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At least 811 records · Page 45Linked to original sources
PubMed1996

Fifteen open reading frames in a 30.8 kb region of the right arm of chromosome VI from Saccharomyces cerevisiae.

The nucleotide sequence of cosmid clone 9765, which contains 30.8 kb of the right arm of chromosome VI, was determined. Both strands were sequenced, with an average redundancy of 8.17 per base pair by both dye primer and dye terminator cycle sequencing methods. The G+C content of the sequence was found to be 40.3%. Fifteen open reading frames (ORFs) greater than 100 amino acids and one tRNA-Tyr gene (SUP6) were detected. Seven of the ORFs were found to encode previously identified genes (HIS2, CDC14, MET10, SMC2, QCR6, PH04 and CDC26). One ORF, 9765orfF010, was found to encode a new member of the Snf2/Rad54 helicase family. Three ORFs (9765orfR002, 9765orfR011 and 9765orfR013) were found to be homologous with Schizosaccharomyces pombe polyadenylate binding protein, Escherichia coli hypothetical 38.1-kDa protein in the BCR 5' region, and transcription regulatory protein Swi3, respectively.

Adenosine Triphosphatases↗
PubMed1994

Light modulation of ferredoxin mRNA abundance requires an open reading frame.

Ferredoxin I (Fed-1) mRNA abundance is modulated by an internal light regulatory element that includes sequences both 5' and 3' of the translational initiation site. To test the hypothesis that the light response mediated by this element might be coupled to translation, we transformed tobacco plants with gene constructs blocked in translational initiation or elongation. Here, we report that such mutations abolish the light response in vivo. A nonsense mutation could be rescued by restoring the open reading frame with a different sequence, even when the new codon caused an amino acid substitution. Our data establish that the light response requires a translatable reading frame and thus provide strong circumstantial evidence for post-transcriptional modulation of Fed-1 mRNA levels. The Fed-1 system is presently the only higher plant example of a developmentally regulated change in mRNA abundance that requires translation of the affected mRNA.

Ferredoxins↗
PubMed1997

Sequence analysis of a 33.2 kb segment from the left arm of yeast chromosome XV reveals eight known genes and ten new open reading frames including homologues of ABC transporters, inositol phosphatases and human expressed sequence tags.

The complete nucleotide sequence of a 33221 bp segment, contained in cosmid pEOA1044, derived from the left arm of chromosome XV of Saccharomyces cerevisiae, appears in public databases between coordinates 177013 and 210234 (http://speedy.mips.biochem.mpg.de/). Computer analysis of that sequence revealed the presence of the previously known genes IRA2, DEC1, NUF2, HST1, RTG1, RIB2 and HAL2, one previously partially sequenced open reading frame (ORF) of unknown function (SCORFAC) and ten newly identified ORFs. One of the new ORFs is similar to the Drosophila melanogaster white gene and other transmembrane ABC transporters, another one has similarities to inositol phosphatases and others are similar to ORFs of unknown function from various organisms, including human Expressed Sequence Tags (ESTs). Potential transmembrane regions, ATP/GTP-binding and WD motifs have also been identified. The existence of yeast ESTs for two of the newly identified ORFs indicates that they are transcribed.

ATP-Binding Cassette Transporters↗
PubMed2003

CD4+ Th2 cell recognition of HLA-DR-restricted epitopes derived from CAMEL: a tumor antigen translated in an alternative open reading frame.

Tumor Ag NY-ESO-1 is an attractive target for immunotherapy of cancer, since both CD8(+) CTL and CD4(+) Th cells against NY-ESO-1 have been described. Moreover, NY-ESO-1 as well as the highly homologous tumor Ag LAGE-1 are broadly expressed in various tumor types. Interestingly, the NY-ESO-1 and LAGE-1 genes also encode for proteins translated in an alternative open reading frame. These alternatively translated NY-ESO-ORF2 and CAMEL proteins, derived from the NY-ESO-1 and LAGE-1 genes, respectively, have been demonstrated to be immunogenic, since CTL specific for these proteins have been isolated from melanoma patients. In this study a panel of advanced melanoma patients was screened for the presence of Th cells specific for the alternatively translated tumor Ags NY-ESO-ORF2 and CAMEL. PBMC of melanoma patients were stimulated for 4 days with mixes of overlapping peptides covering the entire NY-ESO-ORF2 and CAMEL protein sequences and were tested for the release of type 1 (IFN-gamma) and type 2 (IL-13) cytokines in ELISPOT assays. In three of 15 patients, T cells specific for two CAMEL peptides (CAMEL(71-92) and CAMEL(81-102)) could be detected. From one of these patients, CD4(+) T cell clones specific for CAMEL(81-102) could be generated. These clones recognized a naturally processed epitope presented in both HLA-DR11 and HLA-DR12 and produced high levels of IL-4, IL-5, and IL-13. In conclusion, this study shows the presence of Th cells specific for the alternatively translated tumor Ag CAMEL in melanoma patients and is the first report that describes the isolation of tumor Ag-specific CD4(+) Th 2 clones.

Alternative Splicing↗
PubMed2004

Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs.

To find novel short coding sequences from accumulated full-length cDNA sequences, proteomic analysis of small proteins expressed in human leukemia K562 cells was performed using high-resolution nanoflow liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Our analysis led to the identification of 54 proteins not more than 100 amino acids in length, including four novel ones. These novel short coding sequences were all located upstream of the longest open reading frame (ORF) of the corresponding cDNA. Our findings indicate that the translation of short ORFs occurs in vivo whether or not there exists a longer coding region in the downstream of the mRNA. This investigation provides the first direct evidence of translation of upstream ORFs in human cells, which could greatly change the current outline of the human proteome.

Amino Acid Sequence↗
PubMed1994

The DNA polymerase genes of several HMU-bacteriophages have similar group I introns with highly divergent open reading frames.

A previous report described the discovery of a group I, self-splicing intron in the DNA polymerase gene of the Bacillus subtilis bacteriophage SPO1 (1). In this study, the DNA polymerase genes of three close relatives of SPO1: SP82, 2C and phi e, were also found to be interrupted by an intron. All of these introns have group I secondary structures that are extremely similar to one another in primary sequence. Each is interrupted by an open reading frame (ORF) that, unlike the intron core or exon sequences, are highly diverged. Unlike the relatives of Escherichia coli bacteriophage T4, most of which do not have introns (2), this intron seems to be common among the relatives of SPO1.

Amino Acid Sequence↗
PubMed2003

Sixty alleles of the ALS7 open reading frame in Candida albicans: ALS7 is a hypermutable contingency locus.

The ALS (agglutinin-like sequence) gene family encodes proteins that play a role in adherence of the yeast Candida albicans to endothelial and epithelial cells. The proteins are proposed as virulence factors for this important fungal pathogen of humans. We analyzed 66 C. albicans strains, representing a worldwide collection of 266 infection-causing isolates, and discovered 60 alleles of the ALS7 open reading frame (ORF). Differences between alleles were largely caused by rearrangements of repeat elements in the so-called tandem repeat domain (21 different types occurred) and the VASES region (19 different types). C. albicans is diploid, and combinations of ALS7 alleles generated 49 different genotypes. ALS7 expression was detected in samples isolated directly from five oral candidosis patients. ORFs in the opposite direction contained within the ALS7 ORF were also transcribed in all strains tested. Isolates representing a more pathogenic general-purpose genotype (GPG) cluster of strains tended to have more tandem repeats than other strains. Two types of VASES regions were largely exclusive to GPG strains; the remaining types were largely exclusive to noncluster strains. Our results provide evidence that ALS7 is a hypermutable contingency locus and important for the success of C. albicans as an opportunistic pathogen of humans.

Alleles↗
PubMed1996

Isoforms of selenoprotein P in rat plasma. Evidence for a full-length form and another form that terminates at the second UGA in the open reading frame.

Several forms of selenoprotein P that share the same N-terminal sequence have been identified in rat plasma, but only one selenoprotein P mRNA has been characterized. The open reading frame of the mRNA contains 10 UGAs that presumably code for selenocysteine residues. Using heparin-Sepharose, we isolated two of the protein forms from immunoaffinity-purified selenoprotein P. One of the forms, Se-P45B, migrates at 45 kDa on SDS-polyacrylamide gel electrophoresis, and the other, Se-P57B, migrates at 57 kDa. These two forms were cleaved with cyanogen bromide, and both yielded 40-kDa fragments that were consistent with those fragments being an inter-methionine peptide near the N terminus of the predicted polypeptide. A 20-kDa fragment present in the cleavage products of Se-P57B was absent from the products of Se-P45B. This result suggested that Se-P45B lacks the C-terminal region of the predicted polypeptide. Carboxypeptidase P digestion of Se-P45B indicated that its C-terminal amino acid is Ser244, the amino acid immediately upstream from the predicted second selenocysteine. C-terminal analysis of Se-P57B indicated that its final residue is Asn366, the last amino acid predicted by the cDNA sequence. Amino acid composition analyses of the two forms were consistent with both arising from the same mRNA. Immunoaffinity-purified selenoprotein P was digested with proteases, and the resulting peptides were separated and sequenced. Only amino acid sequences predicted by the cDNA were found, and 80% of the predicted amino acid sequence was confirmed. These results are compatible with Se-P45B arising from termination of translation at the second in-frame UGA codon and all of the 10 in-frame UGA codons being read through to produce Se-P57B. These findings demonstrate that selenoprotein P isoforms of differing peptide lengths are present in plasma. They raise the possibility that the second UGA codon in selenoprotein P mRNA can have alternative functions: coding for the incorporation of selenocysteine or coding for termination of translation.

Amino Acid Sequence↗
PubMed2000

An accessory open reading frame (orf-x) of jaagsiekte sheep retrovirus is conserved between different virus isolates.

Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of a contagious lung tumour of sheep known as sheep pulmonary adenomatosis (syn: ovine pulmonary carcinoma, jaagsiekte). JSRV exhibits a simple genetic organization, characteristic of the type D and type B retroviruses, with the canonical retroviral sequences gag, pro, pol and env encoding the structural proteins of the virion. An additional open reading frame (orf-x), of approximately 500 bp overlapping pol, is present in the only two complete sequences of JSRV published to date. Since very little information is available on the biology of JSRV it is important to establish if orf-x is conserved between different virus isolates. In this study we analysed the orf-x region of JSRV isolates collected from the United Kingdom, Italy, Spain and South Africa. In addition we also analysed the presence of orf-x in JSRV-related endogenous sequences (enJSRVs) present in the sheep genome. Orf-x was highly conserved in all the exogenous isolates (n=10) and in most of the endogenous sequences (n=8). Thus orf-x may be an accessory gene of JSRV and haves a biological function which might be advantageous to JSRV. Phenetic analysis conducted on the complete orf-x nucleotide sequences seems to highlight the presence of three distinct groups statistically well supported by bootstrapping: i) exogenous JSRV sequence from the UK; ii) exogenous JSRV sequences from Southern Europe and iii) the exogenous South African strain plus all the endogenous sequences analyzed and collected from Australia, Italy, UK and South Africa.

Animals↗
PubMed1996

Characterization of the tol-pal region of Escherichia coli K-12: translational control of tolR expression by TolQ and identification of a new open reading frame downstream of pal encoding a periplasmic protein.

The TolQ, TolR, TolA, TolB, and Pal proteins appear to function in maintaining the integrity of the outer membrane, as well as facilitating the uptake of the group A colicins and the DNA of the infecting filamentous bacteriophages. Sequence data showed that these genes are clustered in a 6-kb segment of DNA with the gene order orf1 tolQ tolR tolA tolB pal orf2 (a newly identified open reading frame encoding a 29-kD9 protein). Like those containing orf1, bacteria containing an insertion mutation in this gene showed no obvious phenotype. Analysis of beta-galactosidase activity from fusion constructs in which the lac operon was fused to various genes in the cluster showed that the genes in this region constitute two separate operons: orf1 tolQRA and tolB pal orf2. In the orf1 tolQRA operon, translation of MR was dependent on translation of the upstream tolQ region. Consistent with this result, no functional ribosome-binding site for TolR synthesis was detected.

Amino Acid Sequence↗
PubMed2006

Human T-cell leukemia virus open reading frame II encodes a posttranscriptional repressor that is recruited at the level of transcription.

Human T-cell leukemia virus (HTLV) infection is a chronic, lifelong infection that is associated with the development of leukemia and neurological disease after a long latency period, and the mechanism by which the virus is able to evade host immune surveillance is elusive. Besides the structural and enzymatic proteins, HTLV encodes regulatory (Tax and Rex) and accessory (open reading frame I [ORF I] and ORF II) proteins. Tax activates viral and cellular transcription and promotes T-cell growth and malignant transformation. Rex acts posttranscriptionally to facilitate cytoplasmic expression of incompletely spliced viral mRNAs. Recently, we reported that the accessory gene products of HTLV-1 and HTLV-2 ORF II (p30II and p28II, respectively) are able to restrict viral replication. These proteins act as negative regulators of both Tax and Rex by binding to and retaining their mRNA in the nucleus, leading to reduced protein expression and virion production. Here, we show that p28II is recruited to the viral promoter in a Tax-dependent manner. After recruitment to the promoter, p28II or p30II then travels with the transcription elongation machinery until its target mRNA is synthesized. Experiments artificially directing these proteins to the promoter indicate that p28II, unlike HTLV-1 p30II, displays no transcriptional activity. Furthermore, the tethering of p28II directly to tax/rex mRNA resulted in repression of Tax function, which could be attributed to the ability of p28II to block TAP/p15-mediated enhancement of Tax expression. p28II-mediated reduction of viral replication in infected cells may permit survival of the cells by allowing escape from immune recognition, which is consistent with the critical role of HTLV accessory proteins in viral persistence in vivo.

Cell Line↗
PubMed2003

Mutational analysis of open reading frames 62 and 71, encoding the varicella-zoster virus immediate-early transactivating protein, IE62, and effects on replication in vitro and in skin xenografts in the SCID-hu mouse in vivo.

The varicella-zoster virus (VZV) genome has unique long (U(L)) and unique short (U(S)) segments which are flanked by internal repeat (IR) and terminal repeat (TR) sequences. The immediate-early 62 (IE62) protein, encoded by open reading frame 62 (ORF62) and ORF71 in these repeats, is the major VZV transactivating protein. Mutational analyses were done with VZV cosmids generated from parent Oka (pOka), a low-passage clinical isolate, and repair experiments were done with ORF62 from pOka and vaccine Oka (vOka), which is derived from pOka. Transfections using VZV cosmids from which ORF62, ORF71, or the ORF62/71 gene pair was deleted showed that VZV replication required at least one copy of ORF62. The insertion of ORF62 from pOka or vOka into a nonnative site in U(S) allowed VZV replication in cell culture in vitro, although the plaque size and yields of infectious virus were decreased. Targeted mutations in binding sites reported to affect interaction with IE4 protein and a putative ORF9 protein binding site were not lethal. Single deletions of ORF62 or ORF71 from cosmids permitted recovery of infectious virus, but recombination events repaired the defective repeat region in some progeny viruses, as verified by PCR and Southern hybridization. VZV infectivity in skin xenografts in the SCID-hu model required ORF62 expression; mixtures of single-copy recombinant Oka Delta 62 (rOka Delta 62) or rOka Delta 71 and repaired rOka generated by recombination of the single-copy deletion mutants were detected in some skin implants. Although insertion of ORF62 into the nonnative site permitted replication in cell culture, ORF62 expression from its native site was necessary for cell-cell spread in differentiated human skin tissues in vivo.

Animals↗
PubMed2001

Phosphorylation of the protein encoded by the first open reading frame of the MDG4 transposable element (gypsy) by homologous and heterologous casein kinases type 2.

Homogeneous casein kinase type 2 (CK2) was obtained from oocytes of Rana temporaria and cells of Drosophila melanogaster by chromatography on heparin-Sepharose, phosphocellulose, and Mono Q columns using a Pharmacia FPLC system. The procedure was first successfully used for the purification of CK2 from the Drosophila melanogaster cell culture. It has been shown that the protein encoded by the first open reading frame (ORF) of the gypsy transposable element (MDG4) is an effective protein substrate both for homologous and heterologous CK2 from the oocytes of Rana temporaria in vitro. Both enzymes catalyze the incorporation of two moles of phosphate per mole of protein. The Km and Vmax values for the reaction catalyzed by CK2 from the Drosophila cell culture were 32.5 +/- 2.1 nM and 70.97 +/- 1.89 nmol/min per microg, respectively, and for CK2 from oocytes, these values were 37.6 +/- 2.8 nM and 66.02 +/- 2.15 nmol/min per microg, respectively.

Animals↗
PubMed1999

Immortalization of human prostate epithelial cells by HPV 16 E6/E7 open reading frames.

BACKGROUND: The exact pathogenesis for prostate cancer is not known. Progress made in prostate cancer research has been slow, largely due to the lack of suitable in vitro models. Here, we report our work on the immortalization of a human prostate epithelial cell line and show that it can be used as a model to study prostate tumorigenesis. METHODS: Replication-defective retrovirus harboring the human papillomavirus (HPV) type 16 E6 and E7 open reading frames was used to infect primary human prostate epithelial cells. Polymerase chain reaction, followed by Southern hybridization for the HPV 16 E6/E7, Western blot for prostatic acid phosphatase, telomeric repeat amplification protocol assay for telomerase activity, two-dimensional gels for cytokeratins, and cytogenetic analysis were undertaken to characterized the infected cells. RESULTS: The retrovirus-infected cell line, HPr-1, continued to grow in culture for more than 80 successive passages. Normal primary cells failed to proliferate after passage 6. HPr-1 cells bore close resemblance to normal primary prostate epithelial cells, both morphologically and biochemically. However, they possessed telomerase activity and proliferated indefinitely. Cytogenetic analysis of HPr-1 cells revealed a human male karyotype with clonal abnormalities and the appearance of multiple double minutes. CONCLUSIONS: The HPr-1 cells expressed prostatic acid phosphatase and cytokeratins K8 and K18, proving that they were prostate epithelial cells. They were benign in nude mice tumor formation and soft agar colony formation assay. The HPr-1 cell line is an in vitro representation of early prostate neoplastic progression.

Acid Phosphatase↗
PubMed1998

Human T-cell leukemia virus type 1 pX-I and pX-II open reading frames are dispensable for the immortalization of primary lymphocytes.

Human T-cell leukemia virus type 1 (HTLV-1) infects and transforms CD4+ T-lymphocytes both in vivo and in vitro. Although the Tax protein of HTLV-1 has been strongly implicated as a transforming agent, other virally encoded proteins may also play a role in the transformation process. In addition to the rex and tax genes, the pX region of the HTLV-1 genome contains two open reading frames (pX-I and pX-II) which encode the putative viral accessory proteins known as p12I, p30II, and p13II. Mutations in the ACH molecular clone of HTLV-1 that are predicted to abrogate the expression of p12I, p13II and p30II were constructed. These mutations had no effect on viral replication or the immortalization of primary lymphocytes. Although these proteins are dispensable for viral replication and immortalization in vitro, it remains possible that they alter infection in vivo.

Cells, Cultured↗
PubMed2002

RNA 2 of Citrus psorosis virus is of negative polarity and has a single open reading frame in its complementary strand.

Citrus psorosis virus (CPsV) causes a citrus disease occurring worldwide. Isolate CPV 4 has a genome with three single-stranded RNAs. The complete sequence of RNA 2 (1643 nucleotides) is reported here. Northern blot hybridization with strand-specific probes showed that most of the encapsidated RNA 2 is of negative polarity, although a small amount of the complementary strand may also be present in particles. The RNA 2 complementary strand contained a single open reading frame encoding a protein of 476 amino acids, which includes a motif resembling a nuclear localization signal. The sequence of this putative protein shows no significant similarity to any other in the databases. In the 3'-terminal untranslated region there is a putative polyadenylation signal. No subgenomic RNAs derived from RNA 2 were detected.

Amino Acid Sequence↗
PubMed2001

1-Deoxy-D-xylulose 5-phosphate synthase, the gene product of open reading frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus.

In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and D-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-D-xylulose 5-phosphate synthase. In Rhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-D-xylulose 5-phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-D-xylulose 5-phosphate synthase A and B, containing a His(6) tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-D-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-D-xylulose 5-phosphate synthase A and B: K(m)(pyruvate) = 0.61 and 3.0 mM, K(m)(D-glyceraldehyde 3-phosphate) = 150 and 120 microM, and V(max) = 1.9 and 1.4 micromol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-D-xylulose 5-phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11.

Amino Acid Sequence↗
PubMed2026

PKD1 upstream open reading frames affect Polycystin-1 expression and polycystic kidney disease phenotypes.

Autosomal dominant polycystic kidney disease (ADPKD) accounts for 5%-10% of prevalent end-stage kidney failure (ESKD). ADPKD cysts result from a loss of sufficient functional expression of PKD1/Polycystin-1 (PC1) in approximately 80% of families. Kidney disease severity correlates with the extent to which PC1 dosage is reduced below a critical level, and evidence suggests therapeutic benefit from increasing PC1 expression in these conditions. Upstream open reading frame (uORF) translation can reduce translation of a protein's coding sequence. Ribosome profiling data and bioinformatic predictions suggested the presence of conserved PKD1 uORFs, so we sought to explore their biological role. We generated luciferase reporters and two humanized PKD1 5' UTR mouse models with or without single nucleotide edits removing uORF start codons (ΔuORF) to define active uORFs and test their impact on PC1 translation. PKD1 uORF start codons can robustly initiate translation, and ΔuORF conveys a 2-4 fold increase in PC1 protein expression and resultant prevention of kidney cysts in Dnajb11 as well as in Pkd1 missense models. PKD1 uORF1-blocking steric antisense oligonucleotides (ASOs) substantially increase PC1 expression in vitro. PKD1 uORFs play an important role in the low basal expression of WT PKD1, and their inhibition represents an opportunity to therapeutically increase PC1 translation in polycystic kidney and liver disease resulting from reduced dosage of PC1.

Animals↗