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We performed whole-genome resequencing of 12 field isolates and eight commonly studied laboratory strains of the model organism Chlamydomonas reinhardtii to characterize genomic diversity and provide a resource for studies of natural variation. Our data support previous observations that Chlamydomonas is among the most diverse eukaryotic species. Nucleotide diversity is ∼3% and is geographically structured in North America with some evidence of admixture among sampling locales. Examination of predicted loss-of-function mutations in field isolates indicates conservation of genes associated with core cellular functions, while genes in large gene families and poorly characterized genes show a greater incidence of major effect mutations. De novo assembly of unmapped reads recovered genes in the field isolates that are absent from the CC-503 assembly. The laboratory reference strains show a genomic pattern of polymorphism consistent with their origin as the recombinant progeny of a diploid zygospore. Large duplications or amplifications are a prominent feature of laboratory strains and appear to have originated under laboratory culture. Extensive natural variation offers a new source of genetic diversity for studies of Chlamydomonas, including naturally occurring alleles that may prove useful in studies of gene function and the dissection of quantitative genetic traits.

The formation and function of eukaryotic cilia/flagella require the action of a large array of dynein microtubule motor complexes. Due to genetic, biochemical, and microscopic tractability, Chlamydomonas reinhardtii has become the premier model system in which to dissect the role of dyneins in flagellar assembly, motility, and signaling. Currently, fifty-four proteins have been described as components of various Chlamydomonas flagellar dyneins or as factors required for their assembly in the cytoplasm and/or transport into the flagellum; orthologues of nearly all these components are present in other ciliated organisms including humans. For historical reasons, the nomenclature of these diverse dynein components and their corresponding genes, mutant alleles and orthologues has become extraordinarily confusing. Here, we unify Chlamydomonas dynein gene nomenclature and establish a systematic classification scheme based on structural properties of the encoded proteins. Furthermore, we provide detailed tabulations of the various mutant alleles and protein aliases that have been used and explicitly define the correspondence with orthologous components in other model organisms and humans.

Clustering and assembly of expressed sequence tags (ESTs) constitute the basis for most genomewide descriptions of a transcriptome. This approach is limited by the decline in sequence quality toward the end of each EST, impacting both sequence clustering and assembly. Here, we exploit the available draft genome sequence of the unicellular green alga Chlamydomonas reinhardtii to guide clustering and to correct errors in the ESTs. We have grouped all available EST and cDNA sequences into 12 063 ACEGs (assembly of contiguous ESTs based on genome) and generated 15 857 contigs of average length 934 nt. We predict that roughly 3000 of our contigs represent full-length transcripts. Compared to previous assemblies, ACEGs show extended contig length, increased accuracy and a reduction in redundancy. Because our assembly protocol also uses ESTs with no corresponding genomic sequences, it provides sequence information for genes interrupted by sequence gaps. Detailed analysis of randomly sampled ACEGs reveals several hundred putative cases of alternative splicing, many overlapping transcription units and new genes not identified by gene prediction algorithms. Our protocol, although developed for and tailored to the C. reinhardtii dataset, can be exploited by any eukaryotic genome project for which both a draft genome sequence and ESTs are available.

IN BRIEF Individuals with type 2 diabetes have a higher incidence of liver function test abnormalities than individuals who do not have diabetes. Mild chronic elevations of transaminases often reflect underlying insulin resistance. Elevation of transaminases within three times the upper limits of normal is not a contraindication for starting oral antidiabetic or lipid-modifying therapy. In contrast, antidiabetic agents have generally been shown to decrease alanine aminotransferase levels as tighter blood glucose levels are achieved.

The National Science Foundation-funded Chlamydomonas reinhardtii genome project involves (a) construction and sequencing of cDNAs isolated from cells exposed to various environmental conditions, (b) construction of a high-density cDNA microarray, (c) generation of genomic contigs that are nucleated around specific physical and genetic markers, (d) generation of a complete chloroplast genome sequence and analyses of chloroplast gene expression, and (e) the creation of a Web-based resource that allows for easy access of the information in a format that can be readily queried. Phases of the project performed by the groups at the Carnegie Institution and Duke University involve the generation of normalized cDNA libraries, sequencing of cDNAs, analysis and assembly of these sequences to generate contigs and a set of predicted unique genes, and the use of this information to construct a high-density DNA microarray. In this paper, we discuss techniques involved in obtaining cDNA end-sequence information and the ways in which this information is assembled and analyzed. Descriptions of protocols for preparing cDNA libraries, assembling cDNA sequences and annotating the sequence information are provided (the reader is directed to Web sites for more detailed descriptions of these methods). We also discuss preliminary results in which the different cDNA libraries are used to identify genes that are potentially differentially expressed.

Chlamydomonas reinhardtii is a unicellular eukaryotic alga possessing a single chloroplast that is widely used as a model system for the study of photosynthetic processes. This report analyzes the surprising structural and evolutionary features of the completely sequenced 203,395-bp plastid chromosome. The genome is divided by 21.2-kb inverted repeats into two single-copy regions of ∼80 kb and contains only 99 genes, including a full complement of tRNAs and atypical genes encoding the RNA polymerase. A remarkable feature is that >20% of the genome is repetitive DNA: the majority of intergenic regions consist of numerous classes of short dispersed repeats (SDRs), which may have structural or evolutionary significance. Among other sequenced chlorophyte plastid genomes, only that of the green alga Chlorella vulgaris appears to share this feature. The program MultiPipMaker was used to compare the genic complement of Chlamydomonas with those of other chloroplast genomes and to scan the genomes for sequence similarities and repetitive DNAs. Among the results was evidence that the SDRs were not derived from extant coding sequences, although some SDRs may have arisen from other genomic fragments. Phylogenetic reconstruction of changes in plastid genome content revealed that an accelerated rate of gene loss also characterized the Chlamydomonas/Chlorella lineage, a phenomenon that might be independent of the proliferation of SDRs. Together, our results reveal a dynamic and unusual plastid genome whose existence in a model organism will allow its features to be tested functionally.

▪ Abstract The unicellular green alga Chlamydomonas offers a simple life cycle, easy isolation of mutants, and a growing array of tools and techniques for molecular genetic studies. Among the principal areas of current investigation using this model system are flagellar structure and function, genetics of basal bodies (centrioles), chloroplast biogenesis, photosynthesis, light perception, cell-cell recognition, and cell cycle control. A genome project has begun with compilation of expressed sequence tag data and gene expression studies and will lead to a complete genome sequence. Resources available to the research community include wild-type and mutant strains, plasmid constructs for transformation studies, and a comprehensive on-line database.

In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxidase (Protox) by the N-phenyl heterocyclic herbicide S-23142 causes massive protoporphyrin IX accumulation, resulting in membrane deterioration and cell lethality in the light. We have identified a 40.4 kb genomic fragment encoding S-23142 resistance by using transformation to screen an indexed cosmid library made from nuclear DNA of the dominant rs-3 mutant of Chlamydomonas reinhardtii. A 10.0 kb HindIII subclone (Hind10) of this insert yields a high frequency of herbicide-resistant transformants, consistent with frequent non-homologous integration of the complete RS-3 gene. A 3.4 kb XhoI subfragment (Xho3.4) yields rare herbicide-resistant transformants, suggestive of homologous integration of a portion of the coding sequence containing the mutation. Molecular and genetic analysis of the transformants localized the rs-3 mutation conferring S-23142 resistance to the Xho3.4 fragment, which was found to contain five putative exons encoding a protein with identity to the C-terminus of the Arabidopsis Protox enzyme. A cDNA clone containing a 1698 bp ORF that encodes a 563 amino acid peptide with 51% and 53% identity to Arabidopsis and tobacco Protox I, respectively, was isolated from a wild-type C. reinhardtii library. Comparison of the wild-type cDNA sequence with the putative exon sequences present in the mutant Xho3.4 fragment revealed a G→A change at 291 in the first putative exon, resulting in a Val→Met substitution at a conserved position equivalent to Val-389 of the wild-type C. reinhardtii cDNA. A sequence comparison of genomic Hind10 fragments from C. reinhardtii rs-3 and its wild-type progenitor CC-407 showed this G→A change at the equivalent position (5751) within exon 10.

Three classes of cell wall-defective mutants of the unicellular green alga Chlamydomonas reinhardtii have been described in the literature differing with respect to the amounts of cell wall material and its attachment to the plasma membrane, respectively. We have compared the production of the chaotrope-soluble cell wall polypeptides by the different mutants. These experiments have been performed by comparative Western-blot analyses using antibodies raised (1) against the deglycosylation products of the insoluble wall fraction of wild-type cells, (2) against the deglycosylation product of the ‘150 kDa’ chaotrope-soluble cell wall polypeptide and (3) against the carbohydrate side chains of the Chlamydomonas cell wall glycoproteins, respectively. Considerably different levels of cell wall polypeptides were found in the LiCl-extracts from intact cells of the various mutant strains containing the apoplastic, chaotrope-soluble cell wall glycoproteins. No correlation was found between the amounts and the patterns of cell wall glycoproteins present in the LiCl-extracts and the electron microscopical classification of the mutant strains. All the mutant strains were shown to contain the same amounts and patterns of intracellular cell wall precursors as wild-type cells as revealed by Western-blot analyses of urea-SDS lysates of LiCl-pretreated cells. These findings indicate that the different mutant strains produce the same set of cell wall polypeptides at the same relative amounts as wild-type cells. However, in the case of some strains belonging to different classes of cell wall mutants and showing differential seggregation patterns in crosses, alterations were observed for the pattern of extracellular cell wall polypeptides present in the LiCl-extracts from intact cells and in the culture medium, respectively.

A group of highly active N -phenylimide photobleaching herbicides have been synthesized. These N -phenylimide herbicides as well as diphenyl ether herbicides induce protoporphyrin IX accumulation and inhibit protoporphyrinogen oxidase activity at extremely low concentrations in higher plants. The binding of a 14C -labeled N -phenylimide herbicide S-23121 [N-[4-chloro- 2-fluoro-5-[(1-m ethyl-2-propynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide] to the solubilized plastid fractions of greening corn seedlings is competed by the diphenyl ether herbicide acifluorfen-ethyl, but not by diuron, an inhibitor of photosynthetic electron transport. These results indicate a similar mode of action for both N -phenylimide and diphenyl ether herbicides. In order to investigate the mechanism of photobleaching herbicides at the molecular level, a strain of Chlamydomonas reinhardtii RS-3 resistant to N -phenylimide S-23142 [N -(4-chloro- 2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide] was isolated by mutagenesis with N -m ethyl-N′-nitro-N -nitrosoguanidine. The 90% inhibition concentration of N -phenylimide S-23142 for growth of RS-3 was 100 times higher than that for wild type. Maximum accumulation of protoporphyrin IX was reached at 0.03 μᴍ of S-23142 for the wild type and 3 μᴍ for RS-3. RS-3 was resistant to oxadiazon, oxyfluorfen and acifluorfen-ethyl which had been shown to have the same mechanism of action as N -phenylimide herbicides, but not to paraquat, diuron or fluridone. Genetic analysis of RS-3 strain showed that the resistance results from a dominant mutation ( rs-3) in the nuclear genome. The magnesium protoporphyrin IX synthesizing activity from 5-am inolevulinic acid in chloroplast fragments isolated from RS-3 was less sensitive to S-23142 than that from wild type (CC-407). Protoporphyrinogen oxidase activity in Percoll™ -purified chloroplasts from RS-3 was also less sensitive to S-23142 than that from wild type. These results indicate that the resistance of RS-3 is specific for photobleaching herbicides, and that the mutation is related to protoporphyrinogen oxidase, the primary site of the photobleaching herbicide action.

The respiratory deficient dum-1 mutant of Chlamydomonas reinhardtii fails to grow in the dark because of a terminal 1.5 kb deletion in the linear 15.8 kb mitochondrial genome, which affects the apocytochrome b (CYB) gene. In contrast to the wild type where only mitochondrial genomes of monomer length are observed, the dum-1 genomes are present as a mixture of monomer and dimer length molecules. The mutant dimers appear to result from head-to-head fusions of two deleted molecules. Furthermore, mitochondrial genomes of dum-1 were also found to be unstable, with the extent of the deletion varying among single cell clones from the original mutant population. The dum-1 mutant also segregates, at a frequency of ca. 4% per generation, lethal minute colonies in which the original deletion now extends at least into the adjacent gene encoding subunit four of NAD dehydrogenase (ND4). We have used the dum-1 mutant as a recipient to demonstrate stable mitochondrial transformation in C. reinhardtii employing the biolistic method. After 4 to 8 weeks dark incubation, a total of 22 respiratory competent colonies were isolated from plates of dum-1 cells bombarded with C. reinhardtii mitochondrial DNA (frequency 7.3 x 10(-7)) and a single colony was isolated from plates bombarded with C. smithii mitochondrial DNA (frequency 0.8 x 10(-7)). No colonies were seen on control plates (frequency < 0.96 x 10(-9)). All transformants grew normally in the dark on acetate media; 22 transformants were homoplasmic for the wild-type mitochondrial genome typical of the C. reinhardtii donor. The single transformant obtained from the C. smithii donor had a recombinant mitochondrial genome containing the donor CYB gene and the diagnostic HpaI and XbaI restriction sites in the gene encoding subunit I of cytochrome oxidase (COI) from the C. reinhardtii recipient. The characteristic deletion fragments of the dum-1 recipient were not detected in any of the transformants.

Intermolecular recombination of Chlamydomonas chloroplast genes has been analyzed in sexual crosses and following biolistic transformation. The pattern and position of specific exchange events within 15 kb of the 22-kb inverted repeat have been mapped with respect to known restriction fragment length polymorphism markers that distinguish the chloroplast genomes of the interfertile species Chlamydomonas reinhardtii and Chlamydomonas smithii. Recombinant progeny were selected from two- and three-factor crosses involving point mutations conferring herbicide (dr) and antibiotic resistance (er and spr) in the psbA, 23S and 16S ribosomal RNA genes, respectively. Exchange events were not randomly distributed over the 15-kb region, but were found to occur preferentially in a 0.7-kb sequence spanning the 3' end of the psbA gene and were much less common in an adjacent region of ca. 2.0 kb. These findings are corroborated by data showing that the dr mutation is unlinked genetically (3% recombination/kb) to the er and spr rRNA mutations, which are themselves linked and show ca. 1% recombination/kb. This discrepancy is significant since the dr-er and er-spr intervals are about the same length (ca. 7 kb). During chloroplast transformation, the 0.7-kb recombination hotspot also functions as a preferential site for exchange events leading to the integration of donor psbA gene sequences. The 0.7-kb hotspot region contains four classes of 18-37-bp direct repeats also found in other intergenic regions, but no open reading frame. Using deletion constructs in a chloroplast transformation assay, the hotspot was localized to a 500-bp region that lacks most of these repeats, which suggests that the repeats themselves are not responsible for the increased recombination frequency. Within this region, a 400-bp sequence is highly conserved between the chloroplast genomes of C. reinhardtii and C. smithii and includes several structural motifs characteristic of recombination hotspots in other systems.

We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of non-photosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.

Summary Although the chloroplast genome of Chlamydomonas reinhardtii contains essentially the same complement of genes as chloroplast genomes of land plants, the arrangement of these genes within the circular DNA molecule is very different. Studies in several laboratories suggest that comparison of gene order, restriction fragment length polymorphisms, and sequences within chloroplast DNA may be valuable tools in phylogenetic analysis of Chlamydomonas species. We have found that six interfertile strains in the C. reinhardtii group differ by restriction fragment length polymorphisms throughout their chloroplast genomes. In two of these strains the chloroplast genomes are known to be colinear and the differences largely attributable to variations in the size and distribution of clusters of short dispersed repeat sequences occurring in intergenic regions. These repeats, which are marked by Aat II and Kpn I restriction sites, are present in the chloroplast genomes of all six interfertile strains of the C. reinhardtii group, but are absent from the chloroplast genomes of C. eugametos, C. moewusii, C. incerta and C. globosa. The latter two species, which are sensitive to the same vegetative cell autolysin as C. reinhardtii, do not appear to be interfertile with C. reinhardtii. Restriction fragment analysis of chloroplast DNA suggests that C. incerta and C. globosa are very similar to one another, but differ greatly from C. reinhardtii.

The unicellular green alga Chlamydomonas has found widespread use as a model experimental system for diverse studies in cell and molecular biology. The ability of C. reinhardtii to grow heterotrophically with acetate as its sole carbon source has made this species especially useful for investigation of chloroplast biogenesis and function, since mutants unable to carry out photosynthesis are viable. The simple vegetative and sexual cycles are easily manipulated in the laboratory, making this organism a powerful tool for genetic analysis of photosynthesis as well as many other cellular functions. The usual laboratory strain of C. reinhardtii is the descendant ofan isolate made in Massachusetts in 1945. Several additional strains interfertile with this one have been isolated from nature, all from North America, and are providing a useful source of molecular diversity. More than 300 genetic and molecular loci have now been identified in seventeen linkage groups in the nuclear genome. Maps, references, cultures and other resources for Chlamydomonas research are available from the Chlamydomonas Genetics Center and other collections.

Chlamydomonas reinhardtii, the first alga subject to a genome project, has been the object of numerous morphological, physiological, and genetic studies. The organism has two genetically determined mating types (plus and minus) and all stages of the simple life cycle can be evoked in culture. In the nearly 60 years since the first standard laboratory strains were isolated, numerous crosses and exchanges among laboratories have led to some confusion concerning strain genealogy. Here we use analyses of the nuclear internal transcribed spacer regions and other genetic traits to resolve these issues, correctly identify strains currently available, and analyze phylogenetic relationships with all other available similar chlamydomonad types. The presence of a 10-bp indel in ITS2 in some but not all copies of the nuclear ribosomal cistrons of an individual organism, and the changing ratios of these in crosses, provide a tool to investigate mechanisms of concerted evolution. The standard C. reinhardtii strains, plus C. smithii +, plus the new eastern North American C. reinhardtii isolates, comprise one morphological species, one biological species of high sexual intercompatibility, and essentially identical ITS sequences (except the tip of helix I of ITS2). However, variant RFLP patterns characterize strains from each geographic site.

Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival.

The green alga Chlamydomonas reinhardtii is an ideal model in which to study the cooperation of nuclear and chloroplast genes in chloroplast ribosome biogenesis. At least six of the 33 r-proteins identified in the large subunit and 14 of the 31 r-proteins of the small subunit appear to be made in the chloroplast of C. reinhardtii, with the remainder being synthesized in the cytoplasm (Schmidt et al, 1983). Antibodies to 16 chloroplast r-proteins from C. reinhardtii have been examined for cross reactivity with specific r-proteins from Anabaena, E. coli and the chloroplast of spinach (Randolph-Anderson et al, 1989; Schmidt et al, 1984). To date 11 chloroplast-encoded r-protein genes have been identified in C. reinhardtii (Harris, 1992). The completely sequenced chloroplast genomes of tobacco, rice and the liverwort Marchantia have revealed an additional 12 r-protein genes which have not yet been located in Chlamydomonas (Shimada and Sugiura, 1991). With a few exceptions, the same set of r-protein genes is found in all chloroplast genomes examined in green algae and land plants (Palmer, 1991). The exceptions include the location of the rpl5 gene in the chloroplast genome of Chlamydomonas and certain other algae, the rpl21 gene in the chloroplast genome of Marchantia, and the rpsl6 gene in the chloroplast genome of higher plants but not in Marchantia.

Of the more than 400 species of unicellular green algae in the genus Chlamydomonas (Ettl, 1976), only a few have been used for genetic investigations. The small cell size, rapid division time, and short sexual cycles of these green flagellates make them especially favorable organisms for genetic and molecular studies. The haploid heterothallic species C. reinhardtii has been the organism of choice for most molecular, cellular and developmental investigations (see Harris, 1989). Its nuclear genome of ca. 1-2 × 105 kb comprises 18 genetic linkage groups (Harris, 1989,1990), and physical maps are being developed for many of these (Ranum et al, 1988). Detailed molecular maps have also been established for the 196 kb chloroplast and 15.8 kb mitochondrial genomes of C. reinhardtii, and formal genetic studies are possible for both organelles (see Harris, 1989,1990). A fourth genome of 6–9 mb, localized in the basal body has recently been defined (Hall et al., 1989) and correlated with a unique quasi-Mendelian circular linkage group (Ramanis and Luck, 1986). Existence of closely related, interfertile isolates such as C. smithii mt + (CC-1373 = SAG 54.72 and UTEX 1062) and the Minnesota S-1 D-2 isolates (CC-2290 and CC-1952) that differ from C. reinhardtii by numerous restriction fragment length polymorphisms (RFLPs) in two or more genomes (Boynton et al, 1987; Gross et al., 1988; Hall et al., 1989; Ranum et al., 1988) makes this species group especially useful for molecular genetic studies.

The green alga Chlamydomonas reinhardtii is an ideal model organism in which to investigate the cooperation of nuclear and chloroplast genomes in chloroplast ribosome formation from both developmental and evolutionary perspectives. Mutations affecting a variety of ribosomal components have been isolated and mapped genetically (see Boynton et al., 1990a and Harris, 1989 for reviews), and both the chloroplast (Boynton et al., 1988, 1990b; Blowers et al., 1989) and nuclear genomes (Debuchy et al., 1989; Kindle et al., 1989; Mayfield and Kindle, 1990; Diener et al., 1990) can be manipulated using newly developed transformation methods. In this paper we review our recent work on 1) the genetics and molecular biology of mutations to antibiotic resistance affecting the rRNA genes; 2) identification of chloroplast and nuclear genes encoding chloroplast ribosomal proteins (r-proteins); 3) expression of chloroplast genes encoding r-proteins; and 4) the transformation of chloroplast rRNA genes. We also outline the direction our current research is taking to define more precisely the rRNA-r-protein interactions involved in chloroplast ribosome biogenesis and function, as well as the molecular mechanisms involved in regulating expression of chloroplast r-protein genes.

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.

In photosynthetic eukaryotes, genes encoding chloroplast proteins of multimeric complexes involved in photosynthesis and protein synthesis are partitioned between the cell’s nuclear and chloroplast genomes. The chloroplast genome’s relatively small size, high copy number and great evolutionary conservation have led to rapid progress in identifying and characterizing chloroplast genes. In fact, the entire chloroplast genomes of tobacco, liverwort and rice have been sequenced within the last three years (1–3). Somewhat less progress has been made in identifying the nuclear genes for the remainder of the chloroplast polypeptides, due to the larger number of genes and the much larger size and evolutionary diversity of the nuclear genomes themselves. However in many plant species, nuclear genes can be engineered in vitro, the constructs introduced by transformation, and their function studied in vivo.

Six chloroplast gene mutants of Chlamydomonas reinhardtii resistant to spectinomycin, erythromycin, or streptomycin have been assessed for antibiotic resistance of their chloroplast ribosomes. Four of these mutations clearly confer high levels of antibiotic resistance on the chloroplast ribosomes both in vivo. Although one mutant resistant to streptomycin and one resistant to spectinomycin have chloroplast ribosomes as sensitive to antibiotics as those of wild type in vivo, these mutations can be shown to alter the wildtype sensitivity of chloroplast ribosomes in polynucleotide-directed amino acid incorporation in vitro. Genetic analysis of these six chloroplast mutants and three similar mutants (Sager, 1972), two of which have been shown to affect chloroplast ribosomes (Mets and Bogorad, 1972; Schlanger and Sager, 1974), indicates that in Chlamydomonas at least three chloroplast gene loci can affect streptomycin resistance of chloroplast ribosomes and that two can affect erythromycin resistance. The three spectinomycin-resistant mutants examined appear to be alleles at a single chloroplast gene locus, but may represent mutations at two different sites within the same gene. Unlike wild type, the streptomycin and spectinomycin resistant mutants which have chloroplast ribosomes sensitive to antibiotics in vivo, grow well in the presence of antibiotic by respiring exogenously supplied acetate as a carbon source, and have normal levels of cytochrome oxidase activity and cyanide-sensitive respiration. We conclude that mitochondrial protein synthesis in these mutants is resistant to these antibiotics, whereas in wild type it is sensitive. To explain the behavior of these two chloroplast gene mutants as well as other one-step mutants which are resistant both photosynthetically and when respiring acetate in the dark, we have postulated that a mutation in a single chloroplast gene may result in alteration of both chloroplast and mitochondrial ribosomes. Mitochondrial resistance would appear to be the minimal necessary condition for survival of all such mutants, and antibiotic-resistant chloroplast ribosomes would be necessary for survival only under photosynthetic conditions.

We have isolated nonphotosynthetic (acetate-requiring) mutants with physical alterations in chloroplast DNA following growth of haploid cells in the chloroplast specific mutagen 5-fluorodeoxyuridine (FdUrd) or treatment of FdUrd-grown diploid cells with X rays. About one-third of the nonphotosynthetic mutations resulting from FdUrd treatment alone show simple deletions. All eight of the mutants examined so far which were obtained with FdUrd plus X rays have deletions that are accompanied by rearrangements, including inversions or duplications. All the alterations extend into one of the two inverted repeat regions of the chloroplast genome which contain the ribosomal RNA cistrons. However, Southern hybridization experiments reveal that the rRNA cistrons are not deleted but instead are contained in new fragments. The relocated rRNA cistrons appear to be functional, since the mutants have normal levels of chloroplast ribosomes. In most cases the deletions and rearrangements are symmetrical and affect both inverted repeats in a similar fashion. An exception is the mutant ac-u-c-2–43, which lacks one inverted repeat region almost completely, including an entire set of rRNA genes. Three additional mutants, which fail to recombine with ac-u-c-2–43 to give photosynthetically competent cells, have smaller deletions in the same region of the genome. These physical mapping studies have allowed us to place the ac-u-c locus itself in a region of unique sequence DNA in a fragment, Ba10, which also includes the right-hand end of one inverted repeat.

Minute colony mutations in C. reinhardtii are induced with 100% efficiency by intercalating dyes such as acriflavin and ethidium bromide. These mutants form small colonies on petri plates because they undergo only 8-9 mitotic divisions before growth ceases. In liquid media without the dye the mutants show gross alterations in mitochondrial structure and function. Here we demonstrate that induction of minute mutations is accompanied by the specific loss of mitochondrial DNA. We also provide evidence that the transmission of the minute colony phenotype in crosses can be explained in terms of uniparental transmission of mitochondrial DNA by the mt- parent.

Chloroplast gene mutations which confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii have been tested for allelism and mapped by recombination analysis of progeny from biparental zygote clones. Thirty-one independently isolated streptomycin resistant mutants have chloroplast ribosomes which are resistant to this drug in an assay based on misreading of isoleucine in response to a poly U template, and comprise one nuclear and four chloroplast gene loci. Four mutants resistant to spectinomycin, and three mutants resistant to neamine and kanamycin, which have chloroplast ribosomes resistant to their respective antibiotics in poly U directed phenylalanine incorporation, appear to map in a single chloroplast gene locus. Representative alleles of this nr/spr locus, the four streptomycin resistance loci, and two chloroplast gene loci for erythromycin resistance, have been analyzed in a series of parallel crosses to establish the following map order for these seven genes in the chloroplast genome: er-u-la-er-u-37-nr-u-2-1/spr-u-1-H-4-sr-u-2-23-sr-u-2-60-sr-u-sm3-sr-u-sm2. These seven genes may constitute a ribosomal region within the chloroplast genome of Chlamydomonas comparable to the ribosomal gene clusters in bacteria.

Mutations at seven recombinationally distinct chloroplast loci confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii. Assays of polynucleotide-directed amino acid incorporation by ribosomes reconstituted from mutant and wild type subunits demonstrate that streptomycin, neamine/kanamycin and spectinomycin resistance mutations specifically affect the small ribosomal subunit, whereas mutations to erythromycin resistance affect the large subunit. Although in each case the subunit site of antibiotic resistance is the same as that observed in analogous mutations in Escherichia coli, the number of loci conferring resistance to a given antibiotic differs in the two organisms. We have previously shown that streptomycin resistance mutations in Chlamydomonas map at five discrete loci (one nuclear and four chloroplast), and that mutations to neamine/kanamycin and spectinomycin resistance appear to define a single chloroplast locus. Results presented here confirm our previous report that all chloroplast erythromycin resistance mutations isolated to date fall into two recombinationally distinct loci, and indicate that mutants at one of these loci may be further divided on the basis of their level of cross resistance to other macrolide antibiotics.

Bombardment of three mutants of the chloroplast atpB gene of Chlamydomonas reinhardtii with high-velocity tungsten microprojectiles that were coated with cloned chloroplast DNA carrying the wild-type gene permanently restored the photosynthetic capacity of the algae. In most transformants of one of the mutants, a fragment with a 2.5-kilobase deletion was restored to normal size by a homologous replacement event; in about 25 percent of the transformants the restored restriction fragment was 50 to 100 base pairs smaller or larger than that of wild type. About one-fourth of the transformants of this mutant contained unintegrated donor plasmid when first examined. This plasmid persisted in four different transformants after 65 cell generations of continuous liquid culture but was lost from all transformants maintained on plates of selective medium. The restored wild-type atpB gene remains in all transformants as an integral part of the chloroplast genome and is expressed and inherited normally.

Physical differences between organelle genomes of the interfertile species Chlamydomonas reinhardtii and Chlamydomonas smithii have been used to demonstrate that sexual zygotes transmit chloroplast and mitochondrial DNA from opposite mating types. Processes responsible can be separated functionally and genetically, although both are controlled by mating type. In vegetative diploids, chloroplast and mitochondrial genomes are transmitted biparentally, but a 1-kilobase insert present in the C. smithii mitochondrial genome spreads unidirectionally to all C. reinhardtii genomes in a manner reminiscent of the intron found in the mitochondrial 21S rRNA gene of omega + strains of yeast.

Mutants resistant to streptomycin, spectinomycin, neamine/kanamycin and erythromycin define eight genetic loci in a linear linkage group corresponding to about 21 kb of the circular chloroplast genome of Chlamydomonas reinhardtii. With one exception, all of these mutants represent single base-pair changes in conserved regions of the genes encoding the 16S and 23S chloroplast ribosomal RNAs. Streptomycin resistance can result from changes at the bases equivalent to Escherichia coli 13, 523, and 912-915 in the 16S gene, or from mutations in the rps12 gene encoding chloroplast ribosomal protein S12. In the 912-915 region of the 16S gene, three mutations were identified that resulted in different levels of streptomycin resistance in vitro. Although the three regions of the 16S rRNA mutable to streptomycin resistance are widely separated in the primary sequence, studies by other laboratories of RNA secondary structure and protein cross-linking suggest that all three regions are involved in a common ribosomal neighborhood that interacts with ribosomal proteins S4, S5 and S12. Three different changes within a conserved region of the 16S gene, equivalent to E. coli bases 1191-1193, confer varying levels of spectinomycin resistance, while resistance to neamine and kanamycin results from mutations in the 16S gene at bases equivalent to E. coli 1408 and 1409. Five mutations in two genetically distinct erythromycin resistance loci map in the 23S rDNA of C. reinhardtii, at positions equivalent to E. coli 2057-2058 and 2611, corresponding to the rib3 and rib2 loci of yeast mitochondria respectively. Although all five mutants are highly resistant to erythromycin, they differ in levels of cross-resistance to lincomycin and clindamycin. The order and spacing of all these mutations in the physical map are entirely consistent with our genetic map of the same loci and thereby validate the zygote clone method of analysis used to generate this map. These results are discussed in comparison with other published maps of chloroplast genes based on analysis by different methods using many of the same mutants.

Chloroplast mutations in the green alga Chlamydomonas reinhardtii exhibit a predominantly maternal pattern of inheritance and this pattern can be perturbed by UV irradiation of the maternal gametes prior to mating. In a series of crosses over a range of UV doses, the transmission, segregation, and recombination of mutations at three closely linked chloroplast loci have been examined by pedigree analysis of products arising from the first three post-zygotic divisions. Stocks used in these crosses were constructed to permit identification of the nuclear products of each of the two meiotic divisions and the first post-meiotic mitotic division.A bias toward maternal alleles at all three chloroplast loci was observed in all pedigrees and in zygote clones analyzed from the same crosses many generations after meiosis. This bias decreased with increasing UV dose and with each subsequent division. Segregation of chloroplast genes was rapid during the first three post-zygotic divisions. The type of segregation event from which a given heteroplasmic cell arose had a significant effect on its most likely segregation. pattern in the subsequent division. The results presented here have been discussed in terms of published models of chloroplast gene segregation.

Expression of three chloroplast genes encoding proteins of different chloroplast complexes and the rRNA gene has been examined in cells having reduced numbers of chloroplast genomes as a result of growth in the presence of the thymidine analog 5-fluorodeoxyuridine. While accumulation of total mRNA for rpl2 (ribosomal protein L-1), rbcL (ribulose bisphosphate carboxylase large subunit) and atpA (alpha-subunit of ATP synthase) declined with gene copy number, the levels of translatable mRNA and rates of synthesis of these three proteins were largely unaffected. Accumulation of rRNA declined less precipitously than mRNA levels for the three proteins in response to the reduction in chloroplast genome number. Chlamydomonas appears to compensate for reductions in the number of chloroplast genomes at several different levels. Populations of cells with only one-fourth the wild-type amount of chloroplast DNA per cell on average have half the normal level of chloroplast ribosomes and nearly normal rates of CO(2) fixation and levels of specific chloroplast encoded proteins. These results suggest that normal cells accumulate a large excess of transcripts for chloroplast genes and that levels of expression of these genes are regulated by posttranscriptional mechanisms.

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.

Chloroplast ribosomes isolated from a spectinomycin-resistant mutant ( spr-1-27-3 ) of Chlamydomonas reinhardtii that displays non-Mendelian inheritance fail to bind labeled antibiotic, in contrast to ribosomes from wild-type cells. In vitro resistance of this mutant appears to result from the absence of a specific protein in the small subunit of the chloroplast ribosome. However, chloroplast protein synthesis in the mutant and wild type shows identical sensitivity to spectinomycin in short-term in vivo experiments where ribulosediphosphate carboxylase serves as the marker. Long-term experiments demonstrate that the mutant can grow in the presence of spectinomycin only when acetate is supplied as a carbon source. Mitochondrial structure and function of the mutant are not affected by the antibiotic, whereas chloroplast structure and function are. Apparently, the mitochondrion, rather than the chloroplast, of this mutant is resistant to spectinomycin in vivo . We hypothesize that the gene product of the spr locus is a protein common to both chloroplast and mitochondrial ribosomes. The mutant gene product, in vivo , confers resistance on mitochondrial, but not chloroplast, ribosomes. We suppose that the mutant spr protein loosely attaches to chloroplast ribosomes in vivo so that the antibiotic is bound and blocks protein synthesis, but it dissociates during isolation, resulting in loss of the binding site.

IT IS no longer necessary to prove to teachers that remedial teaching is necessary in the intermediate and higher grades. All teachers find themselves being remedial teachers practically every year. Even before they become familiar with the characteristics of their new groups in September of any year, they can, in imagination and with an accuracy that would surprise anyone not a teacher, estimate the problems that will be faced.

In Chlamydomonas, as in higher plants, the chloroplast genome codes for chloroplast ribosomal RNAs, tRNAs, about 1/3 of the ribosomal proteins, and a number of proteins of the photosynthetic apparatus (Table 1, Fig. 1). Other proteins are coded by the nuclear genome and imported, generally as precursors which are processed to mature form within the chloroplast (Table 1). Because C. reinhardtii can dispense with photosynthetic functions if provided acetate as an alternative carbon source, this species is an ideal model system for investigation of mutations affecting these components. In the case of the large subunit of ribulose bisphosphate carboxylase (RUBISCO; Spreitzer & Ogren, 1985), the 32 kd herbicide binding protein (Erickson et al., 1985), and the beta subunit of the CF0CF1 ATPase (Woessner et al., 1986), analysis of chloroplast genes has extended to the DNA sequence level, and to comparison of mutations and reversions within the same gene.

The allelic nuclear mutations of Chlamydomonas reinhardtii, cr-6 and cr-7, result in the loss of two proteins from the large subunit of the chloroplast ribosome. One of these proteins, L-13, is synthesized in the chloroplast and the other, L29, is made in the cytoplasm. The loss of these two proteins is correlated with the inability of the large subunits of the chloroplast ribosomes to form monomers which incorporate labeled phenylalanine at normal rates in response to a polyuridylic acid template. Using antisera raised against L13 and L29, we found that protein L-13 was synthesized in appreciable amounts in pulse labeled cells of cr-6 and cr-7, but protein L-29 was not. We conclude that the inability to synthesize protein L29 is a primary defect in both cr-6 and cr-7 and that this protein is required for the stable assembly of protein L-13 into chloroplast ribosomes. The absence of one or both of these proteins from the large subunit of chloroplast ribosomes of the mutants interferes with the ability of the small and large subunits to associate properly into normal 70S monomers.