Nucleotide sequence and genetic map of the 16-kb vaccinia virus HindIII D fragment

We have determined the nucleotide sequence of the 16,059-bp HindIII D fragment from vaccinia virus strain WR. Translation in all 6 reading frames reveals a set of 22 open reading frames (ORFs), which are capable of encoding proteins ranging from 61 to 844 amino acids in length. With one exception, ORF 12, we have divided them into two primary sets according to their size. The minor group contains eight members ranging in length from 61 to 84 amino acids. The major group has thirteen members varying from 146 to 844 amino acids in length, and, in addition, due to its location on the DNA, one small ORF, 61 amino acids long. The neighboring major ORFs are closely packed along the DNA, being separated by 42 or fewer base pairs. In several instances the ends of adjoining ORFs overlap for up to 11 triplet codons. In three cases, 1 or 2 bases are shared between translation start and stop signals in adjacent ORFs. Regions of both strands of the DNA are transcribed. Two sets of temperature-sensitive mutations, totaling 17, which map to the HindIII D fragment, have been combined into eight complementation groups. The results of marker rescue analysis map one or more member of each group to a site in the HindIII D fragment within a defined open reading frame.     

Cloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins.

Gene psaA, which encodes the Streptococcus pneumoniae 37-kDa protein, was cloned in Escherichia coli, and its complete nucleotide sequence was determined. Analysis of the sequence of the 2.4-kb cloned fragment revealed three open reading frames (ORFs). ORF2, which is 933 bp long, was identified as psaA. The two other ORFs identified flank psaA. ORF1, located upstream of psaA, is 836 nucleotides long and encodes a protein with a calculated molecular mass of 29,843 Da. The sequence for ORF3, located downstream of psaA, was only partially determined. Northern (RNA) blot analysis of pneumococcal RNA suggests that psaA is transcribed as part of a polycistronic message. Analysis of the primary structure of the protein encoded by this gene indicated significant similarity to two previously reported streptococcal proteins, SsaB (80% similarity) and FimA (92.3% similarity), from S. sanguis and S. parasanguis, respectively. These two homologous proteins have been shown to be associated with bacterial adhesion, and the possibility of a similar role for PsaA is hypothesized.     

Genome of the European elk papillomavirus (EEPV)

The genome of the European elk papillomavirus (EEPV) was found to be 8,095 base pairs (bp) long and its genetic organization was similar to that of other papillomaviruses. Ten open reading frames (ORFs), designated E1-E7 and L1-L3, were identified in the genome, all located on one strand. The presence of the L3 ORF is rare among the papillomaviruses and to date has only been identified in the genomes of EEPV, the deer papillomavirus (DPV) and the Cottontail papillomavirus (CRPV). The ORF is well conserved beteeen DPV and EEPV with regard to both length and sequence. Potential promoter regions were identified at the 5-end of the E6 ORF, at the 3-end of the E1 ORF and downstream of the L1 ORF. Furthermore, two potential polyadenylation signals were found, one located in the long control region (LCR), downstream of the L1 ORF, and another preceding the L2 ORF. The EEVP genome is closely related to the genome of the DPV, the most highly conserved regions being ORFs E1 (70%), E5 (69%), and L1 (74%).     

Complete Sequence of a 93.4-kb Contig from Chromosome 3 of Trypanosoma cruzi Containing a Strand-Switch Region

We have initiated large-scale sequencing of the third smallest chromosome of the CL Brener strain of Trypanosoma cruzi and we report here the complete sequence of a contig consisting of three cosmids. This contig covers 93.4 kb and has been found to contain 20–30 novel genes and several repeat elements, including a novel chromosome 3-specific 400-bp repeat sequence. The intergenic sequences were found to be rich in di- and trinucleotide repeats of varying lengths and also contained several known T. cruzi repeat elements. The sequence contains 29 open reading frames (ORFs) longer than 700 bp, the longest being 5157 bp, and a large number of shorter ORFs. Of the long ORFs, seven show homology to known genes in parasites and other organisms, whereas four ORFs were confirmed by sequencing of cDNA clones. Two shorter ORFs were confirmed by a database homology and a cDNA clone, respectively, and one RNA gene was identified. The identified genes include two copies of the gene for alanine-aminotransferase as well as genes for glucose-6-phosphate isomerase, protein kinases and phosphatases, and an ATP synthase subunit. An interesting feature of the sequence was that the genes appear to be organized in two long clusters containing multiple genes on the same strand. The two clusters are transcribed in opposite directions and they are separated by an ~20-kb long, relatively GC-rich sequence, that contains two large repetitive elements as well as a pseudogene for cruzipain and a gene for U2snRNA. It is likely that this strand switch region contains one or more regulatory and promoter regions. The reported sequence provides the first insight into the genome organization of T. cruzi and shows the potential of this approach for rapid identification of novel genes.     

Sequence requirements for translation initiation of Rhopalosiphum padi virus ORF2

Rhopalosiphum padi virus (RhPV) is an aphid-infecting virus with a 10-kb ssRNA genome that contains two large open reading frames (ORFs). ORF1 and ORF2 encode the nonstructural and structural polyproteins, respectively. Both ORFs are preceded by noncoding regions of 500 nt that could function as internal ribosome entry segments (IRESes). The sequence for ORF2 lacks an obvious initiation codon, but an out-of-frame AUG codon is present that could translate ORF2 through a +1 frameshift. To investigate the mechanisms of translation initiation of ORF2, a series of point and deletion mutations were constructed and transcribed and translated in vitro. A bicistronic plasmid containing two copies of the RhPV intergenic region translated both ORFs efficiently, indicating that the region functioned as an IRES in vitro. Deletion analysis showed that the region required for activity of the IRES extended from 178 nt upstream and 6 nt downstream of the 5' border of ORF2. Changes in the out-of-frame AUG codon had little effect on translation initiation, but mutations that included the first and second codons of ORF2 or that disrupted a putative pseudoknot structure near the 3' end of the IRES failed to initiate protein synthesis. Sequence analysis of the in vitro synthesized proteins showed that the first amino acid of the polyprotein corresponded to the second codon of ORF2. These results show that in vitro the noncoding region upstream of RhPV ORF2 functions as an IRES that contains a pseudoknot that directs translation initiation at a non-AUG codon. If the in vitro synthesized proteins have not been processed by an aminopeptidase, translation is initiated at the second (GCA) codon of ORF2.     

Complete cDNA sequence of a South American isolate of potato virus X

The complete cDNA sequence corresponding to the genomic RNA of a South American strain of potato virus X (PVXc) is reported. The sequence (6432 nucleotides) contains five open reading frames coding for polypeptides with molecular weights of 165.3, 24.3, 12.3, 7.6 and 25.0 and displays an overall homology of 77.4% with those previously reported for two European isolates. Comparison of amino acid sequences shows an average homology of 87%. Two major domains of variability, located between amino acids 476-615 of ORF 1 and 64-100 of ORF 5, are identified. Sequence similarities between RNA stretches lying upstream of ORFs 2, 4 and 5, and at the 3'-non coding regions of PVX and other plus-strand RNA viruses are described.     

Sequence analysis of open reading frames (ORFs) 2 to 4 of a U.S. isolate of porcine reproductive and respiratory syndrome virus

Summary The sequence of ORFs 2 to 4 of a U.S. isolate of porcine reproductive and respiratory syndrome virus (PRRSV), ATCC VR2385, was determined by analysis of a cDNA library. The cDNA clones containing PRRSV specific sequences were selected using a VR2385 ORF 4 specific PCR probe and sequenced. The ORFs 2, 3 and 4 overlapped each other and encoded polypeptides with predicted M _r of 29.5 kDa (ORF 2), 28.7 kDa (ORF 3) and 19.5 kDa (ORF 4), respectively. No overlap was found between ORFs 4 and 5, and instead there was a 10 bp sequence which separated these two ORFs. The nucleic acid homology with corresponding ORFs of the European PRRSV isolate Lelystad virus (LV) was 65% for ORF 2, 64% for ORF 3 and 66% for ORF 4. Comparison of the ORF 4 sequences of VR2385 with that of another U.S. isolate MN-1b revealed only 86% amino acid sequence homology and the presence of deletions in the ORF 4 of MN-1b. Our results further strengthen the observation that there is sequence variation between US and European PRRSV isolates.     

Map location of lactate dehydrogenase-elevating virus (LDV) capsid protein (Vp1) gene

Lactate dehydrogenase-elevating virus (LDV) is currently classified within the Togaviridae family. In an effort to obtain further information on the characteristics of this virus, we have begun to sequence the viral RNA genome and to map the virion structural protein genes. A sequence of 1064 nucleotides, which represents the 3' terminal end of the genome, was obtained from LDV cDNA clones. A 3' noncoding region of 80 nucleotides followed by two complete open reading frames (ORFs) were found within this sequence. The two ORFs were in different reading frames and overlapped each other by 11 nucleotides. One ORF encoded a protein of 170 amino acids and the other ORF, located adjacent to the 3' noncoding region of the viral genome, encoded a 114 amino acid protein. Thirty-three N-terminal residues were sequenced directly from purified LDV capsid protein, Vp1, and this amino acid sequence mapped to the ORF adjacent to the 3' noncoding region. The presence of overlapping ORFs and the 3' terminal map position of Vp1 indicate that LDV differs significantly from the prototype alpha togaviruses.