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PubMed2005

SAT: a late NS protein of porcine parvovirus.

The genomes of all members of the Parvovirus genus were found to contain a small open reading frame (ORF), designated SAT, with a start codon four or seven nucleotides downstream of the VP2 initiation codon. Green fluorescent protein or FLAG fusion constructs of SAT demonstrated that these ORFs were expressed. Although the SAT proteins of the different parvoviruses are not particularly conserved, they were all predicted to contain a membrane-spanning helix, and mutations in this hydrophobic stretch affected the localization of the SAT protein. SAT colocalized with calreticulin in the membranes of the endoplasmic reticulum and the nucleus. A knockout mutant (SAT(-)), with an unmodified VP sequence, showed a "slow-spreading" phenotype. These knockout mutants could be complemented with VP2(-) SAT(+) mutant. The SAT protein is a late nonstructural (NS) protein, in contrast to previously identified NS proteins, since it is expressed from the same mRNA as VP2.

Amino Acid Sequence↗

PubMed1998

Developmental and tissue-specific expression of 2-methyl branched-chain enoyl CoA reductase isoforms in the parasitic nematode, Ascaris suum.

The 2-methyl branched-chain enoyl CoA reductase (ECR) plays a pivotal role in the reversal of beta-oxidation operating in anaerobic mitochondria of the parasitic nematode, Ascaris suum. Two-dimensional gel electrophoresis of the purified ECR yielded multiple spots, with two distinct but overlapping N-terminal sequences. These multiple isoforms were not the result of population effects, as the pattern observed on 2-D gels of the purified ECR was identical to those on immunoblots of muscle homogenates isolated from individual worms. A full-length cDNA coding for the major ECR isoform (ECRI) has been cloned and sequenced and compared with that of the minor isoform (ECRII) which has been described previously (Duran et al. J Biol Chem 1993;268:22391-22396). ECRI contained the 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, a 5' untranslated region (UTR) of 13 nucleotides, an open reading frame (ORF) of 1257 nucleotides, a 3'-UTR of 110 nucleotides that included the polyadenylation signal AATAAA downstream of the termination codon and a short poly(A) tail. The ORF predicted a 16 amino acid leader sequence not found in the native protein and a mature protein of 403 amino acids with a molecular weight of 43 698 and a predicted pI of 6.2. ECRI and ECRII were 73% identical at the predicted amino acid level and their mRNAs exhibited significant structural similarity even though they were products of separate genes. Comparison of ECRI and ECRII with the sequences of acyl CoA dehydrogenases from a variety of different sources revealed a high degree of interspecies sequence identity, suggesting that these enzymes may have evolved from a common ancestral gene. This result is surprising since the ascarid enzymes function as reductases, not as dehydrogenases. Both ECRs were tissue-specific and developmentally regulated and were found in transitional third-stage larvae (L3) and adult muscle, but not in early, aerobic larval stages or adult testis, ovary, or intestine. The ratio of ECRII to ECRI was greater in L3 than in adult muscle. Interestingly, both ECRs also appeared to be expressed in pharyngeal muscle, suggesting that branched-chain fatty acid synthesis may not be confined exclusively to body wall muscle.

Amino Acid Sequence↗

PubMed1986

Messenger RNA encoding the phosphoprotein (P) gene of human parainfluenza virus 3 is bicistronic.

The complete nucleotide sequence of the phosphoprotein (P) mRNA of human parainfluenza virus 3 (PIV-3) was derived from two cDNA clones spanning almost the entire P gene. The mRNA, excluding the poly(A) tail, is 2014 nucleotides long and is bicistronic. The first open reading frame (ORF) codes for the phosphoprotein (P) of mol wt 68,860. Seven nucleotides downstream from the first AUG codon, in a +1 reading frame, there is an additional ORF which can code for a polypeptide of mol wt 23,266. The latter protein appears to be similar to the C proteins found in cells infected with several paramyxoviruses. Comparison of the predicted amino acid sequence of the P and C proteins of PIV-3 with the corresponding Sendai virus proteins reveals considerable homology at the C-terminal half. In contrast, the P and C proteins of PIV-3 share very little homology with the measles virus P and C proteins, respectively.

Amino Acid Sequence↗

PubMed2005

The human cytomegalovirus UL78 gene is highly conserved among clinical isolates, but is dispensable for replication in fibroblasts and a renal artery organ-culture system.

The human cytomegalovirus (HCMV) UL78 ORF is considered to encode a seven-transmembrane receptor. However, neither the gene nor the UL78 protein has been characterized so far. The objective of this study was to investigate the UL78 gene and to clarify whether it is essential for replication. UL78 transcription was activated early after infection, was inhibited by cycloheximide but not by phosphonoacetic acid, and resulted in a 1.7 kb mRNA. Later in the replication cycle, a second mRNA of 4 kb evolved, comprising the UL77 and UL78 ORFs. The 5' end of the UL78 mRNA initiated 48 bp upstream of the translation start and the polyadenylated tail started 268 bp downstream of the UL78 translation stop codon within the UL79 ORF. By using bacterial artificial chromosome technology, a recombinant HCMV lacking most of the UL78 coding region was constructed. Successful reconstitution of the UL78-deficient virus proved that the gene was not essential for virus replication in fibroblasts. The deletion also did not reduce virus replication in ex vivo-cultured sections of human renal arteries. Analysis of viral proteins at different stages of the replication cycle confirmed these results. Among clinical HCMV isolates, the predicted UL78 protein was highly conserved. However, an accumulation of different single mutations could be found in the N-terminal region and at the very end of the C terminus. Due to the absence of an in vivo HCMV model, the role of UL78 in the pathogenesis of HCMV infection in humans remains unclear.

Adult↗

PubMed1988

Transformation of rat 3Y1 cells by human papillomavirus type-18 DNA.

Rat 3Y1 cell cultures transfected with human papillomavirus type-18 (HPV 18) DNA produced foci of morphologically transformed cells capable of anchorage-independent growth. Analyses of the transformed cell clones obtained after transfection with EcoRI-cleaved linear HPV 18 DNA showed that, although the viral DNAs integrated in the cell DNA had rearrangements (probably around the sites of integration), they appeared to retain the putative transcriptional control region and the downstream area containing E6E7 open reading frames (ORF) intact, and to be transcriptionally active. The expression plasmids constructed from an SV40-derived expression vector (pSV2-0) and HPV 18 DNA fragments containing either E6 or E7 ORF intact were examined for their transforming capacity. HPV 18 E7 ORF was found to encode a transforming gene for rat 3Y1 cells.

Cell Line↗

PubMed1997

Sequence and analysis of a dnaJ homologue gene in cyanobacterium Synechococcus sp. PCC7942.

The chromosomal region containing a dnaJ gene homologue (dnaJ7942) was sequenced from unicellular cyanobacterium Synechococcus sp. PCC7942. The dnaJ7942 gene as well as following two orfs are located in the region immediately downstream of dnaK3, and they seem to be cotranscribed. The dnaJ7942 gene product shares, like all J homologues, homology for the highly conserved "J-domain" of DnaJ. It does not have, however, a glycine and phenylalanine (G/F)-rich region nor cysteine (Cys)-rich region unlike the Escherichia coli DnaJ protein. When this gene was expressed in E. coli, cells became filamentous in contrast to those expressing the E. coli dnaJ gene. Gene disruption experiments indicated that the dnaJ7942 gene was essential for growth. Analysis of subcellular localization revealed that the DnaJ protein is mainly located on the thylakoid membrane in the cyanobacterium.

Amino Acid Sequence↗

PubMed1995

Differentiation-dependent transcription of the epidermodysplasia verruciformis-associated human papillomavirus type 5 in benign lesions.

Human papillomavirus type 5 (HPV 5) induces cutaneous lesions and persists in skin carcinomas of patients with epidermodysplasia verruciformis (EV). We investigated the expression pattern of HPV 5 in biopsies from benign skin lesions of EV patients by cDNA analysis and in situ hybridization. Nine different cDNAs could be generated from total RNA of one of these lesions by reverse transcription and PCR amplification with HPV 5-specific primers. We could identify two major splice donors: one was found in the E6-proximal part of the noncoding region (NCR), and the other just downstream of the first ATG codon of ORF E1. Each of the characterized transcripts was processed at one or the other donor site and the two corresponding leader exons were found in combination with both 3'-early and late exons. Two transcripts appear to be specific for EV-associated papillomaviruses: one species might encode an E1--E2C fusion protein, and the other mRNA (NCR/E2) is probably encoding for the full-length E2 protein. According to the results of the cDNA analysis, riboprobes were designed for in situ hybridization experiments to study the cell differentiation-dependent expression of the different exons. Only the E7/E1 and E4 probes led to strong signals almost throughout the epithelium. The signals generated by the 5'-E2 and E1 probe increased with cell differentiation and were mainly confined to the nucleus. The NCR, E6, E7, L2, and L1 probes yielded more or less strong signals in the terminally differentiated epidermal layers. The difference in the cell differentiation-dependent expression of the 5'-early region exon (probe E7/1) and L2/L1 exons may point to a differentiation-dependent processing of transcripts.

Base Sequence↗

PubMed1990

Genome structure of tobacco necrosis virus strain A.

An almost complete sequence of the RNA genome of tobacco necrosis virus (TNV) strain A has been determined. The genome organization is very similar to that of carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 5'-proximal open reading frame (ORF) encodes a 23-kDa protein and read-through of its amber codon into the second ORF is presumably used for the translation of a 82-kDa protein. The third large ORF encodes the 30-kDa coat protein. Two small ORFs are located upstream and one immediately downstream of this coat protein cistron. Extensive sequence similarity was found between the TNV 82-kDa protein and the putative polymerases of TCV, CarMV, cucumber necrosis virus (CNV), maize chlorotic mottle virus (MCMV), red clover necrotic mosaic virus (RCNMV), and barley yellow dwarf virus (BYDV). The TNV coat protein is very similar to southern bean mosaic virus (SBMV) capsid protein. Of the predicted small proteins only a 7.9-kDa protein shows some sequence similarity with a corresponding protein of MCMV, CarMV, and TCV. The others are unique to TNV. Except for the first four nucleotides at the 5' end no homology was found with the RNA of STNV (satellite of TNV).

Amino Acid Sequence↗

PubMed2004

Genetic and metabolic analysis of folate salvage in the human malaria parasite Plasmodium falciparum.

Antifolate drugs that target the biosynthesis and processing of essential folate cofactors are widely used for treatment of chloroquine-resistant falciparum malaria. Salvage of pre-formed folate can strongly compromise the efficacy of these drugs in vitro and the availability of folate from the human host in natural infections also influences therapeutic outcomes. To investigate how different parasite lines respond to the presence of exogenous folate, we measured the effect of the latter on the susceptibility of parasites to sulfa-drug blockage of folate biosynthesis, utilising the parents and 22 progeny of the HB3-Dd2 genetic cross of Plasmodium falciparum, together with selected unrelated lines. Complete linkage of the folate utilisation phenotype was observed to a DNA sequence of 48.6 kb lying between nucleotide positions 738,489 and 787,058 of chromosome 4 and encompassing the dihydrofolate reductase-thymidylate synthase (dhfr-ts) gene locus. Examination of the putative ORFs on this fragment upstream (3) and downstream (4) of dhfr-ts revealed no plausible candidate genes for folate processing. Similarly, a marked heterogeneity in the 5'-UTR regions of Dd2 and HB3, manifest as a directly repeated 256 bp sequence in the former, also did not correlate with the folate utilisation phenotype nor apparently influence levels of dhfr-ts transcripts or protein products. By contrast, the nature of the coding sequence of the dhfr domain appeared to play a direct role, with the single mutant (S108N) HB3-type utilising folic acid much less efficiently than other allelic variants. We also compared the processing of exogenous folic acid, folinic acid and p-aminobenzoic acid (pABA) in metabolic labelling studies of HB3 and Dd2. These support the view that DHFR is likely to have a low-level folate reductase activity as well as its normal function of reducing dihydrofolate to tetrahydrofolate, and that a significant hurdle in the utilisation of exogenous folic acid is the initial reduction of fully oxidised folic acid to dihydrofolate, an activity that the single mutant enzyme found in HB3 is postulated to perform particularly poorly. This would mirror earlier studies indicating that the DHFR activity of HB3 is also compromised relative to other variants.

4-Aminobenzoic Acid↗

PubMed1999

Biochemical evidence that Escherichia coli hyi (orf b0508, gip) gene encodes hydroxypyruvate isomerase.

We found a significant activity of hydroxypyruvate isomerase in Escherichia coli clone cells harboring an E. coli gene (called orf b0508 or gip), which is located downstream of the glyoxylate carboligase gene. We newly designated the gene hyi. The enzyme was purified from cell extracts of the E. coli clone. The enzyme had a molecular mass of 58 kDa and was composed of two identical subunits. The optimum pH for the isomerization of hydroxypyruvate was 6.8-7.2. The enzyme required no cofactor. It exclusively catalyzed the isomerization between hydroxypyruvate and tartronate semialdehyde. The apparent K(m) value for hydroxypyruvate was 12.5 mM. The amino acid sequence of E. coli hydroxypyruvate isomerase is highly similar to those of glyoxylate-induced proteins, Gip, found widely from prokaryotes to eukaryotes.

Aldose-Ketose Isomerases↗

PubMed2000

Temporal secretion of a multicellulolytic system in Myxobacter sp. AL-1. Molecular cloning and heterologous expression of cel9 encoding a modular endocellulase clustered in an operon with cel48, an exocellobiohydrolase gene.

The Gram-negative soil micro-organism Myxobacter sp. AL-1 possesses at least five extracellular cellulases, the production of which is regulated by the growth cycle. We cloned the complete gene for one of these cellulases, termed cel9, which encoded a 67-kDa modular family 9 endoglycohydrolase, which was produced during the stationary phase of growth and was strongly enhanced by avicel. The predicted product of cel9 matches the structural architecture of family 9 cellulases such as Thermonospora fusca endo/exocellulase E4. Cel9 protein was synthesized in Escherichia coli from a multicopy plasmid and in Bacillus subtilis from the isopropyl thiogalactoside-inducible Pspac promoter and was purified from the culture medium. Thermal stability, optimum pH and temperature dependence of Cel9 were similar when expressed from either source, and were indistinguishable from related cellulases produced by thermophilic bacteria. Downstream from cel9 was found a partial ORF, designated cel48, the deduced product of which was highly similar to bacterial exocellobiohydrolases and processive endoglucanases belonging to family 48 of the glycosyl hydrolases. The cel9 and cel48 genes appear to be arranged as part of an operon.

Amino Acid Sequence↗

PubMed1993

Nucleotide sequence and transcriptional analysis of the p10 gene of Spodoptera exigua nuclear polyhedrosis virus.

The p10 gene of Spodoptera exigua multiple nuclear polyhedrosis virus (SeMNPV) was localized on the XbaI fragment H (5.1 kb) of the physical map of the viral genome. The coding sequence of the SeMNPV p10 gene is 264 nucleotides (nt) long corresponding to a predicted protein of 88 amino acids with an MHF of 9607. The SeMNPV p10 protein showed only limited amino acid identity (39% and 26%, respectively) to those of Orgyia pseudotsugata MNPV (OpMNPV) and Autographa californica MNPV (AcMNPV) and thus appears less conserved than other viral proteins. The SeMNPV p10 gene was expressed by a transcript of approximately 450 nt, which started in the conserved baculovirus late gene promoter motif TAAG. The leader of the SeMNPV p10 transcript was AT-rich (92%) and at 36 nt was the shortest leader of all baculovirus major late genes reported so far. The SeMPNV p10 transcript terminated 6 nt downstream from a putative poly(A) signal sequence (AATAAA); the latter was 61 nt downstream of the translational stop codon TAA. Upstream and downstream of the p10 gene, partial putative ORFs were found that showed significant amino acid sequence identity to the baculovirus p26 and p74 proteins. It is concluded that the region of SeMNPV DNA containing the p10 gene is collinear with the corresponding regions in the AcMNPV and OpMNPV genomes.

Amino Acid Sequence↗

PubMed2001

Frankia sequences exhibiting RNA polymerase promoter activity.

Frankia are Gram-positive, filamentous bacteria capable of fixing atmospheric dinitrogen either in the free-living state or in symbiosis with a variety of woody plants. Only a few Frankia genes have been sequenced and gene expression is not well characterized. To isolate a segment of Frankia DNA that functions as an RNA polymerase promoter, fragments of Frankia strain ArI5 genomic DNA were cloned upstream of a promoterless, Vibrio harveyi luxAB cassette. Constructs were screened for luminescence in E. coli and positive clones assayed for in vitro transcription activity with a partially purified Frankia RNA polymerase extract. Primer extension analysis of in vitro transcripts produced from one clone, GLO7, identified two major transcription start sites, TSP-1 and TSP-2, 52 bp apart. Deletion analysis then localized sequences essential for promoter activity. The upstream promoter region, GLO7p1, contains sequences resembling the -35 element of a Streptomyces promoter and the -35 and -10 elements of the canonical E. coli promoter. Also within this region are two pentamers identical to sequences near the 5' end of the Frankia strain CpI1 glutamine synthetase gene. The second promoter, GLO7p2, contains a putative NtrC binding site at -145 and a possible sigma(N)-RNA polymerase recognition sequence at -14 suggesting that GLO7p2 may be a nitrogen-regulated promoter. An in vivo transcript representing an ORF of 498 aa starting 64 bp downstream of the distal transcription start, TSP-1, was detected by RT-PCR. This supports the conclusion that this DNA fragment has promoter activity in vivo as well as in vitro.

Actinomycetales↗

PubMed1996

Analysis of putative ABC transporter genes in Mycoplasma hyopneumoniae.

A previously described DNA probe specific for Mycoplasma hyopneumoniae (I-141) was fully sequenced and found to consist of 1618 bp and to contain two tandemly repeated ORFs. The deduced amino acid sequence of the two ORFs showed significant homologies with ATP-binding cassette (ABC) transporter proteins, particularly those of the eukaryotic multidrug resistance (MDR) protein family (up to 21% identity and 47% similarity). A somewhat lower homology was evident with the secretion protein HlyB of the RTX-haemolysin from Escherichia coli. The location of the two ORFs on the M. hyopneumoniae chromosome was downstream of the rrl gene encoding the 23S rRNA, but transcribed in the opposite direction. PCR amplification and subsequent chromosomal analysis by Southern blot hybridization of several M. hyopneumoniae strains showed that all field strains contained the two putative ABC transporter genes. However, some culture collection strains derived from strain J had lost these genes as the result of a 2221 bp deletion.

ATP-Binding Cassette Transporters↗

PubMed2003

Salmonella enterica serovar Typhimurium bla(PER-1)-carrying plasmid pSTI1 encodes an extended-spectrum aminoglycoside 6'-N-acetyltransferase of type Ib.

We have studied the aminoglycoside resistance gene, which confers high levels of resistance to both amikacin and gentamicin, that is carried by plasmid pSTI1 in the PER-1 beta-lactamase-producing strain of Salmonella enterica serovar Typhimurium previously isolated in Turkey. This gene, called aac(6')-Ib(11), was found in a class 1 integron and codes for a protein of 188 amino acids, a fusion product between the N-terminal moiety (8 amino acids) of the signal peptide of the beta-lactamase OXA-1 and the acetyltransferase. The gene lacked a plausible Shine-Dalgarno (SD) sequence and was located 45 nucleotides downstream from a small open reading frame, ORF-18, with a coding capacity of 18 amino acids and a properly spaced SD sequence likely to direct the initiation of aac(6')-Ib(11) translation. AAC(6')-Ib(11) had Leu118 and Ser119 as opposed to Gln and Leu or Gln and Ser, respectively, which were observed in all previously described enzymes of this type. We have evaluated the effect of Leu or Gln at position 118 by site-directed mutagenesis of aac(6')-Ib(11) and two other acetyltransferase gene variants, aac(6')-Ib(7) and -Ib(8), which naturally encode Gln118. Our results show that the combination of Leu118 and Ser119 confers an extended-spectrum aminoglycoside resistance, with the MICs of all aminoglycosides in clinical use, including gentamicin, being two to eight times higher for strains with Leu118 and Ser119 than for those with Gln118 and Ser119.

Acetyltransferases↗

PubMed1994

Nucleotide sequence of the rpoN gene and characterization of two downstream open reading frames in Pseudomonas aeruginosa.

The rpoN gene of Pseudomonas aeruginosa is required for the expression of a number of diverse genes, ranging from several classes of bacterial adhesins to enzymes for amino acid biosynthesis. The nucleotide sequence of the rpoN gene and its flanking region has been determined. The deduced amino acid sequence of the rpoN product is highly homologous to sequences of RpoN proteins of other microorganisms. Moreover, two open reading frames (ORF1 and ORF2) encoding peptides of 103 and 154 amino acids long, respectively, were found downstream of the rpoN gene. These two ORF products have a high degree of amino acid sequence homology with products of similar ORFs located adjacent to the rpoN genes in other microorganisms. Mutations in either ORF lead to a significant increase in P. aeruginosa generation time when propagated on minimal medium. These mutations had no effect on the expression of pilin or flagellin genes, whose expression depends on RpoN. Complementation analysis showed that the two ORFs are in the same transcriptional unit and the growth defects of the two ORF mutants on minimal medium are due to mutational effects on ORF2. The adverse effect of the ORF mutations on the growth of P. aeruginosa in minimal media can be suppressed by the addition of glutamine but not arginine, glutamate, histidine, or proline. Since rpoN mutants of P. aeruginosa display this same amino acid requirement for growth, the ORF2 product very likely functions as a coinducer of some but not all of the RpoN-controlled genes.

Amino Acid Sequence↗

PubMed2005

Construction of a bacterial artificial chromosome library for a myxobacterium of the genus Cystobacter and characterization of an antibiotic biosynthetic gene cluster.

A bacterial artificial chromosome (BAC) library was constructed to isolate the biosynthetic gene cluster for the polyketide/peptide hybrid-type antibiotic cystothiazole A from the myxobacterium Cystobacter fuscus strain AJ-13278. Sequence analysis of a 63.9 kb contiguous region that encompasses the biosynthetic gene cluster (cta) led to the identification of a polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) hybrid gene cluster 32.1 kb in size, which consists of six open reading frames (ORFs), ctaB to ctaG, as well as downstream genes ctaJ and ctaK (1.0 and 0.9 kb, respectively) responsible for the final biosynthetic steps. The genes ctaB, ctaE, and ctaF encode PKSs, the genes ctaC and ctaG encode NRPSs, and ctaD encodes an NRPS-PKS hybrid enzyme. Disruption of ctaD impaired cystothiazole A production. Additionally, two downstream genes, ctaJ and ctaK, which encode a nitrilase and an O-methyltransferase, respectively, must be responsible for the final methyl ester formation in the cystothiazole A biosynthesis.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed1995

Cloning and characterization of the cytotoxin L-encoding gene of Clostridium sordellii: homology with Clostridium difficile cytotoxin B.

Hybridization of an oligodeoxyribonucleotide (oligo) probe, designed from a repeated sequence ('oligo rep') at the C terminus of the Clostridium difficile (Cd) cytotoxin (Cyt), revealed that homologies exist between the Cd cyt gene and the genomes of several other clostridia, including Clostridium sordellii (Cs), suggesting a common ancestral cyt amongst the Clostridium genus. This Cd 'oligo rep' probe was used to clone the Cs (strain 6018) cyt. The sequenced (7095 bp) region encodes 2364 amino acids (aa) and corresponds to a protein of 270,614 Da. Cs Cyt has 76% identity with the Cd Cyt and 47% identity with the Cd enterotoxin (Ent). The latter third of the protein consists of repeated units, similar to those found for Cd Cyt. A highly conserved hydrophobic domain can be delineated. Few structural differences are evident between Cd and Cs Cyt to explain their different cellular and sub-cellular effects. A small open reading frame (ORF) encoding a protein of 16,484 Da is located 210 bp downstream from cyt. No homology was evident with any known sequence. The first 30 aa of this ORF may correspond to a signal peptide.

Amino Acid Sequence↗