PCR amplification of the highly conserved baculovirus genes late expression factor 8 (lef-8), late expression factor 9 (lef-9) and polyhedrin/granulin (polh/gran) combined with molecular phylogenetic analyses provide a powerful tool to identify lepidopteran-specific baculoviruses and to study their diversity. In the present investigation, we have improved the degenerate oligonucleotides and corroborated the approach that was recently described by Lange et al. (Lange, M., Wang, H., Zhihong, H., Jehle, J.A., 2004. Towards a molecular identification and classification system of lepidopteran-specific baculoviruses. Virology 325, 36-47.). Baculovirus DNA was isolated from 71 uncharacterized historic baculovirus samples, and partial gene sequences were amplified by using gene-specific degenerate PCR primers. The obtained PCR products were directly sequenced, and the deduced amino acid sequences were compiled and aligned with published sequences of these target genes. A phylogenetic tree of 117 baculoviruses was inferred using maximum parsimony and distance methods. Based on the comprehensive phylogenetic analysis of the partial lef-8, lef-9 and polh/gran genes, we propose a phylogenetic species criterion for lepidopteran-specific baculoviruses that uses the genetic distances of these genes for species demarcation.

In plants, isoenzymes are present in the cytosol and in plastids. The plastidic enzymes (P1 and P2) are subject to redox regulation, but mechanisms that adjust cytosolic G6PDH activity are largely unknown. We adopted a leaf disc system for monitoring the effects of various conditions on G6PD isoform expression and enzyme activities in potato (Solanum tuberosum). Cytosolic G6PDH activity remained constant during water incubation in the dark. In continuous light or in the presence of metabolizable sugars in the dark, cytosolic G6PDH activity increased 6-fold within 24 h. Cycloheximide incubation demonstrated that enhanced cytosolic G6PDH activity depends on de novo protein synthesis. Osmotic change, phosphate sequestration, or oxidative stress did not affect cytosolic G6PDH activity. Furthermore, enzyme activity and protein contents closely followed the corresponding mRNA levels. Together with the fact that multiple SURE elements are present in the promoter region of the gene, these results suggest that cytosolic G6PDH activity is regulated by sugar availability at the transcriptional level. Plastidic G6PDH activity stayed constant during water incubation in the light and dropped to minimal levels within 6 h in the dark. Conversely, plastidic G6PDH activity of leaf discs incubated on Paraquat rose to 10-fold higher levels, which was not prevented by cycloheximide. Similar increases were found with nitrite, nitrate, or sulfate. No major changes in protein or mRNA contents of the plastidic P1 and P2 isoforms were registered. K m (Glc-6-phosphate) values of plastidic G6PDH activity differed between samples incubated on water or Paraquat, suggesting posttranslational modification of the plastidic enzyme(s). Immunoprecipitation of 32 P-labeled samples with P1 isoform-specific antibodies showed that the chloroplast enzyme is subject to protein phosphorylation. Obviously, in extended dark periods, G6PDH activity in the stroma is restricted but can be stimulated in response to high demands for NADPH.Glc-6-phosphate dehydrogenases (G6PDHs, EC 1.1.1.49) catalyze the oxidation of Glc-6-phosphate (G6P) to 6-phosphogluconolactone concomitant with reducing NADP to NADPH. The product 6-phosphogluconolactone is first converted to 6-phosphogluconate by 6-phosphogluconolactonase (EC 3.1.1.31) and then decarboxylated by 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44), yielding another mole of NADPH and ribulose-5-phosphate. The first enzyme, G6PDH, controls the flux through this nonreversible limb of the oxidative pentose phosphate pathway (OPPP;Williams, 1980; Copeland and Turner, 1987). In higher plants, G6PDH isoenzymes reside in two compartments, the cytosol and plastids (Heber et al., 1967;Schnarrenberger et al., 1973). Reducing power (NADPH) generated by the OPPP sustains reductive biosyntheses (e.g. fatty acids, isoprenoids and aromatic amino acids) in the dark and nitrogen assimilation in heterotrophic tissues (Bowsher et al., 1992). In addition, OPPP intermediates are continuously withdrawn to fuel other metabolic p...

cDNAs encoding for two isoforms of O-acetylserine (thiol) lyase (OAS-TL), which catalyzes the synthesis of cysteine, have been isolated from Arabidopsis thaliana. Secondary structure together with expression patterns derived during photomorphogenesis indicate cellular localizations in the cytosol and plastids, thus allowing a direct comparison of compartment-specific forms within one species. The cytosolic OAS-TL complemented an E. coli auxotrophic mutant lacking cysteine synthesis. Both isoforms are represented by small gene families. They are expressed under all conditions investigated and were observed to increase in expression in plants grown with limited sulfate supply.

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In a recent paper (Wenderoth et al., J Biol Chem 272: 26985-26990, 1997) we reported that the positions of the two redox regulatory cysteines identified in a plastidic G6PD isoform from potato (Solanum tuberosum L.) differ substantially from those conserved in cyanobacterial G6PDH sequences. To investigate the origin of redox regulation in G6PDH enzymes from photoautotrophic organisms, we isolated and characterized several G6PD cDNA sequences from higher plants and from a green and a red alga. Alignments of the deduced amino acid sequences showed that the cysteine residues cluster in the coenzyme-binding domain of the plastidic isoforms and are conserved at three out of six positions. Comparison of the mature proteins and the signal peptides revealed that two different plastidic G6PDH classes (P1 and P2) evolved from a common ancestral gene. The two algal sequences branch off prior to this class separation in higher plants, sharing about similar amino acid identity with either of the two plastidic G6PDH classes. The genes for cytosolic plant isoforms clearly share a common ancestor with animal and fungal G6PDH homologues, whereas the cyanobacterial isoforms branch within the eubacterial G6PDH sequences. The data suggest that cysteine-mediated redox regulation arose independently in G6PDH isoenzymes of eubacterial and eukaryotic lineages.