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The expression of mitochondrial citrate synthase (CS) and the cytosolic enzyme NADP-dependent glutamate dehydrogenase (NADPH-GDH) was examined in Euglena gracilis Klebs strain z Pringsheim in relation to light and glutamate provision. Exposure of dark grown cells to white or red light but not blue light caused a transient increase in CS and NADPH-GDH specific activity over the first 12 hours of regreening. Illumination with white or red light but not blue light also initiated efficient chloroplast development over 72 hours. Provision of L-glutamate as sole nitrogen source caused an increase in the specific activity of both enzymes during the first 48 hours of organotrophic growth. Enhanced enzyme activity was shown to be due to an increase in enzyme protein by immunochemical titration, using monospecific antisera raised against the homogeneously pure proteins. Poly(A)-containing RNA was extracted from regreening and glutamate-grown cells and used to programme a reticulocyte or wheat germ lysate in vitro translation system. The translatable poly(A) RNA profiles remained constant during regreening in white or blue light, and in relation to nitrogen provision. Messenger RNAs encoding CS and NADPH-GDH were detectable at all developmental stages by immunoprecipitation, independent of increases in enzyme synthesis. Results suggest that protein expression operates primarily at a post-transcriptional level in Euglena. In order to study the role of poly(A) RNA abundance in developmental expression, a cDNA library was constructed in E. coli for the isolation of CS and NADPH-GDH cDNAs to probe their target mRNA species. Full length double stranded cDNA was synthesised from Euglena poly(A) RNA, enriched in CS and NADPH-GDH mRNAs by denaturing sucrose gradient fractionation. The cDNA library was screened for CS cDNA with a complementary 17'mer mixed oligodeoxynucleotide probe. Several positive colonies were isolated. The further characterisation of cDNA from two strongly hybridising clones, by Northern-blotting, hybrid-release translation, and SP6-directed in vitro transcription is described. Other positively hybridising clones await further analysis. Heterologous cDNA probes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase (RuBPCase) from pea and Chlamydomonas, and the major light harvesting chlorophyll a/b apoprotein from pea, did not cross-hybridise with Euglena RNA. However, a maize genomic clone encoding the large subunit of RuBPCase was used to measure the relative abundance of large subnit mRNA in Euglena during regreening of cells in white and blue light. Results showed that the lack of a blue light response during proplastid development was apparent at the level of chloroplast gene expression. |
