Biomedical subjects

M Tabler

Publications and source records attributed to M Tabler.

At least 37 records · Page 2Linked to original sources

PubMed1995

Influence of chromosomal position and copy number of a white-directed ribozyme gene on the suppression of eye pigmentation in Drosophila melanogaster.

Different strains of transgenic Drosophila melanogaster carrying one, two, or three copies of a heat-shock promoter 70 (hsp70)-driven catalytic antisense RNA gene, directed against the white gene, were investigated for the expression level of ribozyme RNA. It was found that the steady-state concentrations of the hammerhead ribozyme were proportional to the copy number of the genes and that the suppressive effect on eye pigment accumulation was dosage dependent. In a further experiment, a D. melanogaster strain, deficient in eye pigmentation caused by a deletion of the white gene, was used for P element-mediated germline transformation: the transposon used contained the hsp70-driven, white-directed ribozyme gene and, on the same DNA, the mini-white gene under its own promoter. The spatial coupling of the transcription of ribozyme and target RNA resulted in more effective ribozyme-mediated inhibition of eye pigmentation under heat-shock conditions. These effects were dependent on the chromosomal integration site of the transposon.

Animals↗

PubMed1994

The influence of imperfectly paired helices I and III on the catalytic activity of hammerhead ribozymes.

Several catalytic antisense RNAs directed against different regions of the genomic or antigenomic RNA of Sendai virus were constructed. All RNAs contained the same catalytic domain based on hammerhead ribozymes but some had deletions or mutations resulting in imperfect helices I and III. Pre-annealed substrate/ribozyme complexes were used to determine the rates of the cleavage process for the different ribozymes under single-turnover conditions. It was found that the sequence context surrounding the cleavable motif influenced the cleavage efficiencies. Deletions or mutations of nucleotides 2.1 or 15.1 and 15.2 according to the numbering system for hammerhead ribozymes of Hertel et al. destroyed catalytic activity. Deletions of nucleotide 2.2 or additional nucleotides in the helix I-forming region of the ribozyme did not destruct, but only reduced the cleavage efficiencies. Similar results were observed for a deletion of nucleotide 15.3. Simultaneous deletions within helices I and III resulted in alternative cleavage sites. The potential consequences for the specificity of the ribozyme reaction are discussed.

Base Sequence↗

PubMed1994

Extension of helix II of an HIV-1-directed hammerhead ribozyme with long antisense flanks does not alter kinetic parameters in vitro but causes loss of the inhibitory potential in living cells.

When designed to cleave a target RNA in trans, the hammerhead ribozyme contains two antisense flanks which form helix I and helix III by pairing with the complementary target RNA. The sequences forming helix II are contained on the ribozyme strand and represent a major structural component of the hammerhead structure. In the case of an inhibitory 429 nucleotides long trans-ribozyme (2as-Rz12) which was directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1), helix II was not pre-formed in the single-stranded molecule. Thus, major structural changes are necessary before cleavage can occur. To study whether pre-formation of helix II in the non-paired 2as-Rz12 RNA could influence the observed cleavage rate in vitro and its inhibitory activity on HIV-1 replication, we extended the 4 base pair helix II of 2as-Rz12 to 6, 10, 21, and 22 base pairs respectively. Limited RNase cleavage reactions performed in vitro at 37 degrees C and at physiological ion strength indicated that a helix II of the hammerhead domain was pre-formed when its length was at least six base pairs. This modification neither affected the association rate with target RNA nor the cleavage rate in vitro. In contrast to this, extension of helix II led to a significantly decreased inhibition of HIV-1 replication in human cells. Together with the finding of others that shortening of helix II to less than two base pairs reduces the catalytic activity in vitro, this observation indicates that the length of helix II in the naturally occurring RNAs with a hammerhead domain is already close or identical to the optimal length for catalytic activity in vitro and in vivo.

Base Composition↗

PubMed1994

A three-nucleotide helix I is sufficient for full activity of a hammerhead ribozyme: advantages of an asymmetric design.

Trans-cleaving hammerhead ribozymes with long target-specific antisense sequences flanking the catalytic domain share some features with conventional antisense RNA and are therefore termed 'catalytic antisense RNAs'. Sequences 5' to the catalytic domain form helix I and sequences 3' to it form helix III when complexed with the target RNA. A catalytic antisense RNA of more than 400 nucleotides, and specific for the human immunodeficiency virus type 1 (HIV-1), was systematically truncated within the arm that constituted originally a helix I of 128 base pairs. The resulting ribozymes formed helices I of 13, 8, 5, 3, 2, 1 and 0 nucleotides, respectively, and a helix III of about 280 nucleotides. When their in vitro cleavage activity was compared with the original catalytic antisense RNA, it was found that a helix I of as little as three nucleotides was sufficient for full endonucleolytic activity. The catalytically active constructs inhibited HIV-1 replication about four-fold more effectively than the inactive ones when tested in human cells. A conventional hammerhead ribozyme having helices of just 8 nucleotides on either side failed to cleave the target RNA in vitro when tested under the conditions for catalytic antisense RNA. Cleavage activity could only be detected after heat-treatment of the ribozyme substrate mixture which indicates that hammerhead ribozymes with short arms do not associate as efficiently to the target RNA as catalytic antisense RNA. The requirement of just a three-nucleotide helix I allows simple PCR-based generation strategies for asymmetric hammerhead ribozymes. Advantages of an asymmetric design will be discussed.

Base Sequence↗

PubMed1993

Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1.

The catalytic domain of a hammerhead ribozyme was incorporated into a 413 nucleotides long antisense RNA directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1) (pos. +222 to +634). The resulting catalytic antisense RNA was shown to cleave its target RNA in vitro specifically at physiological ion strength and temperature. We compared the antiviral effectiveness of this catalytic antisense RNA with that of the corresponding unmodified antisense RNA and with a mutated catalytic antisense RNA, which did not cleave the substrate RNA in vitro. Each of these RNAs was co-transfected into human SW480 cells together with infectious complete proviral HIV-1 DNA, followed by analysis of HIV-1 replication. The presence of the catalytically active domain resulted in 4 to 7 fold stronger inhibition of HIV-1 replication as compared to the parental antisense RNA and the inactive mutant. Kinetic and structural studies performed in vitro indicated that the ability for double strand formation was not changed in catalytic antisense RNA versus parental antisense RNA. Together, these data suggest that the ability to cleave target RNA is a crucial prerequisite for the observed increase of inhibition of the replication of HIV-1.

Base Sequence↗

PubMed1993

Attenuation of white gene expression in transgenic Drosophila melanogaster: possible role of a catalytic antisense RNA.

We have transformed Drosophila melanogaster with a DNA construct encoding a catalytic antisense RNA directed against the primary white gene RNA transcript. Total RNA isolated from transformed flies was shown to catalyze the specific cleavage of in vitro transcribed substrate RNA, indicating the expression of a functional ribozyme. Moreover, transgenic lines carrying homozygous copies of this construct depict a further clear-cut reduction in eye pigmentation when present in a genetic background that has a priori reduced levels of white gene expression.

Alleles↗

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

Processing of linear longer-than-unit-length potato spindle tuber viroid RNAs into infectious monomeric circular molecules by a G-specific endoribonuclease.

Different cDNA constructs were used for the in vitro synthesis of RNA transcripts that contain a complete monomeric unit of the potato spindle tuber viroid (PSTVd) plus an additional repeat of a part of the circular RNA genome. These permutated linear longer-than-unit-length PSTVd RNAs were incubated with the G-specific endoribonuclease RNase T1 which generated monomeric circular PSTVd RNA molecules that were infectious when mechanically inoculated to tomato plants. Besides the correct monomeric PSTVd RNA, smaller and larger circular RNAs were also formed during the reaction. The comparison of different transcripts revealed that correct in vitro processing of PSTVd RNA can proceed at alternative sites indicating that circularization is driven by RNA structure and not governed by a particular sequence. Based on these data, we propose a novel model for the processing of multimeric replicative viroid RNA intermediates through RNA cleavage and ligation catalyzed by a host endoribonuclease.

Base Sequence↗

PubMed1991

Catalytic antisense RNAs produced by incorporating ribozyme cassettes into cDNA.

A simple strategy is described for the generation of catalytic hammerhead-type ribozymes (Rz) that can be used as highly specific endoribonucleases to cleave a particular target RNA. The technique requires that a cloned cDNA fragment is available which encodes at least a part of the target RNA. About 25 different restriction recognition sequences can be utilized to incorporate specifically designed DNA cassettes into the cDNA. Besides some nucleotides which are specific for a certain restriction site, the DNA cassettes contain a sequence corresponding to the catalytic domain of the hammerhead Rz and, optionally, selectable marker genes, that are removable. The resulting recombinant DNA constructs permit the in vitro and in vivo synthesis of novel 'catalytic antisense RNAs' or 'antisense Rz (Az)', which combine two features: (i) they bind like antisense RNA to their specific substrate RNA, and (ii) they cleave their target as hammerhead Rz do. The utility of the strategy to generate Rz was demonstrated experimentally by incorporating a synthetic SalI-specific DNA ribozyme (Rz) cassette into a unique SalI site of a cloned cDNA fragment of plum pox virus (PPV), which is a single-stranded positive sense plant RNA virus, belonging to the group of potyviruses. The resulting Az constructs delivered Az that were directed against the PPV (+) or (-) RNA, respectively, which cleaved their corresponding target RNAs in the expected manner. Besides the synthetic Rz cassette, a comparable SalI-specific Rz cassette, that had been prepared from a specifically designed plasmid and that contained the tet gene inserted into the sequence of the catalytic domain of the Rz, was also incorporated into the SalI site of the PPV cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1991

Ribonuclease T1 generates circular RNA molecules from viroid-specific RNA transcripts by cleavage and intramolecular ligation.

A 406 nucleotide long potato spindle tuber viroid (PSTVd)-specific linear RNA transcript was synthesized in vitro and subjected to limited digestion with ribonuclease (RNase) T1. Under certain conditions this guanosine-specific endoribonuclease proved to be capable of processing the longer-than-unit-length, precursor-like viroid RNA transcript by cleaving out a linear 358 nucleotide long product and ligating that to a circular RNA molecule. The new finding that RNase T1 acts as an RNA processing enzyme and, in particular, as an RNA 'circulase' can be explained by the unique structural preconditions inherent in the viroid-specific substrate and by the well characterized two-step cleavage mechanism of the enzyme. These in vitro potentials of RNase T1 suggest that also in vivo procaryotic and eucaryotic RNases with a similar reaction mechanism might not only be involved in RNA degradation and trimming, but also in processing, ligation and recombination of RNA.

Base Sequence↗

PubMed1989

A microscale procedure for isolating and sequencing the viroid RNA present in one gram of infected leaf tissue.

A microscale procedure for the isolation and purification of viroid RNA from one gram of viroid-infected leaf tissue and for its subsequent sequencing at the cDNA level is described using potato spindle tuber viroid (PSTV) as model system. Total nucleic acids are phenol-extracted and salt-fractionated with 2 M LiCl. The viroid-containing fraction is then subjected to bidirectional polyacrylamide gel electrophoresis. This removes all co-fractionated cellular RNAs from the circular viroid RNA which is finally recovered from the gel in pure form by isotachophoresis. Thus, from one gram of PSTV-infected tomato leaf tissue, about 100-250 ng of circular PSTV RNA can be obtained and used as template for several DNA primer-directed reverse transcription reactions. From the primer-extended overlapping cDNAs the entire sequence of the viroid progeny synthesized in an individual plant or plant leaf can thus be established by Maxam-Gilbert sequencing. This renders the procedure especially suited for the routine analysis of the in vivo fate of viroid mutants constructed in vitro.

Autoradiography↗

PubMed1987

Linear oligomeric potato spindle tuber viroid (PSTV) RNAs are accurately processed in vitro to the monomeric circular viroid proper when incubated with a nuclear extract from healthy potato cells.

A nuclear extract for the processing of oligomeric viroid RNA in vitro has been prepared from nuclei isolated from healthy potato cells grown in suspension culture. Linear RNA molecules containing concatameric units of (+) or (-) strands, respectively, of the potato spindle tuber viroid (PSTV) were synthesized in vitro with the aid of the SP6 RNA polymerase and used as substrates for processing. When oligomeric linear PSTV (+)RNAs are incubated with the nuclear extract, monomeric linear molecules are accurately excised from them, and ligated to monomeric PSTV (+)RNA circles representing the viroid proper. Oligomeric PSTV (-)RNAs are likewise processed but with a much lower efficiency. Viroid-processing operates although other nucleolytic activities are still present in the extract. These results substantiate our previous finding that oligomeric PSTV does not process autocatalytically under in vitro conditions where certain introns and other RNAs do. This is the first report of an in vitro RNA processing system derived from higher plants.

Journal Article↗

PubMed1986

Structure of viroid replicative intermediates: physico-chemical studies on SP6 transcripts of cloned oligomeric potato spindle tuber viroid.

The structure and structural transitions of transcripts of cloned oligomeric viroid were studied in physico-chemical experiments and stability calculations. Transcripts of (+) and (-) polarity, from unit up to sixfold length, were synthesized from DNA clones of the potato spindle tuber viroid (PSTV) with the SP6 transcription system. Their structural properties were investigated by optical denaturation curves, high performance liquid chromatography (HPLC), electron microscopy, sedimentation-diffusion equilibrium and velocity sedimentation. Secondary structures of the RNAs and theoretical denaturation curves were calculated using an energy optimization program. The secondary structure of lowest free energy for unit length and oligomeric transcripts is a rod-like structure similar to that of the mature circular viroids. When this structure is used as a model for calculations, there is a large degree of agreement between the theoretical and the experimental denaturation curves. At high temperatures, however, (+) strand transcripts exhibited a transition which was more stable than expected from the calculations or than was known from curves of mature viroids. This transition arises from a rearrangement of the central conserved region of viroids to a helical region of 28 stable base pairs either intermolecularly leading to bimolecular complexes, or intramolecularly giving rise to a branched secondary structure. The rearrangement could be detected by electron microscopy, HPLC, and analytical ultracentrifugation. The helical region serves to divide up the oligomeric (+) strand into structural units which may be recognized by cleavage and ligation enzymes which process the oligomeric intermediates to circular mature viroids.

Microscopy, Electron↗

PubMed1985

Infectivity studies on different potato spindle tuber viroid (PSTV) RNAs synthesized in vitro with the SP6 transcription system.

We have constructed two sets of clones in which one to six head-to-tail connected DNA copies of the potato spindle tuber viroid (PSTV) RNA genome were inserted into the plasmid pSP62- Pl downstream of the promoter for SP6 RNA polymerase. In vitro transcription of these constructs with the promoter-specific SP6 RNA polymerase yielded the corresponding oligomeric single-stranded linear PSTV RNA molecules of (+) and (-) polarity. Except for short vector-derived terminal sequences these in vitro synthesized PSTV RNA forms are equivalent to the RNA intermediates of the PSTV replication cycle which are present in vivo only in extremely low concentrations. From each DNA template molecule up to 600 RNA copies could be transcribed in vitro and yields > 100 microg were obtained. When mechanically inoculated to tomato seedlings the PSTV (+) RNA oligomers were as infectious as the natural PSTV (+) RNA monomers. Surprisingly, the corresponding oligomeric PSTV (-) RNAs wer approximately 10(4)-fold less infectious. However, when these (-) RNAs were partially protected prior to inoculation by mixing or hybridizing them with non-infectious (+) RNA fragments or by 'capping' their 5' terminus, an increase in the number of infections was observed. The in vitro synthesis of infectious RNA from cloned cDNA means that, in principle, it should be possible to develop vector systems from pathogens with RNA genomes.

Cloning, Molecular↗

PubMed1985

Molecular cloning of potato spindle tuber viroid (PSTV) cDNA synthesized by enzymatic elongation of PSTV-specific DNA primers: a general strategy for viroid cloning.

Different cDNAs were synthesized by primer extension from the RNA of the severe strain KF 440 of potato spindle tuber viroid (PSTV) with the aid of reverse transcriptase using three PSTV-specific DNA molecules as primers. The cDNAs were made double-stranded and cloned into plasmid pBR 322. Various overlapping subgenomic DNA fragments were prepared from these clones and recombined in two different ways. In both cases a PSTV DNA copy was obtained which represented the entire PSTV RNA genome. The sequence of the DNA of one of the resulting full-length clones was identical with the original PSTV isolate, whereas the other clone showed one nucleotide change. On the basis of these results the advantages and problems of different strategies for the molecular cloning of the circular viroid RNA genome are discussed.

Base Sequence↗

PubMed1985

Synthesis of (+) and (-) RNA molecules of potato spindle tuber viroid (PSTV) in isolated nuclei and its impairment by transcription inhibitors.

Transcription studies with highly purified potato cell nuclei in combination with a 'transcription-hybridization analysis' unequivocally demonstrate that the nucleus is the subcellular site where the entire process of PSTV replication takes place. Inhibition experiments with actinomycin D and alpha-amanitin furthermore suggest that the nuclear DNA-dependent RNA polymerases I and II are involved in the synthesis of PSTV (+) and (-) RNA, respectively.

Amanitins↗

PubMed1984

Cloned single- and double-stranded DNA copies of potato spindle tuber viroid (PSTV) RNA and co-inoculated subgenomic DNA fragments are infectious.

A set of monomeric and oligomeric potato spindle tuber viroid (PSTV) specific DNA forms representing complete DNA copies of the circular PSTV RNA genome were constructed and cloned in plasmid pBR322 and bacteriophage M13. Both single- and double-stranded PSTV DNAs are capable of initiating viroid replication in mechanically inoculated tomato plants where it normally proceeds via the RNA-RNA pathway without DNA being involved. All dimeric and higher multimeric forms were infectious irrespective of their polarity in the case of single-stranded DNA and regardless of their orientation in the vector DNA in the case of double-stranded DNA. The vector-inserted monomeric PSTV DNA units were also found to be infectious but of low specific infectivity which was increased when these monomers had been excised. Even two subgenomic DNA fragments, representing together the 359 nucleotides of the PSTV RNA genome, initiated the synthesis of viroid RNA progeny when co-inoculated although each fragment by itself is non-infectious. These results are discussed with respect to the infectivity previously observed with certain cloned DNAs of conventional RNA and DNA viruses.

Cloning, Molecular↗

PubMed1981

Extracellular folate deaminase of Dictyostelium discoideum.

Folate deaminase released from cells of Dictyostelium discoideum is heterogeneous with respect to molecular weight and stability at 60 degrees C. The most heat-stable component isoelectrofocuses in a broad band at approx. pH 6. The Km value of this component for folate is approx. 7 x 10(-7)M and Mr approx. 40 000. The major portion if not all of the deaminase binds to immobilized concanavalin A and lentil lectin. Extracellular folate deaminase has a pH-optimum of approx. pH 6.0. This is higher than that of lysosomal enzymes, which are also glycoproteins released into the extracellular medium.

Aminohydrolases↗