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At least 847 records · Page 47Linked to original sources
PubMed2006

Y4xP, an open reading frame located in a type III protein secretion system locus of Sinorhizobium fredii USDA257 and USDA191, encodes cysteine synthase.

Sinorhizobium fredii USDA257, a soybean symbiont, exports several nodulation outer proteins (Nops) into the rhizosphere. These proteins, which are exported by a type III secretion system (TTSS), have a pivotal role in host-specific nodulation. The entire TTSS of S. fredii lies within a 31-kb region that includes conserved genes that code for secretion machinery proteins, Nops, and several open reading frames (ORF) of unknown function. Identifying the functions of these ORF is essential to understand fully the role of TTSS in nodulation. Here, we report the characterization of y4xP, an ORF of previously unknown function. Southern blot analysis revealed that USDA257 contains two copies of y4xP, while a sibling, USDA191, contains a single copy. The amino acid sequence of Y4XP is homologous to both eukaryotic and prokaryotic cysteine synthase, a key enzyme in sulfur assimilation. The coding region of USDA257 y4xP under control of T7 promoter was expressed in Escherichia coli, and the recombinant protein was purified by nickel-affinity chromatography. Antibodies generated against soybean cysteine synthase cross-reacted with the recombinant protein. A nonpolar mutant of y4xP of USDA191 showed a marked reduction in cysteine synthase activity. Enzyme activity was completely restored when the mutant was complemented with a plasmid containing the y4xP sequence. Cysteine synthase activity was confined to the cell cytosol. Extracellular protein fraction from genistein-induced USDA191 showed no cysteine synthase activity. This observation indicates that cysteine synthase, which is located in the TTSS locus, is not a type III secreted protein. A nonpolar cysteine synthase mutant was able to export all the Nops to the rhizosphere, albeit in reduced amounts compared with the wild-type USDA191. Interestingly, USDA191 cysteine synthase mutant was able to initiate nodules on 'McCall' soybean more efficiently than the wild-type. Our results demonstrate that y4xP encodes a cysteine synthase and inactivation of this gene enhances the ability of USDA191 to form nodules on 'McCall' soybean by regulating Nops production.

Amino Acid Sequence↗
PubMed1999

Escherichia coli open reading frame 696 is idi, a nonessential gene encoding isopentenyl diphosphate isomerase.

Isopentenyl diphosphate isomerase catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotes, archaebacteria, and some bacteria, IPP is synthesized from acetyl coenzyme A by the mevalonate pathway. The subsequent isomerization of IPP to DMAPP activates the five-carbon isoprene unit for subsequent prenyl transfer reactions. In Escherichia coli, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pathway. An open reading frame (ORF696) encoding a putative IPP isomerase was identified in the E. coli chromosome at 65.3 min. ORF696 was cloned into an expression vector; the 20.5 kDa recombinant protein was purified in three steps, and its identity as an IPP isomerase was established biochemically. The gene for IPP isomerase, idi, is not clustered with other known genes for enzymes in the isoprenoid pathway. E. coli FH12 was constructed by disruption of the chromosomal idi gene with the aminoglycoside 3'-phosphotransferase gene and complemented by the wild-type idi gene on plasmid pFMH33 with a temperature-sensitive origin of replication. FH12/pFMH33 was able to grow at the restrictive temperature of 44 degrees C and FH12 lacking the plasmid grew on minimal medium, thereby establishing that idi is a nonessential gene. Although the V(max) of the bacterial protein was 20-fold lower than that of its yeast counterpart, the catalytic efficiencies of the two enzymes were similar through a counterbalance in K(m)s. The E. coli protein requires Mg(2+) or Mn(2+) for activity. The enzyme contains conserved cysteine and glutamate active-site residues found in other IPP isomerases.

Carbon-Carbon Double Bond Isomerases↗
PubMed1996

Sequence analysis of the CEN12 region of Saccharomyces cerevisiae on a 43.7 kb fragment of chromosome XII including an open reading frame homologous to the human cystic fibrosis transmembrane conductance regulator protein CFTR.

In the framework of the European Union BIOTECH project for systematically sequencing the Saccharomyces cerevisiae genome, we determined the nucleotide sequence of a 43.7 kb DNA fragment spanning the centromeric region of chromosome XII. A novel approach was the distribution of sublibraries prepared by the DNA coordinator (J. Hoheisel, Heidelberg, FRG), using a new hybridization-based DNA mapping method, in order to facilitate ordered sequencing. The sequence contains 22 open reading frames (ORFs) longer than 299 bp, including the published sequences for ATS/DPS1, SCD25, SOF1, DRS1, MMM1, DNM1 and the centromeric region CEN12. Five putative ORF products show similarity to known proteins: the leucine zipper-containing ABC transporter L1313p to the yeast Ycflp metal resistance protein, to the yeast putative ATP-dependent permease Yhd5p, to the yeast putative proteins Yk83p and Yk84p, to the human cystic fibrosis transmembrane conductance regulator protein (hCFTR) and to the human multidrug resistance-associated protein hMRP1; L1325p to the Drosophila melanogaster Pumilio protein, to the putative yeast regulatory protein Yg13p and to the yeast protein Mpt5p/Htrlp; L1329p to human lipase A and gastric lipase, to rat lingual lipase and to the putative yeast triglyceride lipase Tg11p; L1341p to the putative yeast protein Yhg4p; and the leucine zipper-containing L1361p to the two yeast proteins 00953p and Ym8156.08p and to the Arabidopsis thaliana protein HYP1. Eight ORFs show no homology to known sequences in the database, three small ORFs are internal and complementary to larger ones and L1301 is complementary overlapping the ATS/DPS1 gene. Additionally three equally spaced ARS consensus sequences were found.

Amino Acid Sequence↗
PubMed1998

Capsids are formed in a mutant virus blocked at the maturation site of the UL26 and UL26.5 open reading frames of herpes simplex virus type 1 but are not formed in a null mutant of UL38 (VP19C).

Previously we reported that null mutant viruses of UL19 (VP5) or of UL18 (VP23), essential components of herpes simplex virus type 1 (HSV-1) capsid shells, do not form precursor capsid structures as judged by sedimentation and electron microscope analysis. A goal of the present experiments was to isolate a null mutant virus for the remaining essential component of capsid shells, VP19C, encoded by the UL38 open reading frame (ORF). Furthermore, we wished to determine if a virus altered in the UL26 maturation cleavage site at residues 610 and 611 produced a lethal phenotype. Therefore, we decided to isolate cell lines that encode and express multiple capsid genes. Several cell lines were isolated by transformation of Vero cells and one designated C32 expressed all of the essential capsid proteins. Using this cell line we isolated a null mutant virus in the UL38 ORF and a mutant virus that was altered at residues 610 and 611 of the UL26 and UL26.5 gene products. We found that the null mutant in VP19C did not form a detectable product as judged by sedimentation and electron microscope analyses following infection of nonpermissive cells. The mutant virus altered at the UL26 maturation site resulted in the accumulation of B capsids. Therefore, cleavage at this site was essential for the maturation of B capsids into C capsids. Interestingly, the absence of cleavage at the maturation site was required for the retention of VP24 in the capsid.

Animals↗
PubMed2004

The mouse synemin gene encodes three intermediate filament proteins generated by alternative exon usage and different open reading frames.

We have previously cloned and characterized the human synemin gene, which encodes two intermediate filament proteins (IFPs). We now show that the mouse synemin gene encodes three different synemin isoforms through an alternative splicing mechanism. Two of them, synemin H and M are similar to human alpha and beta synemin, and the third isoform, L synemin, constitutes a new form of IFP. It has a typical rod domain and a short tail (49 residues) with a novel sequence that is produced by a different open reading frame. The synthesis of H/M synemins starts in the embryo, whereas the synemin L isoform is present in adult muscles. The H/M isoforms are bound to desmin or vimentin in the muscle cells of wild-type mice. Using desmin- and vimentin-deficient mice, we have obtained direct evidence that synemin is associated with muscle intermediate filaments in vivo. The organization of the synemin fibril is disrupted in skeletal and cardiac muscle when desmin is absent and in smooth muscle when vimentin is absent. The fact that the three synemin isoforms differ in the sequences of their tail domains as well as in their developmental patterns suggests that they fulfill different functions.

Alternative Splicing↗
PubMed1995

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.

Base Sequence↗
PubMed1995

Characterization of six linked open reading frames necessary for pIP501-mediated conjugation.

pIP501 is a broad-host-range gram-positive conjugative plasmid. Previous transposon mutagenesis of a pIP501 derivative, pVA1702, identified two regions, A and B, that were involved in conjugation proficiency. Recently, we reported that the A region contained a functional oriT site similar to those found on plasmids of gram-negative origin (Wang and Macrina, 1995). We report here the nucleotide sequence of the complete A region. Analysis of this DNA sequence (8121 bp) revealed six contiguous open reading frames (orf1-6) immediately downstream from the oriT site. ORF1 was similar to gram-negative plasmid nickase proteins known to play a role in the initiation of conjugal transfer. The remaining ORF proteins (2-6) were unique. Complementation studies suggested that the gene products of ORFs 3-6 were able to function in trans. A single available insertion mutant in orf2 could not be complemented in trans. Thus, although the conjugative apparatus that initiates transfer (oriT and nickase) appears to be conserved between this class of gram-positive plasmid and gram-negative plasmids, most of the A region genes of pIP501 involved in transfer appear unique and their cellular and molecular roles are undefined.

Amino Acid Sequence↗
PubMed2004

Functional properties of a 16 kDa protein translated from an alternative open reading frame of the core-encoding genomic region of hepatitis C virus.

Hepatitis C virus (HCV) often causes persistent infection in humans. This could be due in part to the effect of viral proteins on cellular gene expression. Earlier observations suggest that the HCV core protein expressed from genotype 1a modulates important cellular genes at the transcriptional level, affects programmed cell death (apoptosis) and promotes cell growth. Recently, different groups of investigators have reported the translation of an approximately 16 kDa protein (named F/ARFP/core+1 ORF) from an alternate open reading frame of the HCV core-encoding genomic region. The functional significance of this F protein is presently unknown. Thus, whether the F and core proteins have both shared and distinct functions was investigated here. The experimental observations suggested that the F protein does not significantly modulate c-myc, hTERT and p53 promoter activities, unlike the HCV core protein. Interestingly, the F protein repressed p21 expression. Further studies indicated that the F protein does not inhibit tumour necrosis factor alpha-mediated apoptosis of HepG2 cells or promote rat embryo fibroblast growth. Taken together, these results suggest that the F protein does not share major properties identified previously for the HCV core protein, other than regulating p21 expression.

Cyclin-Dependent Kinase Inhibitor p21↗
PubMed1994

Mapping and transcriptional analysis of the murine cytomegalovirus homologue of the human cytomegalovirus UL103 open reading frame.

Murine cytomegalovirus (MCMV) has been widely used as a model for human cytomegalovirus (HCMV) infection since the two viruses share many biological and pathogenic properties. To further evaluate the similarities in the genomes of these viruses we have sought to identify MCMV genes that encode immunoreactive proteins by screening a lambda gt11 cDNA library with anti-MCMV monoclonal antibodies (MAb). Clone 430, identified on the basis of its reactivity with the MAb 6/20/1, was completely sequenced and shown to encode the MCMV homologue of the HCMV UL103 open reading frame (ORF). This ORF is encoded by a 1.1-kb transcript that is expressed at late times following infection. Sequencing of the cDNA also revealed the presence of two incomplete ORFs encoding the homologues of the HCMV UL102 and 104 ORFs. These ORFs are encoded by a 2.4-kb leaky late transcript and a 3.0-kb late transcript, respectively. Together these ORFs constitute part of a block of genes conserved in all herpesviruses, and in the MCMV and HCMV genomes this block of genes shows striking positional homology. The high degree of homology between HCMV and MCMV ORFs, at both the nucleotide and amino acid levels, plus the growing evidence of conservation in gene order between the two genomes suggests that the two viruses are very similar at the molecular level.

Amino Acid Sequence↗
PubMed1993

The products of the E5, E6, or E7 open reading frames of RhPV 1 can individually transform NIH 3T3 cells or in cotransfections with activated ras can transform primary rodent epithelial cells.

Rhesus papillomavirus (RhPV) type 1 was recently shown to cooperate with the activated ras oncogene to transform primary rodent epithelial cells at a level comparable to HPV 16. In similar cotransfection studies, subgenomic portions of RhPV 1 driven by either their natural or a strong heterologous promoter were used in primary baby rat kidney cells to demonstrate that transforming properties of RhPV 1 could be localized individually to the E5, E6, and E7 open reading frames. Fully transformed cells were observed when either E5 or E7 were downstream of a strong heterologous promoter. Similarly, either E6 or E6 and E7 downstream of the native promoter fully transformed these cells as determined by immortalization, anchorage independent growth and tumorigenicity studies.

3T3 Cells↗
PubMed1997

Open reading frame 26 of human herpesvirus 8 encodes a tetradecanoyl phorbol acetate- and butyrate-inducible 32-kilodalton protein expressed in a body cavity-based lymphoma cell line.

DNA sequences corresponding to a novel herpesvirus (human herpesvirus 8 [HHV8]) are associated with Kaposi's sarcoma (KS), Castleman's disease, and body cavity-based lymphomas (BCBL). Studies of a BCBL-derived cell line suggest a direct correlation between seropositivity against antigens specifically present in such lines and the development of KS. We have generated recombinant proteins corresponding to open reading frame (ORF) 26 of HHV8 and have produced affinity-purified antibodies. Using these antibodies, we studied the expression of HHV8 ORF26 in a BCBL-derived cell line and found that it encodes a cytoplasmic protein whose expression is induced 16-fold by treatment with phorbol ester or sodium butyrate. This protein induction correlates with a significant induction of viral RNA transcripts. Interestingly, under our experimental conditions minimal increases in viral DNA were observed. No antibodies to the ORF26 protein of HHV8 were found in the sera from two human immunodeficiency virus-positive patients with KS as determined by immunoprecipitation analysis. However, antibodies in the sera from the two KS patients immunoprecipitated a 34-kDa protein found in extracts from induced BCBL1 cells that was not recognized by the control sera.

Antibodies, Viral↗
PubMed2003

Murine cytomegalovirus m41 open reading frame encodes a Golgi-localized antiapoptotic protein.

Viruses have evolved various strategies to prevent premature apoptosis of infected host cells. Some of the viral genes mediating antiapoptotic functions have been identified by their homology to cellular genes, but others are structurally unrelated to genes of known function. In this study, we used a random, unbiased approach to identify such genes in the murine cytomegalovirus genome. From a library of random transposon insertion mutants, a mutant virus that caused premature cell death was isolated. The transposon was inserted within open reading frame m41. An independently constructed m41 deletion mutant showed the same phenotype, whereas deletion mutants lacking the adjacent genes m40 and M42 did not. Apoptosis occurred in different cell types, could be blocked by caspase inhibitors, and did not require p53. Within the murine cytomegalovirus genome, m41, m40, and m39 form a small cluster of genes of unknown function. They are homologous to r41, r40, and r39 of rat cytomegalovirus, but lack sequence homology to UL41, UL40, and UL37 exon 1 (UL37x1) which are located at the corresponding positions of the human cytomegalovirus genome. Unlike UL37x1 of human cytomegalovirus, which encodes a mitochondrion-localized inhibitor of apoptosis that is essential for virus replication, m41 encodes a protein that localizes to the Golgi apparatus. The murine cytomegalovirus m41 product is the first example of a Golgi-localized protein that prevents premature apoptosis and thus extends the life span of infected cells.

Animals↗
PubMed1994

Circadian clock locus frequency: protein encoded by a single open reading frame defines period length and temperature compensation.

The frequency (frq) locus encodes a key component, a state variable, in a cellular oscillator generating circadian rhythmicity. Two transcripts have been mapped to this region, and data presented here are consistent with the existence of a third transcript. Analysis of cDNA clones and clock mutants from this region focuses attention on one transcript encoding a protein. FRQ, which is a central clock component: (i) mutations in all of the semidominant frq alleles are the result of single amino acid substitutions and map to the open reading frame (ORF) encoding FRQ; (ii) deletion of this ORF, or a frameshift mutation within it, results in a strain with a recessive clock phenotype characterized by the loss of rhythm stability and compensation. Single amino acid substitutions within, or disruption of, this single ORF are thus sufficient to drive major alterations in both period length and temperature compensation, two canonical characteristics of circadian systems. The 989-amino acid FRQ protein species the circadian function of frq in the assembly of the Neurospora biological clock.

Alleles↗
PubMed1993

A novel multicopy suppressor of a groEL mutation includes two nested open reading frames transcribed from different promoters.

When present on a multicopy plasmid, a newly discovered gene (sugE) mapping to 94 min on the Escherichia coli chromosome, suppresses a groEL mutation and mimics the effects of groE overexpression. A groEL mutant of E.coli, transformed with the Klebsiella pneumoniae nif gene cluster, failed to accumulate nitrogenase components [Govezensky et al. (1991) J. Bacteriol., 173, 6339-6346]. Transformation with sugE reversed the mutant phenotype. In wild type K.pneumoniae, transformation with sugE accelerated the rate of nitrogenase biogenesis after nif derepression. In E.coli, transformation with sugE enabled bacteriophage T4 growth in a groEL mutant. A continuous 178 codon open reading frame (ORF) in sugE encloses another, in-frame, 105 codon ORF similar to a predicted ORF in Proteus vulgaris. In vivo products of both sugE ORFs were observed in transformants expressing the gene from a T7 promoter. In non-transformed cells, a typical sigma 70-dependent promoter found upstream of the larger ORF directs sugE transcription during growth at 30 degrees C. At elevated temperatures or in stationary phase cells, another promoter, found within the coding sequence upstream of the smaller ORF, is activated independently of sigma 32. The results suggest that sugE encodes a chaperonin-related system whose composition might vary with temperature and growth phase.

Amino Acid Sequence↗
PubMed2006

No evidence for the use of DIR, D-D fusions, chromosome 15 open reading frames or VH replacement in the peripheral repertoire was found on application of an improved algorithm, JointML, to 6329 human immunoglobulin H rearrangements.

Antibody diversity is created by imprecise joining of the variability (V), diversity (D) and joining (J) gene segments of the heavy and light chain loci. Analysis of rearrangements is complicated by somatic hypermutations and uncertainty concerning the sources of gene segments and the precise way in which they recombine. It has been suggested that D genes with irregular recombination signal sequences (DIR) and chromosome 15 open reading frames (OR15) can replace conventional D genes, that two D genes or inverted D genes may be used and that the repertoire can be further diversified by heavy chain V gene (VH) replacement. Safe conclusions require large, well-defined sequence samples and algorithms minimizing stochastic assignment of segments. Two computer programs were developed for analysis of heavy chain joints. JointHMM is a profile hidden Markow model, while JointML is a maximum-likelihood-based method taking the lengths of the joint and the mutational status of the VH gene into account. The programs were applied to a set of 6329 clonally unrelated rearrangements. A conventional D gene was found in 80% of unmutated sequences and 64% of mutated sequences, while D-gene assignment was kept below 5% in artificial (randomly permutated) rearrangements. No evidence for the use of DIR, OR15, multiple D genes or VH replacements was found, while inverted D genes were used in less than 1 per thousand of the sequences. JointML was shown to have a higher predictive performance for D-gene assignment in mutated and unmutated sequences than four other publicly available programs. An online version 1.0 of JointML is available at http://www.cbs.dtu.dk/services/VDJsolver.

Algorithms↗
PubMed1997

The DNA sequence of cosmid 14-13b from chromosome XIV of Saccharomyces cerevisiae reveals an unusually high number of overlapping open reading frames.

This work is part of the effort for sequencing chromosome XIV of Saccharomyces cerevisiae. Cosmid 14-13b contains a 37.8 kb insert derived from a partial Sau3A digestion of the genome, cloned into the BamHI site of the vector Pou6. The strategy used for sequencing is based on the fragmentation of the whole cosmid by sonication, followed by shotgun sequencing on an Applied Biosystem DNA sequencer. The clones with inserts corresponding to the vector were identified by dot-blot hybridization, without the need of sequencing. The analysis of the DNA sequence reveals 29 open reading frames (ORFs) longer than 300 bases. Nine ORFs are internal to some other ORFs. Similarity searches against DNA and protein data banks show that six ORFs correspond to already known yeast genes (OMP1, PSU1, MLS1, RPC19, DBP2, CYB5) and one ORF matches the sequence of a putative yeast gene (ESBP6).

Chromosome Mapping↗
PubMed2002

Isolation and expression of three open reading frames from ovine herpesvirus-2.

Ovine herpesvirus-2 (OvHV-2), a member of the gammaherpesviruses (genus Rhadinovirus), asymptomatically infects its natural host, the sheep, but causes malignant catarrhal fever (MCF) in susceptible hosts, such as cattle, deer and pigs. A permissive cell culture system for virus replication has not been identified but viral DNA is present within lymphoblastoid cell lines (LCLs) established from cases of MCF. During this study, a cDNA expression library generated from LCLs was screened with sheep sera and two cDNAs were isolated. One cDNA contained two open reading frames (ORFs) that show similarity to ORFs 58 and 59 of alcelaphine herpesvirus-1 (AlHV-1), a closely related gammaherpesvirus that also causes MCF. Both ORFs 58 and 59 are conserved throughout the gammaherpesviruses. ORF 58 is predicted to be a membrane protein, while ORF 59 has been shown to be an early lytic gene that functions as a DNA polymerase processivity factor. The second cDNA clone contained a partial ORF showing limited similarity to AlHV-1 ORF 73, a homologue of the latency-associated nuclear antigen of human herpesvirus-8, which is associated with latent infections. The full-length OvHV-2 ORF 73 was cloned subsequently by PCR. The ORFs isolated from the library were cloned into a bacterial expression vector and the recombinant proteins tested for their reactivity to sera from OvHV-2-infected animals. An ORF 59 fusion protein was recognized specifically by sera from OvHV-2-infected cattle and will be used to develop a sero-diagnostic test.

Amino Acid Sequence↗
PubMed2003

Optimum growth temperature and the base composition of open reading frames in prokaryotes.

The purine-loading index (PLI) is the difference between the numbers of purines (A+G) and pyrimidines (T+C) per kilobase of single-stranded nucleic acid. By purine-loading their mRNAs organisms may minimize unnecessary RNA-RNA interactions and prevent inadvertent formation of "self" double-stranded RNA. Since RNA-RNA interactions have a strong entropy-driven component, this need to minimize should increase as temperature increases. Consistent with this, we report for 550 prokaryotic species that optimum growth temperature is related to the average PLI of open reading frames. With increasing temperature prokaryotes tend to acquire base A and lose base C, while keeping bases T and G relatively constant. Accordingly, while the PLI increases, the (G+C)% decreases. The previously observed positive correlation between (G+C)% and optimum growth temperature, which applies to RNA species whose structure is of major importance for their function (ribosomal and transfer RNAs) does not apply to mRNAs, and hence is unlikely to apply generally to genomic DNA.

Bacteria↗