No abstract

A Myceliophthora thermophila laccase and a Rhizoctonia solani laccase were mutated on a pentapeptide segment believed to be near the type-1 Cu site. The mutation L513F in Myceliophthora laccase and the mutation L470F in Rhizoctonia laccase took place at a position corresponding to the type-1 Cu axial methionine (M517) ligand in Zucchini ascorbate oxidase. The triple mutations V509L,S510E,G511A in Myceliophthora laccase and L466V,E467S,A468G in Rhizoctonia laccase involved a sequence segment whose homologue in ascorbate oxidase is flanked by the M517 and a type-1 Cu-ligating histidine (H512). The single mutation did not yield significant changes in the enzymic properties (including any significant increase in the redox potential of the type-1 Cu). In contrast, the triple mutation resulted in several significant changes. In comparison with the wild type, the Rhizoctonia and Myceliophthora laccase triple mutants had a phenol-oxidase activity whose pH optimum shifted 1 unit lower and higher, respectively. Although the redox potentials were not significantly altered, the Km, kcat and fluoride inhibition of the laccases were greatly changed by the mutations. The observed effects are interpreted as possible mutation-induced structural perturbations on the molecular recognition between the reducing substrate and laccase and on the electron transfer from the substrate to the type-1 Cu centre.

The complete nucleotide sequence of the mitochondrial DNA (mtDNA) molecule of the parasitic nematode worm Ascaris suum has been determined. This molecule lacks genes for tRNAs of the standard form. Instead, 21 sequences are found that can be folded into structures that resemble tRNAs in which the TIC arm and variable loop are missing and replaced with a single loop of between 4 and 12 nucleotides. Considerations of various properties of these sequences, including the number, predicted anticodons, conserved nucleotides, direction of transcription, base composition, and relative gene arrangements are consistent with the interpretation that they are genes for a different sort of tRNA. Transfer RNA genes with a similar potential secondary structure are found in mtDNA of the free-living nematode Caenorhabditis elegans, suggesting that this unusual form of tRNA is used by all nematode mitochondria.

Sequence analysis of five gene families that were isolated from tobacco thin cell layer explants initiating floral development [Meeks-Wagner et al. (1989). Plant Cell 1, [25][26][27][28][29][30][31][32][33][34][35] showed that two encode the pathogenesis-related proteins basic chitinase and basic ,B-l,3-glucanase, while a third encodes the cell wall protein extensin, which also accumulates during pathogen attack. Another sequence family encodes the water stress-induced protein osmotin [Singh et al. (1989). Plant Physiol. 90, 1096-11011. We found that osmotin was also induced by vira1 infection and wounding and, hence, could be considered a pathogenesis-related protein. These genes, which were highly expressed in explants during de novo flower formation but not in explants forming vegetative shoots [MeeksWagner et al. (1989). Plant Cell 1, 25-35], were also regulated developmentally in day-neutra1 and photoresponsive tobacco plants with high expression levels in the roots and moderate-to low-leve1 expression in other plant organs including flowers. An unidentified gene family, FB7-4, had its highest leve1 of expression in the basal internodes. Our findings indicate that these genes, some of which are conventionally considered to encode pathogen-related proteins, also have a complex association with normal developmental processes, including the floral response, in healthy plants.

To develop an in vitro phytochrome assembly system, we have expressed an oat phytochrome cDNA in both the yeast Saccharomyces cerevisiae and the bacterium Escherichia col. Analysis of soluble protein extracts showed that the recombinant apophytochromes were full-length and capable of covalently attaching the phytochrome chromophore analogue phycocyanobilin. Difference spectra indicated that in vitroassembled holophytochrome species were photoreversible; however, maxima and minima difference absorption values were blue-shifted relative to those of the native photoreceptor. Extracts containing the recombinant apophytochromes were also incubated with phytochromobilin, the natural chromophore synthesized from biliverdin by cucumber etioplast preparations. In these experiments, the difference spectrum obtained was identical to that of native oat holophytochrome. These results suggest that the recombinant apophytochromes adopt a structure similar to that of the apoprotein biosynthesized in vivo. ELISAs were used to quantitate phytochrome expression levels in both yeast and E. coil extracts. These measurements show that 62-75 % of the phytochrome apoprotein in the soluble protein extract was competent to assemble with bilins to form spectrally active holophytochrome.