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PubMed1993

The 3' half of the mouse mammary tumor virus orf gene is not sufficient for its superantigen function in transgenic mice.

The Mouse Mammary Tumor Virus (MMTV) long terminal repeat contains an open reading frame (orf) of 960 nucleotides encoding a 36 kDa polypeptide with a putative transmembrane domain and five N-glycosylation sites in the N-terminal part of the protein. Transgenic mice bearing either the complete or the 3' terminal half of the orf sequence of MMTV-GR under the control of the SV40 promoter were raised. As shown previously by FACS analysis transgenic mice which express the complete orf gene have a significant deletion of V beta 14 expressing T cells at 6 weeks of age. Here we show that no clonal deletion of V beta 14 bearing T cells takes place in transgenic mice that contain orf sequences from the fifth ATG to the termination codon. The pattern of tissues expressing the truncated transgene was studied by the Polymerase Chain Reaction (PCR) and was very similar to the one obtained in the V beta 14 deleting animals. These data suggest that the amino-terminal portion of the ORF protein (pORF) is required for a superantigen function, while our previous data indicated that determinants from the carboxy-terminus play an important role for TCR V beta specificity.

Amino Acid Sequence↗

PubMed1991

Cloning, sequence and characterization of m5C-methyltransferase-encoding gene, hgiDIIM (GTCGAC), from Herpetosiphon giganteus strain Hpa2.

We have cloned the gene (hgiDIIM) encoding the methyltransferase (MTase) of the SalI isoschizomeric restriction-modification (R-M) system, HgiDII (GTCGAC), into Escherichia coli. The hgiDIIM gene has been isolated from the same plasmid library of Herpetosiphon giganteus strain Hpa2, as was the previously cloned R-M system, HgiDI [AcyI/GRCGYC; Düsterhöft et al., Nucleic Acids Res. 19 (1991) 1049-1056]. Sequencing and functional localization of hgiDIIM revealed an open reading frame (ORF) of 354 codons (39786 Da) with significant homologies to the group of m5C-, rather than the m4C-/m6A-, MTases. Subsequent cloning and analysis of adjacent chromosomal segments led to the identification of two additional ORFs upstream (ORF15, 139 codons) and downstream (ORF68, 611 codons) from hgiDIIM with the same transcriptional orientation as the hgiDIIM gene. However, the expected restriction enzyme function was not found in either of these ORFs.

Amino Acid Sequence↗

PubMed2004

Sequence analysis of pDN571, a plasmid encoding novel bacteriocin production in M-type 57 Streptococcus pyogenes.

Production of the novel bacteriocin streptococcin A-M57 (SA-M57) by Streptococcus pyogenes strains of M-protein type 57 is plasmid-associated. Plasmid pDN571 (3351bp) harbored by S. pyogenes 71-724, the prototype M-type 57 strain, has been completely sequenced and contains three putative open reading frames (repA, scnM57 and ORF3). In addition, the double-strand and single-strand (SSO) origins of replication were identified. Analysis of the replication-associated genetic elements places pDN571 in the ubiquitous pC194/pUB110 family of rolling-circle plasmids. The SSO of pDN571 is of the ssoA type. SA-M57 (encoded by scnM57) is synthesized as a secreted 179-amino acid polypeptide with a 27-residue secretion signal peptide and has no homology to proteins of known function.

Amino Acid Sequence↗

PubMed2003

The genome of herpesvirus saimiri C488 which is capable of transforming human T cells.

Herpesvirus saimiri (HVS), the rhadinovirus prototype, is apathogenic in the persistently infected natural host, the squirrel monkey, but causes acute T cell leukemia in other New World primate species. In contrast to subgroups A and B, only strains of HVS subgroup C such as C488 are capable of transforming primary human T cells to stable antigen-independent growth in culture. Here, we report the complete 155-kb genome sequence of the transformation-competent HVS strain C488. The A+T-rich unique L-DNA of 113,027 bp encodes at least 77 open reading frames and 5 URNAs. In addition to the viral oncogenes stp and tip, only a few genes including the transactivator orf50 and the glycoprotein orf51 are highly divergent. In a series of new primary HVS isolates, the subgroup-specific divergence of the orf50/orf51 alleles was studied. In these new isolates, the orf50/orf51 alleles of the respective subgroup segregate with the stp and/or tip oncogene alleles, which are essential for transformation.

Amino Acid Sequence↗

PubMed2003

Cloning and characterization of boar epididymal secretory proteins by homology to the human.

Northern blot analysis suggested that the boar epididymis produces closely related counterparts to human epididymal proteins HE1, HE3, HE4, HE5 and HE12. 'Full-length' cloning by nucleic acid and amino acid sequence similarity was achieved by RT-PCR methods in the case of the porcine counterparts of HE3 and HE4, while the homologues of HE5 and HE12, despite their cross-hybridization during Northern blot analysis, have not yet been cloned. The two novel porcine cDNAs were derived from moderately abundant epididymal mRNAs that were 75 and 83% identical to HE3 and HE4 cDNAs, respectively. To emphasize their relationship to the corresponding HEs, they were named Se3 and Se4 cDNAs. Their open reading frames predicted small secretory proteins with 55% (Se3) and 76% (Se4) conserved amino acids. Monospecific antipeptide antibodies to HE secretory proteins identified He3- and HE12-related proteins on Western blots of porcine epididymal fluid and semen. Both Northern and Western analyses indicated that the Se proteins were produced in a regionalized pattern and accumulated in the cauda fluid.

Amino Acid Sequence↗

PubMed2003

Inter- and intragenic variations complicate the molecular epidemiology of human cytomegalovirus.

Human cytomegalovirus isolates were analyzed, both by restriction fragment-length polymorphism typing and by sequencing for intra- and intergenic variability at 9 sites on the genome, to determine whether genetic variation influenced disease outcome and whether linkage among genes could be identified. Variation at the UL55 (glycoprotein B [gB]), UL74 (gO), UL75 (gH), UL115 (gL), US9, and US28 gene open-reading frames was studied in relationship to outcome of cytomegalovirus disease. Major findings were that (1) on the basis of analysis of only 9 genomic sites, it is apparent that an almost infinite number of genetic combinations are theoretically possible; (2) genetic linkages are rare; (3) intragenic variability may be a complicating factor in molecular epidemiologic studies; and (4) analysis of only a single gene from a clinical isolate may not reveal the presence of either intragenic variants or mixtures of genotypes.

Cytomegalovirus↗

PubMed2007

Complete sequence and analysis of the ovine herpesvirus 2 genome.

Ovine herpesvirus 2 (OvHV-2) is endemic in sheep populations worldwide and causes malignant catarrhal fever (MCF), a lymphoproliferative disease, in cattle, bison and deer. OvHV-2 has been placed in the gammaherpesvirus subfamily and is related closely to Alcelaphine herpesvirus 1 (AlHV-1). Here, the cloning, sequencing and analysis of the complete OvHV-2 genome derived from a lymphoblastoid cell line from an affected cow (BJ1035) are reported. The unique portion of the genome consists of 130,930 bp, with a mean G+C content of 52 mol%. The unique DNA is flanked by multiple copies of terminal repeat elements 4205 bp in length, with a mean G+C content of 72 mol%. Analysis revealed 73 open reading frames (ORFs), the majority (62) of which showed homology to other gammaherpesvirus genes. A further subset of nine ORFs is shared with only the related AlHV-1. Three ORFs are entirely unique to OvHV-2, including a spliced homologue of cellular interleukin-10 that retains the exon structure of the cellular gene. The sequence of OvHV-2 is a critical first step in the study of the pathogenesis and treatment of MCF.

Animals↗

PubMed1995

Immortalization of human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein-Barr virus DNA.

We have assembled derivatives of Epstein-Barr Virus (EBV) that include 71 kbp of noncontiguous DNA sequences cloned into a prokaryotic F-factor plasmid. These mini-EBVs, when introduced into an EBV-containing lymphoblastoid cell, can be packaged by the endogenous helper virus. One such mini-EBV was found to have a single C residue deleted from its EBNA3a open reading frame. When packaged, this mini-EBV initiates proliferation of infected primary human B lymphocytes only in conjunction with a complementing helper virus. Proliferation of the infected cells, however, was maintained either alone by the mini-EBV containing the mutated EBNA3a open reading frame or alone by its derivative in which the EBNA3a open reading frame had been healed of its lesion by recombination with the helper virus. The mini-EBV with a wild-type EBNA3a open reading frame when packaged alone can both initiate and maintain proliferation upon infection of primary human B lymphocytes. These findings identify 41% of EBV DNA which is sufficient to immortalize primary human B lymphocytes and provide an assay to distinguish virus contributions to initiation or maintenance of cell proliferation or both. They also identify EBNA3a as a transforming gene, which contributes primarily to the initiation of cell proliferation.

Amino Acid Sequence↗

PubMed2003

The alternative open reading frame of LAGE-1 gives rise to multiple promiscuous HLA-DR-restricted epitopes recognized by T-helper 1-type tumor-reactive CD4+ T cells.

The NY-ESO-1 and LAGE-1 genes are expressed by many human cancers, but not by normal tissues, with the exception of testis and placenta. The NY-ESO-1 and LAGE-1 genes give rise to multiple MHC class I and class II-presented epitopes derived from the open reading frames (ORF) 1 and 2. Here, we have investigated whether NY-ESO-1/LAGE-1 ORF2 encodes promiscuous MHC class II-restricted epitopes. Using a set of overlapping peptides from the ORF2 protein sequence and autologous dendritic cells (DCs) from normal donors and melanoma patients, we have identified three HLA-DRB1*0401-restricted peptide sequences from the LAGE-1 ORF2 that are capable of stimulating T-helper 1-type melanoma-reactive CD4+ T cells. From these bulk CD4+ T cells, we have generated CD4+ T-cell clones able to recognize not only peptide-pulsed DCs but also autologous DCs loaded with the LAGE-1 ORF2 protein. We have demonstrated that these peptides not only bind to multiple HLA-DR molecules apart from HLA-DRB1*0401 but also stimulate CD4+ T cells when presented in the context of these HLA-DR molecules. Furthermore, our binding data have delineated two additional sequences capable of broadly binding to multiple HLA-DR molecules. Altogether, these data support the immunogenicity of NY-ESO-1/LAGE-1 ORF2 gene products and clearly demonstrate their capability to stimulate T-helper 1 type CD4+ T cells. Because of the role of these cells in promoting long-lasting antitumor CTL responses, our data provide a rationale for cancer vaccine trials with peptides derived from the NY-ESO-1/LAGE-1 ORF2 for a large fraction of patients with NY-ESO-1/LAGE-1(+) tumors.

Amino Acid Sequence↗

PubMed1996

The sequence of a 21.3 kb DNA fragment from the left arm of yeast chromosome XIV reveals LEU4, MET4, POL1, RAS2, and six new open reading frames.

The nucleotide sequence of a fragment of 21 308 bp from the left arm of Saccharomyces cerevisiae chromosome XIV has been determined. Analysis of the sequence revealed 13 open reading frames (ORFs) longer than 300 bp, four of which correspond to the previously identified genes LEU4, MET4, POL1 and RAS2. One putative protein, N2160, shares considerable homology (32% identity) with a hypothetical protein encoded by a gene located on chromosome XV as well as with human OCRL protein (36% identity), involved in Lowe's syndrome. N2185 contains ten predicted transmembrane segments and is similar to another putative protein (YKL146) from yeast.

Amino Acid Sequence↗

PubMed1994

Molecular cloning of a Drosophila melanogaster gene coding for an homologue of human carboxypeptidase E.

PCR primers derived from two functional domains of vertebrate carboxypeptidase E (CPE) were used to generate a probe for screening a size-selected Drosophila melanogaster genomic library. A sequence representing about 50% of the expected complete sequence was obtained by translation of the two open reading frames present on a 1.6 kb DNA genomic fragment. This partial sequence, homologous to human CPE, CPM, and CPN, contained the conserved arginine and zinc binding domains. Similarities to the human enzymes were found with stretches that were equally divergent from the three vertebrate carboxypeptidases. Northern blot analysis revealed the presence of a 6.9 kb transcript for this gene in Drosophila embryos. I postulate that insects possess a single protein fulfilling CPE, CPM, and CPN functions.

Amino Acid Sequence↗

PubMed1994

Molecular construction of Clostridium botulinum type C progenitor toxin and its gene organization.

The 16S progenitor toxin of Clostridium botulinum type C is made up by conjugation of a neurotoxin with nontoxic components designated as nontoxic-nonHA and hemagglutinin (HA). The HA was found to be composed of subcomponents having 53, 33, 22-23, and 17 kDa molecular masses. Since we previously determined the whole nucleotide sequences of the genes for neurotoxin, nontoxic-nonHA, and HA-33, the cloning and nucleotide sequencing of the genes for the remaining HA subcomponents were performed. Two open reading frames coding for 16.7 kDa (HA-17) and 70.6 kDa proteins were identified. The N-terminal amino acid sequences of HA-53 and HA-22-23 indicated that the 70.6 kDa protein is split into 53 and the 22-23 kDa proteins after translation and that the 22-23 kDa protein consists of at least four proteins showing slightly different molecular weights.

Amino Acid Sequence↗

PubMed1994

A new human gene (DXS1357E) with ubiquitous expression, located in Xq28 adjacent to the adrenoleukodystrophy gene.

We have isolated a new human gene (DXS1357E; laboratory name: CDM) localized in Xq28. This gene is transcribed from the same CpG island as the adrenoleukodystrophy gene (ALD) and oriented in the opposite direction. It encodes a 1.5-kb transcript that exhibits ubiquitous expression and contains a single open reading frame. The 246 deduced amino acid sequence suggests the presence of membrane-associated segments and a weak similarity with the rod-like tail portion of heavy chain myosins from different species. The DXS1357E gene may be a candidate for one of the many diseases mapping to this region. A preliminary analysis did not show rearrangements of the gene in 19 independent patients with Emery-Dreifuss muscular dystrophy.

Adrenoleukodystrophy↗

PubMed1996

Cloning and expression of the Zymomonas mobilis "production of ethanol" genes in Lactobacillus casei.

This study describes the expression of the Zymomonas mobilis genes coding for pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) in Lactobacillus casei 686. To promote transcription, the promoter and ribosome binding site (RBS) from the Lactococcus lactis subsp. lactis-derived vector, pMGE36e, were inserted upstream of the pdc gene. The former sequences were positioned such that translation of pdc was coupled to translation of an 81-base pair open reading frame terminating within the pdc initiation site. The recombinant plasmid (pRSG02) was electroporated into L. casei, and transformants were obtained. Northern analysis confirmed the production of a 3. 1-kb transcript corresponding to the predicted size of the PET operon. Western blot analyses revealed that the recombinant strain expressed both enzymes. The recombinant produced more than twice the ethanol produced by the parental L. casei strain.

Alcohol Dehydrogenase↗

PubMed1990

Sequence and transcription of Raji Epstein-Barr virus DNA spanning the B95-8 deletion region.

The DNA sequence of Raji DNA spanning the deletion found in B95-8 cells has been determined. Three open reading frames and a region of homology with the BamHI-H fragment are found within the deletion. The deletion contains a region of 102-bp repeats which is transcribed into an mRNA. The Raji sequence reported here varies slightly from a smaller M-ABA sequence reported previously. This paper completes the sequence of all parts of the wild-type Epstein-Barr virus genome.

Amino Acid Sequence↗

PubMed1991

Cloning and sequence of an infectious bovine rhinotracheitis virus (BHV-1) gene homologous to glycoprotein H of herpes simplex virus.

A homologue to the glycoprotein H (gH) gene of herpes simplex virus (HSV) has been identified in the genome of infectious bovine rhinotracheitis virus (IBR, BHV-1). The gene is located immediately downstream from the thymidine kinase gene, and codes for an open reading frame (orf) of 842 amino acids. The orf has the characteristics of a membrane glycoprotein, including an N-terminal hydrophobic region resembling a signal sequence, a C-terminal region which is probably a transmembrane domain, and six potential sites for N-linked glycosylation. This orf shows significant homology to the gH sequences of both HSV and pseudorabies virus (PRV). We conclude that this gene encodes BHV-1 gH.

Base Sequence↗

PubMed1993

Nucleotide sequence of the Escherichia coli K-12 transketolase (tkt) gene.

The gene tkt for the enzyme transketolase (TK; EC 2.2.1.1) of Escherichia coli K-12 was subcloned and the complete DNA sequence was determined from both strands. An open reading frame with 1992 bp was detected that could encode a protein with a subunit mass of 72,143. The gene forms a monocistronic operon and is transcribed counterclockwise to the E. coli chromosome. From recombinant strains, the enzyme was purified and 39 N-terminal amino acid residues were determined. The DNA-derived protein sequence showed 49.5% identical amino acid residues with the transketolase of Rhodobacter sphaeroides, 46.8% identity with the enzyme from Saccharomyces cerevisiae, and 28.9% identity with the human transketolase.

Amino Acid Sequence↗

PubMed1993

Analysis of the nucleotide sequence of a 43 kbp segment of the genome of variola virus India-1967 strain.

Sequencing and computer analysis of the nucleotide sequence of the variola virus strain India-1967 (VAR) genome segment (43069 bp) from the region of HindIII C, E, R, Q, K, H DNA fragments has been carried out. Forty-three potential open reading frames (ORFs) have been identified, and the polypeptides encoded by them have been compared with the analogous proteins of vaccinia virus strain Copenhagen (COP). ORF E7R of VAR is much shorter than the COP analog. The other polypeptides coded by the potential ORFs of VAR are highly conserved in comparison with COP. Possible functions of the predicted viral polypeptides are discussed.

African Swine Fever Virus↗