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CHLOROPLAST RIBOSOME BIOGENESIS IN CHLAMYDOMONAS

Chloroplast protein synthesis in Chlamydomonas reinhardtii is dispensable when cells are provided acetate as a carbon source. Mutants defective in synthesis, assembly, or function of chloroplast ribosomes are therefore conditionally viable. Positive selection of nonphotosynthetic cells on arsenate has been combined with a simple screening procedure to isolate mutants with a broad spectrum of defects in chloroplast protein synthesis. Eight new mutants deficient in chloroplast ribosomes have been isolated. Three of these have been characterized genetically and phenotypically, and compared with two previously described ribosome mutants, ac-20 and cr-1. A working model of ribosome assembly is proposed which suggests possible biochemical roles for these five Mendelian gene loci.

ac-20 and cr-1. A working model of ribosome assembly is proposed which suggests possible biochemical roles for these five Mendelian gene loci.
Protein synthesis is generally regarded as an indispensable function, and mutants defective in ribosome structure, assembly, or function would normally be lethal. Under permissive conditions, however, the protein-synthesizing systems of mitochondria of baker's yeast, Saccharomyces cerevisiae, a facultative anaerobe (cf. Mahler, 1973), and of the chloroplast of the green acetate flagellate, Chlamydomonas reinhardtii (Boynton et al., 1972Goodenough and Levine, 1971) appear to be dispensable. In addition, variegated plastome mutants of barley, cotton, and Pelargonium have been described in which the white sectors show a deficiency of chloroplast ribosomes while green sectors are normal and apparently serve to support growth of the chloroplast ribosome-deficient tissue (BiSrner et al., 1973;Hagemann et al., 1973;Katterman and Endrizzi, 1973).
In Chlamydomonas, chloroplast protein synthe-sis appears to be necessary only for photosynthetic growth ; mutants blocked in chloroplast ribosome formation or function are thus conditionally viable when supplied with acetate (Boynton et al., 1972;Goodenough and Levine, 1971). Chlarnydomonas therefore offers a unique potential for identification of genes affecting components of a protein-synthesizing system. For such a model system to be useful experimentally, positive techniques are needed for selection and identification of suitable mutants. Genetic control of a limited number of ribosomal components can be studied by using antibiotic resistance mutants, which are readily isolated in Chlamydomonas Burton, 1972;Gillham et al., 1970;Bogorad, 1971, 1972;Schlanger et al., 1972;Schlanger and Sager, 1974), as they are in bacteria. In this paper we report the use of a positive selection method involving preferential killing of photosynthetic cells by arsenate (Togasaki and Hudock, 1974), and a screening protocol  which yields a broad spectrum of mutants with defects in chloroplast protein synthesis unrelated to antibiotic resistance. These include mutants blocked in formation of chloroplast ribosomes. Three new Mendelian mutants deficient in chloroplast ribosome monomers are compared phenotypically and genetically to the previously described chloroplast ribosome mutants ac-20 and cr-I (Boynton et al., , 1972. From our data we have constructed a formal scheme of ribosome assembly which indicates possible functional roles for each of these five nuclear gene loci. This scheme in turn can be used to suggest biochemical approaches to verify the molecular defects in each of the mutants.

Growth Conditions
Haploid and diploid vegetative clones of C. reinhardtii strain 137c were cultured at 25°C under ~6,000 Ix cool white fluorescent light on 1.5% agar plates of Tris-acetate-phosphate (TAP) medium (Gorman and Levine, 1965), and on TAP or on high salt liquid medium with (HSA) or without (HS) 0.2% acetate (Sueoka, 1960), in 300-ml shake cultures under ~15,000 Ix cool white fluorescent light. No significant differences were found in photosynthetic activity or whole cell ribosome profiles between cells grown to midlog phase on TAP and HSA liquid medium.

Mutagenesis and Arsenate Selection of Acetate-Requiring Mutants
Cells grown in TAP liquid medium to midlog phase in the light were harvested and resuspended at a density of 3.8 × 10 e cells/ml in 0.02 M sodium citrate, pH 5.0, containing 1 #g/ml N-methyl-N'-nitro-N-nitrosoguanidine (Lee and Jones, 1973). The suspension was shaken in darkness for 30 min at 25°C, centrifuged, and washed once with citrate buffer and twice with TAP medium. After the density had been adjusted to 5 × 10 ~ cells/ml, l-ml aliquots were dispensed into 100 individual tubes (13 × 100 mm). These tubes were shaken under 15,000 Ix constant illumination for 24 h to allow about four cell doublings. Cells were then plated on to TAP agar containing sodium arsenate and incubated in light. This procedure allowed expression of the mutant phenotype but ensured that mutants isolated from different plates were of independent origin. The concentration of arsenate must be adjusted for light intensity and for strain differences. Under our conditions, 1 mM arsenate for haploid cells and 3 mM for diploids gave a survival rate of approximately 1 × 10-' in unmutagenized cultures.
Cells unable to grow on minimal medium (acetate requirers) were selected and grown to midlog phase in 300-ml TAP liquid cultures. Photosystem 11 (Hill reaction activity) was assayed as photoreduction of dichlorophenol-indophenol . RuDPCase activity was measured by a modification of the procedure of Boynton et al. (1972).
Cell suspensions (2-4 ml, 5 × 107 cells/ml) in buffer A of Hoober and Blobel (1969) without sucrose were broken at 5,000 lb/in 2 in a 3/s-in diameter French pressure cell of an Aminco model 5-598A motor-driven press (American Instrument Co., Inc., Travenol Laboratories, Inc., Silver Spring, Md.) and centrifuged at 40,000 g for 20 min at 2°C. A 100-#1 aliquot of enzyme ( 1 / 1 -l /8 dilution of supernate with 0.5 mg/ml bovine serum albumin in buffer A without sucrose) was added to 900#1 of reaction mixture containing 50 #mol Tris-HCI, pH 7.5; 2.5 ,mol MgCI2; 1.0 ~tmol dithiothreitol; 0.1 #mol ribulose diphosphate (RuDP) (Sigma Chemical Corp., St. Louis, Mo.); 40 #mol KHCO3; and 8 #Ci NaHI'COs. NaHI'COs (New England Nuclear, Boston, Mass., 50 mCi/mmol) was mixed with KHCOs to a final concentration of 200 ,Ci/mmol HCO3 , frozen, and thawed immediately before use. After incubation for 20 min at 25°C, the reaction was stopped with 0.5 ml of glacial acetic acid. Samples were transferred to open scintillation vials and dried at 80-100°C to drive off unfixed 1'CO2. The residue was dissolved in 0.1 ml water and counted in 10 ml Bray's scintillation fluid in a Beckman LS-233 scintillation spectrometer (Beckman Instruments, Inc., Fullerton, Calif.) equipped with a dpm controller programed to correct for sample quenching and to calculate micromoles CO2 fixed per hour per reaction tube. Our modified procedure has resulted in about a twofold increase in RuDPCase activity over values reported by Boynton et al. (1972Boynton et al. ( , 1973. 10-ml cell samples for chlorophyll and whole cell protein determinations were pelleted at 12,000 g and frozen. The pellet was resuspended and chlorophyll extracted with 80% acetone in dim light for 20 min. After centrifugation for 10 min at 12,000 g, total chlorophyll was determined in the supernate by the method of Arnon (1949). The pellet was dissolved in NaOH and assayed for protein (Boynton et al., 1972).
Whole cell ribosome profiles of putative mutants grown in TAP or HSA liquid cultures were analyzed on 10-30% sucrose gradients (Tris-HC1, pH 7.5, 25 raM; MgCI2, 25 mM; KCI, 25 mM) without Triton addition (method modified from Bourque et al., 1971). Gradients (17 ml) were centrifuged for 15 h at 22,500 rpm, 4°C, in a Spinco SW-27.1 rotor (Beckman Instruments, Inc., Spinco Div., Paio Alto, Calif.), and scanned at 254 nm using ISCO density gradient fractionating equipment. Optical density traces were made over a spread of approximately 22 cm using a Sargent recorder, to facilitate comparison of peak areas by cutting and weighing Xerox copies of such traces.

Electron Microscopy
Cells grown on TAP liquid medium were harvested by centrifugation, washed once with HSA medium, and fixed for 2 h at room temperature in a equal volume mixture of 4% glutaraldehyde (70%, Ladd Research Industries, Inc., Burlington, Vt.) in 0.004 M Na2HPO,-KH~PO,, pH 7.0, and HSA culture medium. After three washes in the phosphate-HSA buffer, they were postfixed in 2% OsO, in 0.1 M phosphate buffer, pH 7.0, for 2 h at room temperature. After dehydration in ethanol, cells were embedded in a low viscosity epoxy resin (Spurr, 1969). Sections cut with a DuPont diamond knife (E. I. DuPont de Nemours & Co., Wilmington, Del.) on a Cambridge ultramicrotome (Cambridge Instrument Co., Ossining, N.Y.) were mounted on naked 75 × 300 mesh grids and contrasted with uranyl acetate (saturated solution in 70% ethanol) and lead citrate.

Genetic Analysis
Standard techniques of crossing, tetrad analysis, and diploid formation were used (Levine and Ebersold, 1960;Levine and Goodenough, 1970;Gillham, 1963;Ebersold, 1967). Gametes were differentiated overnight in Nliquid medium (HSA without NH~C1). Zygotes were matured in light on N-plates containing 4% agar (K. Van Winkle-Swift, manuscript in preparation). Products of crosses were scored for acetate requirement by streak tests on HS plates. Diploids used to assess complementation between ribosome mutants were isolated on HSA plates with the complementary arginine auxotrophic markers arg-2 and arg-7. Complementation for ribosome function was tested with respect to RuDPCase activity, Hill reaction activity, and ribosome profile as described above.
Double mutants were identified among progeny of crosses between two mutant parents by a rapid technique which exploits the observed ability of the five mutants studied here to complement one another in diploids with respect to photosynthetic function (see Results). This quick diploid method has general utility for genotypic identification in complementing mutants without the necessity of backcrossing. In a tetratype tetrad (e.g., 3 : 1 acetate-requiring, acetate-independent), 9ne acetaterequiring product will complement with each parent (the two opposite parental types) and the third will complement with neither. Gametes from each tetrad product and each parent tester stock were mated for 1 h and the suspensions plated on HS agar at a density of 0.5 1 × 105 cells/plate. Plates were incubated in light for 4 days before scoring under a dissecting microscope for diploid formation. Diploids formed between complementing mutants appear at this time as discrete, bright green, raised colonies and are readily distinguished from the background of ungerminated zygotes and unmated gametes. When one oftbe mutant strains tested is leaky, i.e., grows appreciably on HS medium, resolution can be improved by using tester stocks carrying an additional auxotrophic marker, e.g. arg-7, to reduce growth of unmated tester gametes.

Selection of Chloroplast Protein Synthesis Mutants
The protocol used to isolate chloroplast ribosome mutants (Fig. 1) relies on preferential killing of photosynthesizing cells by arsenate, leaving nonphotosynthetic (acetate-requiring) cells as survivors. This principle was suggested by Schneyour and Avron (cf. Schiff et al., 1971) for isolation of photosynthetic mutants of Euglena, and has been tested in reconstruction experiments with Chlamydomonas mutants deficient in photosynthetic electron transport and Calvin cycle enzymes by Togasaki and Hudock (1974).
We have confirmed these results and the validity of the method for isolation of chloroplast protein synthesis mutants in reconstruction experiments using ac-20 and cr-1 . In mutagenesis experiments using 1-5 #g/ml nitrosoguanidine, we find that acetate-requiring colonies comprise about 1-2% of arsenate-tolerant colonies tested (Table I). Among these acetaterequiring colonies (Fig. 1, Class II), mutants with deficiencies in chloroplast protein synthesis can be identified by a syndrome of chloroplast defects including low ribulose diphosphate carboxylase (RuDPCase) activity, and deficiency of Photosystem 1I electron transport (Hill reaction activity), as well as characteristic alterations in lamellar organization (Boynton et al., 1972Goodehough et al., 1971). Mutants specifically deficient in RuDPCase or Photosystem 1I are also recovered in good yields, as are other acetate-requiring mutants with normal RuDPCase and Hill reaction activities.
More than 95% of the arsenate-tolerant colonies picked after mutagenesis are resistant to arsenate under all three growth conditions employed ( Fig.  1, Class I). These may represent either permeability mutants or phenocopies of such mutants. Mutants which grow on minimal medium but die  Wild-type haploid mt÷ and diploid (arg-2 arg-7+/arg-2 ÷ arg-7) stocks were used. NG, nitrosoguanidine.
on TAP in the dark (Fig. 1, Class III) may have altered mitochondrial functions and presumably survive in light by virtue of normal photosynthetic ability (A. Wiseman, personal communication). Two mutants have been found which grow on minimal medium or on TAP-arsenate, but die on TAP alone (Fig. 1, Class IV). Togasaki and Hudock (1974) have advanced several possible hypotheses to account for the preferential killing of photosynthetic cells by arsenate, but consider none of them a fully satisfactory explanation. Arsenate is known to be a competitive inhibitor of phosphate in ATP formation both in photosynthesis and in respiration (Avron and Jagendorf, 1959;Crane and Lipmann, 1953). We might account for the observed arsenate tolerance of mutants of classes II, lII, and IV by postulating an intracellular pool of inorganic phosphate shared by mitochondria and chloroplast. In wild-type cells, arsenate would prevent both respiratory and photosynthetic ATP formation in light, with the result that cells die.
Cells blocked in photosynthesis may be unable to form ATP photosynthetically, or may be prevented from utilizing ATP by a block in the carbon cycle. Either condition would result in accumulation of ADP and phosphate. Chloroplast phosphate pools may then be made available to mito-chondria, thus reducing arsenate toxicity sufficiently for respiratory ATP formation to occur. The observation by Togasaki and Hudock (1974) that arsenate is less toxic to wild-type cells in darkness than in light is consistent with this hypothesis. Conversely, a block in mitochondrial ATP formation may allow more phosphate and ADP to be utilized in the chloroplast. Acetate-sensitive mutants (class IV) could have mitochondrial alterations which make acetate-stimulated respiration lethal, perhaps by accumulation of some toxic byproduct. These cells could survive either on arsenate or on minimal medium by repressing mitochondrial functions in favor of photosynthesis. It is worth noting that wild-type cells grow normally on HSA plates at concentrations of arsenate which are lethal in TAP medium, probably because of the 14-fold greater phosphate concentration in the HSA medium.
When stable vegetative diploid cells are subjected to mutagenesis under the same conditions, recessive Mendelian mutations will not be expressed, and most acetate-requiring isolates recovered should be mutants in chloroplast DNA, which segregate vegetatively . Diploid mutants recovered can be returned to the haploid state by a 2N × N cross (Gillham, 1963). In our experience, such mutants can be isolated only if cells are allowed several generations for segregation and expression before being challenged with arsenate. Only one diploid acetate-requiring mutant was found in the experiment shown in Table I, and this appeared to be segregating in a mixed colony.
Another possible approach to selection of mutations in chloroplast DNA would he mutagenesis of synchronous haploid cultures during the time of chloroplast DNA replication. However, yields of non-Mendelian (chloroplast) streptomycin-resistant mutants were found by Lee and Jones (1973) to be enhanced only about twofold by this means, and the background of Mendelian mutants was high. The use of diploid parent stocks may thus be preferable .

Phenotypic Characterization of Chloroplast Ribosome Mutants
In two experiments to test the efficacy of our selection protocol, more than 100 acetate-requiring mutants were recovered. Out of 63 tested, 13 mutants were identified as potentially deficient in chloroplast protein synthesis. Eight mutants have abnormal ribosome profiles characterized by a deficiency of chloroplast ribosome monomers. Three of these are described in detail in the remainder of the paper. Five mutants have normal ribosomal profiles on sucrose gradients but show the syndrome of defects associated with failure of chloroplast protein synthesis. These may make functionally defective ribosomal components which are nevertheless capable of assembly into particles of normal ribosomal size, or they may have defects in auxiliary components of protein synthesis such as tRNAs, activating enzymes, factors, etc.
We have decided to designate all new chloroplast ribosome genes, with the exception of antibiotic resistant mutants, with the symbol cr°, numbered sequentially following the convention established by Boynton et al. (1970). Typical ribosome profiles of cr-2, cr-3, and cr-4, the three new mutants which have been studied in detail, are shown in Fig. 2 together with those of wild type, ac-20, and cr-1. Such ribosome profiles are highly reproducible over a range of 1-15 A~eo units loaded per gradient, and relative sizes of the various ribosome peaks are absolutely characteristic of an individual mutant (Table II A). Since centrifugation conditions were constant for all gradients, and since the pattern for each genotype was not altered by varying the amount of ribosomes loaded per gradient, the differences in ribosome profile among the mutants cannot be attributed to pressure or concentration effects (Chua et al., 1973;lnfante and Baierlein, 1971).
We have identified ribosome peaks in accordance with the generic classification of Bourque et al. (1971). The major peaks of 83s and 70s represent cytoplasmic and chloroplast ribosome monomers, respectively (Bourque et al., 1971;Chua et al., 1973;Hoober and Blobel, 1969;Surzycki et al., 1970). The 54s particles accumulated by crol were found by Boynton et al. (1970) to contain 23s ribosomal RNA, and are therefore believed to be the large subunit of the chloroplast ribosome. The 54s particles accumulated by cr-2 and cr-3 (Fig. 2, Table II A) are assumed to be similar to those found in cr-l, although RNA analyses have not.yet been done. Since deficiency of 41s particles is associated with deficiency of 16s rRNA in cr-I , these particles are thought to be the small chloroplast ribosomal subunit. Bourque et al. (1971) found 66s particles replacing 70s in their ac-20 stock (an ac-20 cr-I double mutant, Boynton et al., 1970), but did not distinguish 66s particles in wild-type cells. 66s ribosomes are also seen in certain antibiotic-resistant mutants , but are not necessarily identical to those seen in ac-20 . With increased resolution, Boynton et al. (1972) showed that both 66s and 70s ribosomes could also be distinguished in wild type, ac-20 and cr-l, but considered that both classes represented chloroplast ribosome monomers. In the gradients shown here (Fig. 2), the 66s peak is clearly resolved from the 70s. The finding that the 66s ribosome content of mutants does not vary nearly as much as the 70s (Table II A,B) raises the question of whether these 66s particles are really chloroplast ribosomes after all. Further analysis of RNA content, subunit structure, etc., of purified 66s particles will be required to resolve this issue.
We find that the 70s ribosome content of a given mutant can be correlated with its RuDPCase and Hill reaction activities, but not with chlorophyll (Fig. 3). This supports the contention that chloroplast ribosome function in vivo is specifically required for RuDPCase synthesis and for formation of some photosystem II component, but is not necessary for chlorophyll synthesis (Boynton et al., 1972Goodenough and Levin¢, 1970 FIGURE 2 Typical ribosome profiles (A2,) of wild type and five ribosome-deficient mutants. Ribosome peaks are assigned to generic classes based on approximate sedimentation constant as described by Bourque et al. (1971). 66s peak is indicated between 54s and 70s peaks but is not labeled. 18s peak is attributed to the enzyme aggregate ribulose diphosphate earboxylase (RuDPCase), mol wt > 500,000. This peak is seen only in wild type and in mutants with an appreciable level of RaDPCase activity. Traces are enlarged from 4-in ISCO strip chart recordings, where the 17-ml gradient is displayed over approximately 3 cm spread, with top of gradient to left in each case. Ordinate varies from 0.25 to 2.5 A264 full scale setting on chart recorder, depending on the amount of material loaded on individual gradients. Peaks are identified as follows: 4Is, small subunit of chloroplast ribosomes; 54s, large subunit of chloroplast ribosomes; 70s and possibly 66s, chloroplast ribosome monomers; 83s, cytoplasmic ribosome monomers (Bourque et al., 1971). In each class, the percentage of the total ribosome area occupied by the peak is given together with the standard error. also apparent from Fig. 3 that the 70s ribosomes formed by the relatively leaky mutants cr-3 and cr-4 have normal activity and are thus presumably not defective, whereas those of cr-I may be partially defective. Both ac-20 and cr-I are capable of slow phototrophic growth. Under these conditions, R u D P C -ase and ultrastructure become partially normalized (Boynton et al., 1972;Goodenough and Levine, 1970;Togasaki and Levine, 1970), but c h l o r o p l a s t r i b o s o m e deficiency r e m a i n s unchanged (Boynton et al., 1972). We have repeated these studies with cr-2, cr-3, and cr-4, and have extended our analysis to include Hill reaction 64 activity (Table Ill) When chloroplast protein synthesis is impaired, 84 either by mutations such as ac-20 and cr-I (Boyn-77 ton et al., 1972;Goodenough and Levine, 1970) or 67 by antibiotic inhibition 84 41 Goodenough, 1971), organization of the chloro-61

Mean Distribution o f Ribosome Particles After Sucrose Gradient Analysis of Wild Type and Mutants Deficient in Chloroplast Ribosomes
plast lamellar system is altered. Chloroplasts of 74 such cells have lamellae which are either unpaired 70 74 or stacked into giant grana, and in addition lack a 69 pyrenoid. Chloroplasts of cr-2, cr-3, and cr-4 show a similar syndrome of ultrastructural defects (Figs. 4-7), with cr-2 having the most extreme manifestations (Fig. 5) and cr-3 the least (Fig. 6), as compared to wild type (Fig. 4); cr-4 is affected to an intermediate degree (Fig. 7). It is interesting that the severity of the ultrastructural abnormalities directly mirrors the relative deficiencies in 70s ribosomes, RuDPCase, and Hill reaction (Fig. 3). RuDPCas¢ activity, Hill reaction activity, and chlorophyll content expressed on a whole cell protein basis, plotted against the 70s ribosome content of wild type and the five chloroplast ribosome-deficient mutants. Individual points are based on the mean of one to five determinations for mutants and 8-10 determinations for wild type. Standard errors are shown for wild type.

Genetic Characterization of Chloroplast Ribosome Mutants
ac-20 has previously been mapped to linkage group XIII (Hastings et al., 1965). Although cr-1 is a Mendelian mutation , it has not been mapped. In crosses to wild type, cr-2, cr-3, and cr-4 show Mendelian inheritance (Table   IV A). Isolates recovered in the minus mating type' (rot-) retain the abnormal ribosome profile char-acteristic of the plus (mt ÷) parent. Each of the five ribosome mutants has been crossed to arg-2 and arg-7 and the complementary arginine markers used to isolate diploids of each ribosome mutant with its wild-type allele and with each of the other four ribosome mutants. All five mutants complement one another with respect to photosynthetic activity and ribosome profile. The significance of these results will be considered in the Discussion.
Pairwise crosses in all possible combinations have also been made among the five mutants. Wild-type recombinants and double mutants recovered in each cross further support the conclusion that these mutants represent five distinct loci. Preliminary segregation data (Table IV B) suggest that none of the loci are closely linked. The excess of NPD over PD tetrads observed in two of the crosses may be due to the small sample size, or may be comparable to the cases of excess NPD's between nonlinked genes in Chlamydomonas discussed by Gowans (1960). We are now in the process of mapping cr-I and the three new mutants. None of the four appears to be linked to arg-2 or arg-7 (linkage group 1) or to mating type (linkage group VI).
We have repeatedly observed a tendency for stocks of cr-1 to alter their ribosome phenotype over time, probably by the occurrence of secondary mutations. These changes involve a reduction in 54s subunit accun~ulation, in some cases accompanied by an increase in 70s ribosomes so that the profile resembles that of wild type, or in other cases with no increase in 70s ribosomes, producing a profile like that of ac-20. However, isolates of cr-1 with the characteristic ribosome phenotype shown in Fig. 2 and Table ii A can always be recovered by outcrossing such double mutants.
Although cr-2, cr-3, and cr-4 have not shown a tendency to revert or accumulate new mutations, our experience with cr-1 has led us to check frequently the ribosome profiles of all stock cultures, and to verify the ribosome phenotype of all products of at least one tetrad from each cross. All arginine-acetate double mutants were therefore checked for ribosome profile before diploid formation, and all products of at least one tetratype tetrad were checked from all crosses between mutants.
We have compared the individual mutants and all possible double mutants and diploids in terms of amount of chloroplast ribosome monomers, accumulation of large chloroplast ribosomal sub-units, and function of chloroplast ribosomes in vivo (measured as RuDPCase and Hill reaction activities) (Tables II A,B, and V; Fig. 8). Among the five individual mutants, only cr-I and cr-2 are indistinguishable with respect to these parameters, and these two mutants show different responses in diploids and double mutants. The discussion will deal in more detail with these comparisons in terms of a model for ribosome assembly.

D I S C U S S I O N
Our working model for ribosome assembly is similar to formal schemes used to deduce enzymatic pathways from studies of mutants and suggests possible biochemical sites of action of the five mutations which produce 70s ribosome deficiency. The rationale for this model can be summarized as follows: (a) the phenotype of an individual mutant demonstrates whether the mutation blocks ribosome formation completely or partially and whether formation of one ribosomal subunit or both is affected. For example, a mutant unable to assemble small subunits will accumulate large subunits and be deficient in monomers; (b) the phenotype of diploids containing one mutant and one wild-type allele indicates whether the mutation is dominant, partially dominant, or recessive. In general, a mutant at a locus which codes for a product required in stoichiometric amounts, i.e. a structural component, would be expected to show partial dominance, since 50% of the ribosome component made in the diploid will be defective. Thus the total number of functional ribosomes per cell will be subnormal although greater than the haploid mutant level. A mutant gene recessive to wild type is likely to affect a catalytic function rather than a stoichiometric one, since a diploid heterozygous for an allele specifying an enzymatic step will probably have a sufficient level of enzyme to allow normal ribosome assembly. However, a stoichiometric mutant for which the wild-type allele overcompensates in function could be confused with a catalytic mutant. Absolute dominance may indicate mutation in a regulatory gene; (c) the phenotype of diploids made from two haploid mutant parents indicates whether the mutants can complement one another in function. Such complementation tests in a system where macromolecular assembly is being studied can give information not only about allel- Comparison was made after growth of midlog phase under mixotrophic (HSA (HS liquid, 5% CO2 in air, ~ 15,000 Ix) growth conditions. Data are from single determinations in which mixotrophic and phototrophic genotype in the same experiment. DPIP, dichlorophenol-indophenol.
liquid, ~ 15,000 Ix) and phototrophic conditions were compared for a given ism, but also about functional relationships in mutants known from recombination frequencies to be nonallelic. Two nonallelic mutants, neither of which is dominant to wild type, presumably involve interrelated functions if they fail to complement one another in a diploid; (d) haploid double mutants can be used in conjunction with data from diploids to order genes in a biosynthetic pathway. In general, a mutation affecting an early step in a pathway will be epistatic to a mutation in a later step. The principal exceptions to this rule should be dominant mutations in regulatory genes, which would be detected by their behavior in diploids.
We have used these criteria to construct the scheme of ribosome assembly shown in Fig. 9. The phenotypes of the five mutants we have studied place them in two groups (cf. Fig. 8, Tables II A, B, and V): ac-20 and cr-4 appear to be involved in synthesis of components common to both the 4Is and 54s ribosomal subunits, whereas cr-l, cr-2, and cr-3, all of which accumulate 54s particles, are probably mutations in a pathway specific to 41s subunit formation, cr-I is partially dominant in diploids with wild type (Fig. 8, Table II B, Table  V) and may therefore affect production of a component needed in stoichiometric amounts. ac-20, cr-2, cr-3, and cr-4 are all recessive to wild type and may affect catalytic functions.
All four double mutants carrying the ac-20 allele have the ribosome phenotype of ac-20 (Table II B ,Fig. 8). Although neither ac-20 nor cr-4 accumulates 54s subunits, they can be distinguished by the greater percentage of 70s ribosomes in cr-4 (Figs. 2 and 8, Table II A) and correspondingly greater RuDPCase activity (Fig. 3, Table III). cr-4 is partially epistatic to cr-l, cr-2, and cr-3 (Table II B, Fig. 8). We have therefore placed ac-20 and cr-4 earlier in sequence than cr-1, cr-2, and cr-3, generating a forked pathway in which steps common to the synthesis of both subunits precede synthesis of the subunits individually (Fig. 9).
cr-I and cr-2 are epistatic to cr-3 in double mutants, since none of these double mutants has as great a content of 70s ribosomes as cr-3 (Table II B, Fig. 8), but cannot be ordered relative to each other since their ribosome profiles are indistinguishable. We have placed cr-I be[ore cr-2 in Fig.  9 with the rationale that the cr-1 ÷ gene may control production of stoichiometric quantities of a ribosome precursor which is then modified by the product of the cr-2 ÷ gene.
We can use this scheme to infer possible biochemical defects in ribosome formation in each of the individual mutants and to suggest ways in which each mutant should be analyzed further: ac-20 has been studied previously and has been found to be deficient in 16s and 23s rRNA (Bourque et al., 1971;Surzycki, cited in Goodenough et al., 1971), and possibly 5s rRNA (Surzycki and Hastings, 1968). The stock of ac-20 used by Bourque et al. (1971) also contained the cr-I mutation . It is not clear whether this is also true of the stock used by Surzycki. Structural genes for chloroplast rRNA are thought to be coded in chloroplast DNA (Surzycki et al., 1970;Surzycki and Rochaix, 1971). Since ac-20 shows Mendelian inheritance, it is probably not a mutation in an rRNA cistron, but is more probably a mutant in some other gene needed for rRNA formation. Goodenough and Levine (1971) have suggested that ac-20 directly affects chloroplast rRNA synthesis. Our data are consistent with this hypothesis, but do not distinguish between a deficiency of chloroplast RNA polymerase and other defects, for example in methylation or cleavage of precursor RNA (Carritt and Eisenstadt, 1973;Luck, 1973, 1974;Pace, 1973).
cr-4 also appears to be blocked at an early catalytic step in ribosome formation before diversion of the 41s and 54s pathways. Like ac-20, cr-4 may be defective in synthesis or processing of rRNA, but the rRNA content of this mutant has not yet been examined. The slight accumulation of 54s subunits in double mutants of cr-4 with cr-l, cr-2, and cr-3 (Table II B Fig. 8) is the expected result of combination of a leaky mutant at an early step with a stringent mutant at a later step.
cr-l, cr-2, and cr-3 all accumulate 54s particles and are therefore suspected of being defective in FIGURE 4 Median section through mixotrophically grown wild-type cell, showing parts of nucleus (N) with nucleolus, and the cup-shaped chloroplast (C) with a well-developed pyrenoid (P). The chloroplast lamellar system is largely organized into two-disk grana, x 24,000.  A. Segregation of acetate-requiring progeny in initial crosses of cr-2, cr-3, and cr-4 to wild type. Aberrant segregations may be attributed to product lethality and extra mitotic divisions of surviving products. Nitrosoguanidine is known to cause product lethality (N.W. Gillham, unpublished observations). B. Segregation of chloroplast ribosome genes in preliminary crosses made to produce double mutants. PD, parental ditype (four acetate-requiring: none acetate-independent); NPD, nonparental ditype (two acetate-requiring: two acetate-independent); T, tetratype (three acetate-requiring: one acetate-independent).
synthesis of 41s ribosomal subunits. They can be distinguished by comparing their ribosomal phenotypes as haploids, diploids, and double mutants. Each shows partial functional complementation with the others, and all three are capable of recombination to give wild-type progeny. Thus three distinct loci with related functions in formation of the small chloroplast ribosomal subunit are now identified.
cr-I appears to have a stoichiometric defect in 41s subunit synthesis, since diploids of cr-I with its wild-type allele still accumulate some 54s subunits and are slightly deficient in 70S ribosomes (Fig. 8, Table II B). The 54s particles accumulated by cr-1 contain only 23s rRNA, and whole cell preparations are deficient in 16s rRNA . Since cr-I shows Mendelian inheritance, it is presumably not a mutation in a structural gene for 16s rRNA. cr-I might, however, be defective in a core protein which is essential for assembly of the proteins of the 4Is subunit on 16s rRNA and without which 16s rRNA is degraded.
cr-2 and cr-3 are more likely to be defective in catalytic steps in 4Is subunit synthesis, since both these mutations are recessive to wild type. Information on the course of ribosome formation in bacteria indicates there may be certain assembly factors which are not themselves part of the mature ribosome structure (Bryant and Sypherd, 1974); cr-2 and cr-3 could be defective in proteins of this type. Another possibility is that one or both of these mutants is deficient in methylation of rRNA, since they are phenotypically remarkably similar to the "poky" strain of FIGURE 6 Median section through mixotrophically grown cr-3 cell, showing parts of nucleus (N) and chloroplast (C). A deranged pyrenoid (P) can be seen, and chloroplast lamellae are abnormally stacked, x 24,000. FIGURE 7 Median section through mixotrophically grown cr-4 cell showing parts of nucleus (N) and chloroplast (C). The pyrenoid is absent and chloroplast lamellae are either unpaired or stacked into giant grana. The abnormalities seen in this mutant are more extreme than in cr-3 (Fig. 6), but less extreme than in cr-2 (Fig. 5). The ultrastructural phenotype is very similar to that described previously for ac-20 and cr-I (Boynton et al., 1972). x 24,000.
In the last 3 yr, seven Mendelian genes, including two loci conferring erythromycin resistance =hl oropl ast ri bosome pool FIGURE 9 Working model for chloroplast ribosome assembly in Chlamydomonas, based on genetic and phenotypic analysis of five nonallelic mutants deficient in chloroplast ribosomes, ac-20 and cr-4 appear to be blocked early in ribosome assembly, cr-l, cr-2, and cr-3 all accumulate 54s subunits and are therefore thought to be blocked in 41s subunit formation.
described by Mets and Bogorad (1971) and five ribosome loci described here, and at least six non-Mendelian gene loci governing antibiotic resistance Burton, 1972;Gillham et al., !970;Bogorad, 1971, 1972;Schlanger et al., 1972;Schlanger and Sager, 1974), have been implicated in the biogenesis of chloroplast ribosomes. Future studies of ribosome-deficient mutants of the type described in this paper together with antibiotic-resistant mutants should reveal the extent of nucleocytoplasmic interaction in organelle ribosome formation.