EXPRESSION OF THE ARYLSULFATASE REPORTER GENE UNDER THE CONTROL OF THE NIT1 PROMOTER IN CHLAMYDOMONAS-REINHARDTII

In Chlamydomonas reinhardtii, the expression of the nit1 gene encoding nitrate reductase is dependent on the nature of the nitrogen source a nd on other environmental factors. We have fused the nit1 promoter reg ion to the arylsulphatase (ars) reporter gene lacking its own promoter and introduced this chimeric construction (nit1/ars) into a wall-less strain of C. reinhardtii. A new and sensitive method, based on the us e of alpha-naphthylsulphate as a substrate and a diazonium salt as a c hromogenic post-coupling agent, was developed to detect the activity o f arylsulphatase (an enzyme which is almost completely secreted in the culture medium) both in vitro and in agar plates. The transformants c arrying nit1/ars did not express arylsulphatase when grown in ammonium -sufficient medium but readily accumulated the enzyme in ammonium-free medium either supplemented, or not supplemented, with nitrate or nitr ite. The nit1/ars construct, however, was not expressed in the nit2 mu tant lacking a specific transcription regulator controlling the expres sion of nit1. These results, together with the observation that the tr anscription of nit1/ars is initiated at the same sites as the nit1 end ogenous gene, confirms the hypothesis that the regulation of nit1 expr ession takes place mainly at the transcriptional level. The expression of the ars gene from the nit1 promoter was high enough to allow direc t measurements of arylsulphatase activities in pools of transformants without prior isolation of nit1/ars clones. This original procedure ha s permitted the analysis of the effects of nested deletions in the nit 1 promoter region on 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 me diating nit1 expression.