Light and Electron Microscopy

Flagellar number counts were performed using phase optics and a 40× objective with cells at densities of 5 × 10⁵ to 2 × 10⁶ cells/ml to ensure that the cells were not approaching stationary phase. Eyespots were visualized using differential interference contrast (DIC) or brightfield light microscopy (Holmes and Dutcher, 1989). The cells used for obtaining longitudinal images were processed for electron microscopy as described in Porter et al. (1992). Images were examined at a magnification of 21,000× with a CM10 microscope (Philips Electronic Instruments, Mahwah, NJ) operating at 80 kV. The cells used for cross-sectional and tangential images were processed for electron microscopy as described by Goodenough and Weiss (1978).

Southern Blot Analysis and Library Screen

Chlamydomonas DNA was isolated as described by Johnson and Dutcher (1991), except that the DNA was precipitated with polyethylene glycol an additional time. Upon resuspension of the DNA pellet with Tris-EDTA, the salt concentration was adjusted to 0.8 M NaCl, and polyethylene glycol (PEG)-4000 was added to a final concentration of 7.5%. Each sample was incubated on ice for >30 min, centrifuged at 14,000 rpm for 10 min, and resuspended in water or Tris-EDTA. This extra precipitation step increased the purity of the sample and made subsequent enzyme digestions significantly more reliable. Hybridization conditions for Southern blots and library filters were described elsewhere (Johnson and Dutcher, 1991). All probes were labeled using the Multiprime DNA Labeling System (Amersham, Arlington Heights, IL).

Plasmids and Phage DNA

pARG7.8, a pBR329-based plasmid containing the Chlamydomonas argininosuccinate lyase gene (ARG7) (Debuchy et al., 1989) was used in the construction of pγΔ-1, which was used for transformation of arg7–8 cells. pMN56, a pUC119-based plasmid containing the nitrate reductase gene (NIT1) was used for cotransformation/rescue experiments of uni3–1::ARG7 NIT2 nit1–1 cells with λ-phage or genomic subclones (Fernández et al., 1989; Nelson and Lefebvre, 1995). Plasmid and λ-phage DNA preparations were performed using Qiagen (Chatsworth, CA) DNA purification kits. Plasmids pCU-1 and pCU-2 were constructed in the vector pUC18. pCU-1 has a 9.5-kb insert after a digestion of λ-CU-2 with SalI. pCU-2 has a 5.68-kb insert made by digesting pCU-1 with XbaI and EcoNI.

Chlamydomonas Transformations

For all transformations we used the glass bead method (Kindle, 1990). Briefly, logarithmically growing 1L cultures were harvested by centrifugation, incubated with autolysin (Harris, 1989; Dutcher, 1995b) for 20 min at room temperature to remove the cell wall, recentrifuged, and resuspended in selective medium: 525 μl of cells, 500 μl of acid-washed glass beads (710–1, 180 nm in diameter, Sigma Chemical, St. Louis, MO), 175 μl of 20% PEG-4000, and 2 μg of EcoRI linearized pΔγ-1 DNA were vortexed at maximum speed for 30 s. Cells were allowed to recover for 2 h to overnight at 25°C and plated onto selective medium that contained ammonium nitrate and no arginine. Cotransformation experiments used 2 μg of EcoRI-linearized pMN56, and 0.5, 1.0, or 2.0 μg of each genomic λ phage. Cells were plated onto medium with sodium nitrate as the sole nitrogen source and with 0.5% top agarose to improve the transformation efficiency (Gumpel et al., 1994). The transforming DNA, pΔγ-1, which was used to generate the uni3–1 allele, contained short stretches of γ-tubulin. Although most integration events in Chlamydomonas are nonhomologous, it is possible that these γ-tubulin sequences may have provided homology with either α-tubulin or the Uni3 to promote integration in this region of the genome.

Construction of the Size-fractionated Library of uni3–1 DNA

uni3–1 (20 μg) DNA was digested with BamHI and AvaI (Boehringer Mannheim, Indianapolis, IN) and fractionated on a 1% low-melting temperature agarose gel (SeaKem, Rockland, ME). Four fractions that included regions slightly above and slightly below the 2.3-kb band of the λ DNA molecular weight marker were excised and purified using β-agarase (Boehringer Mannheim). One-tenth volume of each fraction was electrophoresed on a 1% agarose gel, transferred to a Zetabind filter (Amersham), and hybridized with a single-copy γ-tubulin probe. The band from the endogenous γ-tubulin gene was significantly smaller than the aforementioned 2.3-kb band and therefore did not pose a contamination problem. The fraction with the most intense signal was ligated to the pBR329 vector and transformed into DH5αF′ electrocompetent cells. A colony containing the hybrid γ-tubulin and flanking genomic DNA sequences was isolated using standard colony hybridization techniques and the single-copy γ-tubulin sequences as the hybridization probe (Sambrook et al., 1989).

Isolation of Genomic and cDNA Clones

Genomic clones were isolated from two independent libraries of MboI partially digested arg7–8 and wild-type DNA cloned into the BamHI cloning site of λDASHII (Stratagene, La Jolla, CA) and λEMBL3, respectively (kind gifts of Anthony Palombella and Dr. David Johnson). Screening with a 870-bp fragment that contained flanking genomic DNA from the cloned BamHI-AvaI DNA fragment yielded phage λCU-1 and λCU-2. The remainder of the walk came from screens using single-copy sequences at the ends of λCU-2 and λCU-3 as hybridization probes.

A cDNA library made from light-grown vegetative NO⁻ cells (a gift from Dr. Jeff P. Woessner, Washington University, St. Louis, MO) was screened with the genomic clone, and no positive clones were isolated from 500,000 plaques. cDNA libraries made from light and dark grown cells (a gift from Dr. Andy Wang, Iowa State University, Ames, IA) was screened, and no positives were isolated in 500,000 plaques. A Novagen custom cDNA library of mRNAs isolated from nitrogen-starved cells cloned into the λEXlox vector (a gift from Dr. Rogene Schnell, University of Minnesota, St. Paul, MN) was screened with a 449-bp UNI3 reverse transcriptase (RT)-PCR fragment (see following section). In 2.5 × 10⁶ plaques screened, a single positive plaque was detected. The 1.75- kb cDNA clone, named Exlox53, is missing 133 bp of coding sequence and the 5′ untranslated region.

Reverse Transcription PCR

Poly-A⁺ RNA was enriched from 10 μg of total RNA isolated from wild-type cells using an mRNA isolation kit (Promega, Madison, WI). The primers used for PCR amplification were designed from DNA sequences obtained from the rescuing genomic clone whose predicted amino acid sequence showed similarity to known tubulins using BLAST searches. The primers varied in length from 18 to 37 bases. u12 and u14 (37 and 36 bases, respectively) each contained a 9-base linker with an EcoRI site to facilitate cloning of PCR fragments. The sequence from the genomic clones is underlined below. These primers amplified a 449-bp fragment that was used as a probe to screen the λEXlox cDNA library. A cDNA probe was generated because attempts to screen other libraries and Northern blots with genomic probes failed to detect a UNI3-specific signal. u24 and u26 (both 18 bases long) were used to extend the cDNA sequences beyond the 5′ end of the 1.75-kb cDNA clone, Exlox53. The 384- bp fragment amplified by the primer combination contained 133 bp of coding sequence and 90 bp of 5′ untranslated sequences not found in Exlox53. The sequence of both RT-PCR fragments was identical to the corresponding positions in the genomic clone.

All PCR reactions were performed using Taq DNA polymerase from Boehringer Mannheim in a Perkin Elmer-Cetus (Norwalk, CT) 480 DNA thermal cycler. mRNA (500 ng) primed with 250 ng of random hexamers (Life Technologies/Bethesda Research Laboratories, Gaithersburg, MD) was reverse transcribed with avian myeloblastosis virus-RT (Promega). One microliter of a 1:10 dilution of the first strand synthesis reaction was used in a 100 μl reaction with 25 pmol of each primer. PCR parameters were as follows: one cycle at 97°C for 10 min and 80°C for 5–10 min before the addition of Taq polymerase; 10 cycles at 95°C for 1 min, 53°C for 2 min, and 72°C for 3 min; 30 cycles at 95°C for 45 s, 57°C for 1 min, and 72°C for 2 min; and one final cycle at 95°C for 45 s, 57°C for 1 min, and 72°C for 10 min. The conditions for the RT-PCR with primers u24 and u26 were similar, except that the annealing temperatures were deceased by 1°C in the first 10 cycles, and by 2°C in the middle 30 cycles and in the final cycle.

u12 (antisense): 5′CCGGAATTCcagcgcctcgttctccagcagcaccagc;

u14 (sense): 5′CCGGAATTCggcgctccgtgctcatcgacatggagc; u24 (antisense): 5′CCTTGTGCCCAGTTGTTG; u26 (sense): 5′AGCACGCTCTGTTACTAG.

DNA Sequencing

Double-stranded DNA was made using a combined alkaline lysis-PEG precipitation method and sequenced by the DNA Sequencing Facility of Iowa State University. The cDNA clone, Exlox53, and the 384- and 449-bp RT-PCR fragments were sequenced on both strands. Sequence data were compiled using GCG Sequence Analysis Software (Madison, WI). GenBank database searches for sequence homologies were initially performed using the BLASTN program (Altschul et al., 1990).

Isolation of the Genomic DNA for γ-Tubulin

The genomic DNA was isolated using degenerate PCR with primers designed to match regions of identity among the known γ-tubulins. These included Aspergillus nidulans, Schizosaccharomyces pombe, Drosophila melanogaster, Xenopus laevis, and Homo sapiens. Underlined sequences indicate the region that hybridize to γ-tubulin, and the remainder contain an EcoRI site to facilitate cloning. I indicates an inosine residue, Y indicates a pyrimidine (C or T), R indicates a purine (A or G), and N indicates any base.

g1 (sense): 5′CCGGAATTCTAYCCNGGITAYATGGAAY; g2 (antisense): 5′CCGGAATTCACYTTRTTRAAIARRTG

Primers g1 and g2 generated a 773-bp PCR product that was used to screen a λEMBL3 library (constructed by Dr. David Johnson in our laboratory) using procedures described above for the cloning of the UNI3 gene. The clone was sequenced by the Iowa State University Sequencing Facility.

Molecular Characterization of the UNI3 Locus

In Chlamydomonas, transforming DNA inserts nonhomologously into the nuclear genome and provides a “tag” that can be used for isolating DNA flanking the insertion site (Tam and Lefebvre, 1993; Pazour et al., 1995; Smith and Lefebvre, 1996). Many insertional events in Chlamydomonas are associated with the partial loss of transforming plasmid DNA as well as genome rearrangements. These genomic rearrangements include deletions as well as more complicated events (Smith and Lefebvre, 1996). The uni3–1 mutation was generated after transformation with the plasmid pγΔ-1, which carries the ARG7 gene in a pBR329-based vector flanked by two short segments of the γ-tubulin gene (Figure ​(Figure3A).3A). These segments of the γ-tubulin gene provided insulation of the ARG7 gene against deletion of transforming DNA and would provide single-copy probes for Southern analysis, if sequences needed for plasmid rescue were lost. In the uni3–1 strain, a single insertion of the transforming DNA was observed using pBR329 DNA as the probe (diagrammed in Figure ​Figure3B).3B). Southern blot analysis of the insertion was performed using pBR329 and γ-tubulin sequences as probes. The 2.5 kb at the right end of the plasmid as diagrammed contain the ampicillin resistance gene and the origin of replication; these sequences were deleted in the uni3–1 strain. This precluded the possibility of using plasmid rescue for cloning the DNA adjacent to the site of insertion. The left end was intact and was used for constructing a size-selected library (see MATERIALS AND METHODS).

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Figure 3

Diagram of the pΔγ-1 vector, the genomic insertion site, the size-selected genomic DNA clone with flanking sequences, and demonstration that uni3–1 is a deletion allele. (A) The vector pΔγ-1 carries the ARG7 gene (Debuchy et al., 1989), which is diagrammed as the striped box, and pBR329 sequences, which are diagrammed as black lines. At the ends, when the plasmid is linearized with EcoRI, are 1049-bp and 750-bp segments of the Chlamydomonas γ-tubulin gene, which are diagrammed as open boxes. (B) The orientation of the plasmid at the insertion site in uni3–1 cells. Most of the pBR329 and all of the γ-tubulin sequences were deleted from the right side as diagrammed. The 1,049 bp of γ-tubulin sequences from the left side of the pΔγ-1 plasmid remained in the transformant. (C) The cloned genomic DNA from the size-selected library made from AvaI–BamHI digested DNA from uni3–1 cells. Probe b was used to screen the phage libraries and contains single-copy DNA from the site of the insertion. (D) Restriction map of uni3–1 and wild-type DNA with the enzyme NheI. The location of probes a–f on the wild-type map are shown. Probe b in parts C and D are the same probe. (E) Southern blots of genomic DNA from uni3–1 and wild-type cells hybridized with probes a–f. The left lane in each panel contains wild-type DNA (+) and the right lane contains uni3–1 DNA (−).

A 2.3-kb fragment with approximately 1.3 kb of flanking genomic DNA was cloned from the size-selected library made from BamHI–AvaI-digested uni3–1 DNA using a short γ-tubulin sequence as the probe (Figure ​(Figure3C).3C). A 870-bp AvaI–SacI fragment derived from the flanking genomic sequence recognized a restriction fragment length polymorphism (RFLP) between the uni3–1 strain and the parental strain. This RFLP was linked to the uni3–1 phenotype in 16 meiotic progeny from a cross of uni3–1 and a wild-type strain. These data support the idea that genomic DNA flanking the insertion site was cloned. This 870-bp genomic DNA was used as a probe to begin a chromosome walk in two independent genomic DNA phage libraries (see MATERIALS AND METHODS).

Four overlapping λ clones that span 54 kb of wild-type DNA were obtained from the walk that began with the 870-bp probe (labeled probe b in Figure ​Figure3,3, C and D). The structure of the uni3–1 mutation was determined by hybridizing genomic DNA from wild-type and uni3–1 cells with single-copy probes from various points along the walk (Figure ​(Figure3E).3E). The uni3–1 hybridization patterns detected by probes a and f were indistinguishable from the wild-type patterns. For probe b, as noted above, and for probe e, RFLPs were observed. Each RFLP presumably occurred because a restriction enzyme site was missing or altered in the mutant DNA compared with the wild-type DNA (probes b and e, Figure ​Figure3E).3E). For probes c and d, no signal was detected in the uni3–1 mutant DNA. Based on these data as well as Southern blots from several other probes between b and c that are not shown, we estimated that a 27-kb deletion is present in the uni3–1 strain compared with the DNA from wild-type or parental strains.

Rescue of the uni3–1 Strains by Transformation

To identify the UNI3 gene, we cotransformed a uni3–1 nit1–1 NIT2 strain with pMN56, a plasmid carrying the NIT1 gene (Nelson and Lefebvre, 1995), and one of the four overlapping phage (λCU-1, λCU-2, λCU-3, and λCU-4) found in the walk (Figure ​(Figure4A).4A). We selected for the presence of the NIT1 gene on medium containing sodium nitrate as the sole nitrogen source; 64, 148, 211, and 31 Nit1⁺ transformants were generated in these experiments with λCU-1, λCU-2, λCU-3, and λCU-4, respectively. Because the frequency of cotransformation is often low (Ferris and Goodenough, 1994), we expected only a subset of transformants to show rescue of the Uni⁻ phenotype. These rescued cells would be biflagellate and able to swim; 4.7% of the cotransformants with λCU-2 and λCU-3 DNAs restored wild-type function, while no rescued strains were observed with λCU-1 or λCU-4 DNAs. Light microscopic analysis of the rescued strains revealed wild-type or nearly wild-type distributions of biflagellate, uniflagellate, and aflagellate cells and wild-type frequencies of cells with cleavage defects (n = 500). The presence of transforming DNA was confirmed by Southern blots of genomic DNA from 17 rescued transformants. Genomic DNA from transformants with the nonrescuing clones, λCU-1 or λCU-4, was also examined by Southern blots. The λ DNA was incorporated at a similar frequency, but without rescue of the mutant phenotype. Phage λCU-2 and λCU-3 overlapped each other by 9.5 kb, and we conclude that this overlapping region is sufficient for rescuing the Uni⁻ phenotype. Furthermore, this result demonstrates that the uni3–1 allele is a null mutation as the region needed for rescue is deleted in the uni3–1 allele.

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Figure 4

The λ phage and plasmids used for transformation rescue and in vivo deletion analysis. (A) Phage λCU-1, λCU-2, λCU-3, and λCU-4 were obtained in the chromosome walk and were used for assaying rescue after transformation. The vertical bars indicate the recognition sites for the enzyme NheI. The subclone pCU-1 showed rescue of the Uni⁻ phenotype. (B) Southern blots of phenotypically rescued uni3–1 and control strains. Probes g, h, and i were hybridized against DNA from the rescued strains (+) and control uni3–1 strains (−). Probes g and h are found in the same NheI fragment as probe d in Figure ​Figure3D.3D. Probe i was present in all strains shown as well as 13 other rescued strains. Probes g and h were present in some, but not all, of the rescued strains. The loss of portions of the transforming DNA in rescued strains was used to further delineate the DNA needed for the restoration of the wild-type phenotype to ~5.5 kb.

We took advantage of in vivo deletions of transforming DNA to further define the sequences needed for rescue. Using probes g, h, and i, we found that only 5.5 kb of DNA were needed to rescue the mutant phenotypes (Figure ​(Figure4B).4B). Probes g and h were present in some, but not all, rescued transformants as shown in Figure ​Figure4B.4B. Signal from probe i was present in all 17 strains with a rescued phenotype. Plasmid pCU-2, which has a 5.68-kb insertion, also rescued the mutant phenotype.

Uni3p Defines a New Member of the Tubulin Superfamily

The UNI3 gene encodes a protein that represents a new class of tubulins, which clearly differs from the well known α-, β-, and γ-tubulins (Figures ​(Figures66 and ​and7).7). The Uni3 tubulin differs from other tubulins in several ways. First, a cell with a deletion of the UNI3 gene is viable; this gene is not essential. Null alleles in single-copy α-, β-, or γ-tubulin genes in a variety of organisms have lethal phenotypes (reviewed by Cabral et al., 1984; Huffaker et al., 1987). Southern blots of genomic DNA with genomic or cDNA UNI3 probes did not reveal any additional hybridization signals, even under conditions of reduced stringency (Preble and Dutcher, work in progress). This result suggests that there are not additional genes with similar sequence that may provide redundant function. A second difference is the presence of two additional exons in the UNI3 gene that encode a region not present in other known tubulins. The function of this region in the protein is unknown. These two exons contain a histidine-rich region and many glycine and proline residues.

The UNI3 gene does not appear to be unique to Chlamydomonas as we have found a sequence in the mouse EST database that is likely to be a homolog. Clone 371244 (Accession no. W53427) from 13.5–14.5 d mouse embryos is 62%/51% similar/identical to Uni3p over 129 amino acids. This clone is also 43% identical to human γ-tubulin, but lacks several motifs conserved in γ-tubulins. We are currently sequencing the entire EST to determine the extent of similarity.

Burns (1995) proposed that new tubulin genes found in the sequencing projects of C. elegans and S. cerevisiae are each new members of the tubulin superfamily and named them δ and ε, respectively. As illustrated in Figure ​Figure7,7, these sequences are found in the branch with other γ-tubulin sequences, but are more divergent. The phylogenetic analysis of Keeling and Logsdon (1996) suggested that these genes represent divergent members of the γ-tubulin family rather than founding members of new subfamilies. Furthermore, functional analysis suggests that the TUB4 gene in S. cerevisiae is essential and encodes γ-tubulins (Spang et al., 1996). Tub4p localizes to the spindle pole body, which is the MTOC equivalent in yeast (Sobel and Synder, 1995; Marschall et al., 1996; Spang et al., 1996). The yeast genome is completely sequenced, and no tubulin genes in addition to the known α- and β-tubulin genes were found (J.M. Cherry, C. Adler, C. Ball, S. Dwight, S. Chervitz, Y. Jia, G. Juvik, S. Weng, and D. Botstein [1996], Saccharomyces genome database, http://genome-www.stanford.edu/Saccharomyces/). In C. elegans, no other γ-tubulin gene has been identified by the sequencing project, which is about 80% completed. The C. elegans γ-like tubulin gene is found in the emb-30 operon, but definitive evidence for an embryonic lethal mutation in the γ-like tubulin gene is not yet available (Tabish, Khan, and Siddiqui, personal communication). The essential nature of the γ-tubulin gene in A. nidulans (Oakley et al., 1990), Schizosaccharomyces pombe (Stearns et al., 1991; Horio et al., 1991), or D. melanogaster (Sunkel et al., 1995), as well as yeast, is in contrast to the nonessential nature of the UNI3 gene. This difference underscores the conclusion that Uni3 tubulin is unique compared with other known tubulins.

Recently another gene with a tubulin motif has been described. The Misato gene in D. melanogaster encodes a protein with both tubulin-like and myosin-like motifs (Miklos et al., 1997). A null mutation in Misato results in flies with irregular chromosome segregation. Misato is no more similar to Uni3p than it is to other tubulins.

We are grateful for the genorosity and kindness of Dr. Ursula Goodenough who volunteered to do electron microscopy on uni3–1 cells. This manuscript would have never seen the light of day without her help. We thank Rogene Schnell, Andy Wang, and Jeff Woessner for cDNA libraries, Jean-David Rochaix and Saul Purton for the pARG7.8 plasmid, David Johnson and Anthony Palombella for genomic phage libraries, and Andrea Preble for Southern blots with cDNA probes. We thank Gary Stormo for advice on sequence and phylogenetic analysis and Shmuel Pietrokovski, Steve Henikoff, and Sean Eddy for their help. We thank Scott Schuyler and Tom Giddings for the electron microscopic analysis of longitudinal sections. We thank Sylvia Fromherz, Anthony Palombella, Andrea Preble, and Gary Stormo for their discussion and critical reading of this manuscript. This work was supported by a grant from the National Institutes of Health (GM-32843). S.K.D. was supported in part by a University of Colorado Faculty Fellowship from the Committee on Creative Work and Research. The Perkin Elmer-Cetus thermal cycler was a gift from the Colorado Chapter of the American Cancer Society.