Expression of the arylsulphatase reporter gene under the control of the nit1 promoter in Chlamydomonas reinhardtii
1997
Ohresser, M. | Matagne, R.F. | Loppes, R.
In Chlamydomonas reinhardtii, the expression of the nit1 gene encoding nitrate reductase is dependent or the nature of the nitrogen source and on other environmental factors. We have fused the nit1 promoter region to the arylsulphatase (ars) reporter gene lacking its own promote and introduced this chimeric construction (nit1/ars) into wall-less strain of C. reinhardtii. A new and sensitive method, based on the use of alpha-naphthylsulphate as a substrate and a diazonium salt as a chromogenic post-coupling agent, was developed to detect the activity of arylsulphatase (an enzyme which is almost completely secreted in th culture medium) both in vitro and in agar plates. The transformants carrying nit1/ars did not express arylsulphatas when grown in ammonium-sufficient medium but readily accumulated the enzyme in ammonium-free medium either supplemented, or not supplemented, with nitrate or nitrite. The nit1/ars construct, however, was not expressed in th nit2 mutant lacking a specific transcription regulator controlling the expression of nit1. These results, together with the observation that the transcription of nit1/ars is initiate at the same sites as the nit1 endogenous gene, confirms th hypothesis that the regulation of nit1 expression takes place mainly at the transcriptional level. The expression of the ars gene from the nit1 promoter was high enough to allow direct measurements of arylsulphatase activities in pools of transformants without prior isolation of nit1/ars clones. This original procedure has permitted the analysis of th effects of nested deletions in the nit1 promoter region or the expression of the reporter gene. The results indicate that the -282 to -198 sequence is required for transcription to occur and that the -751 to -282 region contains several elements mediating nit1 expression.
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