Chlorellanitrate reductase gene structure and regulation
The reduction of nitrate to nitrite catalyzed by Nitrate Reductase (NR) is considered to be the rate-limiting and regulatory step of nitrate assimilation. Nitrate assimilation is a major metabolic pathway and occurs in a wide range of organisms including bacteria, yeast, fungi, algae and higher plants which in turn supply the nutritional nitrogen requirements for other forms of life. Chlorella NR mRNA levels are very responsive to changes in nitrogen source. In the presense of ammonia as the sole N-source, repressed conditions, NR mRNA is undetectable. Under inducing conditions, removal of ammonia and addition of nitrate, rapid NR mRNA synthesis occurs. mRNA synthesis also occurs under derepressed conditions, removal of ammonia and no addition of nitrogen, but the pattern of synthesis differs when compared to inducing conditions. We are studying the elements involved in regulating the expression of this important gene. Two overlapping genomic clones (NRS1 and NRS) were isolated from a cosmid library. The two clones were sequenced and their sequences were aligned with that of a full length NR cDNA. The gene is approximately 8 kb and consists of 19 exons and 18 introns. Using primer extension, RNase protection assay and Race PCR, two transcription start sites were identified and each is surrounded by potential initiator sequences. No TATA, CAAT, or GC rich promoter elements were located. We are currently investigating the 5' UTR using in vivo footprinting and deletion analysis of the promoter.
Dawson, HN; Pendleton, LC; Solomonson, LP; Cannons, AC
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