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PubMed1993

Conserved nucleotide sequences in the open reading frame and 3' untranslated region of selenoprotein P mRNA.

Rat liver selenoprotein P contains 10 selenocysteine residues in its primary structure (deduced). It is the only selenoprotein characterized to date that has more than one selenocysteine residue. Selenoprotein P cDNA has been cloned from human liver and heart cDNA libraries and sequenced. The open reading frames are identical and contain a signal peptide, indicating that the protein is secreted by both organs and is therefore not exclusively produced in the liver. Ten selenocysteine residues (deduced) are present. Comparison of the open reading frame of the human cDNA with the rat cDNA reveals a 69% identity of the nucleotide sequence and 72% identity of the deduced amino acid sequence. Two regions in the 3' untranslated portion have high conservation between human and rat. Each of these regions contains a predicted stable stem-loop structure similar to the single stem-loop structures reported in 3' untranslated regions of type I iodothyronine 5'-deiodinase and glutathione peroxidase. The stem-loop structure of type I iodothyronine 5'-deiodinase has been shown to be necessary for incorporation of the selenocysteine residue at the UGA codon. Because only two stem-loop structures are present in the 3' untranslated region of selenoprotein P mRNA, it can be concluded that a separate stem-loop structure is not required for each selenocysteine residue.

Amino Acid Sequence↗

PubMed2006

Translation of open reading frame in kinetoplast DNA minicircles of clinical isolates of L. donovani.

Till today, it remains an enigma whether the open reading frames said to be transcribed in minicircle sequences are indeed translated into protein products or not. We establish a protein-coding gene in minicircle variable region of kinetoplast DNA from clinical isolates of Leishmania donovani. The protein was expressed as an N-tagged green fluorescent protein (GFP) fusion protein in leishmanial expression system. Fluorescence microscopy of the transfectants carrying recombinant GFP construct showed the protein to be localized on the plasmalemma of the parasite. This shows that the minicircle transcript is indeed translated into a protein product in the parasite cell and further points toward probable biological function of minicircles in kinetoplastids.

Animals↗

PubMed1996

The open reading frame 2 product of cacao swollen shoot badnavirus is a nucleic acid-binding protein.

The function of the open reading frame 2 product (p2) of cacao swollen shoot virus (CSSV) and of other badnaviruses is not yet determined. Their carboxyl-termini are lysine and proline rich and also contain alanine residues, amino acids present at the C-termini of histone-like proteins. Full-length CSSV p2 (132 amino acids) or versions truncated at the C-terminus (128, 113, 103, or 101 amino acids) were expressed in Escherichia coli and partially purified. When assayed in nucleic acid-binding tests, p2 was able to interact with CSSV and other double-stranded DNAs and with CSSV and other single-stranded RNA transcripts in sequence-nonspecific manner. Moreover, this binding activity was progressively lost as the C-terminus was gradually deleted.

Amino Acid Sequence↗

PubMed1994

Maintenance of an open reading frame as an additional level of scrutiny during splice site selection.

Although nonsense mutations have been associated with the skipping of specific constitutively spliced exons in selected genes, notably the fibrillin gene, the basis for this association is unclear. Now, using chimaeric constructs in a model in vivo expression system, premature termination codons are identified as determinants of splice site selection. Nonsense codon recognition prior to RNA splicing necessitates the ability to read the frame of precursor mRNA in the nucleus. We propose that maintenance of an open reading frame can serve as an additional level of scrutiny during exon definition. This process may have pathogenic and evolutionary significance.

Base Sequence↗

PubMed1998

The open reading frame III product of cauliflower mosaic virus forms a tetramer through a N-terminal coiled-coil.

The open reading frame III product of cauliflower mosaic virus is a protein of 15 kDa (p15) that is essential for the virus life cycle. It was shown that the 34 N-terminal amino acids are sufficient to support protein-protein interaction with the full-length p15 in the yeast two-hybrid system. A corresponding peptide was synthesized and a recombinant p15 was expressed in Escherichia coli and purified. Circular dichroism spectroscopy showed that the peptide and the full-length protein can assume an alpha-helical conformation. Analytical centrifugation allowed to determine that p15 assembles as a rod-shaped tetramer. Oxidative cross-linking of N-terminal cysteines of the peptide generated specific covalent oligomers, indicating that the N terminus of p15 is a coiled-coil that assembles as a parallel tetramer. Mutation of Lys22 into Asp destabilized the tetramer and put forward the presence of a salt bridge between Lys22 and Asp24 in a model building of the stalk. These results suggest a model in which the stalk segment of p15 is located at its N terminus, followed by a hinge that provides the space for presenting the C terminus for interactions with nucleic acids and/or proteins.

Amino Acid Sequence↗

PubMed1992

Varicella-zoster virus open reading frame 61 protein is functionally homologous to herpes simplex virus type 1 ICP0.

The varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein is thought to be the homolog of herpes simplex virus type 1 (HSV-1) ICP0, based on gene location and limited amino acid homology. However, HSV-1 ICP0 trans activates HSV-1 genes, while VZV ORF61 protein trans represses the function of VZV trans activators on VZV promoters in transient expression assays. To investigate the functional relatedness of HSV-1 ICP0 and VZV ORF61 protein, we established Vero and MeWo cell lines which stably express VZV ORF61 under the control of a metallothionein promoter and performed complementation studies with an HSV-1 ICP0 deletion mutant (7134). Mutant 7134 is impaired for plaque formation and replication at a low multiplicity of infection in cell culture, but these defects were complemented by up to 200-fold in Vero cell lines expressing VZV ORF61. Likewise, the efficiency of plaque formation was improved by up to 100-fold in MeWo cell lines expressing VZV ORF61. A cell line expressing another VZV immediate-early gene product (ORF62) was unable to complement mutant 7134. HSV-1 mutants which are deleted for other HSV-1 immediate-early gene products (ICP4, ICP27) were unable to grow in VZV ORF61-expressing cell lines. These results indicate that, despite marked differences in their sequences and in effects on their cognate promoters in transient expression assays, VZV ORF61 protein is the functional homolog of HSV-1 ICP0.

Amino Acid Sequence↗

PubMed1991

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells.

Animals↗

PubMed1991

Location of open reading frames coding for equine herpesvirus type-1 glycoproteins with homology to gE and gI of herpes simplex virus.

The DNA fragments representing the entire short unique region and part of the repeat sequences of the equine herpesvirus type-1 genome were cloned into plasmid vectors. The approximate positions of the junctions between the short unique region and the inverted repeats were then located by restriction endonuclease mapping. Two open reading frames coding for potential glycoproteins have been identified within the short unique region, using DNA sequence analysis. The predicted amino acid sequences of these open reading frames had extensive homology to the herpes simplex virus glycoproteins gE and gI and the related glycoproteins of pseudorabies virus and varicella-zoster virus.

Amino Acid Sequence↗

PubMed1994

Sequence of a 28.6 kb region of yeast chromosome XI includes the FBA1 and TOA2 genes, an open reading frame (ORF) similar to a translationally controlled tumour protein, one ORF containing motifs also found in plant storage proteins and 13 ORFs with weak or no homology to known proteins.

The complete DNA sequence of cosmid clone pUKG148 comprising 28,600 base pairs was determined from an ordered set of subclones. The sequence contains 22 open reading frames longer than 100 amino acids of which five are entirely covered by other, longer reading frames. YKL054 exhibits 25% homology at the amino acid level to a number of plant storage proteins of the glutenin type, YKL056 is 40% homologous to a translationally controlled mammalian tumour protein, YKL058 (TOA2) is identical to the small subunit of transcription factor TFIIA from yeast and YKL060 is identical to the FBA1 gene also from yeast, already sequenced but not mapped to chromosome XI. The remaining 13 open reading frames show weak or no homology to known genes.

Amino Acid Sequence↗

PubMed1994

Varicella-zoster virus (VZV) virion-associated transactivator open reading frame 62 protein enhances the infectivity of VZV DNA.

Varicella-zoster virus (VZV) open reading frame (ORF) 62 protein (the homolog of herpes simplex virus type 1 (HSV-1) ICP4) and ORF10 protein (the homolog of HSV-1 VP16) are virion-associated transactivators. To investigate whether these proteins function during the initial stages of VZV infection, human melanoma cells were cotransfected with purified VZV DNA, devoid of any structural proteins, along with a plasmid expressing VZV ORF62 or ORF10 under the control of the human cytomegalovirus major immediate-early promoter. Expression of ORF62 enhanced the infectivity of VZV DNA up to 70-fold. In contrast, expression of ORF10 enhanced the infectivity of VZV DNA only threefold. These results show that high-level expression of ORF62 protein increases the probability that transfected VZV DNA will result in productive infection, suggesting that this virion-associated transactivator (ORF62) has a critical role in initiating infection.

Base Sequence↗

PubMedDate not supplied

Expression of human herpesvirus 8 (HHV-8)-encoded immediate early protein, open reading frame 50, in HHV-8-associated diseases.

OBJECTIVES: To investigate the expression of the open reading frame (ORF) 50 protein, a human herpesvirus 8 (HHV-8)-encoded immediate early protein, in HHV-8-associated diseases. STUDY DESIGN/METHODS: We developed a rabbit anti-ORF50 protein polyclonal antibody, and investigated the ORF50 protein expression by immunohistochemistry using primary effusion lymphoma (PEL) cell lines and tissue sections of Kaposi's sarcoma (KS), HHV-8-associated solid lymphoma, and multicentric Castleman's disease (MCD). RESULTS: Western blot analysis revealed that this antibody reacted with a 110-kd protein in the lysate of phorbolester-stimulated HHV-8-infected PEL cell lines. Immunohistochemistry revealed that a very small population of cells expressed the ORF50 protein in KS and HHV-8-associated solid lymphoma cells, and almost all these cells expressed HHV-8-encoded latency-associated nuclear antigen. The ORF50 protein expression was also rare in the cells of PEL cell lines, and the staining pattern was diffuse or dot-like in the nuclei, overlapping partially with that of promyelocytic leukemia protein. In MCD, however, the ORF50 protein was expressed in some cells of the mantle zone of the lymphoid follicle. CONCLUSIONS: The ORF50 protein expression in vivo was considerably rare in KS, HHV-8-associated solid lymphoma, and PEL, but was more frequent in MCD. Rare expression of this transactivator protein in HHV-8-associated malignancies causes low expression levels of other lytic proteins and may play a role in the maintenance of the latent infection. This is the first report describing the expression of an immediate early protein of HHV-8 in cases of HHV-8-associated diseases.

Castleman Disease↗

PubMed1999

A long open reading frame in the mitochondrial LSU rRNA group-I intron of Cryphonectria parasitica encodes a putative S5 ribosomal protein fused to a maturase.

A 4238-bp intervening sequence within the highly conserved U11 region of the mitochondrial large subunit ribosomal RNA gene of the fungus Cryphonectria parasitica Ep155 has been sequenced and identified to be a group-I intron. This is the largest group-I intron reported to-date for fungal mitochondrial genomes. The intron contains an 851-codon open reading frame encoding a putative, but complete, small-subunit ribosomal protein of 510 amino acids which is fused at its carboxyl terminus to a 311 amino-acid polypeptide representing a typical maturase-like protein. A short open reading frame of 83 amino acids with some similarity to maturases, but lacking a translation-initiation codon, was also noted at the 3' end of the intron. The unusual size of the intron and the arrangement of the open and truncated reading frames suggest that this segment of the mtDNA of C. parasitica has arisen by a fusion of components from two or more different introns, possibly involving the re-location of intronic genes.

Amino Acid Sequence↗

PubMed2005

Role of a cytotoxic-T-lymphocyte epitope-defined, alternative gag open reading frame in the pathogenesis of a murine retrovirus-induced immunodeficiency syndrome.

LP-BM5 murine leukemia virus-infected C57BL/6 mice develop profound immunodeficiency and B-cell lymphomas. The LP-BM5 complex contains a mixture of defective (BM5def) and replication-competent helper viruses among which BM5def is the primary causative agent of disease. The BM5def primary open reading frame (ORF1) encodes the single gag precursor protein (Pr60gag). Our lab has recently demonstrated that a novel immunodominant cytotoxic-T-lymphocyte (CTL) epitope (SYNTGRFPPL) is expressed from a +1-nucleotide translational open reading frame of BM5def during the course of normal retrovirus expression. The SYNTGRFPPL CTL epitope may be generated from either of two initiation methionines present, ORF2a or ORF2b, located downstream of the ORF1 initiation site. This study investigates the role(s) of the alternative ORF2-derived gag protein(s) of BM5def in viral pathogenesis. We have examined the disease-inducing capabilities of mutant viruses in which the translational potential of either the initiating ORF2a or ORF2b AUG has been disrupted. Although these mutated viruses are capable of wild-type ORF1 expression, they are unable to induce disease. Our data strongly suggest the existence of a novel ORF2 product(s) that is required for LP-BM5-induced pathogenesis and have potentially broad implications for other retroviral diseases.

Animals↗

PubMed1996

Endogenous D-type (HERV-K) related sequences are packaged into retroviral particles in the placenta and possess open reading frames for reverse transcriptase.

All primates studied to date produce retroviral-like particles in their placentae. We have purified these particles from two primate species, one Old World (human) and one New World (marmoset), and have identified the retroviral sequences which are packaged into these particles. Three families of sequences have been detected in these particles in human, all of which have the highest homology to B- and D-type retroviruses and to the human endogenous retrovirus HERV-K10. Previous studies have reported that the New World monkeys do not possess sequences with homology to HERV-K10. We have identified a new family of low-copy-number sequences which are present in New World monkeys and which possess 70% homology to the HERV-K family. Particles from both species possess reverse transcriptase activity and we have found that some of these retroviral particles package sequences which encode long open reading frames in pol, as revealed by expression cloning in Escherichia coli. These open reading frames could encode the reverse transcriptase enzyme activity found in the particles.

Animals↗

PubMed2006

The 3' untranslated region of tick-borne flaviviruses originated by the duplication of long repeat sequences within the open reading frame.

Comparative alignment of the 3'untranslated regions (3'UTRs) of tick-borne flaviviruses has previously revealed short direct repeat sequences about 25-70 nucleotides long [Gritsun, T.S., Venugopal, K., Zanotto, P.M., Mikhailov, M.V., Sall, A.A., Holmes, E.C., Polkinghorne, I., Frolova, T.V., Pogodina, V.V., Lashkevich, V.A., Gould, E.A., 1997. Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: analysis and significance of the 5' and 3'-UTRs. Virus Res. 49 (1) 27-39; Wallner, G., Mandl, C.W., Kunz, C., Heinz, F.X., 1995. The flavivirus 3'-noncoding region: extensive size heterogeneity independent of evolutionary relationships among strains of tick-borne encephalitis virus. Virology, 213 (1) 169-178]. We now show that these short sequences appear to have originated from longer repeat sequences (LRSs) that are present both in the 3'UTR and the open reading frame of the genome. We propose that the 3'UTR, and possibly the open reading frame, evolved through multiple duplications, deletions and mutations of a primordial sequence element.

3' Untranslated Regions↗

PubMed2006

The 3' untranslated region of tick-borne flaviviruses originated by the duplication of long repeat sequences within the open reading frame.

3' Untranslated Regions↗

PubMed2003

Two alternative translation mechanisms are responsible for the expression of the HCV ARFP/F/core+1 coding open reading frame.

HCV-1 produces a novel protein, known as ARFP, F, or core+1. This protein is encoded by an open reading frame (ORF) that overlaps the core gene in the +1 frame (core+1 ORF). In vitro this protein is produced by a ribosomal frameshift mechanism. However, similar studies failed to detect the ARFP/F/core+1 protein in the HCV-1a (H) isolate. To clarify this issue and to elucidate the functions of this protein, we examined the expression of the core+1 ORF by the HCV-1 and HCV-1a (H) isolates in vivo, in transfected cells. For this purpose, we carried out luciferase (LUC) tagging experiments combined with site-directed mutagenesis studies. Our results showed that the core+1-LUC chimeric protein was efficiently produced in vivo by both isolates. More importantly, neither changes in the specific 10-A residue region of HCV-1 (codons 8-11), the proposed frameshift site for the production of the ARFP/F/core+1 protein in vitro, nor the alteration of the ATG start site of the HCV polyprotein to a stop codon significantly affected the in vivo expression of the core+1 ORF. Furthermore, we showed that efficient translation initiation of the core+1 ORF is mediated by internal initiation codon(s) within the core/core+1-coding sequence, located between nucleotides 583 and 606. Collectively, our data suggest the existence of an alternative translation initiation mechanism that may result in the synthesis of a shorter form of the core+1 protein in transfected cells.

Amino Acid Sequence↗

PubMed1997

Expression profiles of transcripts from 126 open reading frames in the entire chromosome VI of Saccharomyces cerevisiae by systematic northern analyses.

Chromosome VI of Saccharomyces cerevisiae contains 126 open reading frames (ORFs), and the functions of proteins encoded by 80 ORFs are still unknown. In this report, we have systematically examined the expression profiles of all 126 ORFs on chromosome VI under five kinds of growth conditions by quantitative Northern hybridization. A series of Northern analyses and reverse transcription polymerase chain reactions have revealed that more than 64 novel ORFs are transcribed. Two ORFs (YFL059w and YFR011c) are specifically expressed in the presence of galactose. Two ORFs (YFL012w and YFR032c) are specifically transcribed in sporulation. Six ORFs (YFL049w, YFL035c, YFL010c, YFR006w, YFR010w and YFR017c) are abundantly expressed in many growth conditions.

Blotting, Northern↗