Identification of putative polymerase gene product in cells infected with murine coronavirus A59

The virion RNA of mouse hepatitis virus, strain A59 (MHV-A59) is believed to be the mRNA for the viral RNA-dependent RNA polymerase. The cell-free translation of virion RNA results in the synthesis of two predominant products p220 and p28 (M. R. Denison and S. Perlman, 1986, J. Virol. 60, 12-18). p28 is a basic protein and is readily detected by two-dimensional gel electrophoresis. When infected cells and isolated virions were assayed for this protein by two-dimensional gel electrophoresis, p28 could be detected in infected cells labeled at late times after infection, but not at early times or in purified virions. p28 represents the first protein product of the putative coronavirus polymerase gene to be identified in infected cells.     

Chromosomal localization and complete genomic sequence of the murine autoimmune regulator gene (Aire)

We have recently cloned the murine autoimmune regulator (Aire) gene, the homologue of human AIRE responsible for the autoimmune polyglandular syndrome type 1 (APS1) or autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED). Here, we report the genomic sequence (18,413 bp) for the entire Aire gene and its 5' flanking region, which contains putative regulatory sequences. Comparison of the genomic and cDNA sequences indicates that the Aire gene is composed of 14 exons and the coding sequence shares high similarities between mouse and human. The sizes of the homologous introns in the two species are conserved; however, the introns do not share significant sequence homologies except the sequences near the splice donor and acceptor sites. Sequence analyses of the 5' regulatory region and the complete coding region in three mouse strains (B6, NOD and SJL) did not reveal any sequence variation, suggesting sequence conservation between different inbred mouse strains. Using one of the six microsatellite markers identified by genomic sequencing and a B6 x Cast backcross mapping panel, we mapped the mouse Aire gene to chromosome 10, a syntenic region containing the Cdl18 and Pfkl genes on human chromosome 21q22.     

Genomic localization and sequence analysis of the putative bovine herpesvirus-1 DNA polymerase gene

Summary The bovine herpesvirus-1 (BHV-1) genome was analysed by Southern blot hybridization using the herpes simplex virus type 1 (HSV-1) DNA polymerase gene as a probe. A 2.5 kilobase region which hybridized specifically to the HSV-1 DNA polymerase gene was identified within the Hind III G fragment at approximate map units 0.334–0.352. In order to provide further evidence that this is the location of the BHV-1 DNA polymerase gene, the 2.5 kilobase region was cloned and part of it sequenced. An uninterrupted stretch of over 800 nucleotides was obtained and an open reading frame spanning the entire sequence was identified. The amino acid sequence that it encodes shows striking homology to a region within the C-terminal half of other herpesvirus DNA polymerases.     

Nucleotide sequence of the gene encoding the membrane protein of human coronavirus 229 E

Summary The sequence of the gene encoding the membrane protein of human coronavirus 229 E (HCV 229 E) has been determined. The primary translation product, deduced from the DNA sequence, is a polypeptide of 225 amino acids with a predicted molecular weight of 26,000. The polypeptide has 3 potential N-glycosylation sites. Many structural similarities with the membrane proteins of other coronaviruses can be recognized.     

Genomic RNA of the murine coronavirus JHM.

Genomic RNA extracted from the purified murine coronavirus JHM sediments between 52S and 54S in aqueous sucrose gradients. The RNA is single-stranded and has an apparent mol. wt. of 5.4 to 6.5 X 10(6), as determined by electrophoresis in polyacrylamide agarose gels of different concentrations. The presence of polyadenylate sequences in the RNA is demonstrated by binding to oligo-)dT) cellulose and digestion with ribonucleases A and T1. The purified RNA does not dissociate into subunits at high temperatures or in high concentrations of DMSO and is infectious.     

Identification of the gene encoding the human mitochondrial RNA polymerase (h-mtRPOL) by cyberscreening of the Expressed Sequence Tags database

A gene cloning strategy based on the screening of the Expressed Sequence Tags database (dbEST) using sequences of mitochondrial housekeeping proteins of yeast was employed to identify the cDNA encoding the precursor of the human mitochondrial RNA polymerase (h- mtRPOL). The 3831 bp h-mtRPOL cDNA is located on chromosome 19p13.3 and encodes a protein of 1230 amino acid residues. The protein sequence shows significant homologies with sequences corresponding to mitochondrial RNA polymerases from lower eukaryotes, and to RNA polymerases from several bacteriophages. The mitochondrial RNA polymerase carries out the central activity of mitochondrial gene expression and, by providing the RNA primers for replication- initiation, is also implicated in the maintenance and propagation of the mitochondrial genome. Genes involved in the control of mtDNA replication and gene expression are attractive candidates for human disorders due to abnormalities of nucleo-mitochondrial intergenomic signalling. The availability of the h-mtRPOL cDNA will allow us to test its role in mitochondrial pathology. In addition, we propose the 'cyberscreening' of dbEST, based on yeast/human cross-species comparison, as a powerful, simple, rapid and inexpensive method, that may accelerate several-fold the molecular dissection of the human mitochondrial proteome.      

RNA polymerase β-subunit gene (rpoB) sequence analysis for the identification of Bacteroides spp.

Partial rpoB sequences (317 bp) of 11 species of Bacteroides, two Porphyromonas spp. and two Prevotella spp. were compared to delineate the genetic relationships among Bacteroides and closely related anaerobic species. The high level of inter-species sequence dissimilarities (7.6-20.8%) allowed the various Bacteroides spp. to be distinguished. The position of the Bacteroides distasonis and Bacteriodes merdae cluster in the rpoB tree was different from the position in the 16S rRNA gene tree. Based on rpoB sequence similarity and clustering in the rpoB tree, it was possible to correctly re-identify 80 clinical isolates of Bacteroides. In addition to two subgroups, cfiA-negative (division I) and cfiA-positive (division II), of Bacteroides fragilis isolates, two distinct subgroups were also found among Bacteroides ovatus and Bacteroides thetaiotaomicron isolates. Bacteroides genus-specific rpoB PCR and B. fragilis species-specific rpoB PCR allowed Bacteroides spp. to be differentiated from Porphyromonas and Prevotella spp., and also allowed B. fragilis to be differentiated from other non-fragilisBacteroides spp. included in the present study.     

Characterization of the gene encoding the largest subunit of Plasmodium falciparum RNA polymerase III.

We report here the isolation, sequence analysis, structure, and expression of the gene encoding the largest subunit of RNA polymerase III (RPIII) from Plasmodium falciparum. The P. falciparum RPIII gene consists of 5 exons and 4 introns, is expressed in all of the asexual erythrocytic stages of the parasite as a 8.5-kb mRNA, and is present in a single copy on chromosome 13. The predicted 2339 amino acid residue RPIII subunit contained 5 regions that were conserved between different eukaryotic RPIII subunits, and 4 variable regions that separated the conserved regions. Three of the variable regions were greatly enlarged in comparison to the corresponding variable regions in other RPIII subunits. Variable region C' represented nearly one-third of the P. falciparum RPIII subunit (750 amino acid residues), included a unique repeated decapeptide sequence, and had some homology with yeast DNA topoisomerase II. Noteworthy amino acid sequences and structures were identified in both the conserved regions and in the enlarged variable regions, and their possible role(s) as domains that regulate RPIII enzyme activity is discussed.     

Chromosomal assignment of gene encoding the largest subunit of RNA polymerase II in the mouse

The gene encoding the largest subunit of RNA polymerase II was mapped to mouse chromosome 11 by Southern blotting analysis of mouse-Chinese hamster somatic cell hybrids and by in situ hybridization. This assignment extends the previously defined homology between mouse chromosome 11 and human chromosome 17.     

The complete nucleotide sequence of plum pox potyvirus RNA

The complete nucleotide sequence of the plum pox virus (PPV) RNA genome has been determined. The RNA sequence is 9786 nucleotides in length, excluding the 3'-terminal poly(A) tail. An AUG triplet at position 147-149 was assigned as the initiation codon for the translation of the genome size viral polyprotein which would consist of 3140 amino acid residues. The nucleotide sequence of the non-coding regions and the predicted amino acid sequence of the polyprotein of PPV were compared with those previously reported for two other potyviruses (tobacco etch virus, TEV, and tobacco vein mottling virus, TVMV), with nucleotide and amino acid sequences of other viruses, as well as with sequences from data banks. The potyvirus genomic expression is discussed in relation to the homologies observed, in particular the predicted protease recognition sequences in related viruses.     

The complete nucleotide sequence of Pepper mottle virus-Florida RNA

Summary.  The Pepper mottle virus-Florida (PepMoV-FL) RNA genome was cloned and sequenced, and shown to consist of 9,717 nucleotides (nt) excluding the poly (A) tail. A single open reading frame was identified beginning at nucleotide position 169 encoding a polyprotein of 3068 amino acids. Phylogenetic sequence analysis revealed that of 44 full-length viral RNA genomes analyzed within the family Potyviridae, PepMoV-FL was most closely related to PepMoV-California (PepMoV-CA), Potato virus Y-H (PVY-H), PVY-N, PVY^o and Potato virus V-DV42 (PVV-DV42). Using the PepMoV-FL sequence as a basis for comparison, the overall nucleotide sequence identity was highest between PepMoV-FL and PepMoV-CA at 93%, while the relationship was more distant with PVV-DV42 at 64% and for the PVY strains at 61%. A unique direct repeat sequence of 76 nucleotides was identified in the PepMoV-FL 3′-untranslated region (UTR), and this repeat sequence was confirmed not to occur in the PepMoV-CA sequence. Since the Florida isolate was among the first of the PepMoV isolates described, extensive biological and serological data on this isolate are available, and it has now been cloned and sequenced, we recommend that PepMoV-FL be recognized as the PepMoV type strain. Received March 25, accepted July 16, 2002     

The gene encoding the nucleocapsid protein: sequence analysis in murine hepatitis virus type 3 and evolution inCoronaviridae

Summary The nucleoprotein-encoding gene (N) of murine hepatitis virus type 3 (MHV 3), from the Mill Hill strain, was cloned and sequenced. It was compared to gene N from other murine coronaviruses and was found to share more similarities with N sequences from MHV 1 and MHV JHM strains than with the published MHV 3 N sequence which is almost identical to MHV A59. We suggest that the evolution of some MHV N sequences resulted from a double recombination phenomenon between two ancestors. Furthermore, comparison of protein N from avian and mammalian coronaviruses leads to the hypothesis that horizontal transfer events of the virus from one host species to another have occurred.     

Expression, purification, and characterization of SARS coronavirus RNA polymerase

The RNA-dependent RNA polymerase (RdRp) of SARS coronavirus (SARS-CoV) is essential for viral replication and a potential target for anti-SARS drugs. We report here the cloning, expression, and purification of the N-terminal GST-fused SARS-CoV RdRp and its polymerase catalytic domain in Escherichia coli. During purification, the full-length GST-RdRp was found to cleave into three main fragments: an N-terminal p12 fragment, a middle p30 fragment, and a C-terminal p64 fragment comprising the catalytic domain, presumably due to bacterial proteases. Biochemical assays show that the full-length GST-RdRp has RdRp activity and the p64 and p12 fragments form a complex that exhibits comparable RdRp activity, whereas the GST-p64 protein has no activity, suggesting that the p12 domain is required for polymerase activity possibly via involvement in template-primer binding. Nonnucleoside HIV-1 RT inhibitors are shown to have no evident inhibitory effect on SARS-CoV RdRp activity. This work provides a basis for biochemical and structural studies of SARS-CoV RdRp and for development of anti-SARS drugs.     

The complete nucleotide sequence of bean yellow mosaic potyvirus RNA

Summary The complete nucleotide sequence of an Australian strain of bean yellow mosaic virus (BYMV-S) has been determined from cloned viral cDNAs. The BYMV-S genome is 9 547 nucleotides in length excluding a poly(A) tail. Computer analysis of the sequence revealed a single long open reading frame (ORF) of 9 168 nucleotides, commencing at position 206 and terminating with UAG at position 9 374–6. The ORF potentially encodes a polyprotein of 3 056 amino acids with a deduced Mr of 347 409. The 5 and 3 untranslated regions are 205 and 174 nucleotides in length respectively. Alignment of the amino acid sequence of the BYMV-S polyprotein with those of other potyviruses identified nine putative proteolytic cleavage sites. The predicted consensus cleavage site of the BYMV NIa protease was found to differ from that described for other potyviruses. Processing of the BYMV polyprotein at the designated proteolytic cleavage sites would result in a typical potyviral genome arrangement. The amino acid sequences of the putative BYMV encoded proteins were compared to the homologous gene products of twelve individual potyviruses to identify overall and specific regions of amino acid sequence homology.The nucleotide sequence data reported in this paper has been submitted to GenBank nucleotide sequence database and has been assigned the accession number U47033.