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PubMed1994

Sequence analysis of a 10 kb fragment of yeast chromosome XI identifies the SMY1 locus and reveals sequences related to a pre-mRNA splicing factor and vacuolar ATPase subunit C plus a number of unidentified open reading frames.

We report the DNA sequence analysis of a region on the left arm of chromosome XI of Saccharomyces cerevisiae extending over 10 kb. The region contains five open reading frames (ORFs) of greater than 100 amino acids which do not show significant overlap with other ORFs. YKL408 contains a sequence with strong similarity to the RNA helicase pre-mRNA splicing factors PRP2, PRP16 and PRP22 (Burgess et al., 1990; Company et al., 1991; Ruby et al., 1991). YKL409 corresponds to the gene SMY1, the sequence of which was previously reported by Lillie and Brown (1992). YKL410 is identical to ATPase subunit C (Beltran et al., 1992) except for an N-terminal extension. YKL406 and YKL407 show no significant identity with any sequences in the databases searched.

Adenosine Triphosphatases↗
PubMed1992

Human cytomegalovirus UL97 open reading frame encodes a protein that phosphorylates the antiviral nucleoside analogue ganciclovir.

Human cytomegalovirus (HCMV, a betaherpes virus) is the cause of serious disease in immunologically compromised individuals, including those with acquired immunodeficiency syndrome. One of the compounds used in the chemotherapy of HCMV infections is the nucleoside analogue 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (ganciclovir). The mechanism of action of this drug is dependent on the formation of the nucleoside triphosphate, which is a strong inhibitor of the viral DNA polymerase. Thymidine kinase, which is encoded by many of the herpesviruses, catalyses the initial phosphorylation of ganciclovir. But there is no evidence for the coding of this enzyme by HCMV, and DNA sequence analysis of the HCMV genome has shown that there is no open reading frame characteristic of a herpesvirus thymidine kinase. Here we present biochemical and immunological evidence that the HCMV UL97 open reading frame codes for a protein capable of phosphorylating ganciclovir. This protein seems to be responsible for the selectivity of ganciclovir and will be useful tool in the understanding and refinement of the antiviral activity of new selective anti-HCMV compounds.

Cloning, Molecular↗
PubMed2006

Cytotoxicity of a recombinant fusion protein of adenovirus early region 4 open reading frame 4 (E4orf4) and human epidermal growth factor on p53-deficient tumor cells.

Adenovirus early region 4 open reading frame 4 (E4orf4) protein is a novel cell death factor that selectively induces p53-independent apoptosis in cancer cells, but not in normal human cells. This study presents an approach for inhibiting p53-deficient tumor cell growth by using protein-based E4orf4 that had been genetically fused to epidermal growth factor (EGF) to ensure selective targeting of EGF receptor-overexpressing tumor cells. EGF-E4orf4 enables binding onto the cell surface and is then internalized into Saos-2 cells. The success of the process had been demonstrated by immunofluorescence assay and confocal laser microscopy. After prolonged exposure, E4orf4 remained mostly in the nuclei. EGF-E4orf4 treatment of Saos-2 cells showed dose-dependent cytotoxicity. Nearly 50% of the Saos-2 cells were killed at a concentration of 250 nmol/l. In contrast, EGF-E4orf4 showed no significant inhibitory effect iresn primary cells of human umbilical vein endothelial cells. To confirm the ability of EGF-E4orf4 to induce apoptosis, DNA fragmentation was detected using BrdUTP end-labeling. Flow cytometric analysis revealed a significant increase of apoptotic cells in Saos-2 cells treated with EGF-E4orf4, but not in the case of cells cultured in plain medium (t=0.028, P<0.05). In conclusion, these preliminary results indicate that EGF-E4orf4 could show promise as a new reagent that is more efficient and less toxic in anti-cancer therapy.

Cell Death↗
PubMed1986

Nonsense mutation in open reading frame E2 of bovine papillomavirus DNA.

Oligonucleotide-directed mutagenesis was used to construct a nonsense mutation in open reading frame (ORF) E2 of bovine papillomavirus DNA. A single base substitution mutation was constructed which converted a TAC codon into a TAG amber stop codon at a position in the ORF that did not overlap with any other viral ORFs. Full-length viral DNA containing the mutation induced only approximately 2% of the transformed foci of mouse C127 cells that were induced by wild-type DNA. In a different transformation assay, approximately one-half of the C127 cells which had acquired the mutant DNA gave rise to colonies containing at least some cells with transformed morphology. The constructed mutation was maintained in cell lines derived from cells which had acquired the mutant viral DNA, but the viral DNA appeared to be integrated into the host cell genome. Genetic mapping experiments proved that the constructed amber mutation caused the decrease in focus-forming activity and the integration of the mutant viral DNA. These results suggest that ORF E2 encodes a protein which is involved either directly or indirectly in some aspects of oncogenic transformation by bovine papillomavirus and in maintaining the viral DNA as a plasmid in transformed cells.

Animals↗
PubMed1987

Mutational analysis of the 3' open reading frames and the splice junction at nucleotide 3225 of bovine papillomavirus type 1.

Functional analysis of the 3' open reading frames (ORFs) of bovine papillomavirus type 1 (BPV-1) has been complicated by the organization of that part of the genome. A region between nucleotides (nt) 3173 and 3551 contains three overlapping ORFs (E2, E3, and E4), as well as a 3' splice junction at nt 3225 which is used by many of the BPV-1 transcripts. To more clearly assign functions to specific ORFs in this region, single-base substitution mutations were generated which introduced translational termination codons into each of the three ORFs; a fourth mutation substituted an A with a C at nt 3223, altering the 3' splice junction consensus sequence from AG to CG. The E3- and the E4-specific mutants were wild type in their abilities to transform susceptible mouse C127 cells, to replicate as stable plasmids, and to trans-activate the E2 conditional enhancer. The E2-specific termination mutant was defective for plasmid replication, transformation, and trans-activation and could not be complemented for efficient transformation of a flat cell line which expressed the full-length E2 gene product. The splice junction mutant was defective for transformation of C127 cells and of a flat cell line expressing the full-length E2 gene product. These data extend previous analyses of the 3' ORFs and suggest that a spliced E2 product is involved in cellular transformation. The splice junction mutant could replicate as a stable plasmid, indicating that there is no absolute requirement in plasmid replication for a viral gene product expressed solely from an mRNA using the 3' splice junction at nt 3225.

Animals↗
PubMed1988

Variation of R1 repeated sequence present in open reading frame 11 of varicella-zoster virus strains.

We molecularly cloned the tandem direct reiteration (R1) present in open reading frame (ORF) 11 from three independent strains of varicella-zoster virus. Comparison of the R1 sequences among varicella-zoster virus strains revealed that, although the portion of R1 near the 5' terminus of ORF 11 was conserved among strains, the 3'-terminal portion varied remarkably. This variation was due to the different arrangement of two elements (A and B) and a segment produced by fusion of A and B and to a single-base change in the A element. Since the difference in the size of R1 among strains was a multiple of 3 base pairs, the variation in R1 caused no frame shift in ORF 11.

DNA, Recombinant↗
PubMed1990

Presence of antibody reactive with synthetic peptide derived from L2 open reading frame of human papillomavirus types 6b and 11 in human sera.

Nine oligopeptides corresponding to segments of different open reading frame (ORF) proteins of human papillomavirus (HPV) 6b and HPV-16 were prepared and tested for reactivity with human sera in enzyme-immunoassay (ELISA). Of these only heptadecapeptide derived from L2 ORF of HPV-6b, and encoded also by L2 ORF of HPV 11, was reactive with some human sera. Over 400 human sera of different origin were tested for the presence of antibody to this antigen. While less than 15% of sera from healthy subjects or cervical carcinoma patients were found antibody positive, sera from the majority of condylomata accuminata (CA) patients were reactive. The antibody titres varied from 1:10 (initial serum dilution) to 1:80; in this respect there was no marked difference between sera from CA patients and the other subjects. The prevalence of antibody was higher among promiscuous than nonpromiscuous women. This is in line with the assumption that sexual intercourse is the most important route of HPV 6 and 11 transmission.

Amino Acid Sequence↗
PubMed1995

A 43.5 kb segment of yeast chromosome XIV, which contains MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1, predicts an adenosine deaminase gene and 14 new open reading frames.

A 43,481 bp fragment from the left arm of chromosome XIV of Saccharomyces cerevisiae was sequenced. A gene for tRNA(phe) and 23 non-overlapping open reading frames (ORFs) were identified, seven of which correspond to known yeast genes: MFA2, MEP2, CAP/SRV2, NAM9, FKB1/FPR1/RBP1, MOM22 and CPT1. One ORF may correspond to the yet unidentified yeast adenosine deaminase gene. Among the 15 other ORFs, four exhibit known signatures, which include a protein tyrosine phosphatase, a cytoskeleton-associated protein and two ATP-binding proteins, four have similarities with putative proteins of yeast or proteins from other organisms and seven exibit no significant similarity with amino acid sequences described in data banks. One ORF is identical to yeast expressed sequence tags (EST) and therefore corresponds to an expressed gene. Six ORFs present similarities to human dbESTs, thus identifying motifs conserved during evolution. Nine ORFs are putative transmembrane proteins. In addition, one overlapping and three antisense ORFs, which are not likely to be functional, were detected.

Adenosine Deaminase↗
PubMed1993

Large unidentified open reading frame in plastid DNA (ORF2280) is expressed in chloroplasts.

The chloroplast DNA encodes genes for components of photosynthesis and the transcription-translation machinery; a number of unidentified open reading frames (ORFs) are also present. To determine whether a large ORF in the inverted repeat of chloroplast DNA of tobacco (ORF2280) encodes a chloroplast protein, a conserved region of the ORF was expressed in Escherichia coli. An antibody against the ORF protein was prepared using the purified fusion protein as an antigen. When incubated with proteins from the soluble fraction of tobacco, spinach and Oenothera chloroplasts, the antiserum detects relatively labile polypeptides, which have apparent molecular weights of 170 to 180 kDa. The ORF in tobacco and spinach is large enough to encode a protein of 240-250 kDa, thus it is possible that post-transcriptional or post-translational processing reduces the size of the expression product. Analysis of Oenothera chloroplasts representing four different plastome types revealed endonuclease restriction fragment length polymorphisms in chloroplast DNA indicative of insertion/deletion events in a region of the chloroplast DNA that shared significant sequence similarity with ORF2280. The ORF2280 antiserum was used to demonstrate that there are qualitative differences in the ORF proteins from different Oenothera plastome types.

Blotting, Southern↗
PubMed2004

ORFDB: an information resource linking scientific content to a high-quality Open Reading Frame (ORF) collection.

The ORFDB (http://orf.invitrogen.com/) represents an ongoing effort at Invitrogen Corporation to integrate relevant scientific data with an evolving collection of human and mouse Open Reading Frame (ORF) clones (Ultimate ORF Clones). The ORFDB serves as a central data warehouse enabling researchers to search the ORF collection through its web portal ORFBrowser, allowing researchers to find the Ultimate ORF clones by blast, keyword, GenBank accession, gene symbol, clone ID, Unigene ID, LocusLink ID or through functional relationships by browsing the collection via the Gene Ontology (GO) Browser. As of October 2003, the ORFDB contains 6200 human and 2870 mouse Ultimate ORF clones. All Ultimate ORF clones have been fully sequenced with high quality, and are matched to public reference protein sequences. In addition, the cloned ORFs have been extensively annotated across six categories: Gene, ORF, Clone Format, Protein, SNP and Genomic links, with the information assembled in a format termed the ORFCard. The ORFCard represents an information repository that documents the sequence quality, alignment with respect to public protein sequences, and the latest publicly available information associated with each human and mouse gene represented in the collection.

Animals↗
PubMed1997

Characterization of a domain of the genome of BmNPV containing a functional gene for a small capsid protein and harboring deletions eliminating three open reading frames that are present in AcNPV.

The nucleotide sequence of a 1.5-kb fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been determined. An open reading frame (ORF) has been identified, which encodes a protein of a predicted molecular mass of 15 kDa (p15). Analysis of the p15 sequence revealed that it shares significant similarities with a number of previously characterized viral capsid proteins. Two mRNAs, 0.7- and 1.3-kb, are transcribed from the p15 gene. The 0.7-kb transcript appears before BmNPV DNA replication, while the 1.3-kb transcript is transcribed only after viral DNA replication. The transcription start sites for the 0.7- or 1.3-kb transcripts have been localized into two areas mapping 114 and 557/559 bp upstream of the p15 translation initiation codon, respectively, and the two transcripts appear to share a common poly(A) addition site located 114 bp downstream of the translation termination codon. Comparisons of the nucleotide sequence of the 1.5-kb BmNPV fragment with that of another isolate of BmNPV and with Autographa californica NPV (AcNPV) genomic DNA reveal that the corresponding region of AcNPV encompasses 4.1 kb of DNA and three additional ORFs that are absent from the BmNPV genome. These findings raise questions about the origin and functional relevance of the putative polypeptides encoded by the three ORFs of AcNPV.

Amino Acid Sequence↗
PubMed1993

The p12I, p13II, and p30II proteins encoded by human T-cell leukemia/lymphotropic virus type I open reading frames I and II are localized in three different cellular compartments.

Three protein isoforms are encoded by the human T-cell leukemia/lymphotropic virus type I pX region open reading frames (ORF) I and II through alternative splicing. Both the singly and doubly spliced mRNAs from ORF I encode a single 12-kDa protein (p12I), whereas two distinct proteins of 13 kDa (p13II) and 30 kDa (p30II) are encoded from the ORF II alternatively spliced mRNA. Because the p12I protein is very hydrophobic and poorly immunogenic, we genetically engineered its cDNA by adding a short stretch of amino acids from the highly immunogenic epitope HA1 of influenza virus or the AU1 epitope of bovine papillomavirus. The HA1 epitope was also added to the p13II and p30II proteins, albeit rabbit immune sera raised against synthetic peptides were also available. To determine in which cellular compartments these proteins reside, we transfected the tagged and wild-type cDNAs in HeLa/Tat cells and studied their localization by indirect immunofluorescence. The p12I protein was identified in the cellular endomembranes and, particularly, in the perinuclear area. p13II and p30II were found in the nuclei and nucleoli of the transfected cells, respectively. The presence of the HA1 epitope at the carboxy terminus of p13II and p30II did not interfere with their cellular localization, since the rabbit immune sera demonstrated their presence in the same cellular compartments when the untagged proteins were expressed. The defined localization of these proteins in specific cellular compartments warrants further study of their function.

Alternative Splicing↗
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↗
PubMed2003

Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) open reading frame 4 protein (kaposica) is a functional homolog of complement control proteins.

The genome analysis of Kaposi's sarcoma-associated herpesvirus (KSHV) has revealed the presence of an open reading frame (ORF 4) with sequence homology to complement control proteins. To assign a function to this protein, we have now expressed this ORF using the Pichia expression system and shown that the purified protein inhibited human complement-mediated lysis of erythrocytes, blocked cell surface deposition of C3b (the proteolytically activated form of C3), and served as a cofactor for factor I-mediated inactivation of complement proteins C3b and C4b (the subunits of C3 convertases). Thus, our data indicate that this KSHV inhibitor of complement activation (kaposica) provides a mechanism by which KSHV can subvert complement attack by the host.

Cloning, Molecular↗
PubMed1996

Sequence analysis of a 14.6 kb DNA fragment of Saccharomyces cerevisiae chromosome VII reveals SEC27, SSM1b, a putative S-adenosylmethionine-dependent enzyme and six new open reading frames.

The nucleotide sequence of a fragment from the left arm of Saccharomyces cerevisiae chromosome VII has been determined. Analysis of the 14,607 bp DNA segment reveals nine open reading frames (ORFs) longer than 300 bp. G2827 is the SEC 7 gene, an essential coatomer complex subunit. G2834 encodes SSM1b, a ribosomal protein. The G2838 product shows homology to hypothetical yeast proteins, YIF0 and YE09, of unknown function. The G2830 product shows homology with the cell division protein FtsJ from Escherichia coli, with two hypothetical proteins from yeast, YCF4 and YBR1, and with R74.7, a hypothetical protein from Caenorhabditis elegans. Two of the ORFs are completely internal to longer ones and a third is partially embedded in G2850. The remaining ORFs give no significant homology with proteins in the databases.

Amino Acid Sequence↗
PubMed1997

The internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication.

The coronavirus mouse hepatitis virus (MHV) contains a large open reading frame embedded entirely within the 5' half of its nucleocapsid (N) gene. This internal gene (designated I) is in the +1 reading frame with respect to the N gene, and it encodes a mostly hydrophobic 23-kDa polypeptide. We have found that this protein is expressed in MHV-infected cells and that it is a previously unrecognized structural protein of the virion. To analyze the potential biological importance of the I gene, we disrupted its expression by site-directed mutagenesis using targeted RNA recombination. The start codon for I was replaced by a threonine codon, and a stop codon was introduced at a short interval downstream. Both alterations created silent changes in the N reading frame. In vitro translation studies showed that these mutations completely abolished synthesis of I protein, and immunological analysis of infected cell lysates confirmed this conclusion. The MHV I mutant was viable and grew to high titer. However, the I mutant had a reduced plaque size in comparison with its isogenic wild-type counterpart, suggesting that expression of I confers some minor growth advantage to the virus. The engineered mutations were stable during the course of experimental infection in mice, and the I mutant showed no significant differences from wild type in its ability to replicate in the brains or livers of infected animals. These results demonstrate that I protein is not essential for the replication of MHV either in tissue culture or in its natural host.

Animals↗
PubMed1996

Tomato golden mosaic virus open reading frame AL4 is genetically distinct from its C4 analogue in monopartite geminiviruses.

Tomato golden mosaic virus (TGMV) is a bipartite geminivirus with six well-characterized genes. An additional open reading frame (ORF), AL4, lies within the essential AL1 gene. Recent studies of monopartite, dicot-infecting geminiviruses have revealed that mutations in their analogous C4 ORFs have host-specific effects on infectivity, symptomatology, virus movement and/or viral DNA accumulation. We have investigated whether TGMV has a similar host-specific requirement for AL4. The phenotypes of three TGMV al4 mutants were determined in a range of hosts, which included species that revealed c4 mutant phenotypes for monopartite geminiviruses. Each TGMV al4 mutant was indistinguishable from wild-type TGMV in all hosts tested. Additional analyses of double mutants revealed no evidence for functional redundancy between AL4 and the AL3, or AR1 genes. In contrast to the putative C4 proteins of monpartite geminiviruses, TGMV AL4, if it is expressed, is either non-functional, or functionally redundant with an essential TGMV gene product.

Base Sequence↗