Kissing of the two predominant hairpin loops in the coxsackie B virus 3' untranslated region is the essential structural feature of the origin of replication required for negative-strand RNA synthesis.

Higher-order RNA structures in the 3' untranslated region (3'UTR) of enteroviruses are thought to play a pivotal role in viral negative-strand RNA synthesis. The structure of the 3'UTR was predicted by thermodynamic calculations using the STAR (structural analysis of RNA) computer program and experimentally verified using chemical and enzymatic probing of in vitro-synthesized RNA. A possible pseudoknot interaction between the 3D polymerase coding sequence and domain Y and a "kissing" interaction between domains X and Y was further studied by mutational analysis, using an infectious coxsackie B3 virus cDNA clone (domain designation as proposed by E. V. Pilipenko, S. V. Maslova, A. N. Sinyakov, and V.I. Agol (Nucleic Acids Res. 20:1739-1745, 1992). The higher-order RNA structure of the 3'UTR appeared to be maintained by an intramolecular kissing interaction between the loops of the two predominant hairpin structures (X and Y) within the 3'UTR. Disturbing this interaction had no effect on viral translation and processing of the polyprotein but exerted a primary effect on viral replication, as was demonstrated in a subgenomic coxsackie B3 viral replicon, in which the capsid P1 region was replaced by the luciferase gene. Mutational analysis did not support the existence of the pseudoknot interaction between hairpin loop Y and the 3D polymerase coding sequence. Based on these experiments, we constructed a three-dimensional model of the 3'UTR of coxsackie B virus that shows the kissing interaction as the essential structural feature of the origin of replication required for its functional competence.     

Techniques for the quantitative study of mutation in plant viruses

Summary Hardly any other virus is chemically and ultramicroscopically as well known as TMV. It is not possible to perform genetic recombinations with this object. The phenomenon of mutation is, however, known and an analysis of the dosis-effect relationship was possible by using the characters chlorotic versus necrotic primary symptoms. Taking into account the phenomenon of interference (mutual exclusion), i.e., comparing the induced mutation frequency with that of a control virus sample diluted to the same level of infectivity, on can perform quantitative analyses. In this way the first chemical mutagensis in the test tube was demonstrated 10 years ago with nitrous acid as mutagenic agent. The criticism raised byBawden to the first publication ofMundry andGierer was already inappropriate at that time. In the meantime it has been demonstrated byWittmann-Liebold andWittmann through analysis of amino acid exchanges in spontaneous mutants and in those isolated after incubation with HNO₂ that the difference between spontaneous and induced mutants demanded byBawden, which cannot be postulated for symptoms in plants, lies, as expected, in amino acid exchanges of the protein coat.

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Zusammenfassung Kaum ein anderes Virus ist chemisch und ultramikroskopisch so gut bekannt wie das TMV. Rekombinations-Genetik ist nicht möglich. Das Phänomen der Mutation ist aber bekannt, und eine Analyse der Dosis-Effekt-Beziehung wurde möglich durch Benutzung der Symptomcharaktere chlorotische versus nekrotische Primärsymptome. Bei Berücksichtigung des Phänomens der Interferenz (mutual exclusion), d. h. wenn man die induzierte Mutationsrate mit der auf gleiche Infektiosität durch Verdünnen der Viruslösung gebrachten als Kontrolle vergleicht, kann eine quantitative Analyse durchgeführt werden. So wurde vor 10 Jahren die erste Chemomutagenese im Reagenzglas mit salpetriger Säure als mutagenes Agens nachgewiesen. Die an der ersten Veröffentlichung vonMundry undGierer vonBawden geäußerte Kritik war schon damals unzutreffend. Inzwischen ist durch die Analyse der Aminosäureaustausche von spontanen und nach Inkubation mit HNO₂ isolierten Mutanten vonWittmann-Liebold undWittmann gezeigt worden, daß die vonBawden geforderte Verschiedenheit spontaner und induzierter Mutanten, die für Symptome an den Pflanzen nicht postuliert werden kann, in den Aminosäureaustauschen des Hüllproteins wie zu erwarten vorhanden ist.

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This paper was a first written for Methods in Virology, Academic Press. The editors and the author did not come to an agreement in the question of citation ofBawden's criticism to the work ofMundry andGierer 1958. It is published here on the occasion of the 10th anniversary of the first chemomutagenesis in the test tube.     

Referate

Ohne Zusammenfassung     

Localization of the murine lambda 5 gene on chromosome 16

The chromosomal location of the murine lambda 5 gene was analyzed by Southern hybridization using restriction enzyme-digested DNA from a panel of 15 mouse X hamster somatic cell hybrids. Sequences homologous with those of lambda 5 DNA were detected in DNA of 5 hybrids. In all 5 hybrids lambda 5 was contained in restriction fragments of equal sizes, the lengths of which indicated that the germline configuration of lambda 5 with three exons and the restriction sites expected from its genomic structure were present. Southern hybridization with the murine lambda 1 gene as a probe detected the same 5 hybrids as positive. The only mouse chromosome present on all of the positive hybrids, and absent from negative ones, was number 16. We conclude that lambda 5 is situated on the same chromosome as lambda 1, i.e., on the murine chromosome 16.