[Suppression of nonsense mutations in the Dystrophin gene by a suppressor tRNA gene]. / Ispol'zovanie gena supressornoi tRNK dlia ispravleniia nonsens-mutatsii v gene distrofina.

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10-15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing beta-galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.

15N and 31P solid-state NMR investigations on the orientation of zervamicin II and alamethicin in phosphatidylcholine membranes.

The topologies of zervamicin II and alamethicin, labeled with (15)N uniformly, selectively, or specifically, have been investigated by oriented proton-decoupled (15)N solid-state NMR spectroscopy. Whereas at lipid-to-peptide (L/P) ratios of 50 (wt/wt) zervamicin II exhibits transmembrane alignments in 1,2-dicapryl (di-C10:0-PC) and 1,2-dilauroyl (di-C12:0-PC) phosphatidylcholine bilayers, it adopts orientations predominantly parallel to the membrane surface when the lengths of the fatty acyl chains are extended. The orientational order of zervamicin II increases with higher phospholipid concentrations, and considerable line narrowing is obtained in di-C10:0-PC/zervamicin II membranes at L/P ratios of 100 (wt/wt). In contrast to zervamicin, alamethicin is transmembrane throughout most, if not all, of its length when reconstituted into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. The (31)P solid-state NMR spectra of all phospholipid/peptaibol samples investigated show a high degree of headgroup order, indicating that the peptides do not distort the bilayer structure. The observed differences in peptide orientation between zervamicin and alamethicin are discussed with reference to differences in their lengths, helical conformations, distribution of (hydroxy)proline residues, and hydrophobic moments. Possible implications for peptaibol voltage-gating are also described.

A new T7 RNA polymerase-driven expression system induced via thermoamplification of a recombinant plasmid carrying a T7 promoter-Escherichia coli lac operator.

A new temperature-regulated T7 RNA polymerase-driven transcription system has been developed. This system is based on a hybrid regulatory region: the phage T7 late promoter (PT7) linked to the Escherichia coli lac operator (Olac) [Giordano et al., Gene 84 (1989) 209-219], which was located in an earlier obtained [Mashko et al., Gene 97 (1991) 259-266] temperature-controlled amplifiable plasmid, carrying cat under the control of PT7-Olac and, in addition, lambda major early promoter-operator regions and gene cIts857. Plasmids of the pT7-Olac-cat-tsr series were stably maintained at a low-copy-number when grown at low temperature (28 degrees C). In E. coli BL21(DE3), carrying the Plac-controllable T7 RNA polymerase-encoding gene, efficient repression of cat transcription was observed, that was provided by the LacI repressor and, probably, the thermolabile repressor CIts857. At low and moderate temperatures (28/37 degrees C), this 'cooperative' repression was so tight that cat expression was not observed in the cells carrying PT7-Olac on the plasmids, even after IPTG-inducible T7 RNA polymerase biosynthesis. As a result of the thermo-amplification of the recombinant plasmids and temperature-inactivation of CIts857, expression of the T7 RNA polymerase-encoding gene was derepressed due to the titration of LacI by the increasing copies of Olac which in turn, led to the highly efficient T7 RNA polymerase-driven accumulation of CAT in the cells.