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D Riesner

Publications and source records attributed to D Riesner.

At least 19 recordsLinked to original sources
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

Cyclosporin synthetase is a 1.4 MDa multienzyme polypeptide. Re-evaluation of the molecular mass of various peptide synthetases.

The earlier determined molecular mass of 0.8 MDa for the multifunctional polypeptide, cyclosporin synthetase, was re-evaluated by SDS-PAGE and CsCl density gradient centrifugation. In SDS-PAGE, new molecular mass values as standards were available from sequencing data. In the CsCl density gradient extremely low protein concentrations, such as 10-50 nM could be analysed due to the fluorescence detection system of the analytical ultracentrifuge. Both methods yielded approximately the same value of about 1.4 MDa. Using this molecular mass of cyclosporin synthetase as a reference the molecular masses of various related enzymes could be re-evaluated in SDS-PAGE. The sedimentation coefficient of 26.3 S for cyclosporin synthetase indicates an oblate overall shape of the enzyme.

Cesium↗
PubMed1992

Enzymatic biosynthesis of cyclosporin A and analogues.

The final assembly of the undecapeptide chain of cyclosporin A and its cyclization is accomplished in Beauveria nivea by cyclosporin synthetase. This multienzyme is the largest integrated enzyme structure so far reported. Its size has been estimated at approximately 1,400 kDa by two different methods: 1), by 3% SDS-PAGE using the related multienzymes ACV synthetase and gramicidin S synthetase 2 as references (420 and 556 kDa, respectively); and 2), by CsCl density gradient centrifugation experiments using fluorescence-labeled cyclosporin synthetase. Besides cyclosporin A and a number of cyclosporins known from fermentation studies cyclosporin synthetase is capable of synthesizing some new cyclosporins which are so far unobtainable by fermentation. So, for example the synthesis of [N-methyl-(+)-2-amino-3-hydroxy-4,4-dimethyloctanoic acid1]CyA, dihydro-CyA, [L-norvaline2,5, N-methyl-L-norvaline11]CyA, [L-allo-isoleucine5, N-methyl-L-allo-isoleucine11]CyA, [D-2-aminobutyric acid8]CyA, [beta-chloro-D-alanine8]CyA and some related compounds could be established. By using a related but different enzyme from Cylindrotrichum Bonorden, the peptolide [L-threonine2, L-leucine5,10, D-2-hydroxyisovaleric acid8]CyA could be synthesized in vitro. We were able to synthesize these cyclosporins in sufficient quantities to examine their structure by FAB mass spectroscopy and explore their immunosuppressivity. It was found that all new cyclosporins so far synthesized in the in vitro system are immunosuppressive.

Amino Acid Sequence↗
PubMed1992

Further analysis of nucleic acids in purified scrapie prion preparations by improved return refocusing gel electrophoresis.

Although increasingly unlikely, the possibility of a scrapie-specific nucleic acid carried by infectious prion particles is still unresolved. Return refocusing gel electrophoresis was developed to detect homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. This method was improved with respect to the size range from 13 to 1100 nucleotides (nt) over which analyses could be performed. The yield of nucleic acid, particularly of small DNA oligonucleotides and polyadenylated RNA, was determined after deproteinization and two-phase extraction. Despite extensive nuclease digestions some small polynucleotides remained. Although a scrapie-specific nucleic acid cannot be excluded, the results further define the possible characteristics of a hypothetical molecule. If homogeneous in size, such a molecule would be less than 80 nt in length at a particle-to-infectivity ratio near unity, if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nt.

Animals↗
PubMed1991

Formation of a thermodynamically metastable structure containing hairpin II is critical for infectivity of potato spindle tuber viroid RNA.

The functional relevance of a hairpin II-containing structure of viroid RNA was studied by site-directed mutagenesis, thermodynamic calculations, experimental denaturation curves and infectivity tests. Hairpin II is formed during thermal denaturation of circular viroids or as part of a metastable structure during synthesis of viroid replication intermediates. In potato spindle tuber viroid (PSTVd), eight single-site mutations were generated in the segments which form hairpin II. From the mutated viroid cDNA clones, linear RNA transcripts of PSTVd unit length were synthesized. The relevance of hairpin II for the mechanism of denaturation was confirmed quantitatively by optical denaturation curves and temperature-gradient gel electrophoresis. Infectivity tests showed that the mutations in the core region of hairpin II reverted to the wild type sequence whereas the mutations in the peripheral regions of hairpin II remained genetically stable. These data are in accordance with the natural variance of hairpin II in other viroids of the PSTVd class. Thus, the integrity of the core of hairpin II is critical for infectivity. Hairpin II exhibits a strong similarity in sequence as well as in three-dimensional structure to certain DNA GC-clusters found in the 5'-upstream regions of some genes in man, animals, viruses and plants. A hypothesis about a function of hairpin II as a binding site for host cell transcription factors is proposed.

Base Composition↗
PubMed1991

Search for a putative scrapie genome in purified prion fractions reveals a paucity of nucleic acids.

Scrapie can be transmitted by novel infectious pathogens termed prions. No evidence for a scrapie-specific nucleic acid has been detected to date. To investigate amounts, types and sizes of nucleic acid molecules associated with prions in purified preparations, aliquots were deproteinized, and the nucleic acids analysed by PAGE and silver staining. Digestion with nucleases and exposure to Zn2+ prior to analysis substantially diminished the content of nucleic acids, but did not alter the prion titre indicating that those nucleic acids which were removed are not essential for infectivity. Since a single species of scrapie-specific nucleic acid could not be identified, we explored the unprecedented possibility of scrapie-specific nucleic acids of variable length which are biologically active. If such molecules of variable length exist then they might be hidden within the background smear on silver-stained gels after PAGE. A new procedure designated return refocusing gel electrophoresis (RRGE) was developed to identify heterogeneous nucleic acids in purified prion fractions. The content of variable length nucleic acids was reduced by a factor of 10 by exhaustive Bal 31 exonuclease digestion after dispersion of purified prions into detergent-lipid-protein complexes. For example, a typical sample after Bal 31 digestion contained approximately 4 ng of nucleic acid of variable length and 10(8.7) ID50 units of scrapie prion infectivity. Consideration of different models for a hypothetical scrapie-specific nucleic acid suggests that such a molecule would have to be: (i) quite small (less than 100 nucleotides), (ii) possess a particle-to-infectivity ratio near unity or (iii) heterogeneous in size. Although our results do not eliminate the possibility that prions possess a scrapie-specific nucleic acid of variable length, they narrow considerably the spectrum of features specifying such a candidate molecule.

Animals↗
PubMed1990

Analysis of the conformational transitions of proteins by temperature-gradient gel electrophoresis.

Temperature-gradient gel electrophoresis (TGGE) is a technique for studying the structural transitions of nucleic acids and proteins. A temperature gradient is formed in a horizontal slab gel perpendicular to the direction of the electric field. Whereas the principle of the TGGE method has previously been applied to proteins, we describe in this report the systematic optimization of TGGE as a routine technique for the quantitative analysis of conformational transitions in proteins. Using alpha-amylase as an example we show the kinds of results which may be obtained from such measurements. Buffers suitable for use in gel electrophoresis were analyzed with respect to the dependence of their pH value upon temperature. The correct pH range for TGGE of a given protein is determined by electrophoretic titration curves. The protein bands are detected by silver and/or activity staining. The thermal denaturation of alpha-amylase from Aspergillus oryzae showed a discontinuous transition into the denatured conformation, which exhibited much slower electrophoretic mobility. The discontinuity is due to an irreversible denaturation process under the gel conditions. The transition temperature was measured as a function of several parameters, e.g., the concentration of Ca(+)+, dithiotreithol, urea and the pH value. The structural transition of alpha-amylase is accompanied by a loss of enzymatic activity as determined by activity staining or by an activity assay carried out in solution. The structural transitions of two other alpha-amylases from Bacillus subtilis and Bacillus licheniformis were also studied. The results show that the TGGE method is simple to perform and allows the analysis of conformational transitions of proteins in a wide variety of conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspergillus oryzae↗
PubMed1989

Imaging of viroids in nuclei from tomato leaf tissue by in situ hybridization and confocal laser scanning microscopy.

The intracellular localization of viroids has been investigated by viroid-specific in situ hybridization and analysis by digital microscopy of the distribution of the fluorescent hybridization signals. Isolated nuclei from green leaf tissue of tomato plants infected with potato spindle tuber viroid (PSTVd) were bound to microscope slides, fixed with formaldehyde and hybridized with biotinylated transcripts of cloned PSTVd cDNA. The bound probe was detected with lissamine--rhodamine conjugated streptavidin. Nucleoli were identified by immunofluorescence using the monoclonal antibody Bv96 and a secondary FITC-conjugated antibody. In plants infected with either a lethal or an intermediate PSTVd strain, the highest intensity of fluorescence that arose from hybridization with the probe specific for the viroid (+)strand was found in the nucleoli, confirming results of previous fractionation studies. A similar distribution was found for (-)strand replication intermediates of PSTVd using specific (+)strand transcripts as hybridization probes. In order to determine if viroids are located at the surface or in the interior of the nucleoli, the distribution of the fluorescence hybridization signals was studied with a confocal laser scanning microscope (CLSM). It was shown by three-dimensional reconstruction that viroids are neither restricted to the surface of the nucleoli nor to a peripheral zone, but are instead homogeneously distributed throughout the nucleolus. The functional implications of the intranucleolar location of viroids and their replication intermediates are discussed with respect to proposed mechanisms of viroid replication and pathogenesis.

Cell Nucleus↗
PubMed1988

Analysis of RNA structures by temperature-gradient gel electrophoresis: viroid replication and processing.

The structure and structural transitions of single-stranded RNA were investigated by energy calculations and temperature-gradient gel electrophoresis. Most experiments have been carried out on RNA of mature viroids and their replication intermediates, which are RNA (-) strand oligomers and RNA (+) strand oligomers. The technique of temperature-gradient gel electrophoresis proved to be particularly useful for analysing co-existing structures. The secondary structure of lowest free energy for unit length and oligomeric replication intermediates is an extended rod-like structure similar to that of the mature circular viroid. When this structure is used as a model for calculations, there is a large degree of agreement between theoretical and experimental curves. Under particular solution conditions, however, (+) strand oligomers undergo a rearrangement from the extended structure to a branched structure, in which every two units form a region of three helices, together 28 bp long. This structure is called the tri-helical structure. The process of structure formation during the synthesis of oligomers could be followed: at first, a transient multi-branched structure is formed which is then transformed into the extended and the tri-helical structures. The region of the three stable helices serves to divide up the oligomeric (+) strand into structural units which may be recognized by cleavage and ligation enzymes, and be processed into circular mature viroids. Co-transcription of complementary (+) and (-) strands shows that energetically favored double-strand formation may at least partially be prohibited by stable secondary structures of the single strands. Natural replication intermediates have been analysed in respect to their subcellular location and their size distribution. They are associated with the nucleoli as was found earlier for mature viroids. Natural (-) strand oligomers are larger than (+) strand oligomers; both types show a periodicity in the size distribution of two units. The models of the structures, which are involved in viroid processing, are in accordance with recent infectivity data and with the results on natural replication intermediates.

Base Sequence↗
PubMed1988

Isolation and characterization of high-molecular mass DNA from hair shafts.

Experiments to identify species by DNA analysis of their hair failed so far because no DNA could be isolated from hair shafts. In this work the preparation of DNA from human--as well as from animal hair shafts (alpaca, angora-rabbit, cashmere, cashgora, mohair, merino and yak) is described for the first time. In general the isolated DNA shows a length of more than 20 kbp. The species of the hair shaft samples could be exactly identified by DNA hybridization experiments. The isolated DNA from hair shafts allow new possibilities to identify species and individuals employing techniques from molecular biology.

Animals↗
PubMed1987

Chromatographic separation of DNA restriction fragments.

Reversed-phase liquid chromatography (RPC-5) was the first chromatographic technique to be successfully applied to DNA restriction fragments. Size-exclusion high-performance liquid chromatography (HPLC) has the advantage of wide variability in buffer conditions but is restricted to an upper size limit at 800 base pairs. Anion-exchange HPLC is most versatile and may best be scaled up for the preparation of milligram amounts of specific DNA fragments. The preparation of the sample before and after chromatography is described. Because several applications of DNA fragments will be complemented by RNA fragments, their preparation and purification is included. Disposable small-scale columns are available for processing many samples. Future medical applications of HPLC of DNA fragments concern medical microbiology, human genetics and forensic medicine.

Animals↗
PubMed1987

Double-stranded cucumovirus associated RNA 5: experimental analysis of necrogenic and non-necrogenic variants by temperature-gradient gel electrophoresis.

Cucumber mosaic virus (CMV) and peanut stunt virus (PSV) each contain a fifth major RNA in the size range of 334 to 393 nucleotides. This fifth RNA is a satellite capable of modulating the expression of viral disease symptoms. It is present in infected tissue in single-stranded and double-stranded form. Nucleotide sequence variants of the double-stranded CMV-associated RNA 5 (dsCARNA 5) and PSV-associated RNA 5 (dsPARNA 5) were analysed by temperature-gradient gel electrophoresis. Gels were 5% polyacrylamide, containing 8 M urea in 8.9 mM Tris-borate buffer, with temperature differences of 25-40 degrees C establishing gradients either perpendicular or parallel to the direction of the electric field. For dsCARNA 5 two characteristic transitions were detected with increasing temperature: at temperatures between 40 degrees C and 46 degrees C a drastic retardation in electrophoretic mobility induced by partial dissociation of the duplex structure from the ends and at temperatures above 52 degrees C an abrupt increase in mobility due to complete strand dissociation. dsPARNA 5 exhibited both transitions at up to 10 degrees C higher temperatures and an additional retardation between the transitions mentioned. Seven different variants of dsCARNA 5, 4 necrogenic and 3 non-necrogenic, were analysed. Some showed only one single band, others gave rise to up to six well separated bands corresponding to six molecular species. From all experimental results a correlation between the temperature of the retardation transition and the necrogenicity of CARNA 5 was derived. The diagnostic application of the temperature-gradient gel analysis in agriculture, particularly for the use of non-necrogenic variants as biological control agents to impede CMV-infections, is discussed.

Electrophoresis↗
PubMed1987

Double-stranded cucumovirus associated RNA 5: which sequence variations may be detected by optical melting and temperature-gradient gel electrophoresis?

Sequence variants of the double-stranded form of satellite RNAs of cucumber mosaic virus (dsCARNA 5) were analyzed for the possibility to experimentally detect minor nucleotide sequence changes. Denaturation maps (helix-probability versus position of the nucleotide in the sequence versus temperature) were calculated applying the Poland algorithm. Optical denaturation curves and temperature-gradient gel mobility curves were simulated using the denaturation maps and were compared with experimental results from optical melting and temperature-gradient gel electrophoresis (Tien Po et al., accompanying paper). Melting of the dsRNAs starts from both ends of the molecule in two transitions of low co-operativity, continues in the right part in a highly co-operative transition, and is finished in another highly co-operative transition including strand-separation. Whereas all parts of the molecule contribute uniformly to the optical melting curve, opening of the ends predominates in the retardation transition in gel electrophoresis. Detailed discussion of the influence of base pair changes in the sequence shows that a single base pair change may be detected by temperature-gradient gel electrophoresis, if it is located in certain favorable locations, whereas its detection in optical melting curves is possible only in very special cases. The systematic differences found in the accompanying paper between necrogenic and non-necrogenic dsCARNA 5 could be interpreted on the basis of such nucleotide sequence differences.

Base Composition↗
PubMed1987

Temperature-gradient gel electrophoresis. Thermodynamic analysis of nucleic acids and proteins in purified form and in cellular extracts.

A temperature-gradient gel electrophoresis technique and its application to the study of structural transitions of nucleic acids and protein-nucleic acid complexes are described. The temperature gradient is established in a slab gel by means of a simple ancillary device for a commercial horizontal gel apparatus. The gradient may be freely selected between 10 and 80 degrees C, and is highly reproducible and linear. In a normal application the biopolymers migrate perpendicular to the temperature gradient so that every individual molecule is at constant temperature throughout electrophoresis. The structural transition of a biopolymer is seen as a continuous band which is retarded or speeded up in the temperature range of the transition. Dissociation processes are mostly irreversible under the conditions of electrophoresis and, therefore, show up as discontinuous transitions from a slow-moving to fast-moving band. As examples the conformational transitions of viroids, double-stranded RNA from reovirus, double-stranded satellite RNA from cucumber mosaic virus and repressor-operator complexes have been studied. It could be shown that by this method dsRNA molecules may be differentiated which differ only in one base-pair, or proteins differing in one amino acid only. As a particular advantage, temperature-gradient gel electrophoresis allows the study of conformational transitions of biopolymers which have not been purified. The biopolymer may either be identified by silver staining as a specific band among many others or, if the study is carried out on nucleic acids, these may be recorded by hybridization with a radioactive probe.

DNA, Bacterial↗
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↗
PubMed1986

Mismatches in DNA double strands: thermodynamic parameters and their correlation to repair efficiencies.

The helix-coil transitions of the 16 octadecameric DNA duplexes dCGTCGTTTXACAACGTCG X dCGACGTTGTX1AAACGACG with A, T, G, and C for X and X1 were measured by UV-absorption. This sequence was taken from former studies of in vivo determination of efficiencies of mismatch repair (Kramer, Kramer, and Fritz (1984) Cell 38, 879-887). The thermodynamic parameters for double strand and mismatch formation have been obtained by evaluating the partition function of a stack model which allowed for loop formation. As a result the mismatches could be classified into wobble base pairs (T/G, G/G, C/A, A/A, A/G), open base pairs, i.e. permanent loops (T/T, C/T, T/C, C/C), and intermediate or weak base pairs (G/T, A/C, G/A). There is no correlation between Tm and the biological repair efficiency of X/X1. The structure classes, however, as described above show a close correlation: Open base pairs show the lowest repair efficiencies, whereas mismatches with high repair efficiency always belong to the structural class of wobble base pairs. Because of the palindromic nearest neighbors of the variation site X/X1, the influence of next-nearest neighbor interactions could be detected and be estimated to about 1 kJ/mol for one stack.

Base Sequence↗
PubMed1986

The stiffness of dsRNA: hydrodynamic studies on fluorescence-labelled RNA segments of bovine rotavirus.

The sedimentation coefficients of dsRNA segments of bovine rotavirus were determined in the analytical ultracentrifuge. The eleven segments were separated by preparative gel electrophoresis, and isolated by elution from gel pieces. The RNA was labelled by the intercalating fluorescent dye ethidium bromide at a ratio bound dye per base pair between 0.003 to 0.018. The analytical ultracentrifuge was equipped with a fluorescence recording optics. Sedimentation coefficients could be determined with amounts of RNA as little as 8 ng. All sedimentation coefficients were extrapolated to zero-concentration, zero-dye binding, and zero-impurities from the preparative gel electrophoresis. The hydrodynamic model of flexible cylinders was applied for the interpretation of the sedimentation coefficients. All dsRNA segments of rotavirus (663-3409 base pairs) and the dsRNA5 of cucumber mosaic virus (335 base pairs) fit the model of a "worm-like" or flexible cylinder with a persistence length of 1125 A and a hydrated diameter of 30 A. The results are compared with data from the literature on the persistence lengths of the B- and Z-forms of dsDNA and of viroids.

Animals↗