Biomedical subjects

H L Sänger

Publications and source records attributed to H L Sänger.

At least 19 recordsLinked to original sources

PubMed1999

Heavy de novo methylation at symmetrical and non-symmetrical sites is a hallmark of RNA-directed DNA methylation.

Previous analysis of potato spindle tuber viroid (PSTVd) RNA-infected tobacco plants has suggested that an RNA-DNA interaction could trigger de novo methylation of PSTVd transgene sequences. Using the genomic sequencing technique, the methylation pattern associated with the RNA-directed DNA methylation process has been characterized. Three different PSTVd transgene constructs all showed a similar pattern of methylation. Most of the cytosines at symmetrical as well as non-symmetrical positions appeared to be methylated in both DNA strands of the viroid sequences. Heavy methylation was mostly restricted to the viroid cDNA sequences. Flanking DNA regions immediately adjacent to the viroid cDNA displayed a lower but significant level of cytosine methylation. The observation that the heavy methylation was essentially co-extensive with the length of the PSTVd cDNA sequences provided evidence that a direct RNA-DNA interaction can act as a strong and highly specific signal for de novo DNA methylation. These data also confirmed that de novo methylation was not limited to canonical CpG and CpNpG sites, but can also involve all the cytosine residues located in the genomic region where the RNA-DNA interaction takes place.

Base Sequence↗

PubMed1998

The dimerization domain of potato spindle tuber viroid, a possible hallmark for infectious RNA.

Covalently closed circular (+) RNA of the potato spindle tuber viroid (PSTVd) can efficiently dimerize noncovalently upon heating and slow cooling in the presence of monovalent cations or Mg2+. In vitro transcription of subgenomic fragments reveals that the ability to dimerize resides in the "upper strand" of its self-complementary rod-like structure. Nuclease probing of these fragments, namely, molecules spanning either the upper or the lower strand of PSTVd, confirms the existence of the previously proposed hairpins I-III, of which hairpin I might contain noncanonical G.A and A.A base pairs. In addition, the upper and lower (+) strands contain large hairpin loops consisting of stretches rich in either adenosine or uridine. Dimerization of the upper (+) strand results in a nuclease-resistant core encompassing hairpin I and is inhibited by an antisense oligonucleotide spanning the entire hairpin; this palindromic domain thus represents the dimerization site. When upper and lower strands were heated and cooled together, no annealing to a viroid-like duplex of both molecules occurs, only dimerization of the upper strand. Therefore, the dimerization hairpin of viroid RNA represents a unique conformational signal that is homologous to similar regions in the human immunodeficiency virus and other retroviruses.

Base Sequence↗

PubMed1998

Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato.

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.

Amino Acid Sequence↗

PubMed1996

A single nucleotide substitution converts potato spindle tuber viroid (PSTVd) from a noninfectious to an infectious RNA for nicotiana tabacum.

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.

Base Composition↗

PubMed1996

Secondary structure probing of potato spindle tuber viroid (PSTVd) and sequence comparison with other small pathogenic RNA replicons provides evidence for central non-canonical base-pairs, large A-rich loops, and a terminal branch.

Using PCR and in vitro transcription, linear (non-circular) unit-length (+)strand RNA molecules of a lethal PSTVd variant were produced which are able to initiate typical disease symptoms when inoculated into tomato. Non-denaturing gel electrophoresis shows that these transcripts can adopt the same two conformations as circular PSTVd molecules, namely a fast migrating rod-like and a slowly migrating cruciform structure. The rod-like conformer of two end-labelled transcripts was probed with nucleases and dimethyl sulphate, revealing that in solution its right part is identical to computer prediction. In the left part, however, three unique features could be substantiated. (1) In the central region a UV-cross-linkable loop is closed and thus contains non-canonical base-pairs ("loop E structure"). (2) Three large "pre-melting loops" are present at 25 degrees to 37 degrees C. The structure of the leftmost one, which is A-rich and conserved in most viroids, correlates with pathogenicity. (3) Two small stem-loops instead of an unbranched structure are found at the left terminus. These hairpins can form in all "large" viroids (approximately 300 nucleotides or longer), thus placing the dodecamer conserved among these viroids, GGUUCCUGUGGU, within the upper helix and the branch junction. A large viroid from Iresine lacks one of these hairpins, whereas all "small" viroids (approximately 300 nucleotides or smaller) lack both. In several plant virus satellite RNAs and the newt satellite RNA, the motif GAUUU(U) and dodecamer remnants appear in an equivalent structure comprising two or three hairpins. Using lead- and terbium-induced cleavage of the RNA, metal binding sites were found, mostly in loops. Thus, probing of PSTVd RNA and comparison with other.

Base Composition↗

PubMed1996

Molecular analysis of the gene family of the signal recognition particle (SRP) RNA of tomato.

The sequence variants of the signal recognition particle (SRP) RNA gene family from four tomato cultivars have been isolated and characterized which indicated the existence of SRP RNA pseudogenes. Sequence analysis revealed two conserved sequence motifs in the upstream region, a TATA-like box and an upstream sequence element (USE), 'TCCCACATCG', both located at a conserved distance to the transcription start point. These elements are identical to the DNA-dependent RNA polymerase III (pol III)-specific promoters of U-rich small nuclear RNA (UsnRNA) genes of plants. Moreover, T-rich stretches are found at the 3' end of the coding regions of the SRP RNA genes which could act as typical pol III termination signals. These findings and recent results from site-directed mutation analysis of the SRP RNA genes from Arabidopsis thaliana indicate that, in contrast to mammalian systems, plant pol III SRP RNA genes are most probably regulated by external promoter elements. According to the identical promoter organization between plant U3-, U6snRNA, MRP-like RNA and SRP RNA genes, one can group these genes into the 'pol III(EXT)USE' subclass of externally regulated USE-dependent pol III genes.

Base Sequence↗

PubMed1996

A new sequence variant of Coleus blumei viroid 3 from the Coleus blumei cultivar 'Fairway Ruby'.

A viroid was isolated from symptomless Coleus blumei cultivar (cv) 'Fairway Ruby' plants using the bidirectional PAGE method for analysis of small circular RNA molecules. The viroid was transmitted to viroid-free plants of Coleus blumei cv. 'Scarlet Dragonfly' by mechanical inoculation. Cloning and sequencing revealed that the viroid from the Coleus cv. 'Fairway Ruby' is closely related to the Coleus blumei viroid 3-Bv (CbVd 3-Bv) isolated from the Coleus cv. 'Bienvenue'. Therefore, the new viroid sequence variant has been named Coleus blumei viroid 3-FR (CbVd 3-FR). Coleus blumei viroid 3-FR consists of 364 nucleotides, 202 G + C, 162 A + U with a GC content of 55.5%. The most stable rod-like secondary structure of this viroid has 82 G:C, 50 A:U and 10 G:U base pairs with a minimum free energy of -670.5 kJ/mol (-160.4 kcal/mol). Due to mutations that increase the stability of the rod-like secondary structure, CbVd 3-FR has a lower minimum free energy than CbVd 3-Bv (-598.2 kJ/mol; -143.1 kcal/mol). A base-exchange in the upper strand of the central domain of CbVd 3-FR may stabilize the basal helix of hairpin I.

Base Sequence↗

PubMed1996

A viroid from Solanum pseudocapsicum closely related to the tomato apical stunt viroid.

A viroid was isolated from symptomless Solanum pseudocapsicum cultivar (cv) 'New Patterson' plants using the bidirectional PAGE method for analysis of small circular RNA molecules. The viroid was transmitted to tomato by mechanical inoculation. Infected tomato plants developed symptoms similar to those caused by potato spindle tuber viroid (PSTVd). Cloning and sequencing revealed that the viroid from Solanum pseudocapsicum is closely related to the tomato apical stunt viroid (TASVd). Therefore, the new viroid sequence variant has been named TASVd-S. The circular RNA of TASVd-S consists of 360 nucleotides which can potentially form a rod-like structure with a high degree of base-pairing like all the other known viroids. The nucleotide composition is 99 G, 72 A, 101 C and 88 U, corresponding to 200 G + C, 160 A + U with a GC content of 55.6%. In total 71% of the residues are base-paired and the basepaired residues consist of 73 G:C (57%), 43 A:U (34%) and 11 G:U (9%) base pairs. The most stable rod-like secondary structure of this viroid has 80 G:C, 39 A:U and 11 G:U base pairs with a minimum free energy of -147.5 kcal/mol (-616.6 kJ/mol). The sequence similarity of the left terminal (T1) domain of TASVd-S and the T1 domain of tomato planta macho viroid (TPMVd) is higher than the sequence similarity of these domains of TASVd-S and TASV-d.

Base Sequence↗

PubMed1995

Characterization of the signal recognition particle (SRP) RNA population of tomato (Lycopersicon esculentum).

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.

Base Sequence↗

PubMed1994

An infectious viroid RNA replicon evolved from an in vitro-generated non-infectious viroid deletion mutant via a complementary deletion in vivo.

The 359 nucleotides (nt) long potato spindle tuber prototype viroid (PSTVd) is sensitive to experimentally introduced mutations as the substitution or deletion of a single nucleotide usually abolishes its infectivity, although certain sequence alterations are tolerated. This is illustrated by the fact that viroid progeny can evolve in planta upon inoculation with substitution mutants generated in vitro, and by the existence of genetically stable 356-360 nt long PSTVd field isolates. However, to date, no viable in vitro-generated deletion mutant of PSTVd has been reported. We have now found a 341 nt long infectious PSTVd RNA replicon that evolved in agrotransformed plants transformed with the dimeric form of an in vitro-deleted, non-infectious 350 bp long PSTVd cDNA unit by an additional complementary deletion of 9 nt in vivo. This is the first report that the deletion-abolished infectivity of a viroid is restored by an additional deletion that concurrently restabilized its perturbed secondary structure by abandoning an internal segment of the rod-like molecule. The fact that approximately 5% of the total PSTVd RNA genome was deleted demonstrates that the maintenance of this viroid-specific rod-like structure is not only essential for nuclease protection but also for the infectivity, i.e. transmissibility, replicability, processibility and pathogenicity of these minimal infectious agents.

Base Sequence↗

PubMed1994

Structural and functional characterisation of the signal recognition particle-specific 54 kDa protein (SRP54) of tomato.

Two representative genes for the 54 kDa protein subunit of the signal recognition particle (SRP54) of tomato were cloned. It was shown that both genes are expressed in the tomato cv. Rentita. SRP54 is encoded by nine exons distributed over 10 kb of genomic sequence. The amino acid sequences deduced for the two SRP54 genes are 92% identical and the calculated protein size is 55 kDa. Like the homologous proteins isolated from other eukaryotes, the tomato SRP54 is evidently divided into two domains. As deduced from sequence motif identity, the N-terminally located G-domain can be assumed to have GTPase activity. The C-terminal part of the protein is methionine rich (14% methionine) and represents the M-domain. In in vitro binding experiments, SRP54 of tomato was able to attach to the 7S RNA of tomato, its natural binding partner in the SRP. This interaction can only take place in a trimeric complex consisting of 7S RNA, SRP54 and SRP19. The latter protein subunit of the SRP complex is assumed to induce a conformational change in the 7S RNA. The human SRP19 was able to mediate the binding of the tomato SRP54 to the 7S RNA, irrespective of whether this latter originated from tomato or man.

Amino Acid Sequence↗

PubMed1994

Proteasome-associated RNAs are non-specific.

The RNA isolated from RNase-treated proteasome preparations from human erythrocytes, HeLa cells, the archaeon Thermoplasma acidophilum and also from recombinant proteasomes of T. acidophilum expressed in Escherichia coli was characterized. The RNA associated with structurally similar protein particles, namely with the two molecular chaperones, groEL from E. coli and with the thermosome from T. acidophilum, served as controls. Electrophoretic analysis on polyacrylamide gels of the radioactively end-labelled RNA revealed a very similar size distribution pattern, irrespectively of the protein particles from which they had been isolated. The predominant RNA species were in the size ranges 80 nucleotides and 120 nucleotides, respectively. Partial sequencing of their terminal regions by mobility-shift analysis revealed that, of the proteasomes from human erythrocytes, the approximately 80-nucleotide-long RNA consists of a heterogenous population of mostly tRNA species because they carried the tRNA-specific 3'-terminal sequence motif 5'-CCA-3'. The RNA in the size range 120 nucleotides isolated from the proteasomes of human erythrocytes and of T. acidophilum was also heterogeneous and displayed, in the terminal regions, a remarkable sequence similarity to the corresponding regions of the 5S rRNA from the same and different organisms. The total content of RNA of all the protein particles was quantified and found to be consistently sub-stoichiometric. All these findings strongly suggest that RNA associated with the proteasomes and with the molecular chaperones originate from the abundant cellular pool of the tRNAs and 5S rRNAs which bind non-specifically to these large protein particles.

Bacterial Proteins↗

PubMed1994

RNA-directed de novo methylation of genomic sequences in plants.

One monomeric and three oligomeric potato spindle tuber viroid (PSTVd) cDNA units were introduced into the tobacco genome via the Agrobacterium-mediated leaf-disc transformation. Southern analysis of the integrates revealed that only their PSTVd-specific sequences become fully methylated, whereas the flanking T-DNA and the genomic plant DNA remain unaltered. Viroid cDNA methylation could only be observed after autonomous viroid RNA-RNA replication had taken place in these plants. These findings demonstrate that a mechanism of de novo methylation of genes might exist that can be induced and targeted in a sequence-specific manner by their own mRNA.

Base Sequence↗

PubMed1994

Gel dependence of electrophoretic mobilities of double-stranded and viroid RNA and estimation of the contour length of a viroid by gel electrophoresis.

Double-stranded (ds) RNA normally exhibits a lower electrophoretic mobility than dsDNA having the same number of base pairs. This has been attributed to its net charge density that is lower than that of B-form DNA. But we show here that dsRNA runs faster than corresponding DNA in gels containing either > or = 2.5% agarose or > or = 8% acrylamide with high crosslinking (19:1 acrylamide:N,N'-methylenebisacrylamide). However, the relative mobility of dsRNA as compared with DNA, extrapolated to 0% gel (0%T), remains constant (0.90 +/- 0.03) in all systems, in support of the charge density hypothesis. In comparison to dsRNA standards, the potato spindle tuber viroid, a small approximately 70% base-paired rod-like pathogenic RNA, is strongly retarded, presumably because of greater flexibility and/or stable curvature. Depending on the gel system, nonlinear extrapolation to 0% T leads to an apparent contour length of 140-230 bp, whereas 130 +/- 20 bp can be determined from electron micrographs and 123-126 bp from secondary structure modeling. We attribute the variation of the electrophoretic behavior of both dsRNA and viroid RNA to interactions with the gel matrix. Nevertheless, extrapolation of the apparent contour length (in bp dsRNA) determined from low-crosslinked polyacrylamide gels (2.6%C) is comparable to the determination by alternative methods.

Amino Acids↗

PubMed1993

RNA-directed RNA polymerase from tomato leaves. I. Purification and physical properties.

An RNA-directed RNA polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue was purified to electrophoretic homogeneity. A terminal transferase activity that was found to cofractionate with RdRP from DEAE-Sepharose and DNA-cellulose columns was removed by chromatography on a Mono Q column. The highly purified RdRP exhibits a specific activity of 500 nmol x mg-1 x 30 min-1, which corresponds to a 100,000-fold enrichment of the enzyme. In buffer containing 50% glycerol, its activity decreased by about 15%/month. RdRP activity coincided with the silver staining intensity of a single 128-kDa polypeptide when the fractions eluted from the Mono Q column were analyzed by electrophoresis in a SDS-polyacrylamide gel. Its molecular mass and its sedimentation coefficient of 6.6 S indicate that RdRP is a nearly globular molecule. The catalytic activity of RdRP is resistant to alpha-amanitin and actinomycin D. In tomato leaves systemically infected with potato spindle tuber viroid, the activity of RdRP was found to be increased about 3-fold compared with RdRP isolated from healthy leaf tissue.

Chromatography, Affinity↗

PubMed1993

RNA-directed RNA polymerase from tomato leaves. II. Catalytic in vitro properties.

The catalytic properties of electrophoretically homogeneous RNA-directed RNA polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue were studied with the aid of oligonucleotides of defined sequence. It was found that RdRP catalyzes in vitro the transcription of short single-stranded RNA and DNA molecules into precisely complementary RNA copies up to the full length of these templates. The transcription of RNA- and DNA-oligonucleotide templates was equally effective. Differences in transcription efficiency were found to depend on nucleotide sequence rather than on the RNA or DNA nature of the single-stranded nucleic acid. Double-stranded nucleic acids such as poly(A).poly(U) and a double-stranded DNA 14-mer were not transcribed. The RdRP-directed transcription could be primed because RNA and DNA dinucleotides and trinucleotides complementary to the 3'-terminal nucleotides of the template were extended by the enzyme. The unprimed transcription was shown to start preferentially at the 3'-terminal nucleotides of the template. RdRP is capable of adding a single noncomplementary nucleotide to the 3' terminus of about 50% of the runoff transcripts. AMP was preferred over GMP, whereas CMP and UMP were terminally added at very low frequency.

Base Sequence↗

PubMed1992

Structural requirements for viroid processing by RNase T1.

Viroids are replicated via a rolling circle-like mechanism in which (+) strand oligomeric intermediates have to be cleaved enzymatically to unit-length molecules followed by ligation to mature circles. A transcript of potato spindle tuber viroid, which is still infectious, consists of a monomeric molecule with only 22 additional nucleotides, thus doubling part of the central conserved region of viroids. It was shown that this transcript can be cleaved and ligated in vitro to circles by RNase T1. To elucidate the site and mechanism of processing, 16 different site-specific mutants of this longer-than-unit-length transcript were constructed and analyzed by in vitro processing with RNase T1, infectivity studies, temperature-gradient gel electrophoresis, and structure calculations. The wild-type sequence and several mutated transcripts are able to adopt a particular secondary structure which is the prerequisite for enzymatic cleavage and ligation by RNase T1. This "processing structure" exposes both potential cleavage sites in the nearest spatial neighborhood, thus favoring the subsequent ligation to circles. Those mutated sequences for which the formation of the processing structure is impossible or thermodynamically highly unfavored are not processed. The results demonstrate that the particular structural features of viroids enable them to be cleaved and ligated by one and the same enzyme, RNase T1. The in vitro mechanism may serve as a mechanistic model for cellular processing of viroids.

Base Sequence↗

PubMed1992

Sequence analysis of five new field isolates demonstrates that the chain length of potato spindle tuber viroid (PSTVd) is not strictly conserved but as variable as in other viroids.

The sequence analysis of five new field isolates of potato spindle tuber viroid (PSTVd) of different virulence revealed that the length of their RNA chain is not strictly conserved to 359 nucleotides (nts), as one could have inferred from the previously sequenced PSTVd strains. It was now found that the chain length is strain-specific like in the case of practically all other viroids, and that it may vary, so far, between 356 and 360 nts. Taking our previously sequenced and least virulent mild strain PSTVd KF6-M as standard, the new mild strains PSTVd WA-M and PSTVd F-M differ from it by one or two nts. The new intermediate-severe strains PSTVd F-IS and PSTV-F 88-IS differ from the standard mild strain by eight and nine nts, respectively, whereas the new severe-lethal strain PSTVd F-SL differs in seven nts. Most of these mutations are located within the virulence-modulating (VM) region and within the variable region (VR), and only in two strains a single mutation is found in the right terminal domain.

Base Composition↗