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PubMed1993

Cloning, sequencing and expression of the gene encoding the carboxytransferase subunit of the biotin-dependent Na+ pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans in Escherichia coli.

1. The primary sodium-ion pump glutaconyl-CoA decarboxylase (GCD) from Acidaminococcus fermentans is composed of four subunits: GCDA, the carboxytransferase (65 kDa), GCDB, the carboxylyase (36 kDa), GCDC, the biotin carrier (24 kDa) and GCDD (14 kDa) of unknown function. A genomic library of A. fermentans was screened with an antiserum raised against whole GCD. A clone giving the strongest reaction in an immunoassay contained a 12-kbp genomic fragment from A. fermentans and was analysed further. An oligonucleotide deduced from the N-terminus of GCDA was used for probing the corresponding gene gcdA. It is 1761 bp in length and encodes for a protein of 64.3 kDa. Both partial amino acid sequences obtained from GCDA, the N-terminus as well as an internal tryptic peptide, were detected in the open reading frame (ORF) of gcdA. 2. Sequencing of the flanking regions revealed three adjacent ORF (ORF1-3) which do not code for any of the peptide sequences known of the other GCD subunits. The ORF downstream of gcdA (ORF3) is followed by hgdA and hgdB coding for 2-hydroxyglutaryl-CoA dehydratase, the preceding enzyme of the pathway of glutamate fermentation. Our results suggest that at least these three genes of the hydroxyglutarate pathway are organised in an operon and that the genes of the other GCD subunits from which peptide sequences are known (GCDB and GCDC) are not located adjacent to gcdA. 3. gcdA was amplified from genomic DNA using the polymerase chain reaction and cloned into the expression vector pJF118HE. Active GCDA subunit (up to 2.8 nkat/mg protein), catalysing the biotin-dependent formation of crotonyl-CoA from glutaconyl-CoA, was obtained in cell-free extracts of Escherichia coli DH5 alpha by moderately inducing the tac promoter of pJF118HE with 25-100 microM isopropyl-1-thio-beta-D-galactoside. Strong induction (1 mM isopropyl-1-thio-beta-D-galactoside) led to the formation of inclusion bodies from which GCDA could not be reactivated. The apparent Km = 51 mM for free biotin of the expressed GCDA subunit with V = 1.9 nkat/mg protein is similar to that of butanol-treated GCD composed of GCDA and GCDC (apparent Km = 40 mM). Biocytin was found to be a somewhat better carboxy acceptor for the expressed GCDA subunit (apparent Km = 13 mM; V = 1.0 nkat/mg protein). 4. Native GCD and expressed GCDA were treated with 2 mM N-ethylmaleimide showing different kinetics of inactivation: GCD lost half of its activity within 6 min, whereas expressed GCDA required 21 min.

Amino Acid Sequence↗
PubMed2002

Identification of a DtxR-regulated operon that is essential for siderophore-dependent iron uptake in Corynebacterium diphtheriae.

The diphtheria toxin repressor (DtxR) uses Fe(2+) as a corepressor and inhibits transcription from iron-regulated promoters (IRPs) in Corynebacterium diphtheriae. A new IRP, designated IRP6, was cloned from C. diphtheriae by a SELEX-like procedure. DtxR bound to IRP6 in vitro only in the presence of appropriate divalent metal ions, and repression of IRP6 by DtxR in an Escherichia coli system was iron dependent. The open reading frames (ORFs) downstream from IRP6 and previously described promoter IRP1 were found to encode proteins homologous to components of ATP-binding cassette (ABC) transport systems involved in high-affinity iron uptake in other bacteria. IRP1 and IRP6 were repressed under high-iron conditions in wild-type C. diphtheriae C7(beta), but they were expressed constitutively in C7(beta) mutant strains HC1, HC3, HC4, and HC5, which were shown previously to be defective in corynebactin-dependent iron uptake. A clone of the wild-type irp6 operon (pCM6ABC) complemented the constitutive corynebactin production phenotype of HC1, HC4, and HC5 but not of HC3, whereas a clone of the wild-type irp1 operon failed to complement any of these strains. Complementation by subclones of pCM6ABC demonstrated that mutant alleles of irp6A, irp6C, and irp6B were responsible for the phenotypes of HC1, HC4, and HC5, respectively. The irp6A allele in HC1 and the irp6B allele in HC5 encoded single amino acid substitutions in their predicted protein products, and the irp6C allele in HC4 caused premature chain termination of its predicted protein product. Strain HC3 was found to have a chain-terminating mutation in dtxR in addition to a missense mutation in its irp6B allele. These findings demonstrated that the irp6 operon in C. diphtheriae encodes a putative ABC transporter, that specific mutant alleles of irp6A, irp6B, and irp6C are associated with defects in corynebactin-dependent iron uptake, and that complementation of these mutant alleles restores repression of corynebactin production under high-iron growth conditions, most likely as a consequence of restoring siderophore-dependent iron uptake mediated by the irp6 operon.

Bacterial Proteins↗
PubMed1992

Identification and characterization of an extracellular envelope glycoprotein affecting vaccinia virus egress.

Sequence analysis of the vaccinia virus strain Western Reserve genome revealed the presence of an open reading frame (ORF), SalL4R, which has the potential to encode a transmembrane glycoprotein with homology to C-type animal lectins (G. L. Smith, Y. S. Chan, and S. T. Howard, J. Gen. Virol. 72:1349-1376, 1991). Here we show that the SalL4R gene is transcribed late during infection from a TAAATG motif at the beginning of the ORF. Antisera raised against a TrpE-SalL4R fusion protein identified three glycoprotein species of Mr 22,000 to 24,000 in infected cells. Immunogold electron microscopy demonstrated that SalL4R protein is present in purified extracellular enveloped virus particles but not in intracellular naked virus (INV). A mutant virus was constructed by placing a copy of the SalL4R ORF downstream of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible vaccinia virus promoter within the thymidine kinase locus and subsequently deleting the endogenous SalL4R gene. The growth kinetics of this virus demonstrated that SalL4R was nonessential for the production of infectious INV but was required for virus dissemination. Consistent with this finding, the formation of wild-type-size plaques by this mutant was dependent on the presence of IPTG. Electron microscopy showed that without SalL4R expression, the inability of the virus to spread is due to a lack of envelopment of INV virions by Golgi-derived membrane, a morphogenic event required for virus egress.

Amino Acid Sequence↗
PubMed1994

Characterization of a hemA/hemE mutant of E. coli and regulation of hemE.

Uroporphyrinogen III is the committed intermediate common to heme and siroheme biosynthesis in E. coli. Uroporphyrinogen III decarboxylase is the first enzyme at the branch point which commits to heme synthesis. A hemin-permeable hemA mutant which could grow on 5-aminolevulinic acid (ALA) or hemin, was mutagenized to give a double mutant, 10L2-1. The second mutation which was identified as hemE because it was mapped to 90.1 min. by F' and Hfr mapping and P1 transduction, accumulated uroporphyrin and had no uroporphyrinogen decarboxylase activity. This mutation could be complemented with a plasmid harboring the hemE gene of Synechococcus. The complemented strain could grow on ALA and accumulated coproporphyrin and protoporphyrin but not uroporphyrin. The E. coli hemE gene was cloned by transducing 10L2-1 with an E. coli genomic library in lambda gt11. hemE with upstream regions of various sizes was cloned in front of a promoterless CAT gene. Good growth on chloramphenicol (25-75 micrograms/ml) depended on a promoter within 152 bp upstream of the hemE structural gene start of translation site. In addition, this construct could complement the hemE requirement of 10L2-1 as well as allow it to grow on chloramphenicol. Addition of hemin did not inhibit this growth and therefore it appears that it does not affect the hemE promoter. The hemE structural gene alone allowed good growth on 10 micrograms/ml but poor growth on 25 micrograms/ml chloramphenicol, suggesting that there is a weak promoter within hemE for a downstream ORF. Quantitation of CAT protein in these strains showed a weak promoter within hemE, a promoter 152 bp upstream of hemE and another promoter within 1.3 kb upstream of hemE. The 1.3 kb region contains an ORF 40 bp upstream of hemE, thus suggesting that hemE is part of an operon.

Aldehyde Oxidoreductases↗
PubMed1996

The xanthopsins: a new family of eubacterial blue-light photoreceptors.

Photoactive yellow protein (PYP) is a photoreceptor that has been isolated from three halophilic phototrophic purple bacteria. The PYP from Ectothiorhodospira halophila BN9626 is the only member for which the sequence has been reported at the DNA level. Here we describe the cloning and sequencing of the genes encoding the PYPs from E.halophila SL-1 (type strain) and Rhodospirillum salexigens. The latter protein contains, like the E.halophila PYP, the chromophore trans p-coumaric acid, as we show here with high performance capillary zone electrophoresis. Additionally, we present evidence for the presence of a gene encoding a PYP homolog in Rhodobacter sphaeroides, the first genetically well-characterized bacterium in which this photoreceptor has been identified. An ORF downstream of the pyp gene from E.halophila encodes an enzyme, which is proposed to be involved in the biosynthesis of the chromophore of PYP. The pyp gene from E.halophila was used for heterologous overexpression in both Escherichia coli and R.sphaeroides, aimed at the development of a holoPYP overexpression system (an intact PYP, containing the p-coumaric acid chromophore and displaying the 446 nm absorbance band). In both organisms the protein could be detected immunologically, but its yellow color was not observed. Molecular genetic construction of a histidine-tagged version of PYP led to its 2500-fold overproduction in E.coli and simplified purification of the heterologously produced apoprotein. HoloPYP could be reconstituted by the addition of p-coumaric anhydride to the histidine-tagged apoPYP (PYP lacking its chromophore). We propose to call the family of photoactive yellow proteins the xanthopsins, in analogy with the rhodopsins.

Amino Acid Sequence↗
PubMed1991

Identification of Mycobacterium leprae antigens from a cosmid library: characterization of a 15-kilodalton antigen that is recognized by both the humoral and cellular immune systems in leprosy patients.

Screening of the Mycobacterium leprae cosmid library with pooled sera from lepromatous leprosy (LL) patients by a colony immunoblot technique resulted in the identification of about 100 colonies that produced immunologically reactive proteins. Twenty-four of these clones were purified, analyzed, and found to comprise two groups according to the reactivity of the recombinant proteins with LL sera and to the DNA restriction patterns of the recombinant plasmids and cosmids. Proteins specified by clones from group I reacted strongly with LL patients' sera on a Western blot (immunoblot), demonstrating a 15-kDa protein band designated A15. The A15 antigen also reacted with pooled sera from patients with tuberculoid leprosy from the United States and Brazil. Clones from group II did not show any reactive protein band on a Western blot, when reacted with patients' sera. DNAs from cosmids of group II all contain a 10-kb PstI fragment that hybridized to the unique repetitive M. leprae DNA. Sequence analysis of a 1.2-kb fragment containing the entire coding sequence of A15 revealed three open reading frames (ORFs), only one of which (ORF II) contains sufficient genetic information to encode for A15. Part of the A15 gene was found to exist also in a group of lambda gt11:M. leprae clones previously isolated in our laboratory by immunological screening with LL patients' sera. One of the lambda gt11 clones (L8) expresses a beta-galactosidase fusion protein with 89 amino acids from the C terminus of A15. An important result was that the fusion protein was clearly recognized by T cells from leprosy patients. Interestingly, Mycobacterium tuberculosis-stimulated T cells from M. leprae nonresponder (LL as well as borderline tuberculoid) patients were able to respond to the isolated recombinant M. leprae antigen, indicating that nonresponsiveness to M. leprae antigens can be reversible. The sequence of the M. leprae DNA fused to the beta-galactosidase gene of lambda gt11 clone L8 was identical to that of a lambda gt11:M. leprae clone isolated recently that expresses an immunologically reactive fusion protein (S. Laal, Y. D. Sharma, H. K. Prasad, A. Murtaza, S. Singh, S. Tangri, R. Misra, and I. Nath, Proc. Natl. Acad. Sci. USA 88:1054-1058, 1991). Besides the complete sequence of the A15 gene, sequencing data of two flanking ORFs are presented. Downstream from ORF II (A15), ORF III has a high degree of similarity to the genes for tomato ATP-dependent proteases that are members of a larger class of highly conserved proteases ubiquitous among prokaryotes and eukaryotes.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1996

Circular and linear plasmids of Lyme disease spirochetes have extensive homology: characterization of a repeated DNA element.

We have cloned three copies of a repeated DNA segment from Borrelia burgdorferi sensu stricto strain B31, present on both circular and linear plasmids of this and other B. burgdorferi sensu lato strains. The DNA sequences are characterized by a highly homologous segment containing two open reading frames (ORFs), ORF-A and ORF-B. Five additional ORFs can be found on the slightly less homologous flanking sequences: ORF-G on the opposite strand upstream of ORF-A, and ORF-C, ORF-D, ORF-E, and ORF-F downstream of ORF-B. The 4.6-kb-long element containing ORF-A through ORF-E is flanked by approximately 180-bp-long imperfect inverted repeats (IRs). The putative gene product of ORF-C displays homology to proteins involved in plasmid maintenance in a number of gram-positive and gram-negative bacteria. ORF-E features several short, highly homologous direct repeats. ORF-A, ORF-B, and ORF-D are homologous to three ORFs on a recently described 8.3-kb circular plasmid of Borrelia afzelii Ip21 that are flanked by similar IRs (J. J. Dunn, S. R. Buchstein, L.-L. Butler, S. Fisenne, D. S. Polin, B. N. Lade, and B. J. Luft, J. Bacteriol. 176:2706-2717,1994). ORF-C and ORF-E, however, are missing from this region on the Ip21 plasmid. Furthermore, the repeated DNA element as defined by the IRs is present in opposite orientations relative to the flanking sequences on the B31 and Ip21 plasmids.

Base Sequence↗
PubMed1988

Genome of the European elk papillomavirus (EEPV).

The genome of the European elk papillomavirus (EEPV) was found to be 8,095 base pairs (bp) long and its genetic organization was similar to that of other papillomaviruses. Ten open reading frames (ORFs), designated E1-E7 and L1-L3, were identified in the genome, all located on one strand. The presence of the L3 ORF is rare among the papillomaviruses and to date has only been identified in the genomes of EEPV, the deer papillomavirus (DPV) and the Cottontail papillomavirus (CRPV). The ORF is well conserved between DPV and EEPV with regard to both length and sequence. Potential promoter regions were identified at the 5'-end of the E6 ORF, at the 3'-end of the E1 ORF and downstream of the L1 ORF. Furthermore, two potential polyadenylation signals were found, one located in the long control region (LCR), downstream of the L1 ORF, and another preceding the L2 ORF. The EEVP genome is closely related to the genome of the DPV, the most highly conserved regions being ORFs E1 (70%), E5 (69%), and L1 (74%).

Amino Acid Sequence↗
PubMed1997

Transcriptional and mutational analyses of the rpoN operon in Caulobacter crescentus.

The alternative sigma factor sigma54 is required for the biogenesis of both the flagellum and the stalk in Caulobacter crescentus. The DNA sequence downstream of the sigma54 gene (rpoN) has been determined, revealing three open reading frames (ORFs) encoding peptides of 203, 208, and 159 amino acids. ORF208 and ORF159 are homologous to ORFs found downstream of rpoN in other microorganisms. The organization of this region in C. crescentus is similar to that in other bacteria, with the exception of an additional ORF, ORF203, immediately downstream from rpoN. There is a single temporally regulated promoter that drives the expression of both rpoN and ORF203. Promoter probe analysis indicates the presence of another promoter downstream from ORF203 which exhibits a temporal control that is different from that of the rpoN promoter. Mutational analysis was used to address the function of the proteins encoded by these three downstream ORFs. The mutations have no effect on the transcription of previously known sigma54-dependent flagellar promoters except for a slight effect of an ORF159 mutation on transcription of fljK.

Amino Acid Sequence↗
PubMed1995

Efficiency of reinitiation of translation on human immunodeficiency virus type 1 mRNAs is determined by the length of the upstream open reading frame and by intercistronic distance.

In this study, we examined the mechanism of translation of the human immunodeficiency virus type 1 tat mRNA in eucaryotic cells. This mRNA contains the tat open reading frame (ORF), followed by rev and nef ORFs, but only the first ORF, encoding tat, is efficiently translated. Introduction of premature stop codons in the tat ORF resulted in efficient translation of the downstream rev ORF. We show that the degree of inhibition of translation of rev is proportional to the length of the upstream tat ORF. An upstream ORF spanning 84 nucleotides was predicted to inhibit 50% of the ribosomes from initiating translation at downstream AUGs. Interestingly, the distance between the upstream ORF and the start codon of the second ORF also played a role in efficiency of downstream translation initiation. It remains to be investigated if these conclusions relate to translation of mRNAs other than human immunodeficiency virus type 1 mRNAs. The strong inhibition of rev translation exerted by the presence of the tat ORF may reflect the different roles of Tat and Rev in the viral life cycle. Tat acts early to induce high production of all viral mRNAs. Rev induces a switch from the early to the late phase of the viral life cycle, resulting in production of viral structural proteins and virions. Premature Rev production may result in entrance into the late phase in the presence of suboptimal levels of viral mRNAs coding for structural proteins, resulting in inefficient virus production.

Base Sequence↗
PubMed1994

Acetoin catabolic system of Klebsiella pneumoniae CG43: sequence, expression, and organization of the aco operon.

A cosmid clone which was capable of depleting acetoin in vivo was isolated from a library of Klebsiella pneumoniae CG43 cosmids. The smallest functional subclone contained a 3.9-kb DNA fragment of the cosmid clone. Sequencing of the DNA fragment revealed three open reading frames (ORFs A, B, and C) encoding polypeptides of 34, 36, and 52 kDa, respectively. The presence of these proteins was demonstrated by expression of the recombinant DNA clone in Escherichia coli. Considerable similarities between the deduced amino acid sequences of the ORFs and those of the following enzymes were found: acetoin dissimilation enzymes, pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex, and branched-chain 2-oxo acid dehydrogenase complex of various origins. Activities of these enzymes, including acetoin-dependent dichlorophenolin-dohenol oxidoreductase and dihydrolipoamide acetyltransferase, were detected in the extracts of E. coli harboring the genes encoding products of the three ORFs. Although not required for acetoin depletion in vivo, a possible fourth ORF (ORF D), located 39 nucleotides downstream of ORF C, was also identified. The deduced N-terminal sequence of the ORF D product was highly homologous to the dihydrolipoamide dehydrogenases of several organisms. Primer extension analysis identified the transcriptional start of the operon as an A residue 72 nucleotides upstream of ORF A.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1998

Targeted gene inactivation for the elucidation of deoxysugar biosynthesis in the erythromycin producer Saccharopolyspora erythraea.

The production of erythromycin A by Saccharopolyspora erythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs 17 and 18 have been constructed and shown to accumulate 3-alpha-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV), ORF17 (eryCIV) and ORF7 (eryBII) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates.

Amino Acid Sequence↗
PubMed2005

Detection of low-level promoter activity within open reading frame sequences of Escherichia coli.

The search for promoters has largely been confined to sequences upstream of open reading frames (ORFs) or stable RNA genes. Here we used a cloning approach to discover other potential promoters in Escherichia coli. Chromosomal fragments of approximately 160 bp were fused to a promoterless lacZ reporter gene on a multi-copy plasmid. Eight clones were deliberately selected for high activity and 105 clones were selected at random. All eight of the high-activity clones carried promoters that were located upstream of an ORF. Among the randomly-selected clones, 56 had significantly elevated activity. Of these, 7 had inserts which also mapped upstream of an ORF, while 49 mapped within or downstream of ORFs. Surprisingly, the eight promoters selected for high activity matched the canonical sigma70 -35 and -10 sequences no better than sequences from the randomly-selected clones. For six of the nine most active sequences with orientations opposite to that of the ORF, chromosomal expression was detected by RT-PCR, but defined transcripts were not detected by northern analysis. Our results indicate that the E.coli chromosome carries numerous -35 and -10 sequences with weak promoter activity but that most are not productively expressed because other features needed to enhance promoter activity and transcript stability are absent.

Base Sequence↗
PubMed1995

A lipopolysaccharide-binding domain of the Campylobacter fetus S-layer protein resides within the conserved N terminus of a family of silent and divergent homologs.

Campylobacter fetus cells can produce multiple S-layer proteins ranging from 97 to 149 kDa, with a single form predominating in cultured cells. We have cloned, sequenced, and expressed in Escherichia coli a sapA homolog, sapA2, which encodes a full-length 1,109-amino-acid (112-kDa) S-layer protein. Comparison with the two previously cloned sapA homologs has demonstrated two regions of identity, approximately 70 bp before the open reading frame (ORF) and proceeding 550 bp into the ORF and immediately downstream of the ORF. The entire genome contains eight copies of each of these conserved regions. Southern analyses has demonstrated that sapA2 existed as a complete copy within the genome in all strains examined, although Northern (RNA) analysis has demonstrated that sapA2 was not expressed in the C. fetus strain from which it was cloned. Further Southern analyses revealed increasing sapA diversity as probes increasingly 3' within the ORF were used. Pulsed-field gel electrophoresis and then Southern blotting with the conserved N-terminal region of the sapA homologs as a probe showed that these genes were tightly clustered on the chromosome. Deletion mutagenesis revealed that the S-layer protein bound serospecifically to the C. fetus lipopolysaccharide via its conserved N-terminal region. These data indicated that the S-layer proteins shared functional activity in the conserved N terminus but diverged in a semiconservative manner for the remainder of the molecule. Variation in S-layer protein expression may involve rearrangement of complete gene copies from a single large locus containing multiple sapA homologs.

Amino Acid Sequence↗
PubMed2002

[Molecular cloning, characterization, chromosomal assignment, genomic organization and verification of SFRS12(SRrp508), a novel member of human SR protein superfamily and a human homolog of rat SRrp86].

We have identified and characterized a novel human serine-arginine-rich (SR) splicing regulatory protein 508 (SRrp508) gene that is related to other members of the growing SR superfamily, but only homologous to rat (Rattus norvegicus) serine-arginine-rich splicing regulatory protein 86 (SRrp86) gene. The full-length cDNA of 3811 bp for human SRrp508 was cloned through a blast search of public databases following the identification of a cDNA contig of 658 bp obtained by EST assembly with full robotization in supercomputer in large-scale. Structurally, human SRrp508 encodes a polypeptide of 508 amino acids, which contains a single amino-terminal RNA recognition motif (RRM) and two carboxy-terminal domains rich in serine-arginine dipeptides that are highly conserved among other members of the SR superfamily. The conserved SR and RRM domains emphasize the biological importance of this gene. The SRrp508 gene, which contains 12 exons ranging from 0.096 to 2.093 kb and 11 introns ranging from 0.14 to 5.153 kb, is mapped to the human cytogenetic region 5q11.2-q12.1 using the bioinformatic analysis, and it does not link to any other genes. Furthermore, we have experimentally cloned and sequenced a cDNA fragment of 1680 bp containing the full-length ORF of 1527 bp in this novel human gene by RT-PCR from the single-stranded human pancreas cDNA library (Clontech), which is fully identical with that of the in silico cloning determined by the nucleotide sequencing. Thus, we in silico cloned his gene with GenBank accession number of AF459094 identified solely by bioinformatic analysis of the nucleotide and protein. This novel gene has promotors, TATA-box, several stop codons in the upstream of ORF, and PolyA signal in the downstream of ORF. Based on the above results, it can be concluded that we have obtained a complete novel human gene. The gene sequence exhibits good overall homology to that of rat SRrp86 gene, with 84% and 86% identity over the full-length nucleotide and protein, respectively, and with 96% and 86% identity over the serine-rich domain (RS) or arginine-rich domain (RA), respectively. The full-length sequence exhibits little overall homology to any other known protein at either the nucleotide or the amino acid level. The other two most closely related proteins, with 34% and 35% identity over the full-length protein, respectively, or with 51% and 54% identity over the full-length nucleotide of ORF, respectively, are drosophila serine-arginine-rich protein 54 (SRp54) and human arginine-rich nuclear protein 54 (p54). When comparisons are restricted to the RS or RA domains, the percent identity increased for both SRp54 and p54 are 44% and 54% or 38% and 43%, respectively. These results well demonstrate that only the novel human protein of 508 amino acids cloned is the human homolog of rat SRrp86, thus correcting the standpoint made by Barnard and Patton (Barnard DC, Patton JG. Identification and Characterization of a Novel Serine-Arginine-Rich Splicing Regulatory Protein. Molecular and Cellular Biology, 2000, 20(9): 3049-3057) that human arginine-rich nuclear protein 54 (p54) is the human homolog of the rat SRrp86, and suggesting that human SRrp508 is a new member of this growing superfamily of SR proteins. SRrp508 has an extensive expression profile, and may be a transcriptional factor. On the basis of its sequence and functional properties, we have named this protein SRrp508 for SR-related splicing regulatory protein of 508 amino acids. In summary, by combining bioinformatic analysis with experimental verification, we have successfully cloned the human cDNA homolog of rat SRrp86, which is verified by a series of theoretical and experimental evidence. The HGNC has just given SRrp508 gene entry the nomenclature information containing APPROVED SYMBOL: SFRS12; NAME: splicing factor, arginine/serine-rich 12; and ALIAS: DKFZp564B176, SRrp86. We have cloned this gene for near one year with no person landing the GenBank for registering the same gene. Our newly-established technique line will be helpful in discovering much more novel human genes.

Amino Acid Sequence↗
PubMed1995

Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase.

Rhodospirillum rubrum strains that overexpress the enzymes involved in posttranslational nitrogenase regulation, dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG), were constructed, and the effect of this overexpression on in vivo DRAT and DRAG regulation was investigated. Broad-host-range plasmid constructs containing a fusion of the R. rubrum nifH promoter and translation initiation sequences to the second codon of draT, the first gene of the dra operon, were constructed. Overexpression plasmid constructs which overexpressed (i) only functional DRAT, (ii) only functional DRAG and presumably the putative downstream open reading frame (ORF)-encoded protein, or (iii) all three proteins were generated and introduced into wild-type R. rubrum. Overexpression of DRAT still allowed proper regulation of nitrogenase activity, with ADP-ribosylation of dinitrogenase reductase by DRAT occurring only upon dark or ammonium stimuli, suggesting that DRAT is still regulated upon overexpression. However, overexpression of DRAG and the downstream ORF altered nitrogenase regulation such that dinitrogenase reductase did not accumulate in the ADP-ribosylated form under inactivation conditions, suggesting that DRAG was constitutively active and that therefore DRAG regulation is altered upon overexpression. Proper DRAG regulation was observed in a strain overexpressing DRAT, DRAG, and the downstream ORF, suggesting that a proper balance of DRAT and DRAG levels is required for proper DRAG regulation.

ADP Ribose Transferases↗
PubMed1992

Role of an upstream open reading frame in the translation of polycistronic mRNAs in plant cells.

The influence of an upstream small open reading frame (URF) on the translation of two consecutive coding regions on an eukaryotic mRNA was studied. The cis effects of leader length, URF length, the sequences of the URF and neighboring regions, and the trans effects of the Cauliflower mosaic virus transactivator (TAV) were analyzed. Translation efficiency of the immediate downstream open reading frame (ORF) decreased with increasing URF length. Short URFs did not drastically inhibit translation of immediate downstream ORFs but supported far downstream translation in the presence of TAV. In the latter case, the optimal URF length was 30 codons.

Base Sequence↗
PubMed2004

Sequence and transcriptional analysis of a gene cluster of Pseudomonas putida 86 involved in quinoline degradation.

Although quinoline 2-oxidoreductase (Qor) and 1H-2-oxoquinoline 8-monooxygenase (OxoOR), which catalyse the first two steps of quinoline degradation by Pseudomonas putida 86, and their genes have been investigated in some detail, the genetic organization and regulation of the catabolic pathway are not known yet. A gene cluster involved in quinoline degradation was characterized. Upstream of oxoO encoding the oxygenase component of OxoOR, the gene oxoS coding for a XylS-type protein is located. The DNA region downstream of oxoO comprises potential open reading frames (ORFs) that may code for further catabolic enzymes (an alpha/beta-hydrolase fold protein, and an amidase), and for accessory proteins presumably required for the assembly of metal cofactor containing holoenzymes (XdhC-like protein, MoeC- and MobA-like protein(s), IscS and IscU). The potential iscU gene is followed by the genes qorMSL that encode the structural subunits of Qor. Three potential ORFs (ORFs7-9) are located between qorMSL and oxoR, which codes for the reductase component of OxoOR. ORFs7-9 have counterparts in the cox (CO oxidizing system) and nic (nicotine degradation) gene clusters. Transcription of all these genes and ORFs located downstream of oxoS is induced by quinoline or 1H-2-oxoquinoline. Insertional inactivation of oxoS abolished quinoline-induced transcription. However, weak transcription of ORFs7-9 also occurred independent of quinoline and OxoS. The typical tandem recognition site for a XylS-type transcriptional activator was identified in the putative promoter region of qorM, and archetypal XylS indeed was found to activate synthesis of Qor. Motifs corresponding to single half-sites of a XylS-type binding site are located upstream of oxoO, the xdhC-like gene, and oxoR. Putative quinoline-specific transcriptional start sites were identified for these genes, and for qorM. The gene cluster probably is transcribed from several promoters, resulting in multiple overlapping polycistronic mRNAs.

Base Sequence↗