her3 encodes a zebrafish bHLH protein of the Hairy-E(Spl) family. During embryogenesis, the gene is transcribed exclusively in the developing central nervous system, according to a fairly simple pattern that includes territories in the mesencephalon/rhombencephalon and the spinal cord. In all territories, the her3 transcription domain encompasses regions in which neurogenin 1 (neurog1) is not transcribed, suggesting regulatory interactions between the two genes. Indeed, injection of her3 mRNA leads to repression of neurog1 and to a reduction in the number of primary neurones, whereas her3 morpholino oligonucleotides cause ectopic expression of neurog1 in the rhombencephalon. Fusions of Her3 to the transactivation domain of VP16 and to the repression domain of Engrailed show that Her3 is indeed a transcriptional repressor. Dissection of the Her3 protein reveals two possible mechanisms for transcriptional repression: one mediated by the bHLH domain and the C-terminal WRPW tetrapeptide; and the other involving the N-terminal domain and the orange domain. Gel retardation assays suggest that the repression of neurog1 transcription occurs by binding of Her3 to specific DNA sequences in the neurog1 promoter. We have examined interrelationships of her3 with members of the Notch signalling pathway by the Gal4-UAS technique and mRNA injections. The results indicate that Her3 represses neurog1 and, probably as a consequence of the neurog1 repression, deltaA, deltaD and her4. Moreover, Her3 represses its own transcription as well. Surprisingly, and in sharp contrast to other members of the E(spl)gene family, transcription of her3 is repressed rather than activated by Notch signalling.

Using a temperature-inducible hsp70:Gal4 activator and UAS:myc-notch1a-intra as effector, we determined quantitatively the kinetics of expression of both transgenes and analysed the effects of varying their expressivity on several phenotypic traits in the developing zebrafish. hsp70:Gal4 is transcribed within 15min after temperature-mediated induction, but Gal4 RNA decays rapidly. The Gal4 protein was found to be quite stable, as functional Gal4, which was detectable 1.5h after heat shock (HS), persisted for at least 13h. myc-notch1a-intra RNA is expressed approximately 1.5h after HS, but unlike the Gal4 RNA, it was found to be very stable; it continues to accumulate during the succeeding 17h after HS. Fully penetrant phenotypic effects are obtained after a relatively long activator induction with a 30-min HS.

In search of the precyanobacterial origin of the typical thylakoid lipids found in cyanobacteria and chloroplasts, we analyzed the polar lipids of the anaerobic phototrophic bacterium Rhodopseudomonas viridis. Glycolipids (monogalactosyl-, digalactosyl- and glucuronosyl diacylglycerol), phospholipids (phosphatidyl choline, -ethanolamine, -glycerol and cardiolipin) and an ornithine lipid were isolated and identified by NMR (1H, 13C, 31P) and mass spectrometry. Positional distribution and pairing of fatty acids in molecular species show small, but significant differences between glyco- and phospholipids. In this context, a new enzymatic method is described for assigning the enantiomeric structure of the diacylglycerol moiety in glyco- and phospholipids. 14C-Labelling studies suggest that monogalactosyl diacylglycerol is formed by galactosylation of diacylglycerol as in chloroplasts and not by glucosylation followed by epimerization as in cyanobacteria. The two 1,6-linked galactopyranose residues of digalactosyl diacylglycerol are both in β-linkage and thus differ from the corresponding chloroplast lipid with its α-β-sequence. R. viridis does not contain the sulfolipid, and even phosphate starvation does not induce the synthesis of this most characteristic thylakoid lipid, which on the other hand is present in other anaerobic phototrophic bacteria.

High-resolution, natural abundance 13C-NMR spectroscopy was used to analyze the positional distribution of fatty acids in the predominant plant glycoglycerolipids. Fatty acid profiles attributed spectroscopically to the sn-1 and sn-2 positions were in very good agreement with the enzymatically measured distribution using a lipase from Rhizopus arrhizus. The 13C-NMR spectroscopy analysis makes use of small shift differences of the aryl carbonyl carbons. As in other lipids, in glycolipids sn-1-bound acyl carbonyls are also shifted further downfield than the sn-2-bound carbonyls. Furthermore, in both positions the carbonyl shift depends on the distance to the nearest double bond resulting in identical series of incremental sequence shifts to higher fields from saturated to Δ9, Δ7 and Δ6 double bonds, respectively. These deductions were enabled by analysis of galactolipids isolated from plants belonging to the Apiaceae and Boraginaceae, which contain high proportions of all-cis-δ7,10,13-hexadecatrienoic and all-cis-Δ6.9,12,15-octadecatetraenoic acid, respectively, in the sn-2 position.