Multiple forms of tubulin in Polytomella and Chlamydomonas: evidence for a precursor of flagellar alpha-tubulin.

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.

Transcription of alpha- and beta-tubulin genes in vitro in isolated Chlamydomonas reinhardi nuclei.

Removal of the flagella of Chlamydomonas results in increases in both flagellar protein synthesis and tubulin messenger RNA accumulation. These observations led us to examine whether flagellar protein gene sequences are transcribed differentially in nuclei isolated before and after deflagellation. A nuclear isolation protocol was developed using the cell wall-less strain of Chlamydomonas, CW 15, after cell lysis with 0.5% Nonidet P-40. Transcriptional activity of isolated nuclei was determined by incorporating [32P]UTP into TCA-precipitable and phenol-extractable RNA, and by hybridizing newly transcribed RNA to complementary DNA clones containing alpha- and beta-tubulin sequences. Nuclei from deflagellated cells are more active in transcribing sequences that hybridize with alpha- and beta-tubulin complementary DNA probes than are nuclei from nondeflagellated cells. In addition, while total [32P]UTP incorporation is inhibited 45% by alpha-amanitin concentrations of 1.0 micrograms/ml, tubulin RNA synthesis in this system is completely inhibited by this concentration of alpha-amanitin. This demonstration of differential transcription in nuclei before and after cell deflagellation provides the means to study in vitro the mechanisms that signal and regulate flagellar protein gene activity.

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.

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 two beta-tubulin genes of Chlamydomonas reinhardtii code for identical proteins.

The two beta-tubulin genes of the unicellular green alga Chlamydomonas reinhardtii are expressed coordinately after deflagellation and produce two transcripts of 2.1 and 2.0 kilobases. Full-length cDNA clones corresponding to the transcript of each gene were isolated. DNA sequences were obtained from the cDNA clones and from cloned tubulin gene fragments. Both genes contained 1,332 base pairs of coding sequence, with only 19 nucleotide differences between the genes. Because all the differences occurred at the third base position of a codon and did not change the predicted amino acid sequence, we concluded that both beta-tubulin genes code for the same protein of 443 amino acids. The predicted amino acid sequence is 89 and 72% homologous with beta-tubulins from chicken and yeast cells, respectively. Each gene had three intervening sequences, which occurred at identical positions. Although the first two intervening sequences were not conserved between the two genes, the nucleotide sequence of the third intervening sequence was 89% conserved between the genes. The codon usage in the tubulin genes of C. reinhardtii was very biased: only 37 different codons were used. Striking differences occurred between the codons used in these nuclear genes and C. reinhardtii chloroplast genes.

mRNA abundance changes during flagellar regeneration in Chlamydomonas reinhardtii.

Flagellar amputation in Chlamydomonas reinhardtii induces the accumulation of a specific set of RNAs, many of which encode flagellar proteins. We prepared a cDNA clone bank from RNA isolated from cells undergoing flagellar regeneration. From this bank, we selected clones that contain RNA sequences that display several different patterns of abundance regulation. Based on quantitation of the relative amounts of labeled, cloned cDNAs hybridizing to dots of RNA on nitrocellulose filters, the cloned sequences were divided into five regulatory classes: class I RNAs remain at constant abundance during flagellar regeneration; classes II, III, and IV begin to increase in abundance within a few minutes after deflagellation, reach maximal abundance at successively later times during regeneration, and return to control cell levels within 2 to 3 h; and class V RNA abundance decreases during flagellar regeneration. Alpha- and beta-tubulin mRNAs are included in regulatory class IV. The abundance kinetics of alpha-tubulin mRNAs differ slightly from those of beta-tubulin mRNAs. The availability of these clones makes possible studies on the mechanisms controlling the abundance of a wide variety of different RNA species during flagellar regeneration in Chlamydomonas.

The two alpha-tubulin genes of Chlamydomonas reinhardi code for slightly different proteins.

Full-length cDNA clones corresponding to the transcripts of the two alpha-tubulin genes in Chlamydomonas reinhardi were isolated. DNA sequence analysis of the cDNA clones and cloned gene fragments showed that each gene contains 1,356 base pairs of coding sequence, predicting alpha-tubulin products of 451 amino acids. Of the 27 nucleotide differences between the two genes, only two result in predicted amino acid differences between the two gene products. In the more divergent alpha 2 gene, a leucine replaces an arginine at amino acid 308, and a valine replaces a glycine at amino acid 366. The results predicted that two alpha-tubulin proteins with different net charges are produced as primary gene products. The predicted amino acid sequences are 86 and 70% homologous with alpha-tubulins from rat brain and Schizosaccharomyces pombe, respectively. Each gene had two intervening sequences, located at identical positions. Portions of an intervening sequence highly conserved between the two beta-tubulin genes are also found in the second intervening sequence of each of the alpha genes. These results, together with our earlier report of the beta-tubulin sequences in C. reinhardi, present a picture of the total complement of genetic information for tubulin in this organism.

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.

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.

Mutants resistant to anti-microtubule herbicides map to a locus on the uni linkage group in Chlamydomonas reinhardtii.

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.

Mapping flagellar genes in Chlamydomonas using restriction fragment length polymorphisms.

To correlate cloned nuclear DNA sequences with previously characterized mutations in Chlamydomonas and, to gain insight into the organization of its nuclear genome, we have begun to map molecular markers using restriction fragment length polymorphisms (RFLPs). A Chlamydomonas reinhardtii strain (CC-29) containing phenotypic markers on nine of the 19 linkage groups was crossed to the interfertile species Chlamydomonas smithii. DNA from each member of 22 randomly selected tetrads was analyzed for the segregation of RFLPs associated with cloned genes detected by hybridization with radioactive DNA probes. The current set of markers allows the detection of linkage to new molecular markers over approximately 54% of the existing genetic map. This study focused on mapping cloned flagellar genes and genes whose transcripts accumulate after deflagellation. Twelve different molecular clones have been assigned to seven linkage groups. The alpha-1 tubulin gene maps to linkage group III and is linked to the genomic sequence homologous to pcf6-100, a cDNA clone whose corresponding transcript accumulates after deflagellation. The alpha-2 tubulin gene maps to linkage group IV. The two beta-tubulin genes are linked, with the beta-1 gene being approximately 12 cM more distal from the centromere than the beta-2 gene. A clone corresponding to a 73-kD dynein protein maps to the opposite arm of the same linkage group. The gene corresponding to the cDNA clone pcf6-187, whose mRNA accumulates after deflagellation, maps very close to the tightly linked pf-26 and pf-1 mutations on linkage group V.

Extragenic suppression and synthetic lethality among Chlamydomonas reinhardtii mutants resistant to anti-microtubule drugs.

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.

The beta-tubulin gene family of Arabidopsis thaliana: preferential accumulation of the beta 1 transcript in roots.

The genome of Arabidopsis thaliana (L.) Heynh. was shown to contain a beta-tubulin gene family consisting of at least seven distinct genes and/or pseudogenes. Genomic clones of five different beta-tubulin genes and/or pseudogenes have been isolated and partially characterized. The complete nucleotide sequence of one A. thaliana beta-tubulin gene, designated beta 1, has been determined. A comparison of the predicted amino acid sequence of the A. thaliana beta 1-tubulin with the predicted sequences of beta-tubulins of animals and protists indicated that this plant beta-tubulin shows a high degree of homology with other beta-tubulins. However, the beta 1-tubulin contains a novel single amino acid insertion at position 41. The A. thaliana beta 1-tubulin gene is transcribed, as shown by RNA blot hybridization and S1 nuclease analyses. A 3'-noncoding gene-specific probe was used to examine the expression of the beta 1-tubulin gene in leaves, roots, and flowers by blot hybridization analyses of total RNA isolated from these tissues. The results showed that the transcript of the beta 1 gene accumulates predominantly in roots, with low levels of transcript in flowers, and barely detectable levels of transcript in leaves. A second genomic clone was shown to contain two essentially identical beta-tubulin coding sequences in direct tandem orientation and separated by 1 kb.

Structure of the three beta-tubulin-encoding genes of the unicellular alga, Polytomella agilis.

The quadriflagellate, unicellular, colorless alga, Polytomella agilis, contains several distinct microtubule arrays. To study the genetic basis of microtubule heterogeneity in P. agilis, we characterized its tubulin(Tub)-encoding genes (tub). The three beta tub genes detected in blots of P. agilis DNA were isolated from a genomic library. The structure and organization of the genes were examined by restriction mapping and nucleotide (nt) sequencing. S1 nuclease protection studies showed that all three genes are expressed. The predicted amino acid (aa) sequences are more than 98% conserved with the Chlamydomonas reinhardtii and Volvox carteri beta-Tubs, underscoring the close phylogenetic relationship of these species. Evolutionary divergence among the P. agilis genes is demonstrated by differences in intron number, nt sequences in noncoding regions, and silent nt substitutions in the coding regions. However, the proteins encoded by the beta 1 and beta 3 tub genes are identical; the beta 2 gene product differs by one conservative aa substitution. These results are in striking contrast to the C-terminal aa diversity reported within beta tub gene families in animal, higher plant and fungal systems. The data support the hypothesis that those tub genes whose products assemble into axonemal microtubules are subject

Chlamydomonas reinhardtii tubulin gene structure.

DNA sequencing studies have provided a picture of the total information available at the gene level for tubulin production in C. reinhardtii. The data indicates that diversity at the gene level is very limited and that all the microtubules in the cell are composed of a very similar set of tubulins. These studies contrast with similar studies of S. pombe alpha-tubulin genes and chicken beta-tubulin genes that show much heterogeneity among members of the same gene family. Further studies will be needed to investigate whether the high degree of conservation of tubulin genes is unique or common among lower eukaryotes, and what mechanisms are used to maintain homogeneity in C. reinhardtii tubulin gene families. Our DNA sequence analysis, in addition to the work of Brunke et al., has provided information on the noncoding, and possibly regulatory, portions of the tubulin genes. For example, the promoter regions of the 4 tubulin genes share a consensus sequence of 16 nucleotides upstream of the TATA box. This sequence could be involved in regulating the coordinate expression of the genes. Although little homology exists generally in the noncoding region of the genes, striking homology between the third IVS in each beta-tubulin gene is observed. Small elements homologous to the beta-tubulin IVS 3 also exist in the second IVS of each alpha-tubulin gene. In addition, considerable homology in the 5' noncoding portion of the alpha-tubulin transcripts has been noted. These homologies may be the result of recent gene conversion events, and may not have functional significance. The possibility, however, must also be considered in future experiments that these elements may play a role in regulating the expression of the tubulin genes.

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.

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.

Stability of polysome-associated polyadenylated RNA from soybean suspension culture cells.

The half-life of polysome-associated, poly(A)-RNA in exponentially growing soybean (Glycine max) suspension culture cells was determined with pulse-chase experiments. Based on a best fit from a computer analysis of the data, two decay components for poly(A)-RNA were found. One component had a half-life of approximately 0.6 h, while the other had a half-life of about 30 h, similar to the doubling time of the cultures. At the beginning of the chase period, the short-lived component represented approximately 90% of the total poly(A)-RNA in the polysomes. This percentage decreased with time so that, under steady-state conditions, the long-lived component probably represented the majority of poly(A)-RNA.