Identification of genes associated with copper tolerance in an adhesion-defective mutant of Aeromonas veronii biovar sobria.

TnphoA mutagenesis was used to identify adhesins of Aeromonas veronii biovar sobria 3767, a strain isolated from a diarrhoeal stool specimen. Six mutants, from a library of 154, exhibited significantly reduced levels of adhesion to HEp-2 cells. Primers to the terminal regions of TnphoA were used for inverse PCR and the product from one mutant was cloned into pBluescript and partial sequence data obtained. Scanning GenBank and EMBL data bases revealed DNA sequence similarity to the copA gene of Pseudomonas syringae pv. tomato which confers resistance to copper and other heavy metals. The transposon was located within the copA gene and the mutant exhibited a reduced tolerance to copper. Primer walking, using the inverse PCR product as a template, revealed three open reading frames (ORFs) copA, B and C in A. veronii biovar sobria 3767. The predicted amino acid sequences of ORFs A and B had significant homology (55 and 34% respectively) to the copA and B proteins of P. syringae. No amino acid or DNA sequence homology existed between ORF C of strain 3767 and any other gene in the data bases scanned. Further analysis of the nucleotide sequence failed to reveal the presence of typical copper regulatory genes within the vicinity of the Aeromonas sequence. The association between copper tolerance and adhesion in A. veronii biovar sobria requires further study.

Crystallization and preliminary X-ray analysis of IND, an enzyme with indole oxygenase activity from Chromobacterium violaceum.

IND, a redox flavoprotein from Chromobacterium violaceum has been crystallized in the presence and absence of NADH. The crystals belong to the space group P41212 or its enantiomorph P43212 with a = 73.9 and c = 153.6 A. There is one molecule per asymmetric unit and the crystals diffract beyond 2.1 A resolution.

Comparison of the coat protein, movement protein and RNA polymerase gene sequences of Australian, Chinese, and American isolates of barely yellow dwarf virus transmitted by Rhopalosiphum padi.

Barely yellow dwarf luteovirus-GPV (BYDV-GPV) is a common problem in Chinese wheat crops but is unrecorded elsewhere. A defining characteristic of GPV is its capacity to be transmitted efficiently by both Schizaphis graminum and Rhopaloshiphum padi. This dual aphid species transmission contrasts with those of BYDV-RPV and BYDV-SGV, globally distributed viruses, which are efficiently transmitted only by Rhopaloshiphum padi and Schizaphis graminum respectively. The viral RNA sequences encoding the coat protein (22K) gene, the movement protein (17K) gene, the region surrounding the conserved GDD motif of the polymerase gene and the intergenic sequences between these genes were determined for GPV and an Australian isolate of BYDV-RPV (RPVa). In all three genes, the sequences of GPV and RPVa were more similar to those of an American isolate of BYDV-RPV (RPVu) than to any other luteovirus for which there is data available. RPVa and RPVu were very similar, especially their coat proteins which had 97% identity at the amino acid level. The coat protein of GPV had 76% and 78% amino acid identity with RPVa and RPVu respectively. The data suggest that RPVu and RPVa are correctly named as strains of the same serotype and that GPV is sufficiently different from either RPV strain to be considered a distinct BYDV type. The coat protein and movement protein genes of GPV are very dissimilar to SGV. The polymerase sequences of RPVu, RPVa and GPV show close affinities with those of the sobemo-like luteoviruses and little similarity with those of the carmo-like luteoviruses. The sequences of the coat proteins, movement proteins and the polymerase segments of BYDV serotypes, other than RPV and GPV, form a cluster that is separate from their counterpart sequences from dicot-infecting luteoviruses. The RPV and GPV isolates consistently fall within a dicot-infecting cluster. This suggests that RPV and GPV evolved from within this group of viruses. Since these other viruses all infect dicots it seems likely that their common ancestor infected a dicot and that RPV and GPV evolved from a virus that switched hosts from a dicot to a monocot.

Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types.

Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

A positive screen for cloning PCR products.

We have developed a positive screen for cloning PCR products based on translational activation of lacZ. A vector with a translationally deficient lacZ alpha gene has been made by deletion of the Shine-Dalgarno sequence and initiation codon. The Shine-Dalgarno sequence and initiation codon are incorporated into one of the PCR primers to allow complementation by the PCR product of the inactive lacZ alpha gene, which results in blue transformed bacterial colonies. This screen allows more efficient detection of clones containing inserts made by PCR.

Sequence of subterranean clover stunt virus DNA: affinities with the geminiviruses.

The nucleotide sequences of seven circular, single-stranded DNA components of one isolate of subterranean clover stunt virus (SCSV) have been determined. Each component, of about 1 kb, appears to encode a single open reading frame in the same sense as the encapsidated DNA. Notably, the proteins encoded by two SCSV components are related. Each has a consensus nucleotide binding motif and shares about 40% amino acid identity with the other and with the putative replication proteins of banana bunchy top virus and coconut foliar decay virus. The noncoding regions of the five other SCSV components share a highly conserved noncoding sequence of about 160 nucleotides. All seven components were found to contain a sequence capable of forming a stable stem-loop structure in the noncoding region which contains a conserved 9-nucleotide sequence in the loop, very similar to that of the geminiviruses. In addition, the putative replication proteins of SCSV are similar to those of the geminiviruses. We suggest that the SCSV-like viruses and the geminiviruses share a common ancestor and that this ancestor was more like SCSV in particle structure and genome organisation.

Plant viruses: master explorers of evolutionary space.

Viruses represent the epitome of genetic diversity, encompassing both DNA and RNA genomes. Their variability probably results from high mutation rates, extensive genetic recombination, gene duplication, large populations and ancient origins. In addition, at least a third of all viral genes may have de novo origins. There is also recent evidence that additional novelty may be generated by genetic recombination between viruses and their plant hosts. Viruses are indeed master explorers of evolutionary space.

Putative full-length clones of the genomic DNA segments of subterranean clover stunt virus and identification of the segment coding for the viral coat protein.

Subterranean clover stunt disease is an economically important aphid-borne virus disease affecting certain pasture and grain legumes in Australia. The virus associated with the disease, subterranean clover stunt virus (SCSV), was previously found to be representative of a new type of single-stranded DNA virus. Analysis of the virion DNA and restriction mapping of double-stranded cDNA synthesized from virion DNA suggested that SCSV has a segmented genome composed of 3 or 4 different species of circular ssDNA each of about 850-880 nucleotides. To further investigate the complexity of the SCSV genome, we have isolated the replicative form DNA from infected pea and from it prepared putative full-length clones representing the SCSV genome segments. Analysis of these clones by restriction mapping indicated that clones representing at least 4 distinct genomic segments were obtained. This method is thus suitable for generating an extensive genomic library of novel ssDNA viruses containing multiple genome segments such as SCSV and banana bunchy top virus. The N-terminal amino acid sequence and amino acid composition of the coat protein of SCSV were determined. Comparison of the amino acid sequence with partial DNA sequence data, and the distinctly different restriction maps obtained for the full-length clones suggested that only one of these clones contained the coat protein gene. The results confirmed that SCSV has a functionally divided genome composed of several distinct ssDNA circles each of about 1 kb.

Origins of genes: "big bang" or continuous creation?

Many protein families are common to all cellular organisms, indicating that many genes have ancient origins. Genetic variation is mostly attributed to processes such as mutation, duplication, and rearrangement of ancient modules. Thus it is widely assumed that much of present-day genetic diversity can be traced by common ancestry to a molecular "big bang." A rarely considered alternative is that proteins may arise continuously de novo. One mechanism of generating different coding sequences is by "overprinting," in which an existing nucleotide sequence is translated de novo in a different reading frame or from noncoding open reading frames. The clearest evidence for overprinting is provided when the original gene function is retained, as in overlapping genes. Analysis of their phylogenies indicates which are the original genes and which are their informationally novel partners. We report here the phylogenetic relationships of overlapping coding sequences from steroid-related receptor genes and from tymovirus, luteovirus, and lentivirus genomes. For each pair of overlapping coding sequences, one is confined to a single lineage, whereas the other is more widespread. This suggests that the phylogenetically restricted coding sequence arose only in the progenitor of that lineage by translating an out-of-frame sequence to yield the new polypeptide. The production of novel exons by alternative splicing in thyroid receptor and lentivirus genes suggests that introns can be a valuable evolutionary source for overprinting. New genes and their products may drive major evolutionary changes.

Comparisons of the genomic sequences of erysimum latent virus and other tymoviruses: a search for the molecular basis of their host specificities.

The nucleotide sequence of the genome of erysimum latent tymovirus (ELV) has been determined. It closely resembles those of the other four sequenced tymoviral genomes in its gene organization and composition, but is the smallest (6034 nucleotides) and most distinct of them. Furthermore the 78 non-coding nucleotides at the 3' terminus of the ELV genome are unable to form a complete tRNA-like structure like that reported for other tymoviruses. Comparisons of the five tymovirus genomes and their encoded proteins indicate that they have probably evolved from the progenitor tymovirus by independent progressive mutational change without genetic recombination. Comparisons of the sequences of the two non-virion proteins of five tymoviruses, and virion proteins of 17 tymoviruses, revealed no specific similarities between those of ELV and turnip yellow mosaic virus that could explain why their host ranges and symptoms are so similar, yet differ, in this respect, from ononis yellow mosaic, kennedya yellow mosaic and eggplant mosaic tymoviruses.

The primary structure of the virion protein gene and encoded protein of erysimum latent tymovirus.

The nucleotide sequence of the virion protein (VP) gene of erysimum latent tymovirus (ELV) has been determined and the amino acid sequence of the VP deduced and confirmed by peptide analysis. The ELV VP is larger than the VPs of other tymoviruses because it has, unexpectedly, 11 more amino acid residues at its N terminus. The amino acid sequences of the VPs of ELV and four other tymoviruses align unequivocally and their relationships, as assessed from the percentage of identical residues, correlate well with previously reported serological tests which have shown ELV to be distant from other tymoviruses.

The nucleotide sequence of the genomic RNA of kennedya yellow mosaic tymovirus-Jervis Bay isolate: relationships with potex- and carlaviruses.

The nucleotide sequence of the genomic RNA of kennedya yellow mosaic tymovirus-Jervis Bay isolate (KYMV-JB) has been determined. The genome of KYMV-JB is 6362 nucleotide residues long and encodes three major open reading frames. The genomic organization and the encoded proteins of KYMV-JB are very similar to those of three other tymoviruses that have recently been reported. Sequence comparisons revealed that the possible replicase proteins of tymoviruses are closely related to those of potexviruses and carlaviruses, suggesting a close evolutionary relationship among these viruses, despite differences in their genome organization and particle morphology.

Nucleotide sequences of an Australian and a Canadian isolate of potato leafroll luteovirus and their relationships with two European isolates.

The genomes of an Australian and a Canadian isolate of potato leafroll virus have been cloned and sequenced. The sequences of both isolates are similar (about 93%), but the Canadian isolate (PLRV-C) is more closely related (about 98% identity) to a Scottish (PLRV-S) and a Dutch isolate (PLRV-N) than to the Australian isolate (PLRV-A). The 5'-terminal 18 nucleotide residues of PLRV-C, PLRV-A, PLRV-N and beet western yellows virus have 17 residues in common. In contrast, PLRV-S shows no obvious similarity in this region. PLRV-A and PLRV-C genomic sequences have localized regions of marked diversity, in particular a 600 nucleotide residue sequence in the polymerase gene. These data provide a world-wide perspective on the molecular biology of PLRV strains and their comparison with other luteoviruses and related RNA plant viruses suggests that there are two major subgroups in the plant luteoviruses.

The tymobox, a sequence shared by most tymoviruses: its use in molecular studies of tymoviruses.

The 5'-terminal sequences of the virion protein mRNAs of ononis yellow mosaic and kennedya yellow mosaic tymoviruses were determined, and also the positions in the genomes of the transcription initiation sites of those mRNAs. Comparisons of the available genomic sequences of tymoviruses revealed two conserved regions, one at the initiation site and another longer sequence of sixteen nucleotides to the 5' side of it. The longer sequence, which we call the tymobox, was tested as a target for a designed ribozyme, which cleaved appropriate genomic fragments of three tymoviruses. A synthetic oligonucleotide with sequence complementary to the tymobox was shown to be a tymovirus-specific probe for diagnosing and identifying tymoviruses, except for wild cucumber mosaic tymovirus. The tymobox sequence was also used as a primer for the second strand DNA synthesis of dsDNA representing the virion protein gene of cacao yellow mosaic tymovirus, a tymovirus with unknown sequence. Thus, the tymobox is a useful tool in molecular studies of tymoviruses.

Nucleotide sequence of the genome of an Australian isolate of turnip yellow mosaic tymovirus.

The nucleotide sequence of the Club Lake isolate of turnip yellow mosaic virus (TYMV-CL) genomic RNA has been determined. The genome is 6319 nucleotide residues in length and has three major open reading frames (ORFs), two of which overlap. The smallest ORF is proximal to the 3' terminus and encodes the virion protein gene, which has 98% sequence similarity with the virion protein gene reported for the type strain of TYMV. The largest ORF is from nucleotide residues 96 to 5630, and encodes a protein some parts of which show sequence similarities to the possible RNA replicases and nucleotide binding proteins of other viruses. The third ORF is from nucleotide residues 89 to 1975 and overlaps the 5' end of the largest ORF in a manner similar to that found in several animal viral genomes. The function of the protein encoded by this ORF is unknown. The genomes of tymoviruses have, characteristically, an unusually large cytosine content and small guanosine content. This compositional bias is mirrored in the codon and dinucleotide frequencies of the TYMV-CL genome, but is only partially reflected in the amino acid sequences encoded by the genome.

Nucleotide sequence of the genome of eggplant mosaic tymovirus.

The sequence of the RNA genome of an isolate of eggplant mosaic tymovirus from Trinidad (EMV-Trin) has been determined. The genome is 6330 nucleotide residues in length and contains three open reading frames; two overlapping genes, whose initiation codons are separated by seven nucleotide residues (nucleotide residues 102-2051 and 109-5628) near the 5' terminus, and the virion protein gene, which is near the 3' terminus (nucleotide residues 5633-6199). The genomes of EMV-Trin and turnip yellow mosaic tymovirus have the same genomic organization and similar nucleotide and encoded amino acid sequences. The nucleotide residues adjacent to the initiation codons of tymoviral overlapping genes have closely similar sequences which may form a weak stem-loop secondary structure that regulates their translation.

Nucleotide sequence of the ononis yellow mosaic tymovirus genome.

The nucleotide sequence of the genome of ononis yellow mosaic tymovirus (OYMV) has been determined. The genome is single-stranded RNA, 6211 nucleotides long, and has three main open reading frames (ORFs), two of them overlapping. The largest ORF (nucleotides 179-5509) encodes a polyprotein of 1776 amino acid residues that has sequence similarities with polymerases of other viruses with RNA genomes. The smaller overlapping ORF (nucleotides 172-1965) encodes a protein of 597 amino acids of unknown function. The third ORF located at the 3' end of the genome (nucleotides 5487-6065) is the virion protein gene, and it overlaps by 20 nucleotides the 3' terminus of the largest ORF. The organization of the OYMV genome, its sequence, and the sequences of the protein it encodes are clearly similar to those of two other tymoviruses, turnip yellow mosaic virus and eggplant mosaic virus. The 5' terminal noncoding region of the OYMV genome is much longer than the same region of other tymoviral genomes and includes a direct duplication of a sequence of 21-23 nucleotides.

Coconut tinangaja viroid: sequence homology with coconut cadang-cadang viroid and other potato spindle tuber viroid related RNAs.

The nucleotide sequence of two variants of coconut tinangaja viroid (CTiV) were obtained. Both sequence variants are 254 nucleotide residues in size but differ in sequence at two positions. In comparisons with other viroids, the sequences and proposed secondary structure of CTiV show most homology with the 246 nucleotide residue variant of coconut cadang-cadang viroid (CCCV (246]. Several regions throughout the rod-like molecules of CTiV show significant structural and sequence homology with other RNAs related to potato spindle tuber viroid. These homologies include a centrally positioned uridine bulged G:C helix and a 17 nucleotide residue sequence found in common with hop stunt viroid in the left-hand end loop of both viroids.

Self-cleavage of RNA in the replication of viroids and virusoids.

Viroids are infectious, circular RNA molecules of 246 to 375 nucleotides found in plants. Virusoids are of similar size and structure but they are dependent on, and encapsidated in, a helper virus. A rolling circle mechanism of replication is considered to account for the presence of greater-than-unit-length plus and minus RNAs of both viroids and virusoids found in infected plants. An essential feature of this mechanism is the specific processing or cleavage of high molecular weight intermediates to produce linear monomers which are then ligated to circular monomers. We have investigated the putative processing cleavage reactions using in vitro-synthesized RNA transcripts of dimeric cDNA clones of the 247-nucleotide avocado sunblotch viroid (ASBV) and of partial cDNA clones of the 324-nucleotide virusoid of lucerne transient streak virus (vLTSV). In both cases, there is a specific, non-enzymic, self-cleavage of plus as well as minus transcripts. The plus and minus sites of cleavage are in neighbouring parts of ASBV and of vLTSV and highly conserved two-dimensional structures can be drawn around the cleavage sites as well as around the putative or demonstrated cleavage sites of precursors of the virusoids of three other viruses and of the linear satellite RNA of tobacco ringspot virus. The results also indicate that the sole function of about one-third of the ASBV and vLTSV molecules is provision of sequences that allow the formation of the self-cleavage structures of both 'plus' and 'minus' RNA precursors during the replication cycle. Similar self-cleavage of 'plus' RNA transcripts of a dimeric cDNA clone of citrus exocortis virus (CEV) was not observed. However, the putative processing site for CEV precursors was located within three nucleotides by site-directed mutagenesis. No two-dimensional structures similar to those found for ASBV and vLTSV were found around the processing site. It is possible that a different type of self-cleavage or enzymic processing event occurs during the replication cycle of CEV and related viroids.