The Chlamydomonas reinhardtii chloroplast mutant 68-4PP is phenotypically indistinguishable from wild type at 25°C but fails to grow photosynthetically at 35°C. It had about 30% of the wild-type level of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) holoenzyme and carboxylase activity when grown at 25°C, but less than 15% when grown at 35°C. Pulse-labeling with 35S showed that the decrease in enzyme Ribulose-1,5-bisphosphate (Rbu-P2) carboxylase/oxygenase (EC 4.1.1.39) is a key enzyme in photosynthetic C02 fixation that is assembled from eight chloroplast-encoded large subunits and eight nuclear-encoded small subunits in all plants and most algae (1). It catalyzes both the carboxylation and the oxygenation of Rbu-P2 in the chloroplast and thus initiates the competitive metabolic pathways of photosynthesis and photorespiration, respectively (2). Photorespiration is a nonessential process in plants (3), and the competition of 02 with CO2 at the large-subunit active site (4) ultimately leads to the loss of fixed carbon and a reduction in photosynthetic efficiency (5

Mutants of the unicellular, green alga Chlamydomonas reinhardtii were recovered by screening for the absence of photoautotrophic growth at 35°C. Whereas nonconditional mutants required acetate for growth at both 25 and 35°C, the conditional mutants have normal photoautotrophic growth at 25°C. The conditional mutants consisted of two classes: (a) Temperature-sensitive mutants died under all growth conditions at 35°C, but (b) temperature-sensitive, acetate-requiring mutants were capable of heterotrophic growth at 35°C when supplied with acetate in the dark. The majority of mutants within the latter of these two classes had defects in photosynthetic functions. These defects included altered pigmentation, reduced whole-chain electron-transport activity, reduced ribulosebisphosphate carboxylase activity, or pleiotropic alterations in a number of these photosynthetic components. Both nuclear and chloroplast mutants were identified, and a correlation between light-sensitive and photosynthesis-deficient phenotypes was observed.The unicellular green alga Chlanmydomtonas reinhardtii has proven to be a useful organism for the genetic dissection and analysis of photosynthesis in eukaryotes (16). Since C. reinhardtii maintains a functional photosynthetic apparatus when grown in the dark with acetate, photosynthesis-deficient mutants have been identified as light-sensitive, acetate-requiring strains (10). Genetic analysis can also be performed, and mendelian or uniparental (maternal) inheritance defines nuclear or chloroplast mutations, respectively (5).We have been particularly interested in using chloroplast mutants and their revertants to investigate the structure/function relationships of Rubisco& (13). As in higher plants, Rubisco consists of eight nuclear-encoded small subunits and eight chloroplast-encoded large subunits, and the large subunit appears to contain the active-site domain (7). However, of three Rubisco mutants defined at the level of the large-subunit gene sequence (3, 15), two were found to result from nonsense mutations. Calculating from the large-subunit gene sequence (2), one would expect only about 5% nonsense mutations out of the total number of point mutations that could alter the large-subunit protein sequence. It thus appeared that many mutants with amino-acid substitutions in the large-subunit protein were not being re-

The 69-12Q mutant of Chiamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase activity, but retains holoenzyme protein. It results from a mutation in the chloroplast large-subunit gene that causes an isoleucine-for-threonine substitution at amino-acid residue 173. Considering that lysine-175 is involved in catalysis, it appears that mutations cluster at the active site.The chloroplast-encoded large-subunit of Rubisco3 contains the active site of the holoenzyme (10). CO2 and 02 compete for RuBP at this site, and the ratio of carboxylation and oxygenation determines net CO2 fixation in photosynthesis (13). Attempts have been made to increase carboxylation by exploring the effects of Rubisco mutations generated by directed mutagenesis in vitro (6-8, 12, 23 with the pf-2 mt-centromere-marker strain following standard procedures (20). All strains were maintained in the dark at 25°C on acetate medium (20) containing 1.5% Bacto agar. Liquid cultures, containing acetate medium without agar, were grown on a rotary shaker in the dark at 25°C.Biochemical Analysis. A Hansatech oxygen electrode was used to measure oxygen evolution and whole-chain electron-transport activity (methyl viologen reduction) as described previously (21). Chl (24) and total soluble protein (1) were determined in sonicated cell lysates. Rubisco carboxylase activity was assayed (16) in clarified lysates, and the amount of holoenzyme was estimated from the absorbance at 280 nm of the lysates fractionated on sucrose gradients (19). All values are the average of three independent determinations.Molecular Biology. Total cell DNA was isolated, cloned in bacteriophage lambda, and the R15 fragment (4) containing the Rubisco large-subunit gene was subcloned in pBR329 (3), all by previously described methods (17). The recombinant plasmid, constructed with the 69-12Q large-subunit gene, was designated pLS69-12Q. A HaeIII fragment, containing the large-subunit gene, was isolated from the plasmid, digested with HindlIl and