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Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized1,2. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms. Here, we generated a genome-wide, indexed library of mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii. The 62,389 mutants in the library, covering 83% of nuclear, protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We performed a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphatase-encoding gene CPL3, showed it is important for accumulation of multiple photosynthetic protein complexes. Notably, 21 of the 43 highest-confidence genes are novel, opening new opportunities for advances in our understanding of this biogeochemically fundamental process. This library will accelerate the characterization of thousands of genes in algae, plants and animals., Editorial summary: Generation of a library of 62,389 mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii enables screening for genes required for photosynthesis and the identification of 303 candidate genes.

Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized1,2. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms that complement the use of multicellular plants by enabling higher-throughput studies. Here, we generated a genome-wide, indexed library of mapped insertion mutants for the flagship unicellular algaChlamydomonas reinhardtii(Chlamydomonas hereafter). The 62,389 mutants in the library, covering 83% of nuclear, protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We leveraged this feature to perform a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphataseCPL3, showed it is important for accumulation of multiple photosynthetic protein complexes. Strikingly, 21 of the 43 highest-confidence genes are novel, opening new opportunities for advances in our understanding of this biogeochemically fundamental process. This library is the first genome-wide mapped mutant resource in any unicellular photosynthetic organism, and will accelerate the characterization of thousands of genes in algae, plants and animals.

The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.

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.

Two mutations in LF5, which encodes a protein kinase orthologous to human CDKL5, cause abnormally long flagella in Chlamydomonas. The localization of LF5p to the very proximal region of flagella in WT cells is regulated by three other LF gene products, which make up the cytoplasmic length regulatory complex., The length of Chlamydomonas flagella is tightly regulated. Mutations in four genes—LF1, LF2, LF3, and LF4—cause cells to assemble flagella up to three times wild-type length. LF2 and LF4 encode protein kinases. Here we describe a new gene, LF5, in which null mutations cause cells to assemble flagella of excess length. The LF5 gene encodes a protein kinase very similar in sequence to the protein kinase CDKL5. In humans, mutations in this kinase cause a severe form of juvenile epilepsy. The LF5 protein localizes to a unique location: the proximal 1 μm of the flagella. The proximal localization of the LF5 protein is lost when genes that make up the proteins in the cytoplasmic length regulatory complex (LRC)—LF1, LF2, and LF3—are mutated. In these mutants LF5p becomes localized either at the distal tip of the flagella or along the flagellar length, indicating that length regulation involves, at least in part, control of LF5p localization by the LRC.

Mutations at the APM1 and APM2 loci in the green alga Chlamydomonas reinhardtii confer resistance to phosphorothioamidate and dinitroaniline herbicides. Genetic interactions between apm1 and apm2 mutations suggest an interaction between the gene products. We identified the APM1 and APM2 genes using a map-based cloning strategy. Genomic DNA fragments containing only the DNJ1 gene encoding a type I Hsp40 protein rescue apm1 mutant phenotypes, conferring sensitivity to the herbicides and rescuing a temperature-sensitive growth defect. Lesions at five apm1 alleles include missense mutations and nucleotide insertions and deletions that result in altered proteins or very low levels of gene expression. The HSP70A gene, encoding a cytosolic Hsp70 protein known to interact with Hsp40 proteins, maps near the APM2 locus. Missense mutations found in three apm2 alleles predict altered Hsp70 proteins. Genomic fragments containing the HSP70A gene rescue apm2 mutant phenotypes. The results suggest that a client of the Hsp70–Hsp40 chaperone complex may function to increase microtubule dynamics in Chlamydomonas cells. Failure of the chaperone system to recognize or fold the client protein(s) results in increased microtubule stability and resistance to the microtubule-destabilizing effect of the herbicides. The lack of redundancy of genes encoding cytosolic Hsp70 and Hsp40 type I proteins in Chlamydomonas makes it a uniquely valuable system for genetic analysis of the function of the Hsp70 chaperone complex.

Hydrothermal carbonization is a process in which biomass is heated in water under pressure to create a char product. With higher plants, the chemistry of the process derives primarily from lignin, cellulose and hemicellulose components. In contrast, green and blue-green microalgae are not lignocellulosic in composition, and the chemistry is entirely different, involving proteins, lipids and carbohydrates (generally not cellulose). Employing relatively moderate conditions of temperature (ca. 200 °C), time (

Positive signaling by nitrate in its assimilation pathway has been studied in Chlamydomonas reinhardtii. Among >34,000 lines generated by plasmid insertion, 10 mutants were unable to activate nitrate reductase (NIA1) gene expression and had a Nit− (no growth in nitrate) phenotype. Each of these 10 lines was mutated in the nitrate assimilation–specific regulatory gene NIT2. The complete NIT2 cDNA sequence was obtained, and its deduced amino acid sequence revealed GAF, Gln-rich, Leu zipper, and RWP-RK domains typical of transcription factors and transcriptional coactivators associated with signaling pathways. The predicted Nit2 protein sequence is structurally related to the Nin (for nodule inception) proteins from plants but not to NirA/Nit4/Yna proteins from fungi and yeast. NIT2 expression is negatively regulated by ammonium and is optimal in N-free medium with no need for the presence of nitrate. However, intracellular nitrate is required to allow Nit2 to activate the NIA1 promoter activity. Nit2 protein was expressed in Escherichia coli and shown to bind to specific sequences at the NIA1 gene promoter. Our data indicate that NIT2 is a central regulatory gene required for nitrate signaling on the Chlamydomonas NIA1 gene promoter and that intracellular nitrate is needed for NIT2 function and to modulate NIA1 transcript levels.

Little is known about how cells regulate the size of their organelles. In this study, we find that proper flagellar length control in Chlamydomonas reinhardtii requires the activity of a new member of the cyclin-dependent kinase (CDK) family, which is encoded by the LF2 (long flagella 2) gene. This novel CDK contains all of the important residues that are essential for kinase activity but lacks the cyclin-binding motif PSTAIRE. Analysis of genetic lesions in a series of lf2 mutant alleles and site-directed mutagenesis of LF2p reveals that improper flagellar length and defective flagellar assembly correlate with the extent of disruption of conserved kinase structures or residues by mutations. LF2p appears to interact with both LF1p and LF3p in the cytoplasm, as indicated by immunofluorescence localization, sucrose density gradients, cell fractionation, and yeast two-hybrid experiments. We propose that LF2p is the catalytic subunit of a regulatory kinase complex that controls flagellar length and flagellar assembly.

Flagellar length is tightly regulated in the biflagellate alga Chlamydomonas reinhardtii. Several genes required for control of flagellar length have been identified, including LF1, a gene required to assemble normal-length flagella. The lf1 mutation causes cells to assemble extra-long flagella and to regenerate flagella very slowly after amputation. Here we describe the positional cloning and molecular characterization of the LF1 gene using a bacterial artificial chromosome (BAC) library. LF1 encodes a protein of 804 amino acids with no obvious sequence homologs in other organisms. The single LF1 mutant allele is caused by a transversion that produces an amber stop at codon 87. Rescue of the lf1 phenotype upon transformation was obtained with clones containing the complete LF1 gene as well as clones that lack the last two exons of the gene, indicating that only the amino-terminal portion of the LF1 gene product (LF1p) is required for function. Although LF1 helps regulate flagellar length, the LF1p localizes almost exclusively in the cell body, with

How centrioles and basal bodies assemble is a long-standing puzzle in cell biology. To address this problem, we analyzed a novel basal body-defective Chlamydomonas reinhardtii mutant isolated from a collection of flagella-less mutants. This mutant, bld10, displayed disorganized mitotic spindles and cytoplasmic microtubules, resulting in abnormal cell division and slow growth. Electron microscopic observation suggested that bld10 cells totally lack basal bodies. The product of the BLD10 gene (Bld10p) was found to be a novel coiled-coil protein of 170 kD. Immunoelectron microscopy localizes Bld10p to the cartwheel, a structure with ninefold rotational symmetry positioned near the proximal end of the basal bodies. Because the cartwheel forms the base from which the triplet microtubules elongate, we suggest that Bld10p plays an essential role in an early stage of basal body assembly. A viable mutant having such a severe basal body defect emphasizes the usefulness of Chlamydomonas in studying the mechanism of basal body/centriole assembly by using a variety of mutants.

A time-series analysis of airborne photographs and Landsat thematic mapper (TM and ETM+) images and hydrochemical data were used to examine the effects of land-use change from 1930 to 2001 on solute inputs to Lake Calado, a floodplain lake in the central Amazon. Deforestation from slash-and-burn agricultural activities has dramatically decreased the amount of primary growth upland and flooded forests in the basin. The increasing area that is converted to agricultural plots and pasture in the Lake Calado basin has increased solute loading to the lake from upland tributaries (storm and base flow), bank seepage and overland flow, and decreased throughfall inputs. Whereas solute concentrations in stream water were generally higher in 1992 than 1930, Na+ and Cl− concentrations were also considerably higher in 2001 than 1992, likely because of an increase in the number of humans and cattle in the watershed. Estimates of solute inputs to Lake Calado via throughfall indicate that the mass transfer of some major solutes in the throughfall of undisturbed flooded forests can be larger than that from a combination of all other sources in areas that do not have a strong influence from the Solimoes River. Chemical gains in rain as it passed through the forest canopy occurred for most major ions and relatively large gains were observed for $${\text{PO}}_{\text{4}}^{3--} $$ and Ca2+. Although often neglected in studies of tropical forest ecosystems, throughfall can be an important source of solutes to relatively undisturbed lake environments in the central Amazon.

Simple, experimentally tractable systems such Saccharomyces cerevisiae, Chlamydomonas reinhardtii, and Arabidopsis thaliana are powerful models for dissecting basic biological processes. The unicellular green alga C. reinhardtii is amenable to a diversity of genetic and molecular manipulations. This haploid organism grows rapidly in axenic cultures, on both solid and liquid medium, with a sexual cycle that can be precisely controlled. Vegetative diploids are readily selected through the use of complementing auxotrophic markers and are useful for analyses of deleterious recessive alleles. These genetic features have permitted the generation and characterization of a wealth of mutants with lesions in structural, metabolic and regulatory genes. Another important feature of C. reinhardtii is that it has the capacity to grow with light as a sole energy source (photoautotrophic growth) or on acetate in the dark (heterotrophically), facilitating detailed examination of genes and proteins critical for photosynthetic or respiratory function. Other important topics being studied using C. reinhardtii, many of which have direct application to elucidation of protein function in animal cells (26), include flagellum structure and assembly, cell wall biogenesis, gametogenesis, mating, phototaxis, and adaptive responses to light and nutrient environments (32, 44). Some of these studies are directly relevant to applied problems in biology, including the production of clean, solar-generated energy in the form of H2, and bioremediation of heavy metal wastes. Recent years have seen the development of a molecular toolkit for C. reinhardtii (42, 44, 66, 98, 99). Selectable markers are available for nuclear and chloroplast transformation (4, 5, 12, 13, 30, 44, 56, 82). The Arg7 (22) and Nit1 (30) genes are routinely used to rescue recessive mutant phenotypes. The bacterial ble gene (which codes for zeocin resistance [70, 112]) is an easily scored marker for nuclear transformation, and the bacterial aadA gene (which codes for spectinomycin and streptomycin resistance) is a reliable marker for chloroplast transformation (39). Nuclear transformation can be achieved by

We have prepared a molecular map of the Chlamydomonas reinhardtii genome anchored to the genetic map. The map consists of 264 markers, including sequence-tagged sites (STS), scored by use of PCR and agarose gel electrophoresis, and restriction fragment length polymorphism markers, scored by use of Southern blot hybridization. All molecular markers tested map to one of the 17 known linkage groups of C. reinhardtii . The map covers approximately 1,000 centimorgans (cM). Any position on the C. reinhardtii genetic map is, on average, within 2 cM of a mapped molecular marker. This molecular map, in combination with the ongoing mapping of bacterial artificial chromosome (BAC) clones and the forthcoming sequence of the C. reinhardtii nuclear genome, should greatly facilitate isolation of genes of interest by using positional cloning methods. In addition, the presence of easily assayed STS markers on each arm of each linkage group should be very useful in mapping new mutations in preparation for positional cloning.

Four long-flagella (LF) genes are important for flagellar length control in Chlamydomonas reinhardtii. Here, we characterize two new null lf3 mutants whose phenotypes are different from previously identified lf3 mutants. These null mutants have unequal-length flagella that assemble more slowly than wild-type flagella, though their flagella can also reach abnormally long lengths. Prominent bulges are found at the distal ends of short, long, and regenerating flagella of these mutants. Analysis of the flagella by electron and immunofluorescence microscopy and by Western blots revealed that the bulges contain intraflagellar transport complexes, a defect reported previously (for review see Cole, D.G., 2003. Traffic. 4:435–442) in a subset of mutants defective in intraflagellar transport. We have cloned the wild-type LF3 gene and characterized a hypomorphic mutant allele of LF3. LF3p is a novel protein located predominantly in the cell body. It cosediments with the product of the LF1 gene in sucrose density gradients, indicating that these proteins may form a functional complex to regulate flagellar length and assembly.

Cilia and flagella are among the most ancient cellular organelles, providing motility for primitive eukaryotic cells living in an aqueous environment. During adaptation to life on land, some groups of organisms, including advanced fungi, red algae, cellular slime molds, conifers, and angiosperms

Chlamydomonas reinhardtii is a unicellular green alga, with a genome estimated to be around 100 Mbp ([Harris, 1989][1]). Similar to yeast, C. reinhardtii has well-understood haploid genetics, but unlike yeast it has both a chloroplast and flagella. These unique properties make it a powerful model

Insertional mutagenesis procedures in Chlamydomonas[1] have facilitated the identification and characterization of dozens of genes required for the assembly and motility of flagella in Chlamydomonas[2–6]. Many of these genes have been found to have homologs in animal systems. Here we describe a new gene required for flagellar assembly. Null mutants at the BLD1 locus assemble no flagella, and the flagellar membrane abuts the end of the transition zone distal to the basal body. Unlike mutants with basal body ultrastructural defects, such as bld2[7], bld1 mutants have normal basal bodies and cytoplasmic microtubule rootlets. The wild-type BLD1 gene was cloned by using DNA flanking the site of insertion of plasmid DNA in an insertional mutant; the cloned gene rescues the bld1 mutant phenotype upon transformation. The predicted BLD1 gene product is a 50.4 kDa protein with extensive regions of sequence similarity to the osm-6 gene of Caenorhabditis elegans[8] whose product is necessary for the assembly of a set of sensory cilia [9]. The protein product of the BLD1 gene corresponds to IFT52 [10], a protein component of “raft” particles shown to undergo rapid transport up and down Chlamydomonas flagella between the flagellar membrane and the axoneme in a process known as intraflagellar transport (IFT) [11, 12]. The BLD1 RNA transcript is upregulated upon flagellar amputation, as observed for many other genes encoding flagellar proteins [13]. These results demonstrate that the function of the IFT52 protein in Chlamydomonas is essential for the assembly and/or maintenance of the flagella.

GREAT strides in understanding fundamental processes in eukaryotic cells have come from genetic studies on budding and fission yeasts and other fungi. The exquisite experimental tools developed for use with these systems have made them the undisputed first choice for studying a variety of metabolic

Flagellar length in the biflagellate alga Chlamydomonas reinhardtii is under constant and tight regulation. A number of mutants with defects in flagellar length control have been previously identified. Mutations in the three long-flagella (lf) loci result in flagella that are up to three times longer than wild-type length. In this article, we describe the isolation of long-flagellar mutants caused by mutations in a new LF locus, LF4. lf4 mutations were shown to be epistatic to lf1, while lf2 was found to be epistatic to lf4 with regard to the flagellar regeneration defect. Mutations in lf4 were able to suppress the synthetic flagella-less phenotype of the lf1, lf2 double mutant. In addition, we have isolated four extragenic suppressor mutations that suppress the long-flagella phenotype of lf1, lf2, or lf3 double mutants.

Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns. Out of several conserved genes in a minus gamete cluster, we focused on Cre06.g280600, an ortholog of the fertilization-related Arabidopsis GEX1. Gene disruption, cell biological, and immunolocalization studies show that CrGEX1 functions in nuclear fusion in Chlamydomonas. Moreover, CrGEX1 and its Plasmodium ortholog, PBANKA_113980, are essential for production of viable meiotic progeny in both organisms and thus for mosquito transmission of malaria. Remarkably, we discovered that the genes are members of a large, previously unrecognized family whose first-characterized member, KAR5, is essential for nuclear fusion during yeast sexual reproduction. Our comparative transcriptomics approach provides a new resource for studying sexual development and demonstrates that exploiting the data can lead to the discovery of novel biology that is conserved across distant taxa.

A yellow-green alga

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

Most estimates of regional and global soil carbon stocks are based on extrapolations of mean soil C contents for broad categories of soil or vegetation types. Uncertainties exist in both the estimates of mean soil C contents and the area over which each mean should be extrapolated. Geographic information systems now permit spatially referenced estimates of soil C at finer scales of resolution than were previously practical. We compared estimates of total soil C stocks of the state of Maine using three methods: (1) multiplying the area of the state by published means of soil C for temperate forests and for Spodosols; (2) calculating areas of inclusions of soil taxa in the 1:5,000,000 FAO/UNESCO Soils Map of the World and multiplying those areas by selected mean carbon contents; and (3) calculating soil C for each soil series and map unit in the 1:250,000 State Soil Geographic Data Base (STATSGO) and summing these estimates for the entire state. The STATSGO estimate of total soil C was between 23% and 49% higher than the common coarse scale extrapolations, primarily because STATSGO included data on Histosols, which cover less than 5% of the area of the state, but which constitute over one-third of the soil C. Spodosols cover about 65% of the state, but contribute less than 39% of the soil C. Estimates of total soil C in Maine based on the FAO map agreed within 8% of the STATSGO estimate for one possible matching of FAO soil taxa with data on soil C, but another plausible matching overestimated soil C stocks. We also compared estimates from the 1:250,000 STATSGO database and from the 1:20,000 Soil Survey Geographic Data Base (SSURGO) for a 7.5 minute quadrangle within the state. SSURGO indicated 13% less total soil C than did STATSGO, largely because the attribute data on depths of soil horizons in SSURGO are more specific for this locality. Despite localized differences, the STATSGO database offers promise of scaling up county soil survey data to regional scales because it includes attribute data and estimates of areal coverage of C-rich inclusions within map units. The spatially referenced data also permit examination of covariation of soil C stocks with soil properties thought to affect stabilization of soil C. Clay content was a poor predictor of soil C in Maine, but drainage class covaried significantly with soil C across the state.

We have isolated telomeric DNA and telomere-associated sequences from Chlamydomonas reinhardtii. The terminal telomere sequences of the green alga Chlamydomonas are composed of (TTTTAGGG)n repeats that are similar, but not identical, to those of the higher plant Arabidopsis thaliana. We demonstrate that these repeats are telomeric by their preferential sensitivity to nuclease Bal-31 digestion, their similarity to A. thaliana telomeres, their orientation relative to the end of the chromosome, and the methods used for their isolation. Five independent telomere clones were isolated, and three of these clones include closely related telomere-associated sequences. One of these telomere-associated sequences hybridizes to a number of genomic fragments sensitive to digestion with the exonuclease Bal-31. Like telomere sequences from other organisms, the C. reinhardtii telomeres display a bias for guanine and thymine nucleotides on the 3'-end strand. However, the sequence of Chlamydomonas telomeres is more A + T-rich than any other known telomere sequence. We propose that the common feature of all known telomere is the frequent occurrence of tracts of three or more adjacent guanine residues.

An electromagnetic flowmeter was developed to measure transient flows at a data rate of 60 Hz. The approach taken was to develop suitable electronics to replace the electronics of a commercially available electromagnetic flowmeter normally used for steady-state operation. Use of the commercially available flowmeter body, which includes the magnetic coils, core, and signal electrodes, provided a relatively economical means of fabricating the transient flowmeter. A transient flow calibration facility consisting of a free-falling water column was also designed and built. Results of the calibrations are presented and show that the flowmeter can accurately measure transient flows up to the maximum observed acceleration of approximately 1 g.

Mutations in the UNI2 locus in Chlamydomonas reinhardtii result in a “uniflagellar” phenotype in which flagellar assembly occurs preferentially from the older basal body and ultrastructural defects reside in the transition zones. The UNI2 gene encodes a protein of 134 kDa that shares 20.5% homology with a human protein. Immunofluorescence microscopy localized the protein on both basal bodies and probasal bodies. The protein is present as at least two molecular-weight variants that can be converted to a single form with phosphatase treatment. Synthesis of Uni2 protein is induced during cell division cycles; accumulation of the phosphorylated form coincides with assembly of transition zones and flagella at the end of the division cycle. Using the Uni2 protein as a cell cycle marker of basal bodies, we observed migration of basal bodies before flagellar resorption in some cells, indicating that flagellar resorption is not required for mitotic progression. We observed the sequential assembly of new probasal bodies beginning at prophase. The uni2 mutants may be defective in the pathways leading to flagellar assembly and to basal body maturation.

Chlamydomonas reinhardtii controls flagellar assembly such that flagella are of an equal and predetermined length. Previous studies demonstrated that lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), induced flagellar elongation, suggesting that a lithium-sensitive signal transduction pathway regulated flagellar length (S. Nakamura, H. Takino, and M. K. Kojima, Cell Struct. Funct. 12: 369-374, 1987). Here, we demonstrate that lithium treatment depletes the pool of flagellar proteins from the cell body and that the heterotrimeric kinesin Fla10p accumulates in flagella. We identify GSK3 in Chlamydomonas and demonstrate that its kinase activity is inhibited by lithium in vitro. The tyrosine-phosphorylated, active form of GSK3 was enriched in flagella and GSK3 associated with the axoneme in a phosphorylation-dependent manner. The level of active GSK3 correlated with flagellar length; early during flagellar regeneration, active GSK3 increased over basal levels. This increase in active GSK3 was rapidly lost within 30 min of regeneration as the level of active GSK3 decreased relative to the predeflagellation level. Taken together, these results suggest a possible role for GSK3 in regulating the assembly and length of flagella.

Numerous studies have indicated that the central apparatus plays a significant role in regulating flagellar motility, yet little is known about how the central pair of microtubules or their associated projections assemble. Several Chlamydomonas mutants are defective in central apparatus assembly. For example, mutant pf15 cells have paralyzed flagella that completely lack the central pair of microtubules. We have cloned the wild-type PF15 gene and confirmed its identity by rescuing the motility and ultrastructural defects in two pf15 alleles, the original pf15a mutant and a mutant generated by insertional mutagenesis. Database searches using the 798-amino-acid polypeptide predicted from the complete coding sequence indicate that the PF15 gene encodes the Chlamydomonas homologue of the katanin p80 subunit. Katanin was originally identified as a heterodimeric protein with a microtubule-severing activity. These results reveal a novel role for the katanin p80 subunit in the assembly and/or stability of the central pair of flagellar microtubules.

How centrioles and basal bodies assemble is a long-standing puzzle in cell biology. To address this problem, we analyzed a novel basal body-defective Chlamydomonas reinhardtii mutant isolated from a collection of flagella-less mutants. This mutant, bld10, displayed disorganized mitotic spindles and cytoplasmic microtubules, resulting in abnormal cell division and slow growth. Electron microscopic observation suggested that bld10 cells totally lack basal bodies. The product of the BLD10 gene (Bld10p) was found to be a novel coiled-coil protein of 170 kD. Immunoelectron microscopy localizes Bld10p to the cartwheel, a structure with ninefold rotational symmetry positioned near the proximal end of the basal bodies. Because the cartwheel forms the base from which the triplet microtubules elongate, we suggest that Bld10p plays an essential role in an early stage of basal body assembly. A viable mutant having such a severe basal body defect emphasizes the usefulness of Chlamydomonas in studying the mechanism of basal body/centriole assembly by using a variety of mutants.

Little is known about the molecular basis of organelle size control in eukaryotes. Cells of the biflagellate alga Chlamydomonas reinhardtii actively maintain their flagella at a precise length. Chlamydomonas mutants that lose control of flagellar length have been isolated and used to demonstrate that a dynamic process keeps flagella at an appropriate length [1, 2]. To date, none of the proteins required for flagellar length control have been identified in any eukaryotic organism. Here, we show that a novel MAP kinase is crucial to enforcing wild-type flagellar length in C. reinhardtii. Null mutants of LF4[2], a gene encoding a protein with extensive amino acid sequence identity to a mammalian MAP kinase of unknown function, MOK [3], are unable to regulate the length of their flagella. The LF4 protein (LF4p) is localized to the flagella, and in vitro enzyme assays confirm that the protein is a MAP kinase. The long-flagella phenotype of lf4 cells is rescued by transformation with the cloned LF4 gene. The demonstration that a novel MAP kinase helps enforce flagellar length control indicates that a previously unidentified signal transduction pathway controls organelle size in C. reinhardtii.

In the unicellular alga Chlamydomonas, two anterior flagella are positioned with 180° rotational symmetry, such that the flagella beat with the effective strokes in opposite directions (Hoops, H.J., and G.B. Witman. 1983. J. Cell Biol. 97:902–908). The vfl1 mutation results in variable numbers and positioning of flagella and basal bodies (Adams, G.M.W., R.L. Wright, and J.W. Jarvik. 1985. J. Cell Biol. 100:955–964). Using a tagged allele, we cloned the VFL1 gene that encodes a protein of 128 kD with five leucine-rich repeat sequences near the NH2 terminus and a large α-helical–coiled coil domain at the COOH terminus. An epitope-tagged gene construct rescued the mutant phenotype and expressed a tagged protein (Vfl1p) that copurified with basal body flagellar apparatuses. Immunofluorescence experiments showed that Vfl1p localized with basal bodies and probasal bodies. Immunogold labeling localized Vfl1p inside the lumen of the basal body at the distal end. Distribution of gold particles was rotationally asymmetric, with most particles located near the doublet microtubules that face the opposite basal body. The mutant phenotype, together with the localization results, suggest that Vfl1p plays a role in establishing the correct rotational orientation of basal bodies. Vfl1p is the first reported molecular marker of the rotational asymmetry inherent to basal bodies.

In Chlamydomonas reinhardtii, the genes required for nitrate assimilation, including the gene encoding nitrate reductase (NIT1), are subject to repression by ammonia. To study the mechanism of ammonia repression, we employed two approaches to search for mutants with defective repression of NIT1 gene expression. (1) PF14, a gene required for flagellar function, was used as a reporter gene for expression from the NIT1 promoter. When introduced into a pf14 mutant host, the NZTl:PF14 chimeric construct produced a transformant (T10-10B) with a conditional swimming phenotype. Spontaneous mutants with defective ammonia repression of the NIT1 promoter were screened for by isolating cells that gained constitutive motility. (2) Insertional mutagenesis was performed, followed by screening for chlorate sensitivity in the presence of ammonia ion. One insertional mutant and six spontaneous mutants were allelic and defined a new gene, FAR1 (free from ammonia repression). FAR1 was mapped to Linkage Group I, 7.7 cM to the right of the centromere. The far1-1 mutant strain was used to clone DNA adjacent to the site of plasmid insertion, which was then used as a hybridization probe to clone the FAR1 gene from wild type.

The central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components, we generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen contains an allele of a previously identified mutation, pf20. Mutant cells have paralyzed flagella, and the entire central apparatus is missing in isolated axonemes. We have cloned the wild-type PF20 gene and confirmed its identity by rescuing the pf20 mutant phenotype upon transformation. Rescued transformants were wild type in motility and in axonemal ultrastructure. A cDNA clone containing a single, long open reading frame was obtained and sequenced. Database searches using the predicted 606-amino acid sequence of PF20 indicate that the protein contains five contiguous WD repeats. These repeats are found in a number of proteins with diverse cellular functions including beta-transducin and dynein intermediate chains. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunogold labeling of wild-type axonemes indicates that the PF20 protein is localized along the length of the C2 microtubule on the intermicrotubule bridges connecting the two central microtubules. We suggest that the PF20 gene product is a new member of the family of WD repeat proteins and is required for central microtubule assembly and/or stability and flagellar motility.

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

We have used homologous recombination to disrupt the nuclear gene NIT8 in Chlamydomonas reinhardtii. This is the first report of targeted gene disruption of an endogenous locus in C. reinhardtii and only the second for a photosynthetic eukaryote. NIT8 encodes a protein necessary for nitrate and nitrite assimilation by C. reinhardtii. A disruption vector was constructed by placing the CRY1-1 selectable marker gene, which confers emetine resistance, within the NIT8 coding region. nit8 mutants are unable to grow on nitrate as their sole nitrogen source (Nit-) and are resistant to killing by chlorate. One of 2,000 transformants obtained after selection on emetine-chlorate medium contained a homologous insertion of five copies of the disruption plasmid into the NIT8 gene, producing an emetine-resistant, chlorate-resistant Nit- phenotype. The mutant phenotype was rescued by the wild-type NIT8 gene upon transformation. Seven other mutations at the nit8 locus, presumably resulting from homologous recombination with the disruption plasmid, were identified but were shown to be accompanied by deletions of the surrounding genomic region.

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

Chlamydomonas is a popular genetic model system for studying many cellular processes. In this report, we describe a new approach to isolate Chlamydomonas genes using the cloned nitrate reductase gene (NIT1) as an insertional mutagen. A linearized plasmid containing the NIT1 gene was introduced into nit1 mutant cells by glass-bead transformation. Of 3000 Nit+ transformants examined, 74 showed motility defects of a wide range of phenotypes, suggesting that DNA transformation is an effective method for mutagenizing cells. For 13 of 15 such motility mutants backcrossed to nit- mutant strains, the motility phenotype cosegregated with the Nit+ phenotype, indicating that the motility defects of these 13 mutants may be caused by integration of the plasmid. Further genetic analysis indicated that three of these mutants contained alleles of previously identified loci: mbo2 (move backward only), pf13 (paralyzed flagella) and vfl1 (variable flagellar number). Three other abnormal-flagellar-number mutants did not map to any previously described loci at which mutations produce similar phenotypes. Genomic sequences flanking the integrated plasmid in the mbo2 and vfl1 mutants were isolated and used as probes to obtain wild-type genomic clones, which complemented the motility defects upon transformation into cells. Our results demonstrate the potential of this new approach for cloning genes identified by mutation in Chlamydomonas.

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii was isolated by using the amiprophos-methyl-resistant mutation apm1-18 as a background to select new mutants that showed increased resistance to the drug. The upA12 mutation caused twofold resistance to amiprophos-methyl and oryzalin, and twofold hypersensitivity to the microtubule-stabilizing drug taxol, suggesting that the mutation enhanced microtubule stability. The resistance mutation was semi-dominant and mapped to the same interval on linkage group III as the alpha 1-tubulin gene. Two-dimensional gel immunoblots of proteins in the mutant cells revealed two electrophoretically altered alpha-tubulin isoforms, one of which was acetylated and incorporated into microtubules in the axoneme. The mutant isoforms co-segregated with the drug-resistance phenotypes when mutant upA12 was backcrossed to wild-type cells. Two-dimensional gel analysis of in vitro translation products showed that the non-acetylated variant alpha-tubulin was a primary gene product. DNA sequence analysis of the alpha 1-tubulin genes from mutant and wild-type cells revealed a single missense mutation, which predicted a change in codon 24 from tyrosine in wild type to histidine in mutant upA12. This alteration in the predicted amino acid sequence corroborated the approximately +1 basic charge shift observed for the variant alpha-tubulins. The mutant allele of the alpha 1-tubulin gene was designated tua1-1.

Genetic evidence suggests that the NIT2 gene of Chlamydomonas reinhardtii encodes a positive regulator of the nitrate-assimilation pathway. To learn more about the function of the NIT2 gene product, we isolated the gene using a transposon-tagging strategy. A nit2 mutation caused by the insertion of a transposon was identified by testing spontaneous nit2 mutants for the presence of new copies of Gulliver or TOC1, transposable elements that have been identified in Chlamydomonas. In 2 of the 14 different mutants that were analyzed, a Gulliver element was found to be genetically and phenotypically associated with the nit2 mutation. Using the Gulliver element as a probe, one of the transposon-induced nit2 alleles was isolated, and a sequence adjoining the transposon was used to isolate the corresponding wild-type locus. The NIT2 gene was delimited by mapping DNA rearrangements associated with nit2 mutations and mutant rescue by genetic transformation. The NIT2 gene encodes a 6-kb transcript that was not detected in cells grown in the presence of ammonium. Likewise, NIT2-dependent genes are repressed in ammonium-grown cells. These results suggest that repression of the NIT2 gene may mediate metabolite repression of the nitrate assimilation pathway in Chlamydomonas.

We previously described two types of genetic interactions among recessive mutations in the APM1 and APM2 loci of Chlamydomonas reinhardtii that may reflect a physical association of the gene products or their involvement in a common structure/process: (1) allele-specific synthetic lethality, and (2) unlinked noncomplementation, or dominant enhancement. To further investigate these interactions, we isolated revertants in which the heat sensitivity caused by the apm2-1 mutation is lost. The heat-insensitive revertants were either fully or partially suppressed for the drug-resistance caused by the apm2-1 allele. In recombination tests the revertants behaved as if the suppressing mutation mapped within the APM2 locus; the partial suppressors of apm2-1 herbicide resistance failed to complement apm2-1, leading to the conclusion that they were likely to be intragenic pseudorevertants. The apm2-1 partial suppressor mutations reversed apm1-apm2-1 synthetic lethality in an allele-specific manner with respect both to apm1- alleles and apm2-1 suppressor mutations. Those apm1- apm2-1rev strains that regained viability also regained heat sensitivity characteristic of the original apm2-1 mutation, even though the apm2-1 suppressor strains were fully heat-insensitive. The Hs+ phenotypes of apm2-1 partial suppressors were also reversed by treatment with the microtubule-stabilizing agent deuterium oxide (D2O). In addition to the above interactions, we observed interallelic complementation and phenotypic enhancement of temperature conditionality among apm1- alleles. Evidence of a role for the products of the two genes in microtubule-based processes was obtained from studying flagellar assembly in apm1- and apm2- mutants.

The biflagellate alga Chlamydomonas has been used extensively in the genetic and biochemical analysis of flagellar assembly and motility. We have restored motility to a paralyzed-flagella mutant of Chlamydomonas by transforming with the corresponding wild-type gene. A nitrate reductase-deficient paralyzed-flagella strain, nit1-305 pf-14, carrying mutations in the genes for nitrate reductase and radial spoke protein 3, was transformed with wild-type copies of both genes. Two-thirds of the cells that survived nitrate selection also regained motility, indicating that they had been transformed with both the nitrate reductase and radial spoke protein 3 genes. Transformants typically contained multiple copies of both genes, genetically linked to each other, but not linked to the original mutant loci. Complementation of paralyzed-flagella mutants by transformation is a powerful tool for investigating flagellar assembly and function.

Monoclonal antibody (SU5), prepared from isolated mitotic spindles of sea urchin eggs, stained centrospheres preferentially and recognized a 50K (K = 10(3) Mr) polypeptide on immunoblots. Three positive clones were isolated by screening a lambda gt11 cDNA expression library prepared from sea urchin egg mRNA with SU5. One clone containing a 1.8-kb (1 kb = 10(3) base-pairs) insert was selected for further characterization. The beta-galactosidase fusion protein encoded by the cDNA clone had an apparent relative molecular mass of 150K, indicating that the inserted cDNA produced an estimated 34K of polypeptide. A single 2.2-kb RNA transcript was detected in sea urchin embryos using the cDNA clone as a probe. The cDNA fragment was sequenced and the nucleotide sequence was used to predict the amino acid sequence of the open reading frames in the clone. The putative gene product shows striking similarity to the peptide chain elongation factor (EF-1 alpha) from yeast, fungus, shrimp, insect, mouse and human.

The nitrate reductase structural gene of Chlamydomonas reinhardtii has been isolated from a genomic library by using a nitrate reductase cDNA probe from barley. Restriction fragment length polymorphism analyses mapped the Chlamydomonas clone (B6a) to the nitrate reductase structural gene locus nit-1. Overlapping inserts cover a region of the genome of about 24 kilobases containing the entire gene, which spans approximately 5-8 kilobases. Sequence analysis of DNA fragments from the B6a clone demonstrated a high degree of sequence similarity at the amino acid level with regions corresponding to portions of the heme and FAD/NADH-binding domains of tobacco and Arabidopsis thaliana nitrate reductases and human NADH cytochrome b5 reductase. The identity of the cloned gene as nitrate reductase was confirmed by its ability to complement a nit-1 mutation upon transformation. The nitrate reductase gene produced a 3.4-kilobase transcript in cells derepressed with nitrate; the transcript was undetectable in cells grown in the presence of ammonium. In cells that contain a mutation in the putative regulatory gene nit-2, significantly lower levels of the 3.4-kilobase transcript were found, indicating that the wild-type nit-2 gene is involved in the control of nitrate reductase transcript levels.

The length of the flagella of Chlamydomonas reinhardtii cells is tightly regulated; both short-flagella and long-flagella mutants have been described. This report characterizes ten long-flagella mutants, including five newly isolated mutants, to determine the number of different loci conferring this phenotype, and to study interactions of mutants at different loci. The mutants, each of which was recessive in heterozygous diploids with wild type, fall into three unlinked complementation groups. One of these defines a new gene, lf3, which maps near the centromere of linkage group I. The flagellar length distributions in populations of each mutant were broad, with the longest flagella measuring four times the length of the longest flagella seen on wild-type cells. Each of the ten mutants had defective flagellar regrowth after amputation. Some of the mutants showed no regrowth within the time required for wild-type cells to regenerate flagella completely. Other mutants had subpopulations with rapid regeneration kinetics, and subpopulations with no observable regeneration. The mutants were each crossed to wild type to form temporary quadriflagellate, dikaryon cells; in each case the long flagella were rapidly shortened in the presence of the wild-type cytoplasm, demonstrating that the mutants were recessive, and that length control could be exerted on already assembled flagella.

The quadriflagellate alga polytomella agilis contains several alpha-tubulins with distinct isoelectric points (McKeithan, T.W., and J.L. Rosenbaum, 1981, J. Cell Biol., 91:352-360). While alpha-3 is the major component in flagella, alpha-1 predominates in cytoskeletal microtubules. For determination of whether the differences in alpha- tubulins are due to distinct genes or to posttranslational modification of a common alpha-tubulin precursor, poly A+ RNA was isolated from deflagellated and control (nonregenerating) cells and translated in vitro. Approximately twice as much alpha-1 was synthesized using RNA from deflagellated as compared to control cells; however, there was no detectable synthesis in vitro of alpha-3 in either. These results suggest that alpha -3 tubulin is formed in vivo by posttranslational modification of a form co- migrating with, and possibly identical to, cytoskeletal alpha-tubulin. In the related alga chlamydomonas reinhardii deflagellation greatly stimulates synthesis of tubulin and tubulin mRNA. As in polytomella, the principal alpha-tubulin synthesized both in vivo and in vitro following deflagellation in chlamydomonas is more basic than the major alpha-tubulin and appears to correspond to alpha-1 tubulin in polytomella. The conversion of alpha-1 to alpha-3 receives additional support from in vivo labeling and pulse-chase experiments. In addition, in both polytomella and chlamydomonas some conversion of alpha-1 to alpha-3 appears to occur even when protein synthesis is inhibited.

We have used genetic analysis to study the mode of action of two anti-microtubule herbicides, amiprophos-methyl (APM) and oryzalin (ORY). Over 200 resistant mutants were selected by growth on APM- or ORY-containing plates. The 21 independently isolated mutants examined in this study are 3- to 8-fold resistant to APM and are strongly cross-resistant to ORY and butamiphos, a close analog of APM. Two Mendelian genes, apm1 and apm2, are defined by linkage and complementation analysis. There are 20 alleles of apm1 and one temperature-sensitive lethal (33 degrees) allele of apm2. Mapping by two-factor crosses places apm1 6.5 cM centromere proximal to uni1 and within 4 cM of pf7 on the uni linkage group, a genetically circular linkage group comprising genes which affect flagellar assembly or function; apm2 maps near the centromere of linkage group VIII. Allele-specific synthetic lethality is observed in crosses between apm2 and alleles of apm1. Also, self crosses of apm2 are zygotic lethal, whereas crosses of nine apm1 alleles inter se result in normal germination and tetrad viability. The mutants are recessive to their wild-type alleles but doubly heterozygous diploids (apm1 +/+ apm2) made with apm2 and any of 15 apm1 alleles display partial intergenic noncomplementation, expressed as intermediate resistance. Diploids homozygous for mutant alleles of apm1 are 4-6-fold resistant to APM and ORY; diploids homozygous for apm2 are ts- and 2-fold resistant to the herbicides. Doubly heterozygous diploids complement the ts- phenotype of apM2, but they are typically 1.5-2-fold resistant to APM and ORY. From the results described we suggest that the gene products of apm1 and apm2 may interact directly or function in the same structure or process.

Synthesis of new proteins is required to regenerate full length Chlamydomonas flagella after deflagellation. Using gametes, which have a low basal level of protein synthesis, it has been possible to label and detect the synthesis of many flagellar proteins in whole cells. The deflagellation-induced synthesis of the tubulins, dyneins, the flagellar membrane protein, and at least 20 other proteins which co-migrate with proteins in isolated axonemes, can be detected in gamete cytoplasm, and the times of initiation and termination of synthesis for each of the proteins can be studied. The nature of the signal that stimulates the cell to initiate flagellar protein synthesis is unknown. Flagellar regeneration and accompanying pool depletion are not necessary for either the onset or termination of flagellar protein synthesis, because colchicine, which blocks flagellar regeneration, does not change the pattern of proteins synthesized in the cytoplasm after deflagellation or the timing of their synthesis. Moreover, flagellar protein synthesis is stimulated after cells are chemically induced to resorb their flagella, indicating that the act of deflagellation itself is not necessary to stimulate synthesis. Methods were defined for inducing the cells to resorb their flagella by removing Ca++ from the medium and raising the concentration of K+ or Na+. The resorption was reversible and the flagellar components that were resorbed could be re-utilized to assemble flagella in the absence of protein synthesis. This new technique is used in this report to study the control of synthesis and assembly of flagella.

Mitotic aparatuses have been isolated which can incorporate heterologous tubulin and can assemble this tubulin into birefringent fibres similar to those of mitotic apparatuses of living cells.

The antimicrotubule agents oryzalin (ORY), colchicine (COL) and taxol (TAX) were used to select three recessive, conditional lethal (ts-) mutants which defined two new essential loci, ory1 and cor1. The two ory1 mutants conferred resistance to ORY, TAX, and COL; the cor1 mutant conferred resistance only to COL. Each of the mutants displayed wild-type sensitivity to a number of unrelated inhibitors. Assembly and disassembly of flagellar microtubules in the ory1 mutants displayed wild-type sensitivity to ORY and COL, suggesting that the ory1 gene product either does not participate in these processes or the ory1 gene product alone is not sufficient to confer resistance. The ory1 locus mapped to linkage group X; cor1 was mapped to the left arm of linkage group XII. A synthetic lethal interaction was observed between ory1 and cor1 mutations, i.e., inferred ory1 cor1 double mutants were inviable at the permissive temperature. The conditional lethal phenotype of ory1-1 was used to select many spontaneous TS+ revertants, which arose at high frequencies. Genetic and phenotypic characterization of the revertants demonstrated that (1) the revertants fell into four phenotypic classes, including some which conferred supersensitivity to ORY and others which conferred cold-sensitive lethality, (2) reversion was caused in most or all cases by extragenic suppressors, (3) suppressor mutations displayed complex behavior in heterozygous (sup/+) diploids, (4) many different loci may be capable of suppressing ory1 mutants, and (5) suppressors of ory1-1 efficiently suppressed an independently isolated allele, ory1-2. Taken together the ory1, cor1, and suppressor mutations identify a number of interacting loci involved in essential cellular processes which are specifically susceptible to antimicrotubule agents.