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PubMed1996

Genetic organization and functional analysis of the otn DNA essential for cell-wall polysaccharide synthesis in Vibrio cholerae O139.

In 1992 a new Vibrio cholerae strain, designated V. cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh. Previously, we have shown that this strain arose from a V. cholerae O1 strain by the acquisition of novel DNA. Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains. The mosaic structure of rfaDVCO139 indicated that it was one of the regions involved in recombination between donor and acceptor DNA. However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second recombination site between donor and O1-acceptor DNA is probably located downstream of rfbDVCO139. The DNA region between rfaDVCO139 and rfbQRSVCO139, designated otn, contained seven open reading frames (ORFs). Two ORFs, otnA and otnB, showed homology with genes involved in cell-wall polysaccharide synthesis. Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis. The otn DNA is also found in V. cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain. However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains. No antigenic relationship was found between the different V. cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA. The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide-synthetic genes in several bacterial species. Furthermore, a repeat motif was observed in extragenic regions. A number of observations suggest that these sequences may facilitate gene flow between V. cholerae strains and the assembly of clusters of functionally related genes.

Antigens, Bacterial↗
PubMed1993

Isolation and sequence analysis of the pmi gene encoding phosphomannose isomerase of Streptococcus mutans.

A gene encoding a phosphomannose isomerase from Streptococcus mutans GS-5 was identified immediately downstream from the fructokinase gene, scrK. Nucleotide sequence analysis of this region revealed an open reading frame (ORF) specifying a putative protein of 316 amino acids. The gene cloned in Escherichia coli expressed strong phosphomannose isomerase activity. The deduced amino acid sequence of the pmi gene has no significant similarity with any of the previously reported phosphomannose isomerase enzymes. Insertional inactivation of the upstream gene, scrK, in S. mutans also drastically reduced phosphomannose isomerase activity and the ability of the organism to utilize mannose as a sole carbon source. These results suggest that the S. mutans pmi gene constitutes an operon with the scrK gene.

Amino Acid Sequence↗
PubMed2002

[In silico cloning of glucose-6-phosphate dehydrogenase cDNA from rice (Oryza sativa L.)].

In silico cloning was a new strategy of gene cloning developed with the development of genome, EST projects and bioinformatics. Using wheat glucose-6-phosphate dehydrogenase cDNA (clone: Tagpdl) sequence as a querying probe, one highly homologous BAC clone sequence was obtained from rice sequence database of GenBank and the putative cDNA sequence of rice glucose-6-phosphate dehydrogenase was assembled according to the wheat clone. Furthermore, the full-length cDNA of rice glucose-6-phosphate dehydrogenase was cloned by RT-PCR with two primers designed based on this assembled cDNA sequence. Since this fragment contained a complete ORF of 1515 bp with a stop codon in its upstream and poly(A) signal in its downstream, it could be concluded that a full-length gene (GenBank accession number AY078072), which was named as OsG6PDH. Homology analysis of OsG6PDH showed a 88% identity with wheat and the deduced amino acid showed 89%, 79% and 80% homology with G6PDH from wheat, tomato and tobacco respectively. OsG6PDH was expressed in inflorescence, embryo, root and leaf of rice, with a slightly higher in inflorescence and root. It was also discussed in this paper that the application of in silico cloning in the isolation of functional genes from rice.

Base Sequence↗
PubMed1997

Enhancer element, repetitive sequences and gene organization in an 8-kbp region containing the polyhedrin gene of the Spodoptera littoralis nucleopolyhedrovirus.

The Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV) shows only a distant genetic relationship to other NPVs. In this report we describe the gene organization of an 8-kbp region of the SpliMNPV which contains the polyhedrin gene. The polyhedrin transcription initiation sites were mapped and the sequence and gene organization of an 8-kbp region of SpliMNPV were determined. The sequences downstream of the polyhedrin gene showed colinearity with the gene organization of other NPVs. An anticlockwise 1035-bp open reading frame (ORF), capable of encoding a proline-rich polypeptide, was found at the 3' end of the polyhedrin gene. followed by an 837-bp ORF encoding a putative protein kinase (PK), with an orientation similar to that of the polyhedrin gene. Sequences upstream of the polyhedrin gene were found to be unique to SpliMNPV and contained two regions consisting of highly repetitive sequences. One region, 980 bp in length and termed sequence repeat region 1 (SR1), contained a variety of short direct repeats, SR1 was found to act as a transcriptional enhancer in a transient expression assay. Additional regions containing different repetitive sequences were identified within the proline-rich ORF1035 and in sequences located downstream of the pk gene.

Amino Acid Sequence↗
PubMed2003

Molecular cloning and characterization of Escherichia coli K12 ygjG gene.

BACKGROUND: Putrescine is the intermediate product of arginine decarboxylase pathway in Escherichia coli which can be used as an alternative nitrogen source. Transaminase and dehydrogenase enzymes seem to be implicated in the degradative pathway of putrescine, in which this compound is converted into gamma-aminobutyrate. But genes coding for these enzymes have not been identified so far. RESULTS: The 1.8-kbp DNA fragment containing E. coli K12 ygjG gene with aer-ygjG intergenic region was examined. It was found that the fragment contains sigma54-depended open reading frame (ORF) of 1,380 nucleotides encoding a 459-amino acid polypeptide of approximately 49.6 kDa. The cytidine (C) residue localized 10 bp downstream of the sigma54 promoter sequence was identified as the first mRNA base. The UUG translation initiation codon is situated 36 nucleotides downstream of the mRNA start. The YgjG was expressed as a his6-tag fused protein and purified to homogeneity. The protein catalyzed putrescine:2-oxoglutaric acid (2-OG) aminotransferase reaction (PATase, EC 2.6.1.29). The Km values for putrescine and 2-OG were found to be 9.2 mM and 19.0 mM, respectively. The recombinant enzyme also was able to transaminate cadaverine and, in lower extent, spermidine, and gave maximum activity at pH 9.0. CONCLUSION: Expression of E. coli K12 ygjG coding region revealed sigma54-depended ORF which encodes a 459-amino acid protein with putrescine:2-OG aminotransferase activity. The enzyme also was able to transaminate cadaverine and, in lower extent, spermidine.

5' Flanking Region↗
PubMed1998

Sendai virus Y proteins are initiated by a ribosomal shunt.

The Sendai virus P/C mRNA expresses eight primary translation products by using a combination of ribosomal choice and cotranscriptional mRNA editing. The longest open reading frame (ORF) of the mRNA starts at AUG104 (the second initiation site) and encodes the 568-amino-acid P protein, an essential subunit of the viral polymerase. The first (ACG81), third (ATG114), fourth (ATG183), and fifth (ATG201) initiation sites are used to express a C-terminal nested set of polypeptides (collectively named the C proteins) in the +1 ORF relative to P, namely, C', C, Y1, and Y2, respectively. Leaky scanning accounts for translational initiation at the first three start sites (a non-ATG followed by ATGs in progressively stronger contexts). Consistent with this, changing ACG81/C' to ATG (GCCATG81G) abrogates expression from the downstream ATG104/P and ATG114/C initiation codons. However, expression of the Y1 and Y2 proteins remains normal in this background. We now have evidence that initiation from ATG183/Y1 and ATG201/Y2 takes place via a ribosomal shunt or discontinuous scanning. Scanning complexes appear to assemble at the 5' cap and then scan ca. 50 nucleotides (nt) of the 5' untranslated region before being translocated to an acceptor site at or close to the Y initiation codons. No specific donor site sequences are required, and translation of the Y proteins continues even when their start codons are changed to ACG. Curiously, ATG codons (in good contexts) in the P ORF, placed either 16 nt upstream of Y1, 29 nt downstream of Y2, or between the Y1 and Y2 codons, are not expressed even in the ACGY1/ACGY2 background. This indicates that ATG183/Y1 and ATG201/Y2 are privileged start sites within the acceptor site. Our observations suggest that the shunt delivers the scanning complex directly to the Y start codons.

Base Sequence↗
PubMed1993

Characterization of the left 4 kb of conjugative transposon Tn916: determinants involved in excision.

The rate-limiting step in movement of the conjugative transposon Tn916, originally identified in Enterococcus faecalis, is believed to be an excision event that generates a non-replicative circular intermediate. When present on a plasmid vector in Escherichia coli, Tn916 generally excises at a high frequency. It was reported previously that insertion of Tn5 in a region near the left end of Tn916 eliminated the ability to excise; and the mutation could be complemented in trans. In this communication the nucleotide sequence of 4 kb of Tn916 DNA connecting the recently sequenced tet(M) determinant (Su et al., 1992; Burdett, 1990) with the left end of the transposon. Ten open reading frames (ORFs) were deduced, two of which (ORF3 and ORF4) were encoded in-frame within a third (ORF2). Mutants with Tn5 insertions in the ORF1 or ORF2 (ORF3 and ORF4) were defective in excision, but could be complemented in vivo by a co-resident plasmid containing the ORF1 or ORF2 determinant, respectively. The data support the view that both ORF1 and ORF2 are essential for excision. ORF1 and ORF2 are essentially identical to determinants designated xis-Tn and int-Tn, respectively, in the closely related Tn1545. A Tn5 insertion in ORF5 eliminated conjugative transfer between E. faecalis strains. Functions for the remaining ORFs (ORF6 through ORF10) remain unknown; however, nucleotide sequences within ORF6 and ORF9 had significant homology with sequence downstream of other tet(M) determinants.

Amino Acid Sequence↗
PubMed1996

Molecular analysis of the gene cluster involved in cephalosporin biosynthesis from Lysobacter lactamgenus YK90.

Determination of the nucleotide sequence downstream from the Lysobacter lactamgenus pcbC gene encoding isopenicillin N synthase revealed that five open-reading frames (ORF) including the pcbC gene were tightly linked in the same orientation. Each ORF has the remarkable feature of the protein-coding frame in the DNA sequence with a high G+C content. Expression in Escherichia coli and a comparison of the deduced amino acid sequences with published sequences showed that the gene cluster contained a deacetoxycephalosporin C synthetase (DAOCS) gene (cefE), an ORF having homology with the Cephalosporium acremonium DAOCS/deacetylcephalosporin C synthetase gene (cefEF), an isopenicillin N epimerase gene(cefD), and a beta-lactamase gene. The gene order was pcbC-cefE-ORF3-cefD-beta-lactamase.

Amino Acid Sequence↗
PubMed1992

The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes.

A 3789 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located 1.65 kb downstream of the N gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs) which are bounded by putative consensus (AACAGG) and polyadenylation (CATG[A]7) sequences and are separated by an intergenic region of 53 nucleotides. Discrete mRNAs corresponding to each ORF have been identified. The first ORF encodes a polypeptide comprising 623 residues which was identified by peptide sequencing as the virion G protein. The deduced amino acid sequence of the G protein includes putative signal and transmembrane domains and five potential glycosylation sites. The second ORF encodes a polypeptide of 586 amino acids which also has characteristics of a rhabdovirus glycoprotein, including putative signal and transmembrane domains and eight potential glycosylation sites, and appears to correspond to a 90-kDa nonstructural glycoprotein (GNS) identified in BEFV-infected cells (Walker et al. [1991] J. Gen. Virol. 72, 67-74). A database search indicated that both the G and GNS proteins share significant amino acid sequence homology with other rhabdovirus G proteins and with each other. Highest homology scores for each protein were with sigma virus and vesicular stomatitis virus serotypes.

Amino Acid Sequence↗
PubMed1999

Toluene metabolism by the solvent-tolerant Pseudomonas putida DOT-T1 strain, and its role in solvent impermeabilization.

Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow with toluene as the sole C-source. Tn5 mutagenesis was carried out and a mutant unable to use toluene as the sole C-source was isolated. DNA was sequenced upstream and downstream of the site where the Tn5 was inserted. Analysis of the DNA revealed 13 open reading frames (ORFs) homologous to the tod genes for the toluene dioxygenase pathway of P. putida F1, which are organized in two operons: todXFC1C2BADEGIH and todST. The Tn5 was inserted at the todH gene. The role of the todXFC1C2BADEGIH operon in toluene metabolism was further confirmed in a todC1 mutant (generated by insertional inactivation), which was unable to use toluene as the sole C-source. Primer extension analysis identified a single promoter upstream from the todX gene. The -10 and -35 regions of this promoter showed no significant homology to known promoters. Expression from the todX promoter occurred in response to toluene, ethylbenzene, styrene, xylenes and other aromatic hydrocarbons. Expression from the todS gene was constitutive. Sensitivity to toluene of the todH and todC1 mutants was similar to that of the wild-type strain. This suggests that toluene metabolism is not involved in toluene tolerance.

Base Sequence↗
PubMed2001

The two-component regulatory system BacRS is associated with bacitracin 'self-resistance' of Bacillus licheniformis ATCC 10716.

Bacitracin is a peptide antibiotic produced by several Bacillus licheniformis strains that is most active against other Gram-positive microorganisms, but not against the producer strain itself. Recently, heterologous expression of the bacitracin resistance mediating BcrABC transporter in Bacillus subtilis and Escherichia coli was described. In this study we could determine that the transporter encoding bcrABC genes are localized about 3 kb downstream of the 44-kb bacitracin biosynthetic operon bacABC. Between the bac operon and the bcrABC genes two orfs, designated bacR and bacS, were identified. They code for proteins with high homology to regulator and sensor proteins of two-component systems. A disruption mutant of the bacRS genes was constructed. While the mutant displayed no effects on the bacitracin production it exhibited highly increased bacitracin sensitivity compared to the wild-type strain. Western blot analysis of the expression of BcrA, the ATP-binding cassette of the transporter, showed in the wild-type a moderate BcrA induction in late stationary cells that accumulate bacitracin, whereas in the bacRS mutant cells the BcrA expression was constitutive. A comparison of bacitracin stressed and nonstressed wild-type cells in Western blot analysis revealed increasing amounts of BcrA and a decrease in BacR in the stressed cells. From these findings we infer that BacR acts as a negative regulator for controlling the expression of the bcrABC transporter genes.

ATP-Binding Cassette Transporters↗
PubMed1989

Sequence, transcription and translation of a late gene of the Autographa californica nuclear polyhedrosis virus encoding a 34.8K polypeptide.

A 1.4 kb region downstream of the DNA polymerase gene of Autographa californica nuclear polyhedrosis virus was sequenced. Two open reading frames (ORFs) were identified of 927 and 474 bases in length. The 927 base ORF encodes a 34.8K protein as determined by in vitro translation of both hybrid-selected RNA and RNA synthesized in vitro from a 927 base ORF template. The predicted amino acid sequence of the 34.8K polypeptide (p34.8) reveals a hydrophobic N terminus, two potential N-glycosylation sites, and potential sites for phosphorylation by casein kinase I and protein kinase C. The p34.8 gene has a strong codon usage bias which is strikingly different from that of the polyhedrin gene. The two 5' ends of the 927 base ORF transcripts initiate from an ATAAG sequence and a GTAAG sequence 11 and 87 bases upstream of the ATG codon respectively. A short upstream reading frame is present in the leader sequence of the longer RNA. The transcripts have multiple 3' ends; the most proximal endpoint correlates with a polyadenylation signal overlapping the translational termination codon of the 927 base ORF. Transcripts of the latter were not observed early in the infection cycle but appeared 6 h after infection and were maximally expressed at 12 to 24 h post-infection. The late nature of these transcripts was confirmed by their sensitivity to aphidicolin and cycloheximide, inhibitors of DNA replication and protein synthesis respectively. Attempts to construct viral mutants carrying a deletion of the p34.8 gene and fusion with the beta-galactosidase gene suggest that the former gene is essential for viral replication.

Amino Acid Sequence↗
PubMed1994

Cloning and sequencing of sakP encoding sakacin P, the bacteriocin produced by Lactobacillus sake LTH 673.

Sakacin P is a heat-stable, unmodified peptide bacteriocin produced by Lactobacillus sake LTH 673. The strain was isolated from fermented dry sausages and is well adapted to this habitat. The bacteriocin inhibits the growth of the opportunistic food pathogens Enterococcus faecalis and Listeria monocytogenes and therefore, it may improve the hygienic status of fermented food, i.e. meat products. Oligonucleotide probes were designed from the N-terminal amino acid sequence of sakacin P and used to identify sakP, the structural gene of sakacin P, on the chromosome of L. sake LTH 673. SakP was cloned into Escherichia coli NM554 and the nucleotide sequence of the gene and its adjacent regions were determined. Sakacin P appears to be synthesized as a prepeptide of 61 amino acids which is proteolytically processed to the mature bacteriocin consisting of 43 amino acids. Sequencing of the cloned fragment also revealed the presence of two other open reading frames orfX and orfY, which are located upstream and downstream of sakP, respectively, putatively encoding proteins of 52 and 98 amino acids, respectively. The functions of both ORFs remain unknown. Primer extension analysis revealed a promoter upstream of sakP. Two transcripts of approximately 0.35 and 1.0 kb were detected by Northern hybridization encoding either only sakP, or both sakP and orfY, respectively.

Amino Acid Sequence↗
PubMed2000

A novel lantibiotic, nukacin ISK-1, of Staphylococcus warneri ISK-1: cloning of the structural gene and identification of the structure.

Staphylococcus warneri ISK-1, which we had previously reported as Pediococcus sp. ISK-1, produces a novel bacteriocin, nukacin ISK-1. Edman degradation of the chemically reduced nukacin ISK-1 produced a sequence of 27 amino acids, 7 of which were unidentified. Using single-specific-primer-PCR product as a probe, a 3.6-kb HindIII fragment containing the nukacin ISK-1 structural gene (nukA) was cloned and sequenced. The deduced amino acid sequence of nukacin ISK-1 had 57 amino acids, including a 30-amino acid leader region. The propeptide sequence showed significant similarity to those of lacticin-481 type lantibiotics. In the region upstream of nukA, a part of a long open reading frame (ORF), designated as nukM, encoding a putative modification enzyme was oriented in the opposite direction. In the region downstream of nukA, ORF1 was found in which the sequence of the putative translational product was similar to various response regulatory proteins.

Amino Acid Sequence↗
PubMed1993

LINE-1: a human transposable element.

Among the 10(5) LINE-1 sequences (L1Hs) in the human genome are one or more 6-kb segments that are active retrotransposons. Expression of these retrotransposons appears to be favored in cells of germ line origin, as well as in some other tumor cells of epithelial origin. In such cells, the product of the first L1Hs open reading frame (ORF), a protein called p40, is detectable; p40 has no apparent similarity to gag proteins, but contains a leucine zipper region which may be responsible for the occurrence of p40 multimers. Transcription of L1Hs initiates at residue 1 although the transcriptional regulatory regions are downstream in the first 670 bp of the 5' untranslated region; deletion of a YY1-binding site in the first 20 bp reduces transcription by fivefold. Translation of the second ORF, which encodes reverse transcriptase, is independent of the translation of the frame encoding p40.

Amino Acid Sequence↗
PubMed1999

Regulation of hexuronate utilization in Bacillus subtilis.

We have identified a locus essential for galacturonate utilization in Bacillus subtilis. Genes homologous to Escherichia coli and Erwinia chrysanthemi glucuronate and galacturonate metabolic genes were found in a cluster consisting of 10 open reading frames (ORFs) in the B. subtilis chromosome. A mutant of B. subtilis containing a replacement of the second and third ORFs was unable to grow with galacturonate as its primary carbon source. Galacturonate induced expression from a sigmaA-dependent promoter, exuP1, located upstream from the first ORF. The eighth ORF in this cluster (the exu locus) encodes a LacI and GalR homolog that negatively regulated expression from exuP1. A 26-bp inverted repeat sequence centered 15 bp downstream from the exuP1 start point of transcription acted in cis to negatively regulate expression from exuP1 under noninducing conditions. Expression from the exuP1 promoter was repressed by high levels of glucose, which is probably mediated by CcpA (catabolite control protein A). A sigmaE-dependent promoter, exuP2, was localized between the second and third ORFs and was active during sporulation.

Aldose-Ketose Isomerases↗
PubMed2004

Steroid degradation gene cluster of Comamonas testosteroni consisting of 18 putative genes from meta-cleavage enzyme gene tesB to regulator gene tesR.

Steroid degradation genes of Comamonas testosteroni TA441 are encoded in at least two gene clusters: one containing the meta-cleavage enzyme gene tesB and ORF1, 2, 3; and another consisting of ORF18, 17, tesI, H, A2, and tesA1, D, E, F, G (tesA2 to ORF18 and tesA1 to tesG are encoded in opposite directions). Analysis of transposon mutants with low steroid degradation revealed 13 ORFs and a gene (ORF4, 5, 21, 22, 23, 25, 26, 27, 28, 30, 31, 32, 33, and tesR) involved in steroid degradation in the downstream region of ORF3. TesR, which is almost identical to that of TeiR, a positive regulator of Delta1-dehydrogenase (corresponds to TesH in TA441) and 3alpha-dehydrogenase (currently not identified in TA441), in C. testosteroni ATCC11996 (Pruneda-Paz, 2004), was shown to be necessary for induction of the steroid degradation gene clusters identified in TA441, tesB to tesR, tesA1 to tesG, and tesA2 to ORF18. At least some of the ORFs from ORF3 to ORF33 were suggested to be involved in 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid degradation.

Bacterial Proteins↗
PubMed1999

Regulation of human papillomavirus type 31 polyadenylation during the differentiation-dependent life cycle.

The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed late in the differentiation-dependent life cycle from a promoter located in the E7 open reading frame (ORF) of the early region. These late HPV genes are transcribed by RNA polymerase II which reads through the region containing early polyadenylation signals and proceeds to a poly(A) site downstream of L1. In this study, we have investigated the mechanisms regulating differentiation-dependent polyadenylation and read-through in HPV-31. HPV-31 early transcripts were found to utilize a heterogeneous series of polyadenylation sites in undifferentiated cells. The sites for polyadenylation extended over a range of 100 nucleotides from within the E5 ORF to upstream of L2. Upon differentiation, the transcription of early genes increased, but no change in the heterogeneous distribution of 3' ends was detected. The early polyadenylation region was found to contain a single consensus hexanucleotide sequence, AAUAAA, as well as three weak binding sites for the cleavage stimulatory factor, CstF. In contrast to the heterogeneity at the early site, the 3' ends of late transcripts encoding L1 and L2 were localized to a narrow region downstream of the late AAUAAA element. The late polyadenylation signal was found to contain a single high-affinity site for CstF, as well as one consensus hexanucleotide sequence. By using a reporter assay, it was determined that the HPV-31 early polyadenylation sequences allowed significant levels of read-through into the late region in undifferentiated cells. Upon differentiation, this read-through was increased by approximately 50%, indicating that use of the early site decreased. Differentiation was also found to induce a 40% reduction in the levels of CstF subunits, which may contribute to the increased read-through of the early sequence. The insertion of the late high-affinity binding site for CstF into the early polyadenylation region significantly reduced the level of read-through, suggesting that these factors modulate read-through activity. Our studies demonstrate that HPV-31 late gene expression is regulated in a large part by posttranscriptional mechanisms, including the polyadenylation of early transcripts.

Binding Sites↗