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G Steger

Publications and source records attributed to G Steger.

103 records · Page 6Linked to original sources

Dynamics and interactions of viroids.

Viroids are single stranded circular RNA molecules of 120,000 daltons which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but do replicate only as a part of a larger plant virus. The structure and structural transitions have been investigated by thermodynamic, kinetic and hydrodynamic methods and have been compared to results from calculations of the most favorable native structures and the denaturation process. The algorithm of Zuker et al. was modified for the application to circular nucleic acids. For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids, although described in the literature as viroid-like, show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but sequences generated by a computer. The comparison of viroids, virusoids and circular RNA of random sequences underlines the uniqueness of viroid structure. The interactions of viroids with dye and oligonucleotide-ligands and with RNA-polymerase II from wheat germ, which enzyme replicates viroids in vitro, has been studied in order to correlate viroid structure and its ability for specific interactions. Specificity of the interactions may be interpreted on the basis of the neighbourhood of double stranded and single stranded regions. In the host cell viroids are localized in the cell nucleus; they may be detected as free nucleic acids and in high molecular weight complexes together with other RNA and proteins.

Base Sequence↗

Structural transitions in viroid-like RNAs associated with cadang-cadang disease, velvet tobacco mottle virus, and Solanum nodiflorum mottle virus.

The conformational transitions of viroid-like RNAs associated with cadang-cadang disease, velvet tobacco mottle virus, and solanum nodiflorum mottle virus were studied by melting analysis and fast temperature jump technique in 1 mM sodium-cacodylate, 10 mM NaCl, 0.1 mM EDTA, pH 6.8. The 4 circular RNAs of cadang-cadang show a highly cooperative transition between 45 and 49 degrees C, respectively, and a second transition of less hypochromicity at about 10 degrees C higher temperatures. The data are interpreted quantitatively on the basis of the sequences and secondary structure models. A very similar scheme for the structure and structural transitions as derived earlier for other viroids applies to the cadang-cadang RNAs. In the main transition the total native secondary structure is disrupted and a stable hairpin consisting of 9 base pairs is newly formed which dissociates in the second transition. The thermal denaturation of the circular RNAs from the viruses mentioned above is clearly distinct from viroid RNA in respect to stability and cooperativity. The results on cadang-cadang RNA are discussed in the light of recent hypotheses about the interference of viroids with the splicing process of the host cell.

Kinetics↗

Helix-coil transitions in double-stranded viral RNA. Fine resolution melting and ionic strength dependence.

Helix-coil transitions of double-stranded RNA from reovirus and infectious bursal disease virus were measured optically in aqueous medium of different ionic strengths. In RNA from reovirus four transitions and in RNA from infectious bursal disease virus two transitions were resolved and evaluated quantitatively. The ionic strength dependence of their midpoint temperatures dTm/dlog[Na+] were 13.5 +/- 0.3 degrees C for reovirus RNA and 14.9 +/-0.7 degrees C for infectious bursal disease virus RNA. The midpoint temperatures extrapolated to 1 M ionic strength were 102.9, 104.3, 105.6, and 108.8 +/- 0.3 degrees C for reovirus RNA, and 108.8 +/- 1.8 and 109.6 +/- 1.0 degrees C for infectious bursal disease virus RNA. The G + C content of the regions in reovirus RNA melting in the different transitions were determined from the spectrum of the hypochromicity. The quantitative interpretation of the data is carried out on the basis of the ion condensation theory. It is estimated for double-stranded RNA of 100% G + C, that dTm/dlog[na+] = 8.4 degrees C. The two-dimensional dependence Tm = Tm (ionic strength, G + C content) is given. The ionic strength dependence in different double-stranded RNAs is correlated to the spacing of the phosphate backbone, secondary structure, and tertiary structure.

Animals↗

Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions.

Temperature-gradient gel electrophoresis (TGGE) is applied to analyze conformational transitions and sequence variations of nucleic acids and protein-nucleic acid interactions. A linear and highly reproducible temperature-gradient is established perpendicular or parallel to the direction of the electrophoresis. The instrument consists of an electrically insulated metal plate, which is heated at one edge and cooled at the other edge by two thermostating baths and is used as an ancillary device for commercial horizontal gel electrophoresis instruments. Biopolymers are separated in TGGE according to size, shape and thermal stability of their conformational transitions. If the temperature-gradient is established perpendicular to the electrophoresis, monomolecular conformational transitions of nucleic acids show up as continuous transition curves; strand-separation leads to discontinuous transitions. In the studies on viroid RNA it was shown that natural circular viroid RNA undergoes one highly cooperative transition detected by TGGE as a drastic retardation in mobility. Oligomeric replication intermediates of viroids exhibit coexisting structures which could not be detected by any other technique. Double-stranded satellite RNA from cucumber mosaic virus is a mixture of sequence variants, all of which have the identical length of 335 nucleotides. In TGGE six different strains were resolved. Sequence variants of viroids were analyzed by hybridizing viroid RNA to (-)strand viroid RNA transcripts from viroid cDNA clones. Sequence variations lead to mismatches in the double strands and thereby to a shift of the transition curve to lower temperature. Mutations in plasmids, particularly in cloned inserts, were detected by mixing plasmids of two different clones, linearizing, denaturing, renaturing, and searching for shifts in the transition curves, which are generated by mismatch-formation during the renaturation of (+)- and (-)strands from different clones. Examples are given for different viroid clones and HIV-clones from one and the same patient. In another example, clones with point mutations from site-directed mutagenesis are analyzed and selected by TGGE. TGGE is also applied to study the effect of amino acid exchanges in the Tet repressor from E. coli on the thermal stability of the repressor and on the mode of binding of the repressor to the operator DNA. The results are discussed under the aspect that TGGE may be applied as routine analytical laboratory procedure.

Base Sequence↗

Temperature-gradient gel electrophoresis for the detection of polymorphic DNA and for quantitative polymerase chain reaction.

In order to detect mutations in a gene, either known mutations from human diseases or artificial ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarantees detection of mutations which might occur in every single position of the whole open reading frame (ORF). It will be shown that a combination of polymerase chain reaction (PCR) and temperature gradient gel electrophoresis (TOGE) fulfills these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated in dependence on the position of the mutation. The theoretical results were tested with the mutations known so far. The quantitative determination of the copy number of a specific DNA or RNA sequence in a biological specimen (quantitative PCR) can be performed precisely and easily by combining PCR and TGGE. The system uses a quantification strategy of a new type of internal standardization. TGGE is applied to separate homo- and heteroduplexes which correspond respectively to standard and template sequences. The accuracy of this quantification strategy is very high, with a variability of < 15%. In addition to quantification, PCR/TGGE detects PCR artifacts and template mutants.

DNA↗

Sequential administration of interferon-gamma, GM-CSF, and interleukin-2 in patients with metastatic renal cell carcinoma: results of a phase II trial.

Various cytokine combinations have been tested for efficacy in the treatment of metastatic renal cell carcinoma (MRCC). Because several immunologic synergisms between granulocyte-macrophage colony-stimulating-factor (GM-CSF) and interleukin-2 (IL-2) have been demonstrated, this phase II trial was conducted on the efficacy and toxicity of subcutaneous, sequentially administered, interferon-gamma (IFNgamma), GM-CSF, and IL-2. Fifty-five consecutive patients with MRCC were treated with 100 microg recombinant IFNgamma1b administered thrice weekly during weeks 1 and 4, followed by 400 microg GM-CSF on 5 consecutive days during weeks 2 and 5. In weeks 3 and 6, patients received 4.5 MU recombinant IL-2 from days 1 to 4. The treatment was repeated every 8 weeks. Five (10%) of patients experienced an objective response (complete response [CR]: 2%, partial response [PR]: 8%). Fourteen (26%) patients had stable disease with a median duration of 19 months (6-47+). The median overall survival was 12 months (range: 0.3-44 months). No toxicity greater than World Health Organization grade II was observed, with fever (43%) and erythema (43%) being the most frequent side effects. Compared with other phase II trials with IFN-gamma and IL-2 alone, the addition of GM-CSF failed to improve response or survival in patients with MRCC.

Adult↗

Phase II trial of recombinant interferon alpha-2C in metastatic renal cell carcinoma.

A total of 18 patients with advanced metastatic renal cell cancer were treated with recombinant interferon alpha-2C (rIFN alpha-2C) at daily doses of 10 X 10(6) IU by intramuscular injection. All patients had evaluable metastatic lung, liver, or abdominal disease as measured by radiographic or computerized tomographic scans. In 2 of the 18 patients an objective response (1 CR, 1 PR) with a duration of +28 and 12 months, respectively was achieved. A 25$ to 50$ decrease in tumor measurements (MR) was seen in 2 additional patients; in 3 cases a stabilisation of the disease (SD) was observed, whereas it progressed in 11. 3/4 responding patients (including MR) and all 3 cases with SD had measurable disease in the lungs as predominant site of metastatic disease. Additional clinical characteristics of patients exhibiting response or SD to IFN therapy included prior nephrectomy, favourable initial performance status and limited metastatic disease. No serious haematologic or irreversible organ toxic effects were attributed to interferon. Several patients, however, had constitutional symptoms, and major dose reductions due to CNS toxicity became necessary in two. Further studies are warranted to evaluate the use of interferons in combination with cytotoxic drugs or other biologic response modifiers.

Adult↗