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PubMed2003

Automated identification of putative methyltransferases from genomic open reading frames.

We have analyzed existing methodologies and created novel methodologies for the automatic assignment of S-adenosylmethionine (AdoMet)-dependent methyltransferase functionality to genomic open reading frames based on predicted protein sequences. A large class of the AdoMet-dependent methyltransferases shares a common binding motif for the AdoMet cofactor in the form of a seven-strand twisted beta-sheet; this structural similarity is mirrored in a degenerate sequence similarity that we refer to as methyltransferase signature motifs. These motifs are the basis of our assignments. We find that simple pattern matching based on the motif sequence is of limited utility and that a new method of "sensitized matrices for scoring methyltransferases" (SM2) produced with modified versions of the MEME and MAST tools gives greatly improved results for the Saccharomyces cerevisiae yeast genome. From our analysis, we conclude that this class of methyltransferases makes up approximately 0.6-1.6% of the genes in the yeast, human, mouse, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Escherichia coli genomes. We provide lists of unidentified genes that we consider to have a high probability of being methyltransferases for future biochemical analyses.

Amino Acid Motifs↗

PubMed1991

Characterization of variable regions in the envelope and S3 open reading frame of equine infectious anemia virus.

The polymerase chain reaction was used to amplify and clone parts of the envelope gene and overlapping S3 open reading frame, thought to encode rev, of the virulent in vivo-derived Th-1 isolate of equine infectious anemia virus (EIAV). The results indicated that EIAV consists of a heterogeneous mixture of genotypes present at the first febrile cycle after initial infection. We showed that the Th-1 isolate apparently contains nondefective genotypes as well as types which have transmembrane protein truncations or are rev deficient. Furthermore, we could confirm the presence of a hypervariable region in the gp90 envelope glycoprotein. Taken together with earlier data on the heterogeneity of the regulatory motifs present in the long terminal repeat sequences of viruses from the same in vivo isolate (S. Carpenter, S. Alexandersen, M. J. Long, S. Perryman, and B. Chesebro, J. Virol. 65:1605-1610, 1991), our findings indicate that EIAV uses a complex system of diversity in biological phenotypes together with variation in regulatory and antigenic makeup to evade host response and to cause persistent infection and recurrent chronic disease.

Amino Acid Sequence↗

PubMed1999

Proliferation response to interleukin-2 and Jak/Stat activation of T cells immortalized by human T-cell lymphotropic virus type 1 is independent of open reading frame I expression.

Human T-cell lymphotropic virus type 1 (HTLV-1), a complex retrovirus, encodes a hydrophobic 12-kD protein from pX open reading frame (ORF) I that localizes to cellular endomembranes and contains four minimal SH3 binding motifs (PXXP). We have demonstrated the importance of ORF I expression in the establishment of infection and hypothesize that p12(I) has a role in T-cell activation. In this study, we tested interleukin-2 (IL-2) receptor expression, IL-2-mediated proliferation, and Jak/Stat activation in T-cell lines immortalized with either wild-type or ORF I mutant clones of HTLV-1. All cell lines exhibited typical patterns of T-cell markers and maintained mutation fidelity. No significant differences between cell lines were observed in IL-2 receptor chain (alpha, beta, or gamma(c)) expression, in IL-2-mediated proliferation, or in IL-2-induced phosphorylated forms of Stat3, Stat5, Jak1, or Jak3. The expression of ORF I is more likely to play a role in early HTLV-1 infection, such as in the activation of quiescent T cells in vivo.

Cell Line, Transformed↗

PubMed1996

Adenovirus E4 open reading frame 4 protein autoregulates E4 transcription by inhibiting E1A transactivation of the E4 promoter.

Here we show that the adenovirus early region 4 (E4) open reading frame 4 (ORF4) protein autoregulates its own transcription by inhibiting adenovirus E1A-induced activation of E4 transcription both in transient transfection experiments and during lytic virus growth. The inhibitory activity of E4-ORF4 was selective for E1A-CR3-dependent transactivation and had no effect on CR1 transactivation. The inhibitory activity of E4-ORF4 was relieved by okadaic acid treatment, which inhibits the cellular protein phosphatase 2A (PP2A), suggesting that E4-ORF4 controls the phosphorylated status of transcription factors important for E4 promoter activity. This conclusion agrees with previous demonstrations that E4-ORF4 associates with PP2A and causes a partial dephosphorylation of certain transcription factors, including E1A (U. Müller, T. Kleinberger, and T. Shenk, J. Virol. 66:5869-5878, 1992; T. Kleinberger and T. Shenk, J. Virol. 67:7556-7560, 1993). However, our results indicate that dephosphorylation of E1A itself might not be the primary target for E4-ORF4. Instead, the E4-ORF4-PP2A complex appears to work by dephosphorylation of multiple cellular transcription factors that are involved in E1A transactivation of the E4 promoter.

Adenoviridae↗

PubMed1999

The open reading frame 57 gene product of herpesvirus saimiri shuttles between the nucleus and cytoplasm and is involved in viral RNA nuclear export.

The herpesvirus saimiri open reading frame (ORF) 57 is homologous to genes identified in all classes of herpesviruses. It has previously been shown to regulate gene expression through a posttranscriptional mechanism. We demonstrate in this report that the expression of the ORF 57 protein leads to the cytoplasmic accumulation of glycoprotein B and capsid mRNAs. We also demonstrate that ORF 57 has the ability to specifically bind viral RNA transcripts. Utilizing an interspecies heterokaryon assay, we show that ORF 57 has the ability to shuttle between the nucleus and the cytoplasm. Furthermore, we show that ORF 57 contains a relatively leucine-rich sequence which shares some homology with nuclear export signals (NES) found in a number of proteins with the ability to shuttle between the nucleus and the cytoplasm. Moreover, we demonstrate that the ORF 57 NES enables the nuclear export of a heterologous protein and that mutation of the conserved leucine residues contained within the ORF 57 NES signal abrogates the ability of the ORF 57 protein to shuttle between the nucleus and cytoplasm. These results suggest that ORF 57 is involved in mediating the nuclear export of viral transcripts.

Animals↗

PubMed2003

Regulation of fungal gene expression via short open reading frames in the mRNA 5'untranslated region.

We review how the expression of fungal mRNAs can be controlled by ribosome interactions with short upstream open reading frames (uORFs) within the 5'untranslated region. The efficiency of uAUG recognition modulates the impact of a uORF but steps during and after translation of the uORF also influence uORF function. The post-termination behaviour of ribosomes, therefore, plays a major role in determining the expression level of these main ORFs. Translation of a uORF can produce a cis-acting peptide that causes effector molecule-dependent stalling of the ribosomes at the end of the uORF. In other cases it is the length or position, or other features of the uORF, rather than the peptide it encodes, that determine the efficiency with which ribosomes reinitiate translation downstream of it. Whether the form of the ribosome that resumes scanning after termination is the 40S subunit alone or the entire 80S ribosome is not known. Translation of the uORF can also control gene expression by affecting the stability of the mRNA. Finally, trans-acting factors may participate in the regulatory mechanisms. Future work will need not only to provide more information on the mechanisms underlying the known cases of uORF-mediated control but also to define the full complement of uORF-containing mRNAs in at least one fungal organism.

5' Untranslated Regions↗

PubMed1997

Intron open reading frames as mobile elements and evolution of a group I intron.

Group I introns are proposed to have become mobile following the acquisition of open reading frames (ORFs) that encode highly specific DNA endonucleases. This proposal implies that intron ORFs could behave as autonomously mobile entities. This was supported by abundant circumstantial evidence but no experiment of ORF transfer from an ORF-containing intron to its ORF-less counterpart has been described. In this paper we present such experiments, which demonstrate the efficient mobility of the mitochondrial nad1-i4-orf1 between two Podospora strains. The homing of this mobile ORF was accompanied by a bidirectional co-conversion that did not systematically involve the whole intron sequence. Orf1 acquisition would be the most recent step in the evolution of the nad1-i4 intron, which has resulted in many strains of Podospora having an intron with two ORFs (biorfic) and four splicing pathways. We show that two of the splicing events that operate in this biorfic intron, as evidenced by PCR experiments, are generated by a 5'-alternative splice site, which is most probably a remnant of the monoorfic ancestral form of the intron. We propose a sequential evolution model that is consistent with the four organizations of the corresponding nad1 locus that we found among various species of the Pyrenomycete family; these organizations consist of no intron, an intron alone, a monoorfic intron, and a biorfic intron.

Alternative Splicing↗

PubMed2005

Regions of the varicella-zoster virus open reading frame 63 latency-associated protein important for replication in vitro are also critical for efficient establishment of latency.

Varicella-zoster virus (VZV) open reading frame 63 (ORF63) is one of the most abundant transcripts expressed during VZV latency in humans, and ORF63 protein has been detected in human ganglia by several laboratories. Deletion of over 90% of the ORF63 gene showed that the protein is required for efficient establishment of latency in rodents. We have constructed viruses with a series of mutations in ORF63. While prior experiments showed that transfection of cells with a plasmid expressing ORF63 but lacking the putative nuclear localization signal of the protein resulted in increased expression of the protein in the cytoplasm, we found that ORF63 protein remained in the nucleus in cells infected with a VZV ORF63 nuclear localization signal deletion mutant. This mutant was not impaired for growth in cell culture or for latency in rodents. Replacement of five serine or threonine phosphorylation sites in ORF63 with alanines resulted in a virus that was impaired for replication in vitro and for latency. A series of ORF63 carboxy-terminal mutants showed that the last 70 amino acids do not affect replication in vitro or latency in rodents; however, the last 108 amino acids are important for replication and latency. Thus, regions of ORF63 that are important for replication in vitro are also required for efficient establishment of latency.

Animals↗

PubMed1993

Alterations of the three short open reading frames in the Rous sarcoma virus leader RNA modulate viral replication and gene expression.

The Rous sarcoma virus (RSV) leader RNA has three short open reading frames (ORF1 to ORF3) which are conserved in all avian sarcoma-leukosis retroviruses. Effects on virus propagation were determined following three types of alterations in the ORFs: (i) replacement of AUG initiation codons in order to prohibit ORF translation, (ii) alterations of the codon context around the AUG initiation codon to enhance translation of the normally silent ORF3, and (iii) elongation of the ORF coding sequences. Mutagenesis of the AUG codons for ORF1 and ORF2 (AUG1 and AUG2) singly or together delayed the onset of viral replication and cell transformation. In contrast, mutagenesis of AUG3 almost completely suppressed these viral activities. Mutagenesis of ORF3 to enhance its translation inhibited viral propagation. When the mutant ORF3 included an additional frameshift mutation which extended the ORF beyond the initiation site for the gag, gag-pol, and env proteins, host cells were initially transformed but died soon thereafter. Elongation of ORF1 from 7 to 62 codons led to the accumulation of transformation-defective virus with a delayed onset of replication. In contrast, viruses with elongation of ORF1 from 7 to 30 codons, ORF2 from 16 to 48 codons, or ORF3 from 9 to 64 codons, without any alterations in the AUG context, exhibited wild-type phenotypes. These results are consistent with a model that translation of the ORFs is necessary to facilitate virus production.

Animals↗

PubMed2005

Deletion of M2 gene open reading frames 1 and 2 of human metapneumovirus: effects on RNA synthesis, attenuation, and immunogenicity.

The M2 gene of human metapneumovirus (HMPV) contains two overlapping open reading frames (ORFs), M2-1 and M2-2. The expression of separate M2-1 and M2-2 proteins from these ORFs was confirmed, and recombinant HMPVs were recovered in which expression of M2-1 and M2-2 was ablated individually or together [rdeltaM2-1, rdeltaM2-2, and rdeltaM2(1+2)]. Each M2 mutant virus directed efficient multicycle growth in Vero cells. The ability to recover HMPV lacking M2-1 contrasts with human respiratory syncytial virus, for which M2-1 is an essential transcription factor. Expression of the downstream HMPV M2-2 ORF was not reduced when translation of the upstream M2-1 ORF was silenced, indicating that it is initiated separately. The rdeltaM2-2 mutants exhibited a two- to fivefold increase in the accumulation of mRNA, normalized to the genome template, suggesting that M2-2 has a role in regulating RNA synthesis. Replication and immunogenicity were tested in hamsters. Animals infected intranasally with rdeltaM2-1 or rdeltaM2(1+2) did not have recoverable virus in the lungs or nasal turbinates on days 3 or 5 postinfection and did not develop HMPV-neutralizing serum antibodies or resistance to HMPV challenge. Thus, M2-1 appears to be essential for significant virus replication in vivo. In animals infected with rdeltaM2-2, virus was recovered from only 1 of 12 animals and only in the nasal turbinates on a single day. However, all of the animals developed a high titer of HMPV-neutralizing serum antibodies and were highly protected against challenge with wild-type HMPV. The HMPV rdeltaM2-2 virus is a promising and highly attenuated HMPV vaccine candidate.

Amino Acid Sequence↗

PubMed1995

Expression of the mouse mammary tumor virus long terminal repeat open reading frame promotes tumorigenic potential of hyperplastic mouse mammary epithelial cells.

The retrovirus mouse mammary tumor virus (MMTV) 3' long terminal repeat (LTR) contains an open reading frame (ORF) for a 36-kDa protein and encodes a superantigen activity [pORF(sag)]. We have tested the potential oncogenic activity of pORF(sag) in two immortalized mouse mammary epithelial cells. We subcloned MMTV LTR ORF DNA into the pRc/CMV mammalian expression vector in order to place LTR ORF transcription under the control of the constitutive CMV promoter. Mouse mammary epithelial cell lines TM3 and FSK7e4 were transfected and G418-resistant cell clones were isolated. Reverse transcription-polymerase chain reaction and Northern blot analyses revealed modest overexpression of LTR RNA in several transfected cell clones of each line. Individual cell clones were transplanted into cleared mammary gland fat pads of syngeneic BALB/c mice. The parental cell lines and FSK7e4-derived clones did not form tumors, whereas ORF-transfected clones derived from the TM3 cells formed tumors within 8 weeks in 100% of transplanted fat pads in multiple experiments. The tumor cells expressed exogenous LTR ORF RNA and were proven to be derivatives of TM3 cells based on a marker p53 mutation. Immunohistochemistry using a polyclonal antiserum raised against pORF(sag) expressed in insect cells revealed a cytoplasmic reaction in TM3-CMV-LTR tumor cells; a much weaker cytoplasmic reaction was detected in the transfected tissue culture cells. These observations suggest that MMTV pORF(sag) may act as an oncogene in certain mouse mammary epithelial cells and raise the possibility that pORF(sag) may have a role in mammary tumorigenesis. As the parental FSK7 cell line has produced only ductal outgrowths upon transplantation in vivo and the TM3 cell line produces a nontumorigenic hyperplasia, the results suggest further that pORF(sag) may influence the latter stages of mammary tumorigenesis, namely, the preneoplastic to neoplastic transformation.

Animals↗

PubMed2000

In vitro and in vivo functional analysis of human T cell lymphotropic virus type 1 pX open reading frames I and II.

Human T lymphotropic virus type 1 (HTLV-1) is a complex retrovirus containing regulatory and accessory genes encoded in four open reading frames (ORF I-IV) of the pX region. It is not clear what role pX ORFs I and II-encoded proteins have in the pathogenesis of the lymphoproliferative diseases associated with HTLV-1 infection. The conserved ORF I encodes for a hydrophobic 12-kDa protein, p12, (I) that contains four SH3 binding motifs (PXXP) that localizes to cellular endomembranes when overexpressed in cultured cells. Differential splicing of pX ORF II results in the production of two nuclear proteins, p13(II) and p30(II). p13(II) also localizes to mitochondria. p30(II) shares homology with the POU family of transcription factors. We have identified functional roles of pX ORF I and ORF II in establishment and maintenance of infection in a rabbit model. To functionally study p12(I) we have tested a proviral clone with selective ablation of ORF I (ACH.p12(I)) for its ability to infect quiescent peripheral blood mononuclear cells (PBMC). Our data indicate that T cells infected with the wild-type clone of HTLV-1 (ACH) are more efficient than ACH.p12(I) in infecting quiescent PBMC. These findings parallel our animal model data and suggest a role for p12(I) in the activation of quiescent lymphocytes, a prerequisite for effective viral replication in vivo. To test the ability of p30(II) to function as a transcription factor we have constructed p30(II) as a Gal4-fusion protein. When transfected with Gal4-driven luciferase reporter genes, the p30(II)-Gal4-fusion protein induces transcriptional activity up to 50-fold in both 293 and HeLa-Tat cells. These systems will be useful to identify molecular mechanisms that explain the functional role of pX ORF I and ORF II-encoded proteins in HTLV-1 replication.

Animals↗

PubMed2000

Expression of the Totivirus Helminthosporium victoriae 190S virus RNA-dependent RNA polymerase from its downstream open reading frame in dicistronic constructs.

The undivided double-stranded RNA (dsRNA) genome of Helminthosporium victoriae 190S virus (Hv190SV) (genus Totivirus) consists of two large overlapping open reading frames (ORFs). The 5'-proximal ORF encodes a capsid protein (CP), and the downstream, 3'-proximal ORF encodes an RNA-dependent RNA polymerase (RDRP). Unlike the RDRPs of some other totiviruses, which are expressed as a CP-RDRP (Gag-Pol-like) fusion protein, the Hv190SV RDRP is detected only as a separate, nonfused polypeptide. In this study, we examined the expression of the RDRP ORF fused in frame to the coding sequence of the green fluorescent protein (GFP) in bacteria and Schizosaccharomyces pombe cells. The GFP fusions were readily detected in bacteria transformed with the monocistronic construct RDRP:GFP; expression of the downstream RDRP:GFP from the dicistronic construct CP-RDRP:GFP could not be detected. However, fluorescence microscopy and Western blot analysis indicated that RDRP:GFP was expressed at low levels from its downstream ORF in the dicistronic construct in S. pombe cells. No evidence that the RDRP ORF was expressed from a transcript shorter than the full-length dicistronic mRNA was found. A coupled termination-reinitiation mechanism that requires host or eukaryotic cell factors is proposed for the expression of Hv190SV RDRP.

Capsid↗

PubMed1987

Mutation to male fertility and toxin insensitivity in Texas (T)-cytoplasm maize is associated with a frameshift in a mitochondrial open reading frame.

Tissue culture-derived mutants of male-sterile and disease toxin-sensitive Texas (T)-cytoplasm maize that exhibit male fertility and toxin insensitivity carry numerous alterations in mitochondrial DNA. In these mutants, a 6.7-kilobase Xho I fragment characteristic of parental T cytoplasm has been rearranged. In the mutant T-4, the parental 6.7-kilobase Xho I fragment contains a guanine to adenine transition adjacent to a 5-base-pair insertion not found in T cytoplasm. The insertion, internal to a 345-base-pair open reading frame (T ORF13), generates a frameshift, resulting in a premature stop codon that terminates the open reading frame at base pair 222. In other mutants, the 345-base-pair ORF is part of a 3-kilobase deletion, which extends into a 5-kilobase repeat characteristic of mtDNA from T but not N male-fertile cytoplasm. Clones specific to T ORF13 hybridize to eight transcripts in T and T-4, yet only hybridize to three transcripts in T-7, a deletion mutant. Transcription of the T ORF13 region appears not to be altered in T-4, but the frameshift mutation in the T ORF13 reading frame indicates that a biologically inactive gene product could be associated with the mutational events. The results suggest that cytoplasmic male sterility and disease toxin sensitivity may be associated with presence of T ORF13 in T-cytoplasm maize.

Journal Article↗

PubMed1995

Sequence analysis of a 5.6 kb fragment of chromosome II from Saccharomyces cerevisiae reveals two new open reading frames next to CDC28.

The sequence of a 5653 bp DNA fragment of the right arm of chromosome II of Saccharomyces cerevisiae contains two unknown open reading frames (YBR1212 and YBR1213) next to gene CDC28. Gene disruption reveals both putative genes as non-essential. ORF YBR1212 encodes a predicted protein with 71% similarity and 65% identity (total polypeptide of 376 aa) with the 378 aa Surl protein of S. cerevisiae, while the putative product of ORF YBR1213, which is strongly expressed, has 28% identity with a Lactococcus lactis-secreted 45 kDa protein and 24% identity with the Saccharomyces cerevisiae AGA1 gene product.

Amino Acid Sequence↗

PubMed1996

Sequence analysis of a 12 801 bp fragment of the left arm of yeast chromosome XV containing a putative 6-phosphofructo-2-kinase gene, a gene for a possible glycophospholipid-anchored surface protein and six other open reading frames.

The DNA sequence of a 12,801 bp fragment located near the left telomere of chromosome XV has been determined. Sequence analysis reveals eight open reading frames (ORFs) encoding polypeptides larger than 100 residues. ORFs AOE129 and AOAA121 are in opposite strands and they overlap at their 3' ends. AOE397 has similarity with phosphofructokinase genes from other organisms and may code for a second 6-phosphofructo-2-kinase of Saccharomyces cerevisiae. Sequence of AOA471 shows significant similarity with yeast genes coding for glycophospholipid-containing proteins. AOD1341 would code for a 1341 amino acids long protein with a predicted ATP/GTP-binding site and a transmembrane domain.

Amino Acid Sequence↗

PubMed1995

Sequencing analysis of a 15.4 kb fragment of yeast chromosome XIV identifies the RPD3, PAS8 and KRE1 loci, five new open reading frames.

The DNA sequence of a 15.4 kb region covering the left arm of chromosome XIV from Saccharomyces cerevisiae was determined. This region contains eight open reading frames (ORFs) which code for proteins of more than 100 amino acids. Three ORFs correspond to the RPD3, PAS8 and KRE1 loci, described previously. Three ORFs show limited homology with known proteins: NO330 with the recessive suppressor of secretory defect SAC1, NO325 with YCR094W identified during chromosome III sequencing; whereas NO315 presents a motif conserved in the dnaJ family. Two ORFs (NO320 and NO325) show no homology to known proteins within the databases screened, but NO320 corresponds to a serine-threonine-rich protein. The sequence has been entered in the EMBL data library under Accession Number Z46259.

Amino Acid Sequence↗

PubMed1996

Analysis of the DNA sequence of a 15,500 bp fragment near the left telomere of chromosome XV from Saccharomyces cerevisiae reveals a putative sugar transporter, a carboxypeptidase homologue and two new open reading frames.

We report the sequence of a 15.5 kb DNA segment located near the left telomere of chromosome XV of Saccharomyces cerevisiae. The sequence contains nine open reading frames (ORFs) longer than 300 bp. Three of them are internal to other ones. One corresponds to the gene LGT3 that encodes a putative sugar transporter. Three adjacent ORFs were separated by two stop codons in frame. These ORFs presented homology with the gene CPS1 that encodes carboxypeptidase S. The stop codons were not found in the same sequence derived from another yeast strain. Two other ORFs without significant homology in databases were also found. One of them, O0420, is very rich in serine and threonine and presents a series of repeated or similar amino acid stretches along the sequence.

Amino Acid Sequence↗