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The dystonias are a common clinically and genetically heterogeneous group of movement disorders. More than ten loci for inherited forms of dystonia have been mapped, but only three mutated genes have been identified so far. These are DYT1, encoding torsin A and mutant in the early-onset generalized form, GCH1 (formerly known as DYT5), encoding GTP-cyclohydrolase I and mutant in dominant dopa-responsive dystonia, and TH, encoding tyrosine hydroxylase and mutant in the recessive form of the disease. Myoclonus-dystonia syndrome (MDS; DYT11) is an autosomal dominant disorder characterized by bilateral, alcohol-sensitive myoclonic jerks involving mainly the arms and axial muscles. Dystonia, usually torticollis and/or writer's cramp, occurs in most but not all affected patients and may occasionally be the only symptom of the disease. In addition, patients often show prominent psychiatric abnormalities, including panic attacks and obsessive-compulsive behavior. In most MDS families, the disease is linked to a locus on chromosome 7q21 (refs. 11-13). Using a positional cloning approach, we have identified five different heterozygous loss-of-function mutations in the gene for epsilon-sarcoglycan (SGCE), which we mapped to a refined critical region of about 3.2 Mb. SGCE is expressed in all brain regions examined. Pedigree analysis shows a marked difference in penetrance depending on the parental origin of the disease allele. This is indicative of a maternal imprinting mechanism, which has been demonstrated in the mouse epsilon-sarcoglycan gene.

A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed. INTRODUCTIONRNA-directed RNA polymerase (RdRP) from healthy tomato leaf tissue seems to represent a plant-specific and hence exceptional nucleic acid-synthesizing enzyme because higher plants are the only eukaryotes in which the presence of a cellular RdRP has been unambiguously demonstrated to date (for discussion, see Schiebel et al., 1993aSchiebel et al., , 1993b. RdRP activity has been detected in Chinese cabbage (Astier-Manifacier and Cornuet, 1971), cauliflower (Astier-Manifacier and Cornuet, 1978), tobacco (Duda et al., 1973;Duda, 1979;Takanami and Fraenkel-Conrat, 1982), tomato (Boege and Sänger, 1980), cowpea (Dorssers et al., 1982), and cucumber (Khan et al., 1986), but only the RdRP from tomato leaf tissue has been isolated and characterized with respect to its physicochemical (Schiebel et al., 1993a) and in vitro catalytic (Schiebel et al., 1993b) properties. These cellular RdRPs should not be mistaken for RNA-dependent RNA polymerases (EC 2.7.7.48), which become detectable when bacteria and eukaryotes are infected with RNA viruses. RNA-dependent RNA polymerases mediate viral RNA replication and are therefore much more appropriately called virus RNA replicases.Despite all of these studies, the origin and the actual biological function(s) of plant-encoded RdRP have remained unresolved and are enigmatic because its cognate template(s) and in vivo transcription products remain unknown. Nevertheless, we surmised (Schiebel et al., 1993b) that in the cell, RdRP might be of paramount importance because it transcribes from corresponding RNA sequences small RNA molecules that control the synthesis of nucleic acids and their translation into proteins.Studies on the induction of a highly specific antiviral state in transgenic plants led to a hypothesis that cellular RdRP c...

Mechanical inoculation of Nicotiana tabacum with the PSTVd isolate KF 440-2 from the host plant tomato resulted in the de novo emergence, replication, and accumulation of a new "tobacco variant," designated PSTVd NT. It produces no symptoms in tobacco but, like PSTV KF 440-2, severe ones in tomato. The sequence analysis of PSTVd NT revealed a single nucleotide substitution from C-->U at position 259. Autonomous viroid replication was also induced in tobacco by genomic integration of oligomeric cDNA copies of PSTVd KF 440-2. Although these cDNAs contained the original tomato-specific C259, the circular PSTVd RNA subsequently accumulating in tobacco also exhibited the C259-->U259 substitution. In the secondary structure of PSTVd, nucleotide 259 is part of an internal loop analogous to loop E of eukaryotic 5S rRNA and presumed to be the only bulged extrahelical nucleotide of this loop. The C259 in PSTVd KF 440-2 and in practically all other isolates and the U259 in PSTVd NT of the loop E-like structure might be involved in protein binding and in viroid processing. The new variant PSTVd NT is genetically stable in both tobacco and tomato.

Molecular cloning of 30 cDNAs and subsequent characterization of the corresponding SRP RNA from four cultivars of tomato (Lycopersicon esculentum) revealed altogether 14 sequence variants, which could be ordered into six groups. The expression of five representatives from these groups was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) in different cultivars and different tissues. Although one cultivar-specific SRP RNA variant could be detected in the leaf SRP RNA population, identical SRP RNA populations seem to be present in the four different cultivars as well as in different tissues, such as leaves, flowers, fruits, stems and roots. Sequence comparison revealed that several variants might have evolved by recombination of two different SRP RNA sequences. On the basis of five SRP RNA variants, the current secondary structure model was refined and a new conserved structural element was detected. Comparative sequence analysis of domain II from all known SRP RNA homologues reveals a remarkable conservation of this element. As demonstrated previously, the corresponding area overlaps with a region that interact with the SRPp68/p72 heterodimer and/or with ribosomes. Based on structural and functional considerations, we propose that the domain IV structure together with the highly conserved area of domain II constitutes the essential core of the SRP RNA.