The rbcL locus codes for the large subunit of rubisco. Severa! light-sensitive, uniparental rubisco mutants have been characterized. The first isolated mutant, 10-6C, was shown to produce an inactive rubisco enzyme with a lower large-subunit isoelectric point [Spreitzer and Mets, 1980].
Comparison of the sequences of the rbcL genes of this mutant and wild type reveals a single nucleotide change that replaces the gly residue 171 near the first active site of the large subunit with aspartic acid [Dron et al., 1983].
The significance of this base substitution has been confirmed recently when a revertant of this mutant was found to have the original wild-type nucleotide
32 Rochaix: et al.
a
ii ~"'
"'
D ci ;:!; a:
s
Fig. 3. Chloroplast DNA map of Chlamydomonas reinhardii. The three inner circles from the outside to the inside represent the EcoRI, BamHI, and Bglll restriction maps [Rochaix, 1978]. Dark wedges indicate the positions of the 4S RNA genes [Malnoe and Rochaix, 1978].
The two segments of the inverted repeat are drawn on the outside of the map. They contain the rRNA genes and the gene of the 32 kd membrane polypeptide psbA [Erickson et al., 1984a]. The introns in the 23S rRNA gene and in psbA are drawn in thinner lines relative to the coding sequences. 02 is the gene for another photosystem II polypeptide. The genes for the large subunit of ribulose bisphosphate carboxylase, rbcL [Malnoe et al., 1978], for the a and f3 subunits of the ATP synthase, atpA and atpB, respectively [Woessner et al., 1984;
Kovacic and Rochaix, unpublished results], and for the elongation factor EF-Tu, tufA [Watson and Surzycki, 1982] are also indicated. The other gene locations should be considered as tentative; they are based only on heterologous hybridizations with specific probes for the E.
coti genes of the ribosomal proteins L22 and/or SI9, for S4 and/or Sil and/or SI3 and for the genes of the f3 and {3' subunits of E. coli RNA polymerase [Watson and Surzycki, 1983]. The chloroplast DNA regions whose transcripts are present in large (•), medium (@), and low (!?il) amounts are shown. The eight identified chloroplast ARS sequences are indicated by 01 to 08 [Vallet et al., 1984; Loppes and Denis, 1983]. The four chloroplast DNA sequences promoting autonamous replication in Chlamydomonas are marked by ARCI, ARC2, and AROa,b [Rochaix et al., 1984a]. Two origins of replication, oriA and oriB, are indicated [Waddell et al., 1984].
TABLE II. Protein Genes Localized on the Chloroplast Genome of C. reinhardii
Locus Polypeptide Introns Reference
rbcL Large subunit of rubisco 0 Malnoe et al. [1979]
psbA 32 kd thylakoid polypeptide 4 Malnoe et al. [1979]; Erickson
associated with PSII, et al. [1984]
targe! for herbicides
psbD 02 polypeptide associated 0 Rochaix et al. [ 1984]
with PSU
atpA ex subunit of ATP synthase nd3 Woessner et al. [1984]; Kovacic and Rochaix (unpublished results)
atpB {3 subunit nd Woessner et al. [1984]; Kovacic
and Rochaix (unpublished results)
atpE E subunit nd Woessner et al. [ 1984]
atpH CF 0 subunit III nd Woessner et al. [1984]
tuf A elongation factor Tu nd Watson and Surzycki [1982]
"nd, not determined.
TABLE III. Chloroplast Mutations Characterized at the Gene Level Mutation Polypeptide affected Locus Genetie alteration Reference
J0-6C Large subunit of rbcL Point mutation Spreitzer and Mets [1980]; Oron
rubisco et al. [1983]
18-5B rbcL Point mutation Spreitzer and Ogren [1983]
DCMU4 32 kd membrane psbA Point mutation Erickson et al. [1984]
polypeptide
Dr2 psbA Point mutation Galloway and Mets [1982]
Ar7 psbA Point mutation Galloway and Mets [1984]
11-IA psbA
Dele<;oo}
8-36C psbA Deletion Spreitzer and Mets [1981]; cf.
Figure 4
11-40 psbA Deletion
FuDll-2 psbA
Dold;oo}
FuD7 psbA Deletion Bennoun (unpublished results);
cf. Figure 4
FuDl3 psbA Deletion
FuD-50 {3 subunit of ATP atpB Delction Woessner et al. [1982]
synthase
34 Rochaix et al.
[Spreitzer et al., 1982; Spreitzer, Rahire, and Rochaix, unpublished results].
In that the 10-6C mutation has been shown to be Iinked to other genetic markers in the uniparental linkage group [Mets and Geist, 1983], this muta-tion provides the first correlamuta-tion site between the genetic and physical chloroplast DNA maps in C. reinhardii. Two other uniparental rubisco mutants, 18-5B and 18-7G, were isolated by screening photosynthetic mutants for their inability to recombine with the original 10-6C mutant [Spreitzer and Ogren, 1983]. Both ofthese mutants Jack rubisco enzyme. Although no large subunit can be detected when cells of 18-7G are labeled for a short period with 35S-sulfate, a slightly truncated product (which is immunoprecipitated with antirubisco antibody) is seen in the 18-5B mutant. Indeed, sequencing of the rbcL gene of this mutant has revealed the presence of a termination codon near the end of the gene. Pulse-chase experiments indicate that the truncated product is unstable. The 18-58 and 18-7G mutants are not only useful for correlating large-subunit structure to function and enzyme assem-bl y and for searching for chloroplast suppressors [Spreitzer et al., 1984], they are also of considerable interest for investigations of the coordination of synthesis of the large and small subunits of rubisco in the chloroplast and nucleocytoplasmic compartments, respectively. Hybridizations of RNA from wild type and from mutant cells with DNA probes specific for the genes of the large and small subunit indicate that these two genes are transcribed at nearly the same level in the mutants and in wild type. Immunoprecipitation of pulse-labeled cells with antirubisco antibody shows that the small subunit is synthesized and processed to its mature size in both mutants, suggesting that it is imported into the chloroplast. lt is, however, rapidly degraded in these mutants, indicating that the stoichiometry of the two subunits is achieved at a posttranslational level by some chloroplast-located protease. These re-sults agree with the work of Schmidt and Mishkind [1983], which demon-strated that under conditions of inhibition of chloroplast protein synthesis the small subunit is still synthesized but is rapidly degraded.