A New Wilt of Cyclamen Caused by Phytophthora tropicalis in Germany and the Netherlands.

Unusual symptoms were observed on cyclamen (Cyclamen persicum Mill.) in Germany and the Netherlands in 1997. Symptoms began with a change of leaf color from dark green to olive green and an unspecified flagging of leaves, followed by a yellowing of the margins of older leaves and then yellowing of entire leaves. Corms, sectioned lengthwise, were firm, cream colored, without discoloration of vascular bundles, but with some browning at the base and dark brown discolored roots. Eventually, the plants died. Five isolates (one from Netherlands, two from Northern Germany, and two from Southern Germany) of a Phytophthora species recovered from diseased corms of cyclamen were heterothallic, all A1 mating type, and occasionally formed a few chlamydospores in culture. Sporangia were commonly produced in umbellate clusters. Sporangial shape was naviculate to limoniform with a tapered base. They were papillate, occasionally bipapillate, caducous, with a pedicel of about 1.5 times of the sporangial length. Sporangial dimensions were 52 × 26 µm, with a length to width ratio of 2:1. Cardinal temperatures were 10 and 35°C; optimum growth occurred at 30°C. The pathogen was originally described as a taxonomically nonclassified species closely related to the Phytophthora palmivora complex (2). Based on isolate ATCC No. 76656, received from Uchida (1), we recognized that the above-mentioned cyclamen pathogen exhibits morphological characteristics typical of Phytophthora tropicalis. This taxon has been proposed by Aragaki and Uchida (1) for a number of Phytophthora capsici-like isolates (3) which differ from the type isolate of Phytophthora capsici Leonian. Pathogen identity was further confirmed by cloning subgenomic DNA fragments. Oligonucleotide primers and polymerase chain reaction were employed to amplify the internal transcribed spacer regions ITS1/ITS2 including the 5.8S subunit of the rRNA gene repeat. Both 809-bp products obtained from the American and the European P. tropicalis isolates H 778-1 (Isle of Oahu, Hawaii; host: Dianthus caryophyllus L.) and 066 (Bavaria; host: Cyclamen persicum) respectively, reveal identical nucleotide sequences with the exception of two single bp changes (EMBL accession numbers AJ299734 and AJ299733). In public electronic databases, there were no similar sequences with other Phytophthora spp. Using a sand-corn meal mixture added as inoculum to the planting substrate, pathogenicity of the European P. tropicalis-isolates was shown on Cyclamen persicum, Epipremnum aureum (Linden et André) Bunting, Dianthus caryophyllus, partially on Diascia vigilis Hilliard et B. C. Burtt and Hedera helix L. No pathogenicity was observed on Lycopersicon esculentum Mill., Capsicum annuum L., Cucurbita pepo L., and Cucumis sativus L., representing important hosts of P. capsici, and not on Carica papaya L., a typical host for P. palmivora (Butler) Butler. All data described here confirm the identity of the new cyclamen pathogen as P. tropicalis. The uniformity of the mating type, A1, suggests that the pathogen was inadvertently introduced at one point into Europe and possibly was distributed on seedling plants of cyclamen, since apparently no other host of this pathogen has been observed so far in Europe. References: (1) M. Aragaki and J. Y. Uchida. Phytopathology 82:1164, 1992. (2) E. Idczak et al. Nachrichtenbl. Deut. Pflanzenschutzd. 50:1, 1998. (3) G. R. A. Mchau and M. D. Coffey. Mycol. Res. 99:89, 1995.

Catalytic nucleic acids: from lab to applications.

Since the discovery of self-cleavage and ligation activity of the group I intron, the expansion of research interest in catalytic nucleic acids has provided a valuable nonprotein resource for manipulating biomolecules. Although a multitude of reactions can be enhanced by this class of catalyst, including trans-splicing activity of the group I intron (which could be applied to gene correction), RNA-cleaving RNA enzymes or "ribozymes" hold center stage because of their tremendous potential for mediating gene inactivation. This application has been driven predominantly by the "hammerhead" and "hairpin" ribozymes as they induce specific RNA cleavage from a very small catalytic domain, allowing delivery either as a transgene expression product or directly as a synthetic oligonucleotide. Although advances in the development of RNA modifications have improved the biological half-life of synthetic ribozymes, their use is restricted by the mechanistic dependence on conserved 2'OH-moieties. Recently a new class of catalytic nucleic acid made entirely of DNA has emerged through in vitro selection. DNA enzymes or deoxyribozyme with extraordinary RNA cleavage activity has already demonstrated their capacity for gene suppression both in vitro and in vivo. These new molecules, although rivaling the activity and stability of synthetic ribozymes, are limited equally by inefficient delivery to the intracellular target RNA. The challenge of in vivo delivery is being addressed with the assessment of a variety of approaches in animal models with the aim of bringing these compounds closer to the clinic.

The use of ribozyme gene therapy for the inhibition of HIV replication and its pathogenic sequelae.

Human immunodeficiency virus (HIV) is a lentivirus, a separate genus of the Retroviridae which are RNA viruses that integrate as DNA copies into the genomes of host cells and replicate intracellularly through various RNA intermediates. Several of these RNA molecules can be targeted by ribozymes and a number of investigators, including our group, have demonstrated the ability of ribozymes to suppress HIV replication in cultured cells. It is argued that the use of this ribozyme gene therapy approach for the treatment of HIV infection may act as an adjunct to chemotherapeutic drugs and may affect not just viral suppression, but also immune restoration. This approach can be tested in Clinical Trials, several of which are currently under way.

Suppression of smooth muscle cell proliferation by a c-myc RNA-cleaving deoxyribozyme.

A small catalytic DNA molecule targeting c-myc RNA was found to be a potent inhibitor of smooth muscle cell (SMC) proliferation. The catalytic domain of this molecule was based on that previously derived by in vitro selection (Santoro, S. W., and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4262-4266) and is known as the "10-23" general purpose RNA-cleaving deoxyribozyme. In addition to inhibiting SMC proliferation at low concentration, this molecule (targeting the translation initiation region of c-myc RNA) was found to efficiently cleave its full-length substrate in vitro and down-regulate c-myc gene expression in smooth muscle cells. The serum nuclease stability of this molecule was enhanced without substantial loss of kinetic efficiency by inclusion of a 3'-3'-internucleotide inversion at the 3'-terminal. The extent of SMC suppression was found to be influenced by the length of the substrate binding arms. This correlated to some extent with catalytic activity in both the short substrate under multiple turnover conditions and the full-length substrate under single turnover conditions, with the 9 + 9 base arm molecule producing the greatest activity.

Preclinical characterization of an anti-tat ribozyme for therapeutic application.

A hammerhead ribozyme retroviral construct, denoted RRz2, targeting the coding region of the human immunodeficiency virus type 1 (HIV-1) tat gene, has shown itself to be effective in a range of test systems. Inhibition of the replication of HIV-1 IIIB and primary drug-resistant strains in pooled transduced CEMT4 cells was consistently found to be more than 80% compared with the control-vector transduced cells, whereas a mutant RRz2 gave approximately 45% inhibition. A multiple HIV-1 passage assay showed the absence of emergence of mutations within the specific viral RNA ribozyme target sequences. This lack of generation of ribozyme "escape mutants" occurred despite the almost complete disappearance of a HIV-1 quasi-species in the testing virus. When RRz2 was tested in peripheral blood lymphocytes (PBLs) from HIV-1-infected patients, paired analysis showed that cell viability in the ribozyme-transduced HIV-1-infected PBLs was significantly higher than that in the vector-transduced cells. This difference in viability (vector versus RRz2) was not observed in PBLs from non-HIV-1-infected donors. Taken together, these results indicate that the transfer of an anti-HIV-1 ribozyme gene into human T lymphocytes could have major impact on viral replication and T cell viability in the HIV-1-infected individual.

Anti-HIV ribozymes.

HIV is an RNA virus that replicates intracellularly through various RNA intermediates. Several of these can be targeted by ribozymes (catalytic RNA molecules), and a number of investigators, including this group, have demonstrated the ability of ribozymes to suppress HIV replication in this way. It is argued that this gene therapy approach may be viewed as an adjunct to chemotherapeutic drugs, which may allow not just viral suppression, but also immune restoration. This can only finally be tested in clinical trials, and several are planned. The basic ribozyme unit, the potential of which was described less than 10 years ago, is about to be tested in an amunable disease state.

A ribozyme gene and an antisense gene are equally effective in conferring resistance to tobacco mosaic virus on transgenic tobacco.

Ribozymes of the hammerhead class can be designed to cleave a target RNA in a sequence-specific manner and can potentially be used to specifically modulate gene activity. We have targeted the tobacco mosaic virus (TMV) genome with a ribozyme containing three catalytic hammerhead domains embedded within a 1 kb antisense RNA. The ribozyme was able to cleave TMV RNA at all three target sites in vitro at 25 degrees C. Transgenic tobacco plants were generated which expressed the ribozyme or the corresponding antisense constructs directed at the TMV genome. Six of 38 independent transgenic plant lines expressing the ribozyme and 6 of 39 plant lines expressing the antisense gene showed some level of protection against TMV infection. Homozygous progeny of some lines were highly resistant to TMV; at least 50% of the plants remained asymptomatic even when challenged with high levels of TMV. These plants also displayed resistance to infection with TMV RNA or the related tomato mosaic virus (ToMV). In contrast, hemizygous plants of the same lines displayed only very weak resistance when inoculated with low amounts of TMV and no resistance against high inoculation levels. Resistance in homozygous plants was not overcome by a TMV strain which was altered at the three target sites to abolish ribozyme-mediated cleavage, suggesting that the ribozyme conferred resistance primarily by an antisense mechanism.

Resistance to human immunodeficiency virus type 1 infection conferred by transduction of human peripheral blood lymphocytes with ribozyme, antisense, or polymeric trans-activation response element constructs.

Human peripheral blood lymphocytes (PBLs) were transduced with a number of recombinant retroviruses including RRz2, an LNL6-based virus with a ribozyme targeted to the human immunodeficiency virus (HIV) tat gene transcript inserted within the 3' region of the neomycin-resistance gene; RASH5, and LNHL-based virus containing an antisense sequence to the 5' leader region of HIV-1 downstream of the human cytomegalovirus promoter; and R20TAR, an LXSN-based virus with 20 tandem copies of the HIV-1 trans-activation response element sequence driven by the Moloney murine leukemia virus long terminal repeat. After G418 selection, transduced PBLs were challenged with the HIV-1 laboratory strain IIIB and a primary clinical isolate of HIV-1, 82H. Results showed that PBLs from different donors could be transduced and that this conferred resistance to HIV-1 infection. For each of the constructs, a reduction of approximately 70% in p24 antigen level relative to the corresponding control-vector-transduced PBLs was observed. Molecular analyses showed constitutive expression of all the transduced genes from the retroviral long terminal repeat, but no detectable transcript was seen from the internal human cytomegalovirus transcript was seen from the internal human cytomegalovirus promoter for the antisense construct. Transduction of, and consequent transgene expression in, PBLs did not impact on the surface expression of either CD4+/CD8+ (measured by flow cytometry) or on cell doubling time (examined by [3H]thymidine uptake). These results indicate the potential utility of these anti-HIV-1 gene therapeutic agents and show the preclinical value of this PBL assay system.

Target sequence-specific inhibition of HIV-1 replication by ribozymes directed to tat RNA.

The structural motif formed between a hammerhead ribozyme and its substrate consists of three RNA double helices in which the sequence 5' to the XUY is termed helix I and the sequence 3' to the XUY helix III. Two hammerhead ribozymes targeted to the tat gene of HIV-1SF2 were designed to study target specificity and the potential effect of helix I mismatch on ribozyme efficacy both in vitro and in vivo. The first ribozyme (Rz1) targeted to the 5' splicing region of the tat gene was designed to cleave GUC*A. In HIV-1IIIB the A is changed to a G. The second ribozyme (Rz2) was targeted to the translational initiation region of the tat gene which is highly conserved among a variety of HIV-1 isolates, including both HIV-1SF2 and HIV-1IIIB. In vitro cleavage studies demonstrated that Rz1 efficiency cleaved HIV-1SF2 substrate RNA, but not HIV-1IIIB, presumably due to the base change from A to G. In contrast, Rz2 cleaved HIV-1SF2 or HIV-1IIIB substrate with equal efficiency. Both ribozymes were cloned into the 3' untranslated region of the neomycin gene (neo) within the pSV2neo vector and transfected into the SupT1 human CD4+ T cell line. Following selection, stable transfectants were challenged with either HIV-1SF2 or HIV-1IIIB virus. While Rz1-expressing cells were significantly protected from HIV-1SF2 infection, they exhibited no protection when infected with HIV-1IIIB virus. In contrast, Rz2 was effective in inhibiting the replication of both HIV-1SF2 and HIV-1IIIB in SupT1 cells. Expression of both ribozymes in these cells was demonstrated by Northern analysis. RT-PCR sequencing analysis confirmed the respective HIV-1 target sequence integrity. These data demonstrate the importance of the first base pair distal to the XUY within helix I of the hammerhead structure for both in vitro and in vivo ribozyme activities and imply that the effectiveness of the anti-HIV-1 ribozymes against appropriate target sequences is due to their catalytic activities rather than any antisense effect.

The expression of antisense and ribozyme genes targeting citrus exocortis viroid in transgenic plants.

Four ribozyme and antisense genes targeting citrus exocortis viroid (CEVd) positive- and negative-strand RNA molecules were constructed and used to transform the tomato Lycopersicon lycopersicum cv. UC82B. The tomato is a readily transformable plant and will support replication of CEVd following mechanical inoculation. The ribozyme genes contained three hammerhead catalytic motifs with long hybridizing arms and synthetic RNA transcripts were shown to cleave the target CEVd RNA molecule in vitro. Homozygous transgenic plants were produced from independent transformants expressing either ribozymes or antisense constructs. Inoculation of transgenic seedlings expressing antisense constructs targeting the negative-strand CEVd RNA molecule with CEVd resulted in a moderate reduction in the accumulation of CEVd RNA. In contrast, similarly inoculated transgenic plants expressing constructs targeting the positive-strand CEVd RNA molecule resulted in an increase in the rate of CEVd RNA accumulation. Addition of the ribozyme motifs to the antisense genes did not enhance their efficiency in the suppression of viroid replication and a moderation or elimination of the observed antisense effects was seen in plants expressing the corresponding catalytic RNA-encoding genes.

Characterisation of the subgenomic RNAs of an Australian isolate of barley yellow dwarf luteovirus.

We have characterised the subgenomic RNAs of an Australian isolate of BYDV-PAV. Northern blot analyses of infected plants and protoplasts have shown that this isolate synthesizes three subgenomic RNAs. Precise mapping of the transcription start sites of all three subgenomic RNAs and translational analyses of subgenomic RNA 2 and 3 have revealed a number of features. First, the transcription start site of subgenomic RNA 1 in this isolate differs markedly from the start site determined for an Illinois isolate of BYDV-PAV. Second, the start sites of subgenomic RNA 1 and 2 occur at a sequence that closely resembles the 5' end sequence of the genomic RNA (5'AGUGAAGA). Third, subgenomic RNA 2 appears to express ORF 6 of BYDV-PAV but the gene product is truncated due to the appearance of a new stop codon in the sequence. Last, subgenomic RNA 3, which is abundantly transcribed and encapsidated by the virus particle, appears to have no coding ability. We postulate that this novel subgenomic RNA has a regulatory function.

Artificial ribozyme and antisense gene expression in Saccharomyces cerevisiae.

A sensitive, quantitative reporter gene-based experimental system for the in vivo analysis of hammerhead ribozyme and antisense gene function in Saccharomyces cerevisiae is described. The system was constructed to test the activity of ribozyme and antisense genes targeting the chloramphenicol acetyltransferase gene (CAT) in both a cis and trans configuration relative to the target. When both target and ribozyme or antisense genes were transcribed in the same mRNA from an expression vector, CAT expression was reduced by up to 90%. Although the cis-positioned ribozyme molecule cleaved the target RNA in vitro, the steady state RNA levels of these chimeric transcripts were increased several fold relative to control mRNAs. This observation indicates a mechanism of suppression of CAT gene expression other than duplex-dependent degradation of mRNA. When the ribozyme or antisense genes were transcribed in trans from a plasmid-based expression vector, expression of a CAT gene integrated into a chromosome was unaffected. The effect of the cis-located RNA molecules may be dependent on an interaction requiring co-localization of ribozyme or antisense and target mRNAs during or immediately after target gene transcription. The failure of such a co-localization of these RNAs when synthesized in trans may contribute to the lack of efficacy seen in the trans-acting ribozymes or antisense RNAs. These observations are consistent with other studies reporting inefficient trans-acting ribozyme and antisense activity in S. cerevisiae.

Evolution and replication of tobacco ringspot virus satellite RNA mutants.

The replication properties of linker insertion-deletion mutants of tobacco ringspot virus satellite RNA have been studied by amplification in plants infected with the helper virus. Sequence analysis of the cDNAs corresponding to the replicated forms shows that only one of the original mutated molecules replicates unaltered, and in general new variants accumulate. Depending on the location of the original mutation three types of sequence modifications were observed: (i) deletion of the mutated region followed by sequence duplication, (ii) sequence duplication and deletion outside of the mutated region and (iii) limited rearrangements at the site of mutation. The mutant that replicates without sequence changes accumulates linear multimeric forms suggesting that self-cleavage is affected although the sequence alteration does not involve the hammerhead catalytic domain. Alternative RNA conformations are likely to play a role in the origin of this phenotype and in the formation of sequence duplications. These results demonstrate the great structural flexibility of this satellite RNA.

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.

A ribozyme that enhances gene suppression in tobacco protoplasts.

Site-directed mutagenesis has been used to produce two hammerhead ribozyme molecules targeting the chloramphenicol acetyltransferase gene (CAT). One ribozyme has a single catalytic domain between two 12-nucleotide arms that can hybridize 5' and 3' of the GUC target site of the CAT RNA transcript. The second ribozyme is a full-length antisense RNA with four catalytic domains inserted along the length, each targeting a specific GUC site within the CAT mRNA. Our results show that both ribozymes can produce almost equivalent rates of cleavage of the CAT mRNA in vitro (T1/2 of 18 or 15 min, respectively). In tobacco protoplasts we show consistently greater gene suppression in the presence of the long ribozyme molecule, compared with the equivalent antisense (22% gene reduction for antisense compared with 44% with the long ribozyme). These results suggest that hammerhead ribozymes may be developed for the inactivation of gene activity in plant cells.

Extended target-site specificity for a hammerhead ribozyme.

In vitro mutagenesis has been used to systematically mutate the GUC target site cleaved by a synthetic ribozyme based on the catalytic domain of the satellite RNA of tobacco ringspot virus. Amongst the spectrum of changes, it is found that GUC, UUC, CUC, GUA and GUU targets show equivalent rates of cleavage. An AUC target does not cleave, in contrast to observations from other studies. For a GUG target site, the normal ribozyme cannot induce cleavage, but an alteration of the stem-loop in the catalytic domain leads to the formation of a weakly active ribozyme. Certain double mutations, not previously studied, showed slow but discernable cleavage. This mutational approach shows that general rules for cleavage at NUY triplets for the target site of hammerhead ribozymes should be modified. Not all NUY targets cleave under all circumstances, and there are some targets with nucleotides other than U in the centre position which show significant, discernable cleavage.

Breast reconstruction using latissimus dorsi island flap without an endoprosthesis.

Breast reconstruction using a modified latissimus dorsi island flap is presented. The whole muscle is raised on its vascular pedicle, folded beneath the skin island and sutured in layers, thus substituting the volume of an endoprosthesis. The method may give satisfactory results in carefully selected cases (mostly in women with a small contralateral breast). It can also be applied in reconstruction after subcutaneous mastectomy.