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PubMed2001

Open reading frame III of borna disease virus encodes a nonglycosylated matrix protein.

The open reading frame III of Borna disease virus (BDV) codes for a protein with a mass of 16 kDa, named p16 or BDV-M. p16 was described as an N-glycosylated protein in several previous publications and therefore was termed gp18, although the amino acid sequence of p16 does not contain any regular consensus sequence for N glycosylation. We examined glycosylation of p16 and studied its membrane topology using antisera raised against peptides, which comprise the N and the C termini. Neither an N- nor a C-terminal peptide is cleaved from p16 during maturation. Neither deglycosylation of p16 by endoglycosidases nor binding of lectin to p16 was detectable. Introduction of typical N-glycosylation sites at the proposed sites of p16 failed in carbohydrate attachment. Flotation experiments with membranes of BDV-infected cells on density gradients revealed that p16 is not an integral membrane protein, since it can be dissociated from membranes. Our experimental data strongly suggest that p16 is a typical nonglycosylated matrix protein associated at the inner surface of the viral membrane, as is true for homologous proteins of other members of the Mononegavirales order.

Attachment Sites, Microbiological↗
PubMed2000

In vitro translation of the upstream open reading frame in the mammalian mRNA encoding S-adenosylmethionine decarboxylase.

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive element that suppresses translation of the associated downstream cistron in vivo. In this paper, we provide the first direct evidence of peptide synthesis from the S-adenosylmethionine decarboxylase uORF using an in vitro translation system. We examine both the influence of cation concentration on peptide synthesis and the effect of altering the uORF sequence on peptide synthesis. Synthesis of wild type and altered peptides was similar at all concentrations of magnesium tested. In contrast, synthesis of the wild type peptide was more sensitive than that of altered peptides to elevated concentrations of the naturally occurring polyamines, spermidine and spermine, as well as several polyamine analogs. The sensitivity of in vitro synthesis to spermidine was influenced by both the amino acid sequence and the length of the peptide product of the uORF. Findings from the present study correlate with the effects of the uORF and polyamines on translation of a downstream cistron in vivo and support the hypothesis that polyamines and the structure of the nascent peptide create a rate-limiting step in uORF translation, perhaps through a ribosome stalling mechanism.

Adenosylmethionine Decarboxylase↗
PubMed2000

African swine fever virus EP153R open reading frame encodes a glycoprotein involved in the hemadsorption of infected cells.

The open reading frame EP153R, located within the EcoRI E' fragment of the African swine fever (ASF) virus genome, is predicted to encode a membrane protein of 153 amino acids that presents significant homology to the N-terminal region of several CD44 molecules. EP153R contains multiple putative sites for N-glycosylation, phosphorylation, and myristoylation, a central transmembrane region, a C-type animal lectin-like domain, and a cell attachment sequence. Transcription of EP153R takes place at both early and late times during the virus infection. The disruption of the gene, achieved by insertion of the marker gene LacZ within EP153R, did not change either the in vitro virus growth rate or the virus-sensitive/resistant condition of up to 17 established cell lines, but abrogated the hemadsorption phenomenon induced in ASF virus-infected cells. As the sequence and expression of the ASF virus protein pEP402R, a CD2 homolog responsible for the adhesion of erythrocytes to susceptible cells, was unaffected in cultures infected with the EP153R deletion mutant, we conclude that the gene EP153R is needed to induce and/or maintain the interaction between the viral CD2 homolog and its corresponding cell receptor.

African Swine Fever Virus↗
PubMed2004

Myc-ARF (alternate reading frame) interaction inhibits the functions of Myc.

The tumor suppressor protein ARF (alternate reading frame) inhibits MDM2 to stabilize and activate the functions of p53. Here we provide evidence for an additional activity of ARF that attenuates cell cycle progression independently of p53 activation. We show that ARF interacts with c-Myc independently of MDM2 or p53. Consequently, ARF relocalizes c-Myc from the nucleoplasm to the nucleolus. Binding and relocalization by ARF correlate with an inhibition of the c-Myc-activated transcription in both p53-positive and -negative cells. Using inducible cell lines, we show that the wild type ARF, but not a mutant, inhibits expression of the c-Myc-induced genes before inhibiting S phase. Moreover, ARF inhibits Myc-induced progression into S phase in cells lacking p53 or expressing a defective p53, indicating that ARF inhibits the S phase stimulatory function of c-Myc independently of p53. Our results strongly suggest that cMyc is a bona fide target of ARF and that ARF attenuates c-Myc independently of the ARF-p53 axis.

Alternative Splicing↗
PubMed1983

Bunyavirus nucleoprotein, N, and a non-structural protein, NSS, are coded by overlapping reading frames in the S RNA.

It has been shown previously, by sequence analysis of the S RNA segment of snowshoe hare (SSH) bunyavirus, that two overlapping open reading frames in the viral complementary sequence code for proteins with molecular weights of 26.8 X 10(3) and 10.5 X 10(3) respectively. In addition to the viral nucleocapsid (N) protein, which is coded by the S RNA, analyses of parental and reassortant bunyavirus-infected cell extracts have shown that the viral S RNA and M RNA species each code for non-structural proteins (NSS and NSM, respectively). In the present report, in vitro translation analyses of the S mRNA species recovered from virus-infected cells indicate that a single size class of mRNA directs the synthesis of N and NSS. Compositional analyses of selected tryptic peptides of N and NSS have provided proof that N is the product of the first open reading frame, and NSS the product of the second.

Bunyaviridae↗
PubMedDate not supplied

A complex organization of the gene encoding cytochrome oxidase subunit 1 in the mitochondrial genome of the dinoflagellate, Crypthecodinium cohnii: homologous recombination generates two different cox1 open reading frames.

In the course of investigating mitochondrial genome organization in Crypthecodinium cohnii, a non-photosynthetic dinoflagellate, we identified four EcoRI fragments that hybridize to a probe specific for cox1, the gene that encodes subunit 1 of cytochrome oxidase. Cloning and sequence characterization of the four fragments (5.7, 5.1, 4.1, 3.5 kilobase pairs) revealed that cox1 exists in four distinct but related contexts in C. cohnii mtDNA, with a central repeat unit flanked by one of two possible upstream (flanking domain 1 or 2) and downstream (flanking domain 3 or 4) regions. The majority of the cox1 gene is located within the central repeat; however, the C-terminal portion of the open reading frame extends into flanking domains 3 and 4, thereby creating two distinct cox1 coding sequences. The 3'-terminal region of one of the cox1 reading frames can assume an elaborate secondary structure, which potentially could act to stabilize the mature mRNA against nucleolytic degradation. In addition, a high density of small inverted repeats (15-22 base pairs) has been identified at the 5'-end of cox1, further suggesting that hairpin structures could be important for gene regulation. The organization of cox1 in C. cohnii mtDNA appears to reflect homologous recombination events within the central repeat between different cox1 sequence contexts. Such recombining repeats are a characteristic feature of plant (angiosperm) mtDNA, but they have not previously been described in the mitochondrial genomes of protists.

Animals↗
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↗
PubMed1990

Proteins encoded by the bovine papillomavirus E1 open reading frame: expression in heterologous systems and in virally transformed cells.

The E1 open reading frame (ORF) of bovine papillomavirus type 1 is required for the persistence of viral genomes as multicopy plasmid molecules in transformed rodent fibroblasts. E1 has been reported to contain two separate complementation groups (M and R, corresponding to N- and C-terminal domains, respectively) which regulate viral replication. However, E1 behaves as a single gene with respect to cell transformation and viral transcription. We examined the proteins translated from the entire ORF by using three antisera raised against E1 peptide or bacterial fusion proteins. The capacity of the whole ORF to encode a 72-kDa protein was demonstrated by translation of synthetic RNA in a reticulocyte lysate system, by microinjection of RNA into Xenopus oocytes, and by expression in recombinant baculoviruses and vaccinia viruses. In eucaryotic cells, this protein was found to be phosphorylated and targeted to the cell nucleus. In vitro translation also produced shorter peptides, containing only the E1 C-terminal domain, because of internal translation starts on the third and fourth methionine codons within E1 ORF. On the other hand, mammalian cells infected by vaccinia E1 recombinant virus contained additional larger E1 phosphoproteins (transient 85-kDa and stable 88-kDa species), likely representing processed forms of the 72-kDa species. The E1 72-kDa nuclear phosphoprotein was detected in bovine papillomavirus type 1-transformed cells. We report the biochemical characteristics of full-sized and truncated E1 proteins: (i) the C-terminal half of E1 ORF contains a phosphorylation site(s); (ii) the full-sized E1, but not the C-terminal protein, binds DNA, without indication for recognition of defined sequences, and critical determinants for this activity are likely confined to an N-terminal domain of the protein; (iii) covalent affinity labeling experiments performed on vaccinia virus-encoded E1 proteins with an ATP analog confirmed our previous observation of sequence similarities between the E1 C-terminal domain and the ATPase domain of simian virus 40 large T antigen.

Animals↗
PubMed1997

Multiple human endogenous retrovirus (HERV-K) loci with gag open reading frames in the human genome.

Human endogenous retrovirus HERV-K is present in 25-30 copies per haploid human genome. At least one of these loci is capable of producing full-length Gag protein, high amounts of which have been detected in germ cell tumors and derived cell lines. The latter display HERV-K Gag-encoded retroviral particles. Here, we employed the protein truncation test (PTT) in combination with a monochromosomal hybrid mapping panel to identify the human chromosomes harboring HERV-K gag genes with an open reading frame for Gag protein. Eight human chromosomes were found to contain intact HERV-K gag genes. PTT results were corroborated by partial sequencing of subregions from different HERV-K gag genes. The high number of HERV-K Gag open reading frames supports the idea of retroviral sequences retaining a biological benefit in the human genome.

Animals↗
PubMed1991

The 120 kilodalton outer membrane protein (rOmp B) of Rickettsia rickettsii is encoded by an unusually long open reading frame: evidence for protein processing from a large precursor.

A Rickettsia rickettsii outer surface membrane protein (rOmp B), of an apparent molecular mass of 120 kilodaltons, is a major surface antigen of the Rickettsiae that displays genus, species, and sub-species specific antigenic determinants. The 5' portion of this gene was found to be unstable in plasmids, but was stably cloned in a lambda vector. The nucleotide sequence of the 5' terminus has been determined, thus completing the DNA sequence of the entire gene. Genetic analysis revealed an unusually large open reading frame with the capacity to encode a product much larger than the mature protein. A 32 kilodalton peptide from purified rickettsiae was isolated and the amino terminus was sequenced, which revealed that the peptide is encoded by the 3' portion of this large open reading frame. This suggests a role for post-translational processing of rOmp B from a large precursor molecule.

Amino Acid Sequence↗
PubMed1998

Cryptic open reading frames in plasmid vector backbone sequences can provide highly immunogenic cytotoxic T-lymphocyte epitopes.

Murine tumor cells obtained through transfection of expression plasmids carrying activated cellular and/or viral oncogenes constitute formidable tools for immunological tumor research. As reported previously, mouse embryo cells of C57BL/6 origin, transformed by mutated p53 or human papilloma virus type 16 (HPV16), present, at their surface, MHC-bound peptides that are derived from the p53 and the HPV16 E7 oncoproteins, respectively, which can serve as a target for a highly effective antitumor T-cell response. Here, we describe the identification, through molecular cloning, of an additional, highly immunodominant peptide that is presented by the aforementioned HPV16- and p53-transformed cells. This peptide is encoded by a cryptic open reading frame in the backbone sequences of the plasmids that had been used to generate these cells. Considerable amounts of transcripts encompassing this open reading frame were detected in the cells concerned. These transcripts were the result of the bidirectional nature of the retroviral long terminal repeat (LTR) present in the expression plasmids used for transfection, which resulted in transcription of the gene of interest, as well as in transcription of the vector sequences positioned at the other side of the LTR. Due to this mechanism, all tumor cells harboring LTR-driven expression plasmids expressed the highly immunogenic peptide, whereas cells containing plasmids driven by more unidirectional promoters exhibited lower levels of this peptide. LTR-driven expression plasmids were also shown to encode this peptide epitope when used for DNA vaccination, as mice vaccinated with such a plasmid developed a CTL response against this peptide. Our data show that awareness of plasmid backbone-derived epitopes is of crucial importance for the correct interpretation of preclinical experiments and for the design of DNA vaccines.

Amino Acid Sequence↗
PubMed1992

Biased reading frames of pre-existing DH--JH coding joints and preferential nucleotide insertions at VH--DJH signal joints of excision products of immunoglobulin heavy chain gene rearrangements.

During B cell differentiation immunoglobulin (Ig) DH segments join to JH segments, followed by joining of VH to DJH. Although circular excision products of DH--JH rearrangements have been characterized, excision products of VH to DJH joining have never been isolated. We selectively denatured chromosomal DNA of mouse splenocytes and enriched circular DNA spanning the long distance between VH and DH. Subsequent PCR amplifications allowed the identification of signal joints of VH to DJH. Sequence analysis indicated that preexisting DH--JH coding joints of excision products showed a strong bias for reading frame 1, and the absence of reading frame 2, which would allow the expression of a truncated mu chain called D mu protein. When comparing the joints of the VH--DJH and DH--JH rearrangements we observed N-nucleotide insertions to be abundant at the VH--DH signal joint, but very sparse at the DH--JH signal joint, while the coding joints of both contained abundant N-insertions. These differences in N region insertions at the signal joints suggest a differential control of the D--J and V--DJ rearrangements.

Animals↗
PubMed1993

Intervening sequence with conserved open reading frame in eubacterial 23S rRNA genes.

An intervening sequence (IVS) occurred in the 23S rRNA genes (rrl) of some, but not all, strains of four species of the spirochete genus Leptospira and was absent from strains in three other species. The IVS varied in size from 485 to 759 base pairs and replaced bases 1224-1245 in both copies of rrl. The two ends of each IVS shared 22-35 bases of complementarity that could form a stable double helix. The presence of an IVS correlated with a cleaved mature 23S rRNA that probably results from removal of the IVS without religation. The 3' site of cleavage was mapped within the inverted repeat of the IVS. An open reading frame of 121-133 amino acids was conserved in the IVS in all four species, oriented so that the sense strand was in the rRNA transcript. When the open reading frames were compared between species, they predicted polypeptides that showed between 51% and 78% amino acid conservation and similar DNA sequence conservation, indicating selection for protein function.

Amino Acid Sequence↗
PubMed1991

Identification, cloning, and sequencing of a fragment of Amsacta moorei entomopoxvirus DNA containing the spheroidin gene and three vaccinia virus-related open reading frames.

Entomopoxvirus virions are frequently contained within crystalline occlusion bodies, which are composed of primarily a single protein, spheroidin, which is analogous to the polyhedrin protein of baculovirus. The spheroidin gene of Amsacta moorei entomopoxvirus was identified following the microsequencing of polypeptides generated from cyanogen bromide treatment of spheroidin and the subsequent synthesis of oligonucleotide hybridization probes. DNA sequencing of a 6.8-kb region of DNA containing the spheroidin gene showed that the spheroidin protein is derived from a 3.0-kb open reading frame potentially encoding a protein of 115 kDa. Three copies of the heptanucleotide, TTTTTNT, a sequence associated with early gene transcription in the vertebrate poxviruses, and four in-frame translational termination signals were found within 60 bp upstream of the putative spheroidin gene promoter (TAAATG). The spheroidin gene promoter region contains the sequence TAAATG, which is found in many late promoters of the vertebrate poxviruses and which serves as the site of transcriptional initiation, as shown by primer extension. Primer extension experiments also showed that spheroidin gene transcripts contain 5' poly(A) sequences typical of vertebrate poxvirus late transcripts. The 92 bases upstream of the initiating TAAATG are unusually A + T rich and contain only 7 G or C residues. An analysis of open reading frames around the spheroidin gene suggests that the colinear core of "essential genes" typical of the vertebrate poxviruses is absent in A. moorei entomopoxvirus.

Amino Acid Sequence↗
PubMed2001

T-cell receptor gamma chain alternate reading frame protein (TARP) expression in prostate cancer cells leads to an increased growth rate and induction of caveolins and amphiregulin.

Previously, we showed that prostate and prostate cancer cells express a truncated T-cell receptor gamma chain mRNA that uses an alternative reading frame to produce a novel nuclear T-cell receptor gamma chain alternate reading frame protein (TARP). TARP is expressed in the androgen-sensitive LNCaP prostate cancer cell line but not in the androgen-independent PC3 prostate cancer cell line, indicating that TARP may play a role in prostate cancer progression. To elucidate the function of TARP, we generated a stable PC3 cell line that expresses TARP in a constitutive manner. Expression of TARP in PC3 cells resulted in a more rapid growth rate with a 5-h decrease in doubling time. cDNA microarray analysis of 6538 genes revealed that caveolin 1, caveolin 2, amphiregulin, and melanoma growth stimulatory activity alpha were significantly up-regulated, whereas IL-1beta was significantly down-regulated in PC3 cells expressing TARP. We also demonstrated that TARP expression is up-regulated by testosterone in LNCaP cells that express a functional androgen receptor. These results suggest that TARP has a role in regulating growth and gene expression in prostate cancer cells.

Amphiregulin↗
PubMed2006

Analysis of genetic mutations in the 7a7b open reading frame of coronavirus of cheetahs (Acinonyx jubatus).

OBJECTIVE: To analyze the 7a7b genes of the feline coronavirus (FCoV) of cheetahs, which are believed to play a role in virulence of this virus. SAMPLE POPULATION: Biologic samples collected during a 4-year period from 5 cheetahs at the same institution and at 1 time point from 4 cheetahs at different institutions. PROCEDURES: Samples were first screened for FCoV via a reverse transcription-PCR procedure involving primers that encompassed the 3'-untranslated region. Samples that yielded positive assay results were analyzed by use of primers that targeted the 7a7b open reading frames. The nucleotide sequences of the 7a7b amplification products were determined and analyzed. RESULTS: In most isolates, substantial deletional mutations in the 7a gene were detected that would result in aberrant or no expression of the 7a product because of altered reading frames. Although the 7b gene was also found to contain mutations, these were primarily point mutations resulting in minor amino acid changes. The coronavirus associated with 1 cheetah with feline infectious peritonitis had intact 7a and 7b genes. CONCLUSIONS AND CLINICAL RELEVANCE: The data suggest that mutations arise readily in the 7a region and may remain stable in FCoV of cheetahs. In contrast, an intact 7b gene may be necessary for in vivo virus infection and replication. Persistent infection with FCoV in a cheetah population results in continued virus circulation and may lead to a quasispecies of virus variants.

Acinonyx↗
PubMed2001

Mutations in the open reading frame of the beta-site APP cleaving enzyme (BACE) locus are not a common cause of Alzheimer's disease.

Amyloid beta-peptide (Abeta) plays a central role in the pathogenesis of Alzheimer's disease (AD). The gene encoding the beta-site APP cleaving enzyme (BACE), one of two enzymes that sequentially cleave the beta-amyloid precursor protein to generate Abeta, has recently been cloned. We tested the hypothesis that BACE might be genetically associated with AD by linkage analysis (56 pedigrees), by direct nucleotide sequencing of the entire open reading frame (20 subjects with familial AD, and 10 subjects with sporadic AD) and by allelic association analysis (155 AD cases and 173 non-demented controls). Our results revealed no evidence for either genetic linkage or allelic association between BACE and AD, and no coding sequence mutations were detected in the open reading frame of the BACE gene. These data suggest that while BACE protein plays an important role in the pathogenesis of AD, and may be a robust therapeutic target, it is unlikely to be a major AD susceptibility locus.

Aged↗
PubMed1990

Cross talk to the phosphate regulon of Escherichia coli by PhoM protein: PhoM is a histidine protein kinase and catalyzes phosphorylation of PhoB and PhoM-open reading frame 2.

Transcription of the genes in the phosphate regulon in Escherichia coli is activated by PhoB protein, which is phosphorylated by PhoR protein under phosphate-limiting conditions. In the absence of the phoR function, the genes in the phosphate regulon are expressed constitutively and the expression is dependent on the function of phoM and phoB. We constructed a plasmid with a lacZ'-'phoM fusion gene, which encoded a hybrid protein (PhoM1206) in which the hydrophobic amino-terminal half of the native PhoM was replaced by beta-galactosidase. The phoM1206 gene could complement the phoM mutation in vivo. We purified PhoM1206 from the overproducing strain carrying the plasmid; it was autophosphorylated at a histidine residue in the presence of ATP, and the phospho-PhoM1206 phosphorylated PhoB. PhoM1206 could also transphosphorylate the product of phoM-orf2, which is structurally homologous to phoB and located immediately upstream of phoM. Although PhoR1084 that lacked the hydrophobic amino-terminal region of the native PhoR protein transphosphorylated PhoB, it could not phosphorylate PhoM-open reading frame 2. Therefore, cross talk by protein phosphorylation appears to occur from PhoM to PhoB but not from PhoR to PhoM-open reading frame 2.

Adenosine Triphosphate↗