The bld1 mutation identifies the Chlamydomonas osm-6 homolog as a gene required for flagellar assembly.

Insertional mutagenesis procedures in Chlamydomonas have facilitated the identification and characterization of dozens of genes required for the assembly and motility of flagella in Chlamydomonas. 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, 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 whose product is necessary for the assembly of a set of sensory cilia. The protein product of the BLD1 gene corresponds to IFT52, 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). The BLD1 RNA transcript is upregulated upon flagellar amputation, as observed for many other genes encoding flagellar proteins. These results demonstrate that the function of the IFT52 protein in Chlamydomonas is essential for the assembly and/or maintenance of the flagella.

The Vfl1 Protein in Chlamydomonas localizes in a rotationally asymmetric pattern at the distal ends of the basal bodies.

In the unicellular alga Chlamydomonas, two anterior flagella are positioned with 180 degrees 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 NH(2) terminus and a large alpha-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.

Gamma-tubulin in Chlamydomonas: characterization of the gene and localization of the gene product in cells.

In addition to their role in nucleating the assembly of axonemal microtubules, basal bodies often are associated with a microtubule organizing center (MTOC) for cytoplasmic microtubules. In an effort to define molecular components of the basal body apparatus in Chlamydomonas reinhardtii, genomic and cDNA clones encoding gamma-tubulin were isolated and sequenced. The gene, present in a single copy in the Chlamydomonas genome, encodes a protein with a predicted molecular mass of 52,161 D and 73% and 65% conservation with gamma-tubulin from higher plants and humans, respectively. To examine the distribution of gamma-tubulin in cells, a polyclonal antibody was raised against two peptides contained within the protein. Immunoblots of Chlamydomonas proteins show a major cross-reaction with a protein of Mr 53,000. In Chlamydomonas cells, the antibody stains the basal body apparatus as two or four spots at the base of the flagella and proximal to the microtubule rootlets. During cell division, two groups of fluorescent dots separate and localize to opposite ends of the mitotic apparatus. They then migrate during cleavage to positions known to be occupied by basal bodies. Changes in gamma-tubulin localization during the cell cycle are consistent with a role for this protein in the nucleation of microtubules of both the interphase cytoplasmic array and the mitotic spindle. Immunogold labeling of cell sections showed that gamma-tubulin is closely associated with the basal bodies. The flagellar transition region was also labeled, possibly indicating a role for gamma-tubulin in assembly of the central pair microtubules of the axoneme.

SF-assemblin in Chlamydomonas: sequence conservation and localization during the cell cycle.

Previously, SF-assemblin has been identified as the filament-forming component of the striated microtubule-associated fibers (SMAFs), which emerge from the basal bodies in several green flagellates. We have sequenced cDNAs coding for SF-assemblin from Chlalmydomonas reinhardtii and C. eugametos. Comparison of the deduced amino acid sequences with the previously described green algal SF-assemblins shows identities between 54 and 71%, indicating a strong drift in sequence. Cells of C. reinhardtii were analyzed by double immunofluorescence using polyclonal anti-SF-assemblin and anti-alpha-tubulin. In interphase cells, SF-assemblin is associated with all four microtubular flagellar roots. During mitosis the SF-assemblin-based cytoskeleton is reorganized; it divides in prophase and is reduced to two dot-like structures at each spindle pole in metaphase. During anaphase, the two dots present at each pole are connected again. In telophase we observed an asymmetrical outgrowth of new fibers. These observations suggest a role for SF-assemblin in reestablishing the microtubular root system characteristic of interphase cells after mitosis.

Isolation and characterization of glutamine synthetase genes in Chlamydomonas reinhardtii.

To elucidate the role of glutamine synthetase (GS) in nitrogen assimilation in the green alga Chlamydomonas reinhardtii we used maize GS1 (the cytosolic form) and GS2 (the chloroplastic form) cDNAs as hybridization probes to isolate C. reinhardtii cDNA clones. The amino acid sequences derived from the C. reinhardtii clones have extensive homology with GS enzymes from higher plants. A putative amino-terminal transit peptide encoded by the GS2 cDNA suggests that the protein localizes to the chloroplast. Genomic DNA blot analysis indicated that GS1 is encoded by a single gene, whereas two genomic fragments hybridized to the GS2 cDNA probe. All GS2 cDNA clones corresponded to only one of the two GS2 genomic sequences. We provide evidence that ammonium, nitrate, and light regulate GS transcript accumulation in green algae. Our results indicate that the level of GS1 transcripts is repressed by ammonium but induced by nitrate. The level of GS2 transcripts is not affected by ammonium or nitrate. Expression of both GS1 and GS2 genes is regulated by light, but perhaps through different mechanisms. Unlike in higher plants, no decreased level of GS2 transcripts was detected when cells were grown under conditions that repress photorespiration. Analysis of GS transcript levels in mutants with defects in the nitrate assimilation pathway show that nitrate assimilation and ammonium assimilation are regulated independently.

Identification of intrinsic dimer and overexpressed monomeric forms of gamma-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas gamma-tubulin sequence.

A new member of the tubulin superfamily, gamma-tubulin, is localized at microtubule-organizing centers (MTOCs) in a variety of organisms. Chlamydomonas cDNA coding for the full-length sequence of gamma-tubulin was expressed in insect ovarian Sf9 cells using the baculovirus expression system. Approximately half of the induced 52 kDa gamma-tubulin was recovered in the supernatant after centrifugation of Sf9 cell lysates at 18,000 g for 15 minutes. When the cell supernatant was analyzed by FPLC on a Superdex 200 sizing column, Chlamydomonas gamma-tubulin separated into two major peaks. The lagging peak contained a monomeric form of gamma-tubulin with a sedimentation coefficient of 2.5 S, which interacted with the Superdex column in a salt-dependent manner. The leading peak, with an apparent molecular mass of 900 kDa, corresponded to a molecular chaperonin complex, and TCP1 chaperonin released folded gamma-tubulin polypeptide from the complex in the presence of MgATP. The released gamma-tubulin monomers were capable of binding to microtubules in vitro and biochemical quantities of active monomers were further purified using a combination of size-exclusion and ion-exchange column chromatography. The endogenous Sf9 cell gamma-tubulin migrated faster than Chlamydomonas gamma-tubulin with an apparent molecular mass of 49 kDa on gels. Analyses on gel filtration and sucrose density gradient centrifugation showed that, while overexpressed Chlamydomonas gamma-tubulin was present in a monomeric form, endogenous gamma-tubulin from Sf9 and HeLa cells exists as a dimer. These results may suggest the possibility that gamma-tubulin could form a heterodimer with hitherto unknown molecule(s).

gamma-Tubulin in Arabidopsis: gene sequence, immunoblot, and immunofluorescence studies.

gamma-Tubulin is a protein associated with microtubule (Mt)-organizing centers in a variety of eukaryotic cells. Unfortunately, little is known about such centers in plants. Genomic and partial cDNA clones encoding two gamma-tubulins of Arabidopsis were isolated and sequenced. Comparisons of genomic and cDNA sequences showed that both genes, TubG1 and TubG2, contain nine introns at conserved locations. The sequences of the two genes both predict proteins containing 474 amino acids, with molecular masses of 53,250 and 53,280 D, respectively. The predicted gamma 1- and gamma 2-tubulins exhibit 98% amino acid identity with each other and approximately 70% amino acid identity with the gamma-tubulins of animals and fungi. RNA gel blot results demonstrated that both genes are transcribed in suspension culture cells, seedlings, and roots and flowers of mature plants. Immunoblots of Arabidopsis proteins using an antibody specific to a conserved peptide of gamma-tubulin showed a major cross-reacting polypeptide with an M(r) of 58,000. The same antibody stained all Mt arrays in tissue and suspension culture cells of this species. Binding was inhibited by the homologous oligopeptide in the gamma-tubulins predicted by the two Arabidopsis gene sequences. Antibody staining avoided the plus ends of Mts at the kinetochores and cell plate, but unlike the case in animal cells, seemed to be localized over broad stretches of the kinetochore fibers and phragmoplast toward the minus ends. We concluded that at least two gamma-tubulin protein homologs are present in Arabidopsis and that at least one of them is localized along Mt arrays. Its distribution is correlated with and may help explain unique characteristics of Mt organization in plants.

Characterization of four new beta-tubulin genes and their expression during male flower development in maize (Zea mays L.).

Four different beta-tubulin coding sequences were isolated from a cDNA library prepared from RNA from maize seedling shoots. The four genes (designated tub4, tub6, tub7 and tub8) represented by these cDNA clones together with the tub1 and tub2 genes reported previously encode six beta-tubulin isotypes with 90-97.5% amino acid sequence identity. Results from phylogenetic analysis of 17 beta-tubulin genes from monocot and dicot plant species indicated that multiple extant lines of beta-tubulin genes diverged from a single precursor after the appearance of the two major subfamilies of alpha-tubulin genes described previously. Hybridization probes from the 3' non-coding regions of six beta-tubulin clones were used to quantify the levels of corresponding tubulin transcripts in different maize tissues including developing anthers and pollen. The results from these dot blot hybridization experiments showed that all of the beta-tubulin genes were expressed in most tissues examined, although each gene showed a unique pattern of differential transcript accumulation. The tub1 gene showed a high level of transcript accumulation in meristematic tissues and almost no accumulation in the late stages of anther development and in pollen. In contrast, the level of tub4 transcripts was very low during early stages of male flower development but increased markedly (more than 100 times) during the development of anthers and in pollen. Results from RNAse protection assays showed that this increased hybridization signal resulted from expression of transcripts from one or two genes closely related to tub4. The tub4-related transcripts were not present in shoot tissue. Transcripts from the tub2 gene accumulated to very low levels in all tissues examined, but reached the highest levels in young anthers containing microspore mother cells. RNAse protection assays were used to measure the absolute levels of alpha- and beta-tubulin transcripts in seedling shoot and in pollen. The alpha-tubulin gene subfamily I genes (tua1, tua2, tua4) contributed the great majority of alpha-tubulin transcripts in both shoot and pollen. Transcripts from the beta-tubulin genes tub4, tub6, tub7, and tub8 were predominant in shoot, but were much less significant than the tub4-related transcripts in pollen.

Differential expression of six glutamine synthetase genes in Zea mays.

The maize genome has been shown to contain six glutamine synthetase (GS) genes with at least four different expression patterns. Noncoding 3' gene-specific probes were constructed from all six GS cDNA clones and used to examine transcript levels in selected organs by RNA gel blot hybridization experiments. The transcript of the single putative chloroplastic GS2 gene was found to accumulate primarily in green tissues, whereas the transcripts of the five putative GS1 genes were shown to accumulate preferentially in roots. The specific patterns of transcript accumulation were quite distinct for the five GS1 genes, with the exception of two closely related genes.

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii confers resistance to anti-microtubule herbicides.

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.

Semi-constitutive expression of an Arabidopsis thaliana alpha-tubulin gene.

In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple alpha-tubulin and beta-tubulin genes. Previous evidence suggested that the TUA2 alpha-tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5'-flanking DNA fused to the beta-glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active.

Mapping of beta-tubulin genomic sequences in hexaploid oat (Arena sativa L.).

The allohexaploid nature of Avena sativa L. (2n=6x=42) and the availability of aneuploid lines was exploited in designing a strategy for mapping beta-tubulin sequences in the oat genome. Evidence for a minimum of eight beta-tubulin genes was obtained by Southern-blot analysis. Three betatubulin sequences were localized to chromosomes using DNA from monosomic and nullisomic lines in the variety Sun II. One sequence was localized to the chromosome missing in nullisome I. Two other sequences were mapped to satellite chromosome 2, the chromosome that is missing in nullisome VIII and to which one ribosomal RNA gene cluster had previously been mapped. Restriction fragments carrying these two beta-tubulin genomic sequences and the cluster of ribosomal RNA sequences were missing in DNA from nullisomics VIII, IX and X, suggesting that all three nullisome classes are deficient for an identical chromosomal segment that includes these three loci. This study demonstrates how molecular analyses can be used to characterize aneuploid stocks and to better define their genetic constitution.

Alpha-tubulin gene family of maize (Zea mays L.). Evidence for two ancient alpha-tubulin genes in plants.

Among 81 alpha-tubulin cDNA clones prepared from RNA from maize seedling shoot, endosperm and pollen, we identified six different alpha-tubulin coding sequences. The DNA sequence analysis of coding and non-coding regions from the clones showed that they can be classified into three different alpha-tubulin gene subfamilies. Genes within each subfamily encode proteins that are 99 to 100% identical in amino acid sequence. Deduced amino acid sequence identity between genes in different subfamilies ranges from 89 to 93%. The results of hybridizations of genomic DNAs to alpha-tubulin coding region probes and to 3' non-coding region probes constructed from six different alpha-tubulin cDNA clones indicated that the maize alpha-tubulin gene family contains at least eight members. Comparison of deduced alpha-tubulin amino acid sequences from maize and the dicot species Arabidopsis thaliana showed that alpha-tubulin isotypes encoded by genes in maize subfamilies I and II are more similar to specific Arabidopsis gene products (96 to 97% amino acid identity) than to isotypes encoded by genes in the other maize subfamilies. Phylogenetic analyses revealed that genes in these two subfamilies were derived from two ancient alpha-tubulin genes that predate the divergence of monocots and dicots. These same analyses revealed that the gene in maize subfamily III is more closely related to sequences from subfamily I genes than to those from subfamily II genes. However, the subfamily III gene has no close counterpart in Arabidopsis. We found evidence of a transposable element-like insertion in the subfamily III gene in some maize lines.

Tubulin gene expression in maize (Zea mays L.). Change in isotype expression along the developmental axis of seedling root.

Two-dimensional gel/western blot analysis was used to characterize alpha- and beta-tubulin isotype expression along the developmental axis of the maize (Zea mays) seedling primary root. We identified four distinct alpha-tubulin isotypes and a minimum of six beta-tubulin isotypes. This analysis showed differences between the alpha- and beta-tubulin isotypes expressed in rapidly dividing tissue at the root tip and differentiated root tissues proximal to the tip. The alpha 1 and alpha 4 isotypes predominated in samples from immature rapidly dividing tissues such as root tips, whereas in mature tissues such as differentiated root and pollen, alpha 2, alpha 3 and alpha 4 isotypes predominated. The beta 1 and beta 2 isotypes were more abundant in protein samples from root cortex than in samples from the root tip or vascular cylinder. In contrast, the beta 4 and beta 5 isotypes appeared to be more abundant in root tip and vascular cylinder samples than in root cortex samples. Hybridization probes from the 3' non-coding region of six alpha-tubulin cDNA clones were used to quantify the levels of corresponding tubulin transcripts in selected tissues, from embryonic to mature and from largely undifferentiated to highly differentiated. The results from these hybridization experiments showed that all of the alpha-tubulin genes were expressed in all tissues examined, although each gene showed a unique pattern of differential transcript accumulation. A transcript produced from cDNA clone representing the tua5 alpha-tubulin gene was translated in vitro and produced an alpha-tubulin that comigrated with the alpha 2 isotype.

The small genome of Arabidopsis contains at least nine expressed beta-tubulin genes.

The small genome of Arabidopsis contains at least nine expressed beta-tubulin (TUB) genes, in contrast to the large genomes of vertebrate animals, which contain a maximum of seven expressed beta-tubulin genes. In this study, we report the structures of seven new TUB genes (TUB2, TUB3, TUB5, TUB6, TUB7, TUB8, and TUB9) of Arabidopsis. The sequences of TUB1 and TUB4 had been reported previously. Sequence similarities and unique structural features suggest that the nine TUB genes evolved by way of three branches in the plant beta-tubulin gene evolutionary tree. Two genes (TUB2 and TUB3) encode the same beta-tubulin isoform; thus, the nine genes predict eight different beta-tubulins. In contrast to the alpha-tubulin (TUA) genes with their divergent intron patterns, all nine TUB genes contain 2 introns at conserved positions. Noncoding 3' gene-specific hybridization probes have been constructed for all nine TUB genes and used in RNA gel blot analyses to demonstrate that all nine genes are transcribed. Two-dimensional protein immunoblot analyses have resolved at least seven different beta-tubulin isoforms in Arabidopsis, indicating that most, if not all, of the TUB transcripts are translated.