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PubMed2005

Sequence analysis of two cryptic plasmids from an agricultural isolate of Campylobacter coli.

As part of a study identifying plasmids in Campylobacter, we isolated and sequenced two novel cryptic plasmids from an agricultural isolate of Campylobacter coli. The larger of the two plasmids, p3384, is 3316 bp in length and has a G+C content of 31.18%. A typical origin of replication consisting of five iterons was observed directly upstream of the first of three putative ORFs. The smaller plasmid, p3386, is 2426 bp in length and has a G+C content of 26.22%. Of the three putative ORFs detected on p3386, one shared homology with a putative protein from Campylobacter upsaliensis. The unique sequence of p3386 makes it attractive for further study concerning the evolutionary relationship of this plasmid to other Campylobacter plasmids, and to other Campylobacter isolates.

Base Composition↗
PubMed1997

Mutation of D. radiodurans in a gene homologous to ruvB of E. coli.

Following the digestion of chromosomal DNA of Deinococcus radiodurans with a restriction enzyme a partial genomic library was constructed using lambda phage as a vector. A phage clone whose DNA can complement the deficiency in a radiation-sensitive mutant of D. radiodurans was isolated. Following the subcloning using phasmid vector, a hybrid plasmid containing 1.2 kb inserted DNA was obtained. After the determination of nucleotide sequence, the deduced amino acid sequence showed close homology to RuvB protein of Escherichia coli; approximately 81% of the amino acids (310 residues in total) was homologous (152 were identical and 100 amino acids were similar). The putative protein has a conserved ATP binding domain characteristic of DNA helicases. However, we could not find an SOS promoter and ORF for RuvA protein in the sequence upstream of ruvB in contrast to the E. coli homologue. The mutant was transformed with exogenous DNA at the same rate as the wild-type cells, but it was moderately sensitive to UV, gamma-rays and to interstrand cross-linking reagents.

Amino Acid Sequence↗
PubMed2003

Cloning and expression of the gene encoding catalytic subunit of thermostable glucose dehydrogenase from Burkholderia cepacia in Escherichia coli.

We have cloned a 1620-nucleotide gene encoding the catalytic subunit (alpha subunit) of a thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia. The FAD binding motif was found in the N-terminal region of the alpha subunit. The deduced primary structure of the alpha subunit showed about 48% identity to the catalytic subunits of sorbitol dehydrogenase (SDH) from Gluconobacter oxydans and 2-keto-D-gluconate dehydrogenases (2KGDH) from Erwinia herbicola and Pantoea citrea. The alpha subunit of B. cepacia was expressed in Escherichia coli in its active water-soluble form, showing maximum dye-mediated GDH activity at 70 degrees C, retaining high thermal stability. A putative open reading frame (ORF) of 507 nucleotides was also found upstream of the alpha subunit encoding an 18-kDa peptide, designated as gamma subunit. The deduced primary structure of gamma subunit showed about 30% identity to the small subunits of the SDH from G. oxydans and 2KGDHs from E. herbicola and P. citrea.

Amino Acid Sequence↗
PubMed1999

Differential expression of Myc1 and Myc2 isoforms in cells transformed by eIF4E: evidence for internal ribosome repositioning in the human c-myc 5'UTR.

eIF4E is essential for translation initiation, but its overexpression causes malignant transformation. Recent work demonstrated that eIF4E/F participates in exposing and locating alternate translation start codons during scanning. Translation initiation of several important protooncogenes and growth-regulators, such as Myc and FGF-2, can start at CUG start codon(s) upstream of the normal open reading frame (ORF). The resulting amino-terminal extension alters the properties of these proteins and their intracellular distribution. In cells overexpressing eIF4E, c-myc is overexpressed and particularly the larger, CUG-initiated form (Myc1). Recent reports suggest that synthesis of Myc2, the normally expressed AUG-initiated form, is mediated by an IRES. To determine what role eIF4E might play in c-myc expression, the c-myc 5' untranslated region (UTR) was fused in-frame to CAT reporters, and several more derivative constructs were made. In vitro translation experiments (with and without eIF4E/F); expression in CHO cells transformed with eIF4E; and deletion/mutation analysis demonstrated that Myc1 is translated by a scanning mechanism, while Myc2 is translated by Internal Ribosome Repositioning. Moreover, the existence of a true IRES in the 5'UTR was contradicted by its failure to direct translation of a circular transcript, in contrast to hsp70. The c-myc 5'UTR also failed to engage in translation in the absence of functional eIF4F, after cleavage of the eIF4G component with CVB4 protease-2A. The Internal Repositioning Element (IRPE) in c-myc 5'UTR was delimited to nucleotides (nt) 394-440 from the P1 transcription start site.

5' Untranslated Regions↗
PubMed1994

Partial characterization of the lettuce infectious yellows virus genomic RNAs, identification of the coat protein gene and comparison of its amino acid sequence with those of other filamentous RNA plant viruses.

Purified virions of lettuce infectious yellows virus (LIYV), a tentative member of the closterovirus group, contained two RNAs of approximately 8500 and 7300 nucleotides (RNAs 1 and 2 respectively) and a single coat protein species with M(r) of approximately 28,000. LIYV-infected plants contained multiple dsRNAs. The two largest were the correct size for the replicative forms of LIYV virion RNAs 1 and 2. To assess the relationships between LIYV RNAs 1 and 2, cDNAs corresponding to the virion RNAs were cloned. Northern blot hybridization analysis showed no detectable sequence homology between these RNAs. A partial amino acid sequence obtained from purified LIYV coat protein was found to align in the most upstream of four complete open reading frames (ORFs) identified in a LIYV RNA 2 cDNA clone. The identity of this ORF was confirmed as the LIYV coat protein gene by immunological analysis of the gene product expressed in vitro and in Escherichia coli. Computer analysis of the LIYV coat protein amino acid sequence indicated that it belongs to a large family of proteins forming filamentous capsids of RNA plant viruses. The LIYV coat protein appears to be most closely related to the coat proteins of two closteroviruses, beet yellows virus and citrus tristeza virus.

Amino Acid Sequence↗
PubMed1999

Transcription-modulatory activities of differentially spliced cDNAs encoding the E2 protein of human papillomavirus type 16.

Human papillomavirus (HPV) type 16 expresses a variety of alternatively spliced polycistronic mRNAs encoding the E2 transcription-regulatory protein. These mRNAs initiate at the p97 promoter and contain the 880/2708 (a-type), 880/2581 (a'-type) and 226/2708 (d-type) splice sites upstream from the E2 open reading frame (ORF). Recent studies investigating the translational capacities of partial cDNAs representing three of these mRNAs indicated their abilities to function in E2 protein translation, although at different efficiencies. In the present study, the transcription-regulatory activities of the E2 cDNAs towards the virus long control region (LCR) have been examined. LCR regulation was evaluated in transient transfection assays by using the chloramphenicol acetyltransferase reporter gene linked to the HPV-16 LCR. Transfections were carried out into fibroblast (Cf2Th) and epithelial (C33A) cell lines. It is shown that all three E2 cDNAs transrepressed the virus LCR in a dose-dependent manner. Transrepression was mainly dependent on the function of the E2 ORF and was abolished or markedly reduced by premature termination or truncation of the E2 ORF. Transrepression activities exhibited by the various E2 cDNAs correlated with the previously defined efficiencies of E2 protein translation from the respective templates. The truncated E2 cDNAs exhibited variable low regulatory activities that correlated with the activities of the 5' ORFs contained in each cDNA. The E6I and E1C ORFs transactivated the virus LCR whereas the E6IV cDNA transrepressed LCR activity. Thus, the 5' ORFs contribute in different manners to the overall activities of the polycistronic cDNAs.

Animals↗
PubMed2000

2-Oxoacid dehydrogenase multienzyme complexes in the halophilic Archaea? Gene sequences and protein structural predictions.

All Archaea catalyse the conversion of pyruvate to acetyl-CoA via a simple pyruvate oxidoreductase. This is in contrast to the Eukarya and most aerobic bacteria, which use the pyruvate dehydrogenase multienzyme complex [PDHC], consisting of multiple copies of three component enzymes: E1 (pyruvate decarboxylase), E2 (lipoate acetyl-transferase) and E3 (dihydrolipoamide dehydrogenase, DHLipDH). Until now no PDHC activity has been found in the Archaea, although DHLipDH has been discovered in the extremely halophilic Archaea and its gene sequence has been determined. In this paper, the discovery and sequencing of an operon containing the DHLipDH gene in the halophilic archaeon Haloferax volcanii are reported. Upstream of the DHLipDH gene are 3 ORFs which show highest sequence identities with the E1alpha, E1beta and E2 genes of the PDHC from gram-positive organisms. Structural predictions of the proposed protein product of the E2 gene show a domain structure characteristic of the E2 component in PDHCs, and catalytically important residues, including the lysine to which the lipoic acid cofactor is covalently bound, are conserved. Northern analyses indicate the transcription of the whole operon, but no PDHC enzymic activity could be detected in cell extracts. The presence in the E2 gene of an insertion (equivalent to approximately 100 aa) not found in bacterial or eukaryal E2 proteins, might be predicted to prevent multienzyme complex assembly. This is the first detailed report of the genes for a putative 2-oxoacid dehydrogenase complex in the Archaea, and the evolutionary and metabolic consequences of these findings are discussed.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1995

The genomic region of rbcLS in Synechococcus sp. PCC 7942 contains genes involved in the ability to grow under low CO2 concentration and in chlorophyll biosynthesis.

Several genes involved in the ability of Synechococcus sp. PCC 7942 to grow under different CO2 concentrations were mapped in the genomic region of rbcLS (the operon encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase). Insertion of a cartridge encoding kanamycin resistance within open reading frame (ORF) 78, designated ccmJ, located 7 kb upstream of rbcLS, resulted in a kanamycin-resistant, high-CO2-requiring mutant, M3, which does not contain normal carboxysomes. ccmJ shows significant homology to csoS1 encoding a carboxysomal shell polypeptide in Thiobacillus neopolitanus. Analysis of the polypeptide pattern of a carboxysome-enriched fraction indicated several differences between the wild type and the mutant. The amount of the ribulose-1,5-bisphosphate carboxylase/oxygenase subunits was considerably smaller in the carboxysomal fraction of the mutant when compared to the wild type. On the basis of the sequence analyses, ORF286 and ORF466, located downstream of ccmJ, were identified as chlL and chlN, respectively, which are involved in chlorophyll biosynthesis in the dark.

Algal Proteins↗
PubMed1994

Helicobacter pylori hspA-hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity.

All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel-dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat-shock protein of the GroEL class. The gene encoding the GroEL-like protein of H. pylori (HspB) was cloned (pILL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli, the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C-terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel-binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the pILL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (pILL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in the chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose-binding protein (MBP) and purified in large scale. The MBP-HspA and MBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori-infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.

Amino Acid Sequence↗
PubMed1990

Analysis and sequence of the speB gene encoding agmatine ureohydrolase, a putrescine biosynthetic enzyme in Escherichia coli.

The speB gene of Escherichia coli encodes the enzyme agmatine ureohydrolase (AUH). AUH catalyzes the hydrolysis of agmatine to urea and putrescine in one of the two polyamine biosynthetic pathways in E. coli. Sequencing of a 2.97-kilobase-pair fragment of the E. coli chromosome containing speB revealed the presence of three intact open reading frames (ORFs), ORF1 and ORF2 on one strand and ORF3 on the opposite strand, as well as a truncated ORF, ORF4, which terminated 92 kilobase pairs upstream from ORF3. ORF3 contained the coding sequence of the speB gene, as confirmed by complementation analysis. Two ORF3 transcripts were detected: a shorter transcript that included only ORF3 and a longer transcript that included both ORF3 and ORF4. The short transcript was abundantly expressed when the ORF4 sequences were deleted, but when ORF4 and its upstream sequences were present, the polycistronic message predominated and the amount of the monocistronic message was drastically reduced. The promoter from which the shorter transcript was produced contained a TATACT sequence at position -12, but sequences upstream from the -12 position seemed to be irrelevant for promoter activity. The predicted amino acid sequence of AUH contained three regions of high homology to the arginases of yeasts, rats, and humans.

Amino Acid Sequence↗
PubMed1995

Transcriptional analysis of the Pseudomonas aeruginosa exoenzyme S structural gene.

The transcriptional regulation of the Pseudomonas aeruginosa exoS gene was investigated. Expression of exoS in P. aeruginosa PA103 was dependent upon growth in a low-cation environment and the presence of a functional exsA gene. Promoter fusion analysis indicated that a 285-bp PstI-NsiI fragment, located 5' of the exoS coding region, contained a functional promoter for exoS. Expression of the reporter gene was inducible in a low-cation growth environment and required a functional copy of exsA. Divergent promoters, coordinately regulated with exoS transcription, were identified within the PstI-NsiI fragment. A fusion derivative of ExsA, MALA3A2, was shown to bind directly to the PstI-NsiI probe. DNase I protection analysis demonstrated that MALA3A2 bound to the intergenic region between the postulated -35 boxes of each promoter region. Northern (RNA) blot analysis with probes internal to and upstream of exoS demonstrated that separate, coordinately regulated mRNAs were expressed in P. aeruginosa. These data suggested that a locus, coregulated with exoS transcription, was located upstream of exoS. DNA sequence analysis of the exoS upstream region revealed three open reading frames, ORF 1, ORF 2, and ORF 3. ORF 1 demonstrated significant homology to the SycE/YerA protein of Yersinia sp. SycE/YerA is postulated to function as a chaperone for the YopE cytotoxin. The loci encoding YopE and ExoS show similarities in genetic organization, protein composition, and regulation.

ADP Ribose Transferases↗
PubMed1996

Structure of the low-affinity penicillin-binding protein 5 PBP5fm in wild-type and highly penicillin-resistant strains of Enterococcus faecium.

Among its penicillin-binding proteins (PBPs), Enterococcus faecium possesses a low-affinity PBP5, PBP5fm, which is the main target involved in beta-lactam resistance. A 7.7-kb EcoRI chromosomal fragment of E. faecium D63r containing the pbp5fm gene was cloned and sequenced. Two open reading frames (ORFs) were found. A 2,037-bp ORF encoded the deduced 73.8-kDa PBP5fm, the amino acid sequences of which were, respectively, 99.8, 78.5, and 62% homologous to those of the low-affinity plasmid-encoded PBP3r of Enterococcus hirae S185r and the chromosome-encoded PBP5 of E. hirae R40 and Enterococcus faecalis 56R. A second 597-bp ORF, designated psrfm, was found 2.3 kb upstream of pbp5fm. It appeared to be 285 bp shorter than and 74% homologous with the regulatory gene psr of E. hirae ATCC 9790. Different clinical isolates of E. faecium, for which a wide range of benzylpenicillin MICs were observed, showed that the increases in MICs were related to two mechanisms. For some strains of intermediate resistance (MICs of 16 to 64 micrograms/ml), the increased level of resistance could be explained by the presence of larger quantities of PBP5fm which had an affinity for benzylpenicillin (second-order rate constant of protein acylation [k+2/K] values of 17 to 25 M(-1) s(-1)) that remained unchanged. For the two most highly resistant strains, EFM-1 (MIC, 90 micrograms/ml) and H80721 (MIC, 512 micrograms/ml), the resistance was related to different amino acid substitutions yielding very-low-affinity PBP5fm variants (k+2/K < or = 1.5 M(-1) s(-1)) which were synthesized in small quantities. More specifically, it appeared, with a three-dimensional model of the C-terminal domain of PBP5fm, that the substitutions of Met-485, located in the third position after the conserved SDN triad, by Thr in EFM-1 and by Ala in H80721 were the most likely cause of the decreasing affinity of PBP5fm observed in these strains.

Amino Acid Sequence↗
PubMed1999

Cloning and expression in Escherichia coli of 2-hydroxypropylphosphonic acid epoxidase from the fosfomycin-producing organism, Pseudomonas syringae PB-5123.

The fosfomycin resistance gene, fosC, has been cloned from the fosfomycin-producing organism, Pseudomonas syringae PB-5123. Sequence analysis upstream of this gene found a new ORF showing significant homology to 2-hydroxypropylphosphonic acid epoxidase from fosfomycin-producing Streptomyces wedmorensis. The purified recombinant protein of this ORF converted 2-hydroxypropylphosphonic acid to fosfomycin. This result clearly showed the ORF to encode 2-hydroxypropylphosphonic acid epoxidase in PB-5123.

Amino Acid Sequence↗
PubMed2001

Increased virulence of Marek's disease virus type 1 vaccine strain CV1988 after adaptation to qt35 cells.

CVI988/Rispens strain of Marek's disease virus type I (MDV-1) is widely used as efficient vaccine to control Marek's disease (MD) in chicken flocks. Similarly to other live MD vaccine viruses it is propagated in freshly prepared chicken embryo fibroblasts (CEF). In this study, MDV-1 CVI988/Rispens strain was adapted to QT35 cells. The adapted virus, designated QT-CVI, exhibited similar cytopathic effect (CPE) in vitro to that of parental virus propagated in CEE In contrast, QT-CVI induced MD symptoms typical for mild MDV-1 strains after injection to birds. For identification of differentially expressed transcripts that might be involved in increased virulence of QT-CVI, we performed subtractive suppression hybridization (SSH). Subtracted PCR products mapped within MDV-1 BamHI-A and -H fragments and differential gene expression was also confirmed by Northern blot analysis with probes derived from these regions. To examine possible divergence at the virus genome level, PCR analysis was carried out. The BamHI-H fragment- specific 132 bp repeats were present at variable copy number, ranging from 2 to more than 30 copies in both CVI988/ Rispens and QT-CVI DNAs. PCR assays with primers mapping at the US/lRS junction identified CVI988/ Rispens-specific insertion of 116 bp in the region upstream of the ICP4 open reading frame (ORF). PCR analysis was positive also for DNA from non-infected QT35 cells and was consistent with the observation of Yamaguchi et al. (J Virol. 74, 10176-10186, 2000) who have found that QT35 cells carry a latent MDV-1 genome. It is likely, that adaptation of CVI988/Rispens to QT35 cells resulted in reactivation of an endogenous MDV-1 or at least in induction of expression of virulence-related transcripts that have consequently led to QT-CVI pathogenicity for chickens.

Animals↗
PubMed1986

[Primary structure of the 3'-terminal region of the cloned DNA of the bovine leukemia virus].

The nucleotide sequence of the 3'-terminal region of the cloned bovine leukaemia virus cDNA (1474 bp) was elucidated using both Sanger and Maxam-Gilbert techniques. This DNA region contains U3 and R parts of the BLV LTR and an upstream sequence with four open reading frames (ORF) of unknown function. The comparison of the nucleotide substitutions in these ORF with the two variants of proviral BLV DNA suggests that the only pX1 ORF possesses a coding function. The role of the pX1 protein is discussed.

Animals↗
PubMed2004

Extending the classification of bacterial transcription factors beyond the helix-turn-helix motif as an alternative approach to discover new cis/trans relationships.

Transcription factors (TFs) of bacterial helix-turn-helix superfamilies exhibit different effector-binding domains (EBDs) fused to a DNA-binding domain with a common feature. In a previous study of the GntR superfamily, we demonstrated that classifying members into subfamilies according to the EBD heterogeneity highlighted unsuspected and accurate TF-binding site signatures. In this work, we present how such in silico analysis can provide prediction tools to discover new cis/trans relationships. The TF-binding site consensus of the HutC/GntR subfamily was used to (i) predict target sites within the Streptomyces coelicolor genome, (ii) discover a new HutC/GntR regulon and (iii) discover its specific TF. By scanning the S.coelicolor genome we identified a presumed new HutC regulon that comprises genes of the phosphotransferase system (PTS) specific for the uptake of N-acetylglucosamine (PTS(Nag)). A weight matrix was derived from the compilation of the predicted cis-acting elements upstream of each gene of the presumed regulon. Under the assumption that TFs are often subject to autoregulation, we used this matrix to scan the upstream region of the 24 HutC-like members of S.coelicolor. orf SCO5231 (dasR) was selected as the best candidate according to the high score of a 16 bp sequence identified in its upstream region. Our prediction that DasR regulates the PTS(Nag) regulon was confirmed by in vivo and in vitro experiments. In conclusion, our in silico approach permitted to highlight the specific TF of a regulon out of the 673 orfs annotated as 'regulatory proteins' within the genome of S.coelicolor.

Acetylglucosamine↗
PubMed1992

Analysis of upstream sequences of the human estrogen receptor gene.

An upstream region of the human estrogen receptor gene (ER) spanning from -2776 to -38 in relation to a previously described exon 1 was sequenced and analyzed for the possible presence of open reading frames (ORF) and regulatory motifs. We identified a long 459 bp ORF beginning at -2301. Most of the transcriptional regulatory motifs present within the ER upstream sequence are distributed either upstream or downstream of this long ORF which displays a TATA box, a CACC motif and a canonical cap site in the 5' flanking region.

Base Sequence↗
PubMed1997

Organization and expression of a gene cluster involved in the biosynthesis of the lantibiotic lactocin S.

Some 8.8 kb of the Lactobacillus sake plasmid pCIM1 was sequenced, revealing eight tightly clustered open reading frames (ORFs) downstream from lasA, which encodes pre-lactocin S. Transcription analyses demonstrated that the genes are expressed as an operon, with transcription initiating upstream of lasA and terminating immediately 3' to the ninth ORF x lasA is also represented by two small RNAs (RNAI and RNAII) which differ in size by approximately 90 nucleotides, and primer extension experiments demonstrated a corresponding difference in the 5' termini. A palindromie sequence constitutes the 3' terminus of both RNAI and RNAII, and we propose that this sequence has a dual regulatory function in controlling the expression of las operon, acting both as a barrier to 3'-5' exonuclease degradation of the lasA-specific transcript(s), and as a "leaky" transcriptional terminator which limits the expression of down-stream genes. Three of the genes in the las operon have identifiable counterparts in other lantibiotic systems: lasM is likely to be involved in prepeptide modification, lasT, which encodes an ATP-dependent transport protein, is probably involved in the secretion of lactocin S, while lasP specifies a subtilisin-type serine protease which may be the lactocin S leader peptidase. Insertional mutation of either lasT or lasM by the resident transposable element IS1163 abolishes lactocin S production. The remaining five ORFs in the las operon are apparently unique, and their significance with respect to the lactocin S phenotype is presently not known.

Amino Acid Sequence↗