribosome-binding sequence of AGGAG was found at about 10 nucleotides upstream the ORF which can encode a polypeptide of molecular weight 34 kDa consisting of 309 amino acid residues. The deduced amino acid sequence of C23O from A. eutrophus 335 exhibited the highest 59% identity with those of corresponding enzymes from Pseudomonas sp. CF600 (p VI150), P. putida HS1 (pDK1), and P. putida PpG7 (NAH7). An alignment of amino acid sequences of extradiol-type dioxygenases including C23O from A. eutrophus 335 has revealed that catalytically and structurally important amino acid residues of the enzymes were conserved during evolution.

Alcaligenes↗
PubMed1997

Positive regulation of Clostridium difficile toxins.

The toxigenic element of Clostridium difficile VPI 10463 contains a small open reading frame (ORF) immediately upstream of the toxin B gene (G. A. Hammond and J. L. Johnson, Microb. Pathog. 19:203-213, 1995). The deduced amino acid sequence of the ORF, which we have designated txeR, encodes a 22-kDa protein which contains a helix-turn-helix motif with sequence identity to DNA binding regulatory proteins. We used a DNA fragment containing the C. difficile toxin A repeating units (ARU) as a reporter gene to determine if txeR regulates expression from the toxin A and toxin B promoters in Escherichia coli. To test the affect of txeR on expression, we fused the ARU gene fragment in frame with the toxin promoters. The fusions expressed a 104-kDa protein that contained the epitopes for monoclonal antibody PCG-4, which we used to measure levels of recombinant ARU by enzyme-linked immunosorbent assay. When txeR was expressed in trans with the toxin B promoter-ARU fusion contained on separate low-copy-number plasmid, expression of ARU increased over 800-fold. Furthermore, when we tested the toxin A promoter fused to ARU, expression increased over 500-fold with txeR supplied in trans. Our results suggest that TxeR is a positive regulator that activates expression of the C. difficile toxins.

Amino Acid Sequence↗