ABSTRACTwe have investigated regulatory DNA elements in the expression of the drug metabolizing P-450c gene of rats. After combining the 5' flanking and upstream untranslated regions of the isolated P-450c gene with structural gene for chloramphenicol acetyltransferase (CAT), the fusion genes were transfected into cultured cells (Hepa-1 and L cells) for the assay oL transient expression of CAT activity. CAT activity was expressed inducibly in response to 3-methylcholanthrene only in Hepa-1 cells. At least three regions containing regulatory DNA elements were indentified; one, which is present in the sequence from -44b to -0.2kb immediately upstream of the TATA box, functions in the basal level of transcription, and the other two which were located in the sequence from -0.8kb to -1.0kb and from -1.0kb to -6.3kb, enhance in combination, transcription in response to inducers in a manner independent of their orientation.

In the biosphere, sucrose is mainly synthesized in oxygenic photosynthetic organisms, such as cyanobacteria, green algae and land plants, as part of the carbon dioxide assimilation pathway. Even though its central position in the functional biology of plants is well documented, much less is known about the role of sucrose in cyanobacteria. In those prokaryotes, sucrose accumulation has been associated with salt acclimation, and considered as a compatible solute in low-salt tolerant strains. In the last years, functional characterizations of sucrose metabolizing enzymes, metabolic control analysis, cellular localization of gene expressions, and reverse genetic experiments have revealed that sucrose metabolism is crucial in the diazotrophic growth of heterocystic strains, and besides, that it can be connected to glycogen synthesis. This article briefly summarizes the current state of knowledge of sucrose physiological functions in modern cyanobacteria and how they might have evolved taking into account the phylogenetic analyses of sucrose enzymes.

Anabaena sp. PCC 7120 is a filamentous cyanobacterium able to fix atmospheric nitrogen in semi-regularly spaced heterocysts. For correct heterocyst function, a special cell envelope consisting of a glycolipid layer and a polysaccharide layer is essential. We investigated the role of the genes hgdB and hgdC, encoding domains of a putative ABC transporter, in heterocyst maturation. We investigated the subcellular localization of the fusion protein HgdC-GFP and followed the differential expression of the hgdB and hgdC genes during heterocyst maturation. Using a single recombination approach, we created a mutant in hgdB gene and studied its phenotype by microscopy and analytical chromatography. Although heterocysts are formed in the mutant, the structure of the glycolipid layer is aberrant and also contains an atypical ratio of the two major glycolipids. As shown by a pull-down assay, HgdB interacts with the outer membrane protein TolC, which indicates a function as a type 1 secretion system. We show that the hgdB-hgdC genes are essential for the creation of micro-oxic conditions by influencing the correct composition of the glycolipid layer for heterocyst function. Our observations confirm the significance of the hgdB-hgdC gene cluster and shed light on a novel mode of regulation of heterocyst envelope formation.

The presence of two alkaline/neutral invertases (Inv-A and Inv-B) in the filaments of Nostoc (also named Anabaena) sp. strain PCC 7120 and the involvement of sucrose metabolism in nitrogen fixation led us to investigate the physiological function of those isoforms in cells growing under different nitrogen sources. The highest expression level of each encoding gene was obtained in the presence of ammonium. These results were paralleled by polypeptide and enzyme activity level. In cells of N(2)-fixing filaments, localization of gene expression and subcellular enzyme activity assays demonstrated that invA gene (alr1521) expresses only in vegetative cells, whereas for invB (alr0819), expression is detected in both vegetative cells and heterocysts. In contrast to invA, when invB was knocked out, the filaments were unable to grow on diazotrophic conditions and the accumulation of sucrose and glycogen was altered. Our results demonstrate an essential role for Inv-B for diazotrophic growth and that Inv-B plays a key role in the coordination of sucrose and glycogen metabolism. We can also suggest that invB is likely to integrate the repertoire of genes regulated by a cyanobacterial transcription factor (NtcA) that plays a central role in global nitrogen control.