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PubMed1996

A novel DNA damage-inducible transcript, gadd7, inhibits cell growth, but lacks a protein product.

gadd7 cDNA was isolated from Chinese hamster ovary (CHO) cells on the basis of increased levels of RNA following treatment with UV radiation. The transcript for gadd7, as well as for four other gadd genes, was found to increase rapidly and coordinately following several different types of DNA damage and more slowly following other stresses that elicit growth arrest. Agents that induce gadd7 RNA include alkylating agents, such as methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and mechlorethamine HCl (HN2), oxidizing agents, such as hydrogen peroxide, and growth arrest signals, such as medium depletion (starvation). Since growth arrest is a cellular consequence of many types of DNA damage in normal cells, it was thought that gadd7 may play a role in the cellular response to DNA damage. Indeed, overexpression of gadd7 led to a decrease in cell growth. Interestingly, gadd7 cDNA does not contain an appreciable open reading frame and does not appear to encode a protein product, but instead may function at the RNA level.

Alkylating Agents↗

PubMed2000

Ubiquinone (coenzyme Q) biosynthesis in Escherichia coli: identification of the ubiF gene.

Ubiquinone (coenzyme Q; abbreviation, Q) plays an essential role in electron transport in Escherichia coli when oxygen or nitrate is the electron acceptor. The biosynthesis of Q involves at least nine reactions. Three of these reactions involve hydroxylations resulting in the introduction of hydroxyl groups at positions C-6, C-4, and C-5 of the benzene nucleus of Q. The genes encoding the enzymes responsible for these hydroxylations, ubiB, ubiH, and ubiF are located at 87, 66, and 15 min of the E. coli linkage map. The ubiF encoded oxygenase introduces the hydroxyl group at carbon five of 2-octaprenyl-3-methyl-6-methoxy-1,4-benzoquinol resulting in the formation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1, 4-benzoquinol. An ubiF mutant failed to carry out this conversion. Based on the homology to UbiH, an open reading frame (orf391) was identified at the 15 min region of the chromosome, amplified using PCR, and cloned into pUC18 plasmid. The ubiF mutants, when complemented with this plasmid, regained the ability to grow on succinate and synthesize Q.

Amino Acid Sequence↗

PubMed1991

Isolation of an active human transposable element.

Two de novo insertions of truncated L1 elements into the factor VIII gene on the X chromosome have been identified that produced hemophilia A. A full-length L1 element that is the likely progenitor of one of these insertions was isolated by its sequence identity to the factor VIII insertion. This L1 element contains two open-reading frames and is one of at least four alleles of a locus on chromosome 22 that has been occupied by an L1 element for at least 6 million years.

Alleles↗

PubMed1999

Cloning, sequencing, and characterization of the cgmB gene of Sinorhizobium meliloti involved in cyclic beta-glucan biosynthesis.

Periplasmic cyclic beta-glucans of Rhizobium species provide important functions during plant infection and hypo-osmotic adaptation. In Sinorhizobium meliloti (also known as Rhizobium meliloti), these molecules are highly modified with phosphoglycerol and succinyl substituents. We have previously identified an S. meliloti Tn5 insertion mutant, S9, which is specifically impaired in its ability to transfer phosphoglycerol substituents to the cyclic beta-glucan backbone (M. W. Breedveld, J. A. Hadley, and K. J. Miller, J. Bacteriol. 177:6346-6351, 1995). In the present study, we have cloned, sequenced, and characterized this mutation at the molecular level. By using the Tn5 flanking sequences (amplified by inverse PCR) as a probe, an S. meliloti genomic library was screened, and two overlapping cosmid clones which functionally complement S9 were isolated. A 3.1-kb HindIII-EcoRI fragment found in both cosmids was shown to fully complement mutant S9. Furthermore, when a plasmid containing this 3.1-kb fragment was used to transform Rhizobium leguminosarum bv. trifolii TA-1JH, a strain which normally synthesizes only neutral cyclic beta-glucans, anionic glucans containing phosphoglycerol substituents were produced, consistent with the functional expression of an S. meliloti phosphoglycerol transferase gene. Sequence analysis revealed the presence of two major, overlapping open reading frames within the 3.1-kb fragment. Primer extension analysis revealed that one of these open reading frames, ORF1, was transcribed and its transcription was osmotically regulated. This novel locus of S. meliloti is designated the cgm (cyclic glucan modification) locus, and the product encoded by ORF1 is referred to as CgmB.

Base Sequence↗

PubMed2005

Endo-1,4-beta-xylanase B from Aspergillus cf. niger BCC14405 isolated in Thailand: purification, characterization and gene isolation.

During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of 55 degrees C. When tested using xylan from birchwood, it showed K(m) and V(max) values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by CuSO(4) EDTA, and by FeSO(4) The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1,4-beta-xylanase. The full-length gene encoding endo-1,4-beta-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1,4-a-xylanase B previously identified in A. niger.

Amino Acid Sequence↗

PubMed1992

Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer.

Transfer genes of the IncP plasmid RP4 are grouped in two separate regions, designated Tra1 and Tra2. Tra2 gene products are proposed to be mainly responsible for the formation of mating pairs in conjugating cells. To provide information relevant to understanding the function of Tra2 gene products, the nucleotide sequence of the entire RP4 Tra2 region is presented here. Twelve open reading frames were identified in the Tra2 core region, being essential for intraspecific Escherichia coli matings. Predicted sizes of 11 of the 12 Tra2 polypeptides could be verified by expression in E. coli. Based on hydropathy plot analysis, most of the Tra2 open reading frames encode proteins that may interact with membranes. Interestingly, six of the predicted Tra2 gene products exhibited significant sequence similarities to gene products encoded by the VirB operon of the Agrobacterium Ti plasmid. VirB proteins are thought to function in the formation of a transmembrane structure that mediates the passage of T-DNA molecules from bacteria into plant cells. Because of this analogy and the hydropathy of Tra2 gene products, we assume that the DNA transfer machineries acting in bacterial conjugation and T-DNA transfer are structurally and functionally similar. Therefore, the data presented here, support the hypothesis that Ti vir and IncP tra genes evolved from a common ancestor. This suggestion is favored by previous findings of sequence similarities between the IncP and Ti DNA transfer system.

Amino Acid Sequence↗

PubMed1996

The complete nucleotide sequence of barley mild mosaic virus RNA1 and its relationship with other members of the Potyviridae.

The complete nucleotide sequence of RNA1 of a French barley mild mosaic bymovirus isolate has been determined. The molecule is composed of 7261 nucleotides and contains a single large open reading frame encoding a protein of 2258 amino acids with a calculated Mr of 256,375. Amino acid sequence comparison with poty- and rymoviruses reveals eight domains corresponding to the potyviral P3, 6K1, Cl, 6K2, Nla-VPg, Nla-Pro, Nlb and capsid proteins, respectively. Seven putative cleavage sites, similar to those mediated by potyvirus Nla proteinases, were identified. The presence of two, so far undescribed putative cleavage sites (6K2/Nla-VPg and Nla-VPg/Nla-Pro) in the polyproteins encoded by RNA1 of barley yellow mosaic virus and wheat spindle streak mosaic virus, was predicted. These data indicate that the bymovirus RNA1 fits the consensus potyviral genetic map downstream of the helper component gene.

Amino Acid Sequence↗

PubMed1991

The nucleotide sequence of the gene encoding the attachment protein H of canine distemper virus.

The sequence of the H gene and flanking sequences in the F and L genes of canine distemper virus (CDV) have been determined. The H gene of CDV (1946 nucleotides) contains one large open reading frame starting at position 21 and terminating at position 1835, encoding a protein of 604 amino acid residues. This protein contains three potential glycosylation sites in the extracellular domain and, like all other paramyxoviruses, a N-terminal membrane-spanning hydrophobic anchor domain. The deduced H protein sequence shows an identity of 36% with rinderpest virus (RPV) and measles virus (MV). The identities at the nucleotide level are higher (RPV 52% and MV 53%). The amino acid sequence shows conservation of all the structural determinants with the H proteins of MV and RPV. The data also show that CDV is evolutionarily equidistant to RPV and MV with respect to the H gene.

Amino Acid Sequence↗

PubMed1990

Occurrence of a copia-like transposable element in one of the introns of the potato starch phosphorylase gene.

The gene coding for starch phosphorylase (EC 2.4.1.1) was isolated from a potato genomic library constructed in lambda EMBL3. It is an unusually long plant gene (16.4 kb) which encodes a preprotein of 966 amino acids. The phosphorylase coding sequence is interrupted by 14 introns whose positions do not match those of the introns in the human glycogen phosphorylase gene. A 78 amino acid central peptide unique to plant plastidial phosphorylases is hypothesized to have arisen through the mis-splicing of an intron-exon junction site in an ancestral gene. The fifth intron of the phosphorylase is very large (approximately 7 kb) and contains a copia-like transposable element inserted in the opposite orientation to that of the phosphorylase gene. This element has been named Tst1; it is bordered on the 5' and 3' sides by long terminal repeats of 285 and 283 bp respectively, which define an internal domain of 4492 bp. Tst1 contains 4 open reading frames (ORFs) that encode protein domains for a reverse transcriptase, an integrase, an RNA-binding site and a protease. Transcription of the phosphorylase gene appears to proceed unimpaired through the copia element.

Amino Acid Sequence↗

PubMed1996

Genetic analysis of the chromosomal region encoding lysophospholipase L2 of Vibrio cholerae O1.

From the Tn5-inserted mutant library of Vibrio cholerae O1, we found a mutant, NF404, which lost the production of both hemolysin and cholera toxin (CT) even though the Tn5-insertion site was out side from the structural genes for hemolysin and CT. Cloning and sequencing analysis of the homologous region from the wild-type strain, revealed that the sequence spanning the coding region of an ORF1 nominated as lypA, encoding a 39.5 kDa protein. Deduced amino acid sequence of the lypA gene had 37.6% identity to the lysophospholipase L2 (EC 3.1.1.5) of Escherichia coli. In the downstream of lypA, a second open reading frame (ORF2) encoding an unknown protein with molecular weight of 19.9 kDa was found. Assaying the lysophospholipase L2 activity in the cell extract of E.coli harboring lypA in an expression vector showed clear increase in the enzymatic activity.

Amino Acid Sequence↗

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↗

PubMed2002

An open reading frame in intron seven of the sea urchin DNA-methyltransferase gene codes for a functional AP1 endonuclease.

Analysis of the genome structure of the Paracentrotus lividus (sea urchin) DNA methyltransferase (DNA MTase) gene showed the presence of an open reading frame, named METEX, in intron 7 of the gene. METEX expression is developmentally regulated, showing no correlation with DNA MTase expression. In fact, DNA MTase transcripts are present at high concentrations in the early developmental stages, while METEX is expressed at late stages of development. Two METEX cDNA clones (Met1 and Met2) that are different in the 3' end have been isolated in a cDNA library screening. The putative translated protein from Met2 cDNA clone showed similarity with Escherichia coli endonuclease III on the basis of sequence and predictive three-dimensional structure. The protein, overexpressed in E. coli and purified, had functional properties similar to the endonuclease specific for apurinic/apyrimidinic (AP) sites on the basis of the lyase activity. Therefore the open reading frame, present in intron 7 of the P. lividus DNA MTase gene, codes for a functional AP endonuclease designated SuAP1.

Amino Acid Sequence↗

PubMed2003

Unusual multiple recoding events leading to alternative forms of hepatitis C virus core protein from genotype 1b.

In addition to its involvement in the formation of the capsid shell of the virus particles, the core protein of hepatitis C virus (HCV) is believed to play an important role in the pathogenesis and/or establishment of persistent infection. We describe here alternative forms of genotype 1b HCV core protein identified after purification of various products of core protein segment 1-169 expressed in Escherichia coli and their analysis by proteolysis, mass spectrometry, and amino acid sequencing. These proteins all result from a +1 frameshift at codon 42 (a different position than that previously reported in genotype 1a) and, for some of them, from a rephasing in the normal open reading frame at the termination codon 144 in the +1 open reading frame. To test the relevance of these recoding events in a eukaryotic translational context, the nucleotide sequences surrounding the two shift sites were cloned in the three reading frames into expression vectors, allowing the production of a C-terminally fused green fluorescent protein, and expressed both in a reticulocyte lysate transcription/translation assay and in culture cells. Both recoding events were confirmed in these expression systems, strengthening the hypothesis that they might occur in HCV-infected cells. Moreover, sera from HCV-positive patients of genotype 1a or 1b were shown to react differently against synthetic peptides encoded in the +1 open reading frame. Together, these results indicate the occurrence of distinct recoding events in genotypes 1a and 1b, pointing out genotype-dependent specific features for F protein.

Amino Acid Sequence↗

PubMed2003

Genetic analysis of the capsule locus of Haemophilus influenzae serotype f.

A 19-kb DNA region containing genes involved in the biosynthesis of the capsule of Haemophilus influenzae serotype f (Hif) has been cloned and characterized. The Hif cap locus organization is typical of group II capsule biosynthetic loci found in other H. influenzae serotype b bacteria and other gram-negative bacteria. However, the Hif cap locus was not associated with an IS1016 element. Three new open reading frames, Fcs1, Fcs2, and Fcs3, were identified in the Hif capsule-specific region II. The chromosomal location of the Hif cap locus and the organization of the flanking sequences differed significantly from previously described division I H. influenzae serotypes.

Bacterial Capsules↗

PubMed1999

A novel unenveloped DNA virus (TT virus) associated with acute and chronic non-A to G hepatitis.

In 1997, a novel DNA virus was isolated from the serum of a patient with posttransfusion hepatitis of unknown etiology in Japan, and it was named TT virus (TTV) after the initials of the index patient. TTV is a nonenveloped, single-stranded and circular DNA virus, and its entire sequence of approximately 3.9 kb has been determined. For being a DNA virus, TTV has a wide range of sequence divergence, allowing the classification into at least 16 genotypes separated by a sequence difference of >30% from one another. The nucleotide sequence of the noncoding region of the TTV genome is conserved, whereas that of the coding region is highly variable. TTV strains with extremely high sequence divergence are common in the same individuals, thereby indicating a mixed infection of TTV strains of different genotypes. An association is found between hepatitis of unknown etiology and the TTV genotypes which are detectable by PCR with primers deduced from the N22 region (genotype 1) in the open reading frame 1 encoding the capsid protein. It would be important to select the primers for specific detection of the TTV genotypes associated with clinical diseases, to further evaluate the capacity of TTV to induce acute and chronic liver disease as well as extrahepatic manifestations.

Base Sequence↗

PubMed1993

Utilization of autopsy RNA for the synthesis of the nucleocapsid antigen of a newly recognized virus associated with hantavirus pulmonary syndrome.

A newly recognized hantavirus was recently found to be associated with an outbreak of acute respiratory illness in the southwestern United States. The disease, which has become known as hantavirus pulmonary syndrome, has an unusually high mortality (64%). Virus isolation attempts have been unsuccessful thus far, resulting in a lack of homologous antigen for use in diagnostic assays. For this reason, a molecular approach was initiated to produce recombinant homologous antigen. The virus nucleocapsid (N) protein was selected, since N has been shown to be a sensitive antigenic target in other hantavirus systems. The N protein open reading frame of the virus S genome segment was synthesized from frozen autopsy tissue by polymerase chain reaction amplification, followed by cloning and expression in Hela cells (vaccinia-T7 RNA polymerase system) and Escherichia coli. N protein-expressing Hela cells served as excellent antigens for an improved indirect immunofluorescence assay. Use of the E. coli-expressed N protein in an enzyme-linked immunosorbent assay improved the sensitivity and specificity when compared with heterologous antigens used previously. Preliminary analysis also indicates that the higher sensitivity could result in earlier detection of infected persons. These data demonstrate that even in the absence of a virus isolate, the necessary homologous antigen can be produced and can serve to improve the detection and diagnostic capabilities needed to combat this newly recognized fatal respiratory illness in the United States.

Amino Acid Sequence↗

PubMed1994

Human GTP cyclohydrolase I: only one out of three cDNA isoforms gives rise to the active enzyme.

GTP cyclohydrolase I catalyses the first and rate-limiting step of tetrahydrobiopterin biosynthesis. Its expression is regulated by interferon-gamma or kit ligand in a tissue-specific manner. Three different cDNA forms have been reported for human GTP cyclohydrolase I [Togari, Ichinose, Matsumoto, Fujita and Nagatsu (1992) Biochem. Biophys. Res. Commun. 187, 359-365]. We have isolated, from a human liver cDNA library, two clones which contained inserts identical with two of the cDNAs reported by Togari et al. (1992). The three open reading frames corresponding to all reported cDNA sequences were expressed in Escherichia coli. Only the recombinant protein corresponding to the longest reading frame catalysed the conversion of GTP into dihydroneopterin triphosphate. The proteins corresponding to the shorter reading frames failed to catalyse not only the generation of dihydroneopterin triphosphate but also the release of formate from GTP, an intermediate step of the reaction. Recombinant human GTP cyclohydrolase I showed sigmoidal substrate kinetics and maximum activity at 60 degrees C. These findings are well in line with the published properties of the enzyme isolated from rat liver. The data indicate that cytokine-mediated induction of GTP cyclohydrolase I is not due to the expression of enzyme isoforms.

Amino Acid Sequence↗

PubMed2001

Characterization of 16 novel human genes showing high similarity to yeast sequences.

The entire set of open reading frames (ORFs) of Saccharomyces cerevisiae has been used to perform systematic similarity searches against nucleic acid and protein databases: with the aim of identifying interesting homologies between yeast and mammalian genes. Many similarities were detected: mostly with known genes. However: several yeast ORFs were only found to match human partial sequence tags: indicating the presence of human transcripts still uncharacterized that have a homologous counterpart in yeast. About 30 such transcripts were further studied and named HUSSY (human sequence similar to yeast). The 16 most interesting are presented in this paper along with their sequencing and mapping data. As expected: most of these genes seem to be involved in basic metabolic and cellular functions (lipoic acid biosynthesis: ribulose-5-phosphate-3-epimerase: glycosyl transferase: beta-transducin: serine-threonine-kinase: ABC proteins: cation transporters). Genes related to RNA maturation were also found (homologues to DIM1: ROK1-RNA-elicase and NFS1). Furthermore: five novel human genes were detected (HUSSY-03: HUSSY-22: HUSSY-23: HUSSY-27: HUSSY-29) that appear to be homologous to yeast genes whose function is still undetermined. More information on this work can be obtained at the website http://grup.bio.unipd.it/hussy

Amino Acid Sequence↗