No abstract

On the basis of available nucleotide sequence and genetic data; we present a model for RNA splicing in fungal mitochondria. Seven intron RNAs of two fungal species can form identical secondary structures, involving four conserved sequences, which bring the ends of each intron together and allow an internal guide RNA sequence to pair with exon bases adjacent to the splice junctions. The splicing sites are thus aligned precisely within a conserved structure, which we suggest could present specific recognition signals to the proteins that catalyse the splicing reaction.

Tomato plants were transformed with a chimaeric polygalacturonase (PG) gene, designed to produce a truncated PG transcript constitutively. In these plants expression of the endogenous PG gene was inhibited during ripening, resulting in a substantial reduction in PG mRNA and enzyme accumulation. This inhibition was comparable to that achieved previously using antisense genes. The expression of the truncated gene in ripe fruit was substantially lower than its expression in green fruit. Thus expression of both the endogenous and truncated genes is reduced in ripe fruit in which both are active. The implication of this observation is discussed in relation to the possible mechanism whereby sense constructs inhibit gene expression.

Polygalacturonase (PG) is the major cell wall degrading enzyme of tomato fruit. It is developmentally regulated and is synthesised de novo in ripening fruit. Genomic clones encoding a PG gene of tomato (Lycopersicon esculentum Mill cv. Ailsa Craig) have been isolated, mapped and sequenced. The sequence of the protein-coding region is identical to that of a PG cDNA [20]. Comparison of the cloned restriction fragments with genomic Southern data suggests that there may only be one gene for PG per haploid genome. The PG gene, which covers approximately 7 kb, is interrupted by 8 intervening sequences ranging in size from 99 bp to 953 bp. The transcription start point was identified by S1 mapping and primer extension analysis. About 1.4 kb of 5' flanking DNA has been sequenced. This contains putative TATA and CAAT boxes and also direct repeat sequences. A transcriptional fusion has been constructed between the putative 1.4 kb promoter fragment and the chloramphenicol acetyl transferase (CAT) gene. Constructs containing this gene have been transferred to tomato using binary vectors. Regenerated transgenic plants express CAT in ripe tomato fruit, but not in unripe tomatoes, leaves, or roots.

We have investigated the effects of volatile anaesthetics on electron transport chain activity in the mammalian heart. Halothane, isoflurane and sevoflurane reversibly increased NADH fluorescence (autofluorescence) in intact ventricular myocytes of guinea‐pig, suggesting that NADH oxidation was impaired. Using pig heart submitochondrial particles we found that the anaesthetics dose‐dependently inhibited NADH oxidation in the order: halothane > isoflurane = sevoflurane. Succinate oxidation was unaffected by either isoflurane or sevoflurane, indicating that these agents selectively inhibit complex I (NADH:ubiquinone oxidoreductase). In addition to inhibiting NADH oxidation, halothane also inhibited succinate oxidation (and succinate dehydrogenase), albeit to a lesser extent. To test the hypothesis that complex I is a target of volatile anaesthetics, we examined the effects of these agents on NADH:ubiquinone oxidoreductase (EC 1.6.99.3) activity using the ubiquinone analogue DBQ (decylubiquinone) as substrate. Halothane, isoflurane and sevoflurane dose‐dependently inhibited NADH:DBQ oxidoreductase activity. Unlike the classical inhibitor rotenone, none of the anaesthetics completely inhibited enzyme activity at high concentration, suggesting that these agents bind weakly to the ‘hydrophobic inhibitory site’ of complex I. In conclusion, halothane, isoflurane and sevoflurane inhibit complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain. At concentrations of ≈2 MAC (minimal alveolar concentration), the activity of NADH:ubiquinone oxidoreductase was reduced by about 20 % in the presence of halothane or isoflurane, and by about 10 % in the presence of sevoflurane. These inhibitory effects are unlikely to compromise cardiac performance at usual clinical concentrations, but may contribute to the mechanism by which volatile anaesthetics induce pharmacological preconditioning.