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PubMed1990

The T open reading frame of human immunodeficiency virus type 1.

Sequence analysis of multiple isolates of the human immunodeficiency virus type 1 (HIV-1) reveals the existence of a conserved open reading frame, designated T, that partially overlaps the tat, rev, and vpu coding sequences. Here we show that in vitro translation of RNA derived from this region of the viral genome yields a 17 kDa fusion protein, the result of a minus one frameshift event in the overlap between the tat and T open reading frames. It is also shown that messenger RNA species accumulate in HIV-1 infection from which the 17 kDa protein can be made. These observations suggest that ribosomal frameshift events may result in the biosynthesis of viral regulatory as well as viral structural proteins.

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↗

PubMed1993

Mutagenesis of the virion-sense open reading frames of tomato leaf curl geminivirus.

A series of frame shift, deletion, and inversion mutants in the virion-sense open reading frames (ORFs) of the monopartite geminivirus tomato leaf curl virus have been constructed and their ability to replicate, produce single-stranded DNA, spread, and cause symptoms in tomato plants has been investigated. Disruptions in the V1 ORF lead to symptomless, systemic infections with a reduced titer of all viral DNA forms while interruptions in the V2 (coat protein) ORF disrupted spread of the virus. Mutagenesis of the virion-sense ORFs did not affect the replication of viral double-stranded DNA, although both V1 and V2 products appear to play a role in the accumulation of viral single-stranded DNA.

Blotting, Southern↗

PubMed1994

A 4.6 kb DNA region of Rhizobium meliloti involved in determining urease and hydrogenase activities carries the structural genes for urease (ureA, ureB, ureC) interrupted by other open reading frames.

A 4.6 kb DNA region of the Rhizobium meliloti strain AK631 was found to contain seven open reading frames (ORFs), all oriented in the same direction. The putative gene products of four of these ORFs were highly homologous to UreA, UreB and UreC of Klebsiella aerogenes, Proteus mirabilis, Proteus vulgaris and Canavalia ensiformis. The overall organisation of the DNA region analysed was ORF1, ureA (ORF2), ORF3, ureB (ORF4), ORF5, ORF6 and ureC (ORF7), indicating that the organisation of the urease structural genes in R. meliloti differs from that of other urease genes so far characterized. ORF1 was incomplete; only the 3' end of the coding region was present. The six complete ORFs coded for polypeptides of 11.1 (UreA), 8.9 (ORF3), 10.8 (UreB), 15.0 (ORF5), 13.8 (ORF6) and 60.7 kDa (UreC). No sequence homology to known polypeptides could be detected for the gene products of ORF1, ORF3, ORF5 and ORF6. Using a lacZ fusion and insertional mutagenesis it was shown that the seven ORFs identified were all located in the same transcription unit. For mutational analysis a resistance gene cassette was introduced into each of the complete ORFs resulting in apolar mutations. Mutations in ureA, ureB and ureC, but not in ORF3, ORF5 and ORF6, abolished urease activity in R. meliloti. The determination of hydrogen uptake in these R. meliloti mutants revealed that only ORF6 and ureB are necessary for hydrogen uptake.

Amino Acid Sequence↗

PubMed1991

T2 open reading frame from the Shope fibroma virus encodes a soluble form of the TNF receptor.

A transcriptionally active open reading frame (T2) from Shope Fibroma Virus was recently shown to have striking sequence homology with members of a new superfamily of cell surface proteins, including a receptor for human tumor necrosis factor. Here we report that recombinant T2 protein expressed in COS cells is a soluble, secreted glycoprotein which specifically binds human TNF alpha and beta, and inhibits binding of these cytokines to native TNF receptors on cells. T2 binding of TNF is not inhibited by nerve growth factor, although the nerve growth factor receptor is also a member of the same family, nor by nine other recombinant cytokines. Further, the repeating domain structure of T2 most closely resembles that of the type I TNF receptor (p75) and is significantly different from other family members, including the type II TNF receptor (p55). Since T2 possesses a leader sequence but lacks a transmembrane domain, these results confirm the original suggestion (1) that T2 represents a soluble form of the type I TNF receptor which is secreted from virally infected cells, and whose function is to immunosuppress the host by abrogating the potentially destructive effects of TNF. This is the first such virally-encoded soluble cytokine receptor to be identified, and may represent a more general mechanism by which viruses subvert the host immune system.

Amino Acid Sequence↗

PubMed2024

Mapping Start Codons of Small Open Reading Frames by N-Terminomics Approach.

sORF-encoded peptides (SEPs) refer to proteins encoded by small open reading frames (sORFs) with a length of less than 100 amino acids, which play an important role in various life activities. Analysis of known SEPs showed that using non-canonical initiation codons of SEPs was more common. However, the current analysis of SEP sequences mainly relies on bioinformatics prediction, and most of them use AUG as the start site, which may not be completely correct for SEPs. Chemical labeling was used to systematically analyze the N-terminal sequences of SEPs to accurately define the start sites of SEPs. By comparison, we found that dimethylation and guanidinylation are more efficient than acetylation. The ACN precipitation and heating precipitation performed better in SEP enrichment. As an N-terminal peptide enrichment material, Hexadhexaldehyde was superior to CNBr-activated agarose and NHS-activated agarose. Combining these methods, we identified 128 SEPs with 131 N-terminal sequences. Among them, two-thirds are novel N-terminal sequences, and most of them start from the 11-31st amino acids of the original sequence. Partial novel N-termini were produced by proteolysis or signal peptide removal. Some SEPs' transcription start sites were corrected to be non-AUG start codons. One novel start codon was validated using GFP-tag vectors. These results demonstrated that the chemical labeling approaches would be beneficial for identifying the start codons of sORFs and the real N-terminal of their encoded peptides, which helps better understand the characterization of SEPs.

Open Reading Frames↗

PubMed2006

Varicella-zoster virus open reading frame 10 is a virulence determinant in skin cells but not in T cells in vivo.

The open reading frame 10 (ORF10) of varicella-zoster virus (VZV) encodes a tegument protein that enhances transactivation of VZV genes and has homology to herpes simplex virus type 1 (HSV-1) VP16. While VP16 is essential for HSV replication, ORF10 is dispensable for vaccine OKA (VOKA) growth in vitro. We used parent OKA (POKA) cosmids to delete ORF10, producing POKA delta10; point mutations that disrupted the acidic activation domain and the putative motif for binding human cellular factor 1 (HCF-1) in ORF10 protein yielded POKA10-Phe28Ala, POKA10-Phe28Ser, and POKA10-mHCF viruses. Deleting ORF10 or mutating these two functional domains had no effect on VZV replication, immediate-early gene transcription, or virion assembly in vitro. However, deleting ORF10 reduced viral titers and the extent of cutaneous lesions significantly in SCIDhu skin xenografts in vivo compared to POKA. Epidermal cells infected with POKA delta10 had significantly fewer DNA-containing nucleocapsids and complete virions compared to POKA; extensive aggregates of intracytoplasmic viral particles were also observed. Altering the activation or the putative HCF-1 domains of ORF10 protein had no consequences for VZV replication in vivo. Thus, the decreased pathogenic potential of POKA delta10 in skin could not be attributed to absence of these ORF10 protein functions. In contrast to skin cells, deleting ORF10 did not impair VZV T-cell tropism in vivo, as assessed by infectious virus yields. We conclude that ORF10 protein is required for efficient VZV virion assembly and is a specific determinant of VZV virulence in epidermal and dermal cells in vivo.

Animals↗

PubMed2002

The Herpesvirus Saimiri open reading frame 73 gene product interacts with the cellular protein p32.

The role of the gamma-2 herpesvirus open reading frame (ORF) 73 gene product has become the focus of considerable interest. It has recently been shown that the Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is expressed during a latent infection and can modulate both viral and cellular gene expression. The herpesvirus saimiri (HVS) ORF 73 gene product has some sequence homology to LANA; however, the role of HVS ORF 73 is unknown. We have previously demonstrated that HVS ORF73 is expressed in a stably transduced human carcinoma cell line, where HVS genomes persist as nonintegrated circular episomes. This implies that there may be some functional homology between these proteins. To further investigate the role of the HVS ORF 73 protein, the yeast two-hybrid system was employed to identify interacting cellular proteins. We demonstrate that ORF 73 interacts with the cellular protein p32 and triggers the accumulation of p32 in the nucleus. Using reporter gene-based transient-transfection assays, we demonstrate that ORF 73 can transactivate a number of heterologous promoter constructs and also upregulate its own promoter. Moreover, ORF 73 and p32 act synergistically to transactivate these promoters. The binding of ORF 73 to p32 is mediated by an amino-terminal arginine-rich domain, which contains two functionally distinct nuclear localization signals. The p32 binding domains are required for ORF 73 transactivating abilities and for ORF 73 to induce nuclear accumulation of p32. These results suggest that ORF 73 can function as a regulator of gene expression and that p32 is involved in ORF 73-dependent transcriptional activation.

Amino Acid Sequence↗

PubMed1993

The herpes simplex virus type 2 UL3 open reading frame encodes a nuclear localizing phosphoprotein.

The herpes simplex virus type 2 (HSV-2, strain 333) UL3 open reading frame (ORF) codes for a protein with a predicted molecular weight of 29,681. Comparisons of the UL3, strain 333 ORF show essentially complete amino acid identity with HSV-2 (strain HG52) and a 75% amino acid identity with HSV type 1 (strain 17). To characterize the expression of this gene, a hydrophilic region of the HSV-2 UL3 gene was cloned into a bacterial expression vector. The resulting fusion protein was used to generate antibodies in rabbits. In vitro translation of HSV-2-derived mRNA followed by immunoprecipitation with the rabbit antisera reveals a major 28,000-Da protein as judged by SDS-polyacrylamide gel electrophoresis. This is consistent with the predicted molecular weight of an unmodified UL3 protein. Pulse-labeling of infected cells, with [35S]methionine, followed by immunoprecipitation and electrophoretic analysis reveals three distinct bands of 28,000, 30,500, and 33,000 Da. Labeling infected cells with [32P]orthophosphate shows that the 30,500- and the 33,000-Da species are post-translationally phosphorylated. The 30,500-Da species can be converted to the 28,000-Da species with alkaline phosphatase treatment. Interestingly, the 33,000-Da species is resistant to this treatment. Immunohistochemical analysis of infected cells reveals that the UL3 protein has a perinuclear location early in infection and at later times becomes associated with the nucleus as discrete particles. A mutant of HSV, which has a major deletion of the UL3 coding region does not show any immunohistochemical staining with the UL3 antisera. The wild-type virus-infected cell staining pattern remains the same subsequent to DNAse and RNAse treatment, indicating that the UL3 protein product is not directly associated with nucleic acid.

Amino Acid Sequence↗

PubMed1985

Expression in Escherichia coli of open reading frame gene segments of HTLV-III.

Human T-cell lymphotropic virus type III (HTLV-III), the causative agent of the acquired immune deficiency syndrome (AIDS), was recently isolated and its genomic structure analyzed by DNA cloning methods. In the studies reported here a combined cloning and expression system was used to identify HTLV-III encoded peptides that react immunologically with antibodies in sera from AIDS patients. Cloned HTLV-III DNA was sheared into approximately 500-base-pair fragments and inserted into an "open reading frame" expression vector, pMR100. The inserted DNA was expressed in Escherichia coli transformants as a polypeptide fused to the lambda CI protein at its amino terminus and to beta-galactosidase at its carboxyl terminus. Sera from AIDS patients containing antibodies to HTLV-III were then used to screen for immunoreactive fusion proteins. Twenty clones, each specifying a fusion protein strongly reactive with AIDS serum, were identified. DNA sequence analysis indicated that the HTLV-III fragments were derived from the open reading frame DNA segments corresponding to the gag and pol gene coding regions and also the large open reading frame region (env-lor) located near the 3' end of the viral genome.

Acquired Immunodeficiency Syndrome↗

PubMed1989

A conserved open reading frame that overlaps the herpes simplex virus thymidine kinase gene is important for viral growth in cell culture.

Of 18 mutants containing clustered point mutations within UL24 (an open reading frame that overlaps the herpes simplex virus thymidine kinase gene on the opposite strand), 15 formed small plaques and were substantially impaired for virus growth in cell culture. Mutations conferring the small plaque phenotype disrupt regions of UL24 that share considerable sequence similarity with open reading frames common to herpesviruses of mammals and birds. We infer that UL24 is expressed and important for virus growth in cell culture and suggest that possible effects on UL24 should be considered in studies of thymidine kinase-deficient mutants.

Amino Acid Sequence↗

PubMed1991

Characterization of a novel open reading frame, urf a, in the mitochondrial genome of fission yeast: correlation of urf a mutations with a mitochondrial mutator phenotype and a possible role of frameshifting in urf a expression.

Between the genes for tRNA(gin) and tRNA(ile) an open reading frame of 227 amino acids has been identified which is unique among known mitochondrial genomes and which has been termed urf a (Lang et al. 1983; Kornrumpf et al. 1984). It uses the "mitochondrial" genetic code, i.e., it contains a TGA codon, whereas all other protein-encoding genes, and all but one intronic open reading frame, use the "standard" genetic code (UGG for tryptophan). A previous paper has demonstrated that "mutator" strains show an increased formation of mitochondrial drug-resistant and respiration-deficient mutants (including deletions). In this paper we show that the mutator activity is correlated with mutations in urf a. A detailed analysis of one urf a mutant is presented (anar-6), where the deletion of an A residue leads to a frameshift mutation and consequently to premature termination of the putative protein. The phenotype of colonies originating from a single mutant clone varies from no growth up to full growth on non-fermentable substrate. This phenomenon of phenotypic segregation can be explained by the ability of the cell to perform translational frameshifting. A detailed analysis of the DNA sequence and the putative urf a protein will be presented and a possible function of the protein will be discussed.

Amino Acid Sequence↗

PubMed1995

A 37.5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes, a TCP-1-related gene, an open reading frame similar to the DAL80 gene, and a tRNA(Arg).

The complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromosome X was determined from an ordered set of subclones. The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons. Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to the SME1 gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents the MEF2 gene probably encoding a second mitochondrial elongation factor-like protein, D678 is identical to the yeast GSH1 gene encoding gamma-glutamylcysteine synthetase and B746 is identical to the CSD3 gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation. The deduced amino acid sequence of A550 is 63% identical to the Cc eta subunit of a murine TCP-1-containing chaperonin and more than 35% identical to thermophilic factor 55 from Sulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP-1 family. Open reading frame F551 exhibits homology to two regions of the DAL80 gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein. In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3 and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI. Finally, the sequence contained a tRNA(Arg3) (AGC) gene.

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↗

PubMed1993

Sequence of a 7.8 kb segment on the left arm of yeast chromosome XI reveals four open reading frames, including the CAP1 gene, an intron-containing gene and a gene encoding a homolog to the mammalian UOG-1 gene.

We report here the DNA sequence of a segment of chromosome XI of Saccharomyces cerevisiae extending over 7.8 kb. The segment contains four long open reading frames, YKL150, YKL153, YKL155 and YKL156, YKL155 corresponds to the CAP1 gene. YKL153 contains an intron and shows an extremely biased codon usage suggestive of a highly expressed protein. YKL156 is a homolog to UOG-1, an open reading frame associated with the cDNA clone of the mammalian growth/differentiation factor 1. YKL150 reveals common motifs to both the RNA polymerase II elongation factor of Drosophila melanogaster and to the yeast PPR2 gene product.

Actin Capping Proteins↗

PubMed2004

Open reading frame sequencing and structure-based alignment of polypeptides encoded by RT1-Bb, RT1-Ba, RT1-Db, and RT1-Da alleles.

MHC class II genes are major genetic components in rats developing autoimmunity. The majority of rat MHC class II sequencing has focused on exon 2, which forms the first external domain. Sequence of the complete open reading frame for rat MHC class II haplotypes and structure-based alignment is lacking. Herein, the complete open reading frame for RT1-Bbeta, RT1-Balpha, RT1-Dbeta, and RT1-Dalpha was sequenced from ten different rat strains, covering eight serological haplotypes, namely a, b, c, d, k, l, n, and u. Each serological haplotype was unique at the nucleotide level of the sequenced RT1-B/D region. Within individual genes, the number of alleles identified was seven, seven, six, and three and the degree of amino-acid polymorphism between allotypes for each gene was 22%, 16%, 19%, and 0.4% for RT1-Bbeta, RT1-Balpha, RT1-Dbeta, and RT1-Dalpha, respectively. The extent and distribution of amino-acid polymorphism was comparable with mouse and human MHC class II. Structure-based alignment identified the beta65-66 deletion, the beta84a insertion, the alpha9a insertion, and the alpha1a-1c insertion in RT1-B previously described for H2-A. Rat allele-specific deletions were found at RT1-Balpha76 and RT1-Dbeta90-92. The mature RT1-Dbeta polypeptide was one amino acid longer than HLA-DRB1 due to the position of the predicted signal peptide cleavage site. These data are important to a comprehensive understanding of MHC class II structure-function and for mechanistic studies of rat models of autoimmunity.

Alleles↗

PubMed2000

Role of a short open reading frame in ribosome shunt on the cauliflower mosaic virus RNA leader.

The pregenomic 35 S RNA of cauliflower mosaic virus (CaMV) belongs to the growing number of mRNAs known to have a complex leader sequence. The 612-nucleotide leader contains several short open reading frames (sORFs) and forms an extended hairpin structure. Downstream translation of 35 S RNA is nevertheless possible due to the ribosome shunt mechanism, by which ribosomes are directly transferred from a take-off site near the capped 5' end of the leader to a landing site near its 3' end. There they resume scanning and reach the first long open reading frame. We investigated in detail how the multiple sORFs influence ribosome migration either via shunting or linear scanning along the CaMV leader. The sORFs together constituted a major barrier for the linear ribosome migration, whereas the most 5'-proximal sORF, sORF A, in combination with sORFs B and C, played a positive role in translation downstream of the leader by diverting scanning ribosomes to the shunt route. A simplified, shunt-competent leader was constructed with the most part of the hairpin including all the sORFs except sORF A replaced by a scanning-inhibiting structure. In this leader as well as in the wild type leader, proper translation and termination of sORF A was required for efficient shunt and also for the level of shunt enhancement by a CaMV-encoded translation transactivator. sORF A could be replaced by heterologous sORFs, but a one-codon (start/stop) sORF was not functional. The results implicate that in CaMV, shunt-mediated translation requires reinitiation. The efficiency of the shunt process is influenced by translational properties of the sORF.

5' Untranslated Regions↗

PubMed1994

A new open reading frame, encoding a putative regulatory protein, in Agrobacterium rhizogenes T-DNA.

We report the presence of an open reading frame, named ORF13a, encoding a putative regulatory protein on the T-DNA of Agrobacterium rhizogenes 8196 Ri plasmid. Homologous ORFs are present at the same location in two other types of Ri plasmids. We present evidence that ORF13a is transcriptionally active. Expression of ORF13a was investigated by analysis of glucuronidase (GUS) activity in transgenic tobacco containing an ORF13aGUS fusion. The gene fusion was expressed at higher level in roots than in leaves. The putative protein encoded by ORF13a has an isoelectric point of 11.55 and carries SPXX repeated motifs suggesting a possible regulatory function for this gene.

Amino Acid Sequence↗