STRUCTURE AND EXPRESSION OF THE CHLAMYDOMONAS-REINHARDTII ALAD GENE ENCODING THE CHLOROPHYLL BIOSYNTHETIC ENZYME, DELTA-AMINOLEVULINIC-ACIDDEHYDRATASE (PORPHOBILINOGEN SYNTHASE)

cDNA clones for the alad gene encoding the chlorophyll biosynthetic en zyme ALA dehydratase (ALAD) from Chlamydomonas reinhardtii were isolat ed by complementation of an Escherichia coli ALAD mutant (hemB). The C . reinhardtii alad gene encodes a protein that has 50 to 60% sequence identity with higher plant ALADs, and includes a putative Mg2+-binding domain characteristic of plant ALADs. Multiple classes of ALAD cDNAs were identified which varied in the length of their 3'-untranslated re gion. Genomic Southern analysis, using an ALAD cDNA as a probe, indica tes that it is a single-copy gene. This suggests that the differently sized ALAD cDNAS are not the products of separate genes, but that a pr imary ALAD transcript is polyadenylated at multiple sites. A time cour se determination of ALAD mRNA levels in 12-h light: 12-h dark synchron ized cultures shows a 7-fold increase in ALAD mRNA at 2 h into the lig ht phase. The ALAD mRNA level gradually declines but continues to be d etectable up to the beginning of the dark phase. ALAD enzyme activity increases 3-fold by 6 h into the light phase and remains high through 10 h. Thus, there is an increase in both ALAD mRNA level and ALAD enzy me activity during the light phase, corresponding to the previously ob served increase in the rate of chlorophyll accumulation.