CM1-driven assembly and activation of yeast γ-tubulin small complex underlies microtubule nucleation.

Microtubule (MT) nucleation is regulated by the γ-tubulin ring complex (γTuRC), conserved from yeast to humans. In Saccharomyces cerevisiae, γTuRC is composed of seven identical γ-tubulin small complex (γTuSC) sub-assemblies, which associate helically to template MT growth. γTuRC assembly provides a key point of regulation for the MT cytoskeleton. Here, we combine crosslinking mass spectrometry, X-ray crystallography, and cryo-EM structures of both monomeric and dimeric γTuSCs, and open and closed helical γTuRC assemblies in complex with Spc110p to elucidate the mechanisms of γTuRC assembly. γTuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent γTuSCs. By providing the highest resolution and most complete views of any γTuSC assembly, our structures allow phosphorylation sites to be mapped, surprisingly suggesting that they are mostly inhibitory. A comparison of our structures with the CM1 binding site in the human γTuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan γTuRC.

SPECIFIC MOLECULAR DETECTION OF PIROPLASMS AND CHARACTERIZATION OF ß-TUBULIN FOR A NOVEL BABESIA SPECIES IN SIKA DEER (CERVUS NIPPON YESOENSIS).

Piroplasms, which include Babesia spp. and Theileria spp., are protozoan parasites carried by ticks and commonly cause disease in animals and humans. Those caused by Babesia spp. manifest as fever, anemia, and hemoglobinuria, while Theileria spp. can lead to high fever, diarrhea, and lymphadenopathy. Recently, Theileria capreoli and an undescribed Babesia sp. were detected for the first time in sika deer (Cervus nippon yesoensis) from Hokkaido; however, there is limited information available on their epidemiology in Japan. Here, a touchdown polymerase chain reaction and reverse line blot hybridization were used to perform an epidemiological survey of T. capreoli and Babesia sp. using blood samples from 82 sika deer in Hokkaido, Japan. This was followed by partial sequencing and phylogenetic analysis of the 18S rRNA and ß-tubulin genes to characterize both piroplasm species. A total of 43 (52.4%) and 3 (3.7%) of the sika deer were positive for T. capreoli and Babesia sp., respectively. The ß-tubulin gene partial sequences for Babesia sp. were distinct from those of Babesia spp. in GenBank. Phylogenetic analysis showed that the unknown Babesia sp. is more closely related to B. bigemina and B. ovata than other Babesia spp. based on the ß-tubulin gene. Further studies are required to understand the ecology of these tick-borne pathogens in Japan.

Stage-specific functional relationships between Tub1 and Tub2 beta-tubulins in the wheat scab fungus Fusarium graminearum.

The filamentous ascomycete Fusarium graminearum contains two ß-tubulin genes TUB1 and TUB2 that differ in functions during vegetative growth and sexual reproduction. To further characterize their functional relationship, in this study we determined the co-localization of Tub1 and Tub2 and assayed their expression levels in different mutants and roles in DON production. Tub1 co-localized with Tub2 to the same regions of microtubules in conidia, hyphae, and ascospores. Whereas deletion of TUB1 had no obvious effect on the transcription of TUB2 and two α-tubulin genes (TUB4 and TUB5), the tub2 mutant was up-regulated in TUB1 transcription. To assay their protein expression levels, polyclonal antibodies that could specifically detect four α- and ß-tubulin proteins were generated. Western blot analyses showed that the abundance of Tub1 proteins was increased in tub2 but reduced in tub4 and tub5 mutants. Interestingly, protein expression of Tub4 and Tub5 was decreased in the tub1 mutant in comparison with the wild type, despite a lack of obvious changes in their transcription. In contrast, deletion of TUB2 had no effect on translation of TUB4 and TUB5. Ectopic expression of Tub2-mCherry partially recovered the growth defect of the tub1 mutant but did not rescue its defect in sexual reproduction. Expression of Tub1-GFP in the tub2 mutant also partially rescued its defects in vegetative growth, suggesting that disturbance in the balance of α- and ß-tubulins contributes to mutant defects. The tub2 but not tub1 mutant was almost blocked in DON biosynthesis. Expression of TRI genes, toxisome formation, and DON-related cellular differentiation were significantly reduced in the tub2 mutant. Overall, our results showed that Tub1 and Tub2 share similar subcellular localization and have overlapping functions during vegetative growth but they differ in functions in DON production and ascosporogenesis in F. graminearum.

Genome-wide identification, phylogenetic classification, and exon-intron structure characterization of the tubulin and actin genes in flax (Linum usitatissimum).

Flax (Linum usitatissimum L.) is a valuable food and fiber crop cultivated for its quality fiber and seed oil. α-, ß-, γ-tubulins and actins are the main structural proteins of the cytoskeleton. α- and γ-tubulin and actin genes have not been characterized yet in the flax genome. In this study, we have identified 6 α-tubulin genes, 13 ß-tubulin genes, 2 γ-tubulin genes, and 15 actin genes in the flax genome and analyzed the phylogenetic relationships between flax and Arabidopsis thaliana tubulin and actin genes. Six α-tubulin genes are represented by three paralogous pairs, among 13 ß-tubulin genes 7 different isotypes can be distinguished, 6 of which are encoded by two paralogous genes each. γ-tubulin is represented by a paralogous pair of genes one of which may be not functional. Fifteen actin genes represent seven paralogous pairs-seven actin isotypes and a sequentially duplicated copy of one of the genes of one of the isotypes. Exon-intron structure analysis has shown intron length polymorphism within the ß-tubulin genes and intron number variation among the α-tubulin gene: three or four introns are found in two or four genes, respectively. Intron positioning occurs at conservative sites, as observed in numerous other plant species. Flax actin genes show both intron length polymorphisms and variation in the number of intron that may be two or three. These data will be useful to support further studies on the specificity, functioning, regulation, and evolution of the flax cytoskeleton proteins.

Insights of the tubulin code in gametes and embryos: from basic research to potential clinical applications in humans†.

Microtubules are intracellular filaments that define in space and in time a large number of essential cellular functions such as cell division, morphology and motility, intracellular transport and flagella and cilia assembly. They are therefore essential for spermatozoon and oocyte maturation and function, and for embryo development. The dynamic and functional properties of the microtubules are in large part defined by various classes of interacting proteins including MAPs (microtubule associated proteins), microtubule-dependent motors, and severing and modifying enzymes. Multiple mechanisms regulate these interactions. One of them is defined by the high diversity of the microtubules themselves generated by the combination of different tubulin isotypes and by several tubulin post-translational modifications (PTMs). This generates a so-called tubulin code that finely regulates the specific set of proteins that associates with a given microtubule thereby defining the properties and functions of the network. Here we provide an in depth review of the current knowledge on the tubulin isotypes and PTMs in spermatozoa, oocytes, and preimplantation embryos in various model systems and in the human species. We focus on functional implications of the tubulin code for cytoskeletal function, particularly in the field of human reproduction and development, with special emphasis on gamete quality and infertility. Finally, we discuss some of the knowledge gaps and propose future research directions.

Paecilomyces Rot: A New Apple Disease.

Paecilomyces niveus is an important food spoilage fungus that survives thermal processing in fruit products, where it produces the mycotoxin patulin. Spoilage of products has been attributed to soil contamination; however, little is known about the ecology of this organism. In this study, orchard soils and culled apple fruit were surveyed and the ability of P. niveus to infect apple was tested on two popular apple varieties. P. niveus was found in 34% of sampled orchard soils from across New York. Completing Koch's postulates, P. niveus was demonstrated to cause postharvest disease in Gala and Golden Delicious apple. Symptoms of this disease, named Paecilomyces rot, resemble several other apple diseases, including black rot, bitter rot, and bull's-eye rot. External symptoms of Paecilomyces rot include brown, circular, concentrically ringed lesions, with an internal rot that is firm and cone-shaped. Both Gala and Golden Delicious apple fruit inoculated with P. niveus developed lesions ≥43 mm in size at 22 days after inoculation. There is some evidence that the size of lesions and rate of infection differ between Gala and Golden Delicious, which may indicate differing resistance to P. niveus. This work shows that P. niveus is common in New York orchard soil and can cause a novel postharvest fruit disease. Whether infected fruit can serve as an overlooked source of inoculum in heat-processed apple products requires further study.

The Tubulin Superfamily in Archaea.

In comparison with bacteria and eukaryotes, the large and diverse group of microorganisms known as archaea possess a great diversity of cytoskeletal proteins, including members of the tubulin superfamily. Many species contain FtsZ, CetZ and even possible tubulins; however, some major taxonomic groups do not contain any member of the tubulin superfamily. Studies using the model archaeon, Halferax volcanii have recently been instrumental in defining the fundamental roles of FtsZ and CetZ in archaeal cell division and cell shape regulation. Structural studies of archaeal tubulin superfamily proteins provide a definitive contribution to the cytoskeletal field, showing which protein-types must have developed prior to the divergence of archaea and eukaryotes. Several regions of the globular core domain - the "signature" motifs - combine in the 3D structure of the common molecular fold to form the GTP-binding site. They are the most conserved sequence elements and provide the primary basis for identification of new superfamily members through homology searches. The currently well-characterised proteins also all share a common mechanism of GTP-dependent polymerisation, in which GTP molecules are sandwiched between successive subunits that are arranged in a head-to-tail manner. However, some poorly-characterised archaeal protein families retain only some of the signature motifs and are unlikely to be capable of dynamic polymerisation, since the promotion of depolymerisation by hydrolysis to GDP depends on contributions from both subunits that sandwich the nucleotide in the polymer.

Gene duplication, tissue-specific gene expression and sexual conflict in stalk-eyed flies (Diopsidae).

Gene duplication provides an essential source of novel genetic material to facilitate rapid morphological evolution. Traits involved in reproduction and sexual dimorphism represent some of the fastest evolving traits in nature, and gene duplication is intricately involved in the origin and evolution of these traits. Here, we review genomic research on stalk-eyed flies (Diopsidae) that has been used to examine the extent of gene duplication and its role in the genetic architecture of sexual dimorphism. Stalk-eyed flies are remarkable because of the elongation of the head into long stalks, with the eyes and antenna laterally displaced at the ends of these stalks. Many species are strongly sexually dimorphic for eyespan, and these flies have become a model system for studying sexual selection. Using both expressed sequence tag and next-generation sequencing, we have established an extensive database of gene expression in the developing eye-antennal imaginal disc, the adult head and testes. Duplicated genes exhibit narrower expression patterns than non-duplicated genes, and the testes, in particular, provide an abundant source of gene duplication. Within somatic tissue, duplicated genes are more likely to be differentially expressed between the sexes, suggesting gene duplication may provide a mechanism for resolving sexual conflict.

Archaeal origin of tubulin.

Tubulins are a family of GTPases that are key components of the cytoskeleton in all eukaryotes and are distantly related to the FtsZ GTPase that is involved in cell division in most bacteria and many archaea. Among prokaryotes, bona fide tubulins have been identified only in bacteria of the genus Prosthecobacter. These bacterial tubulin genes appear to have been horizontally transferred from eukaryotes. Here we describe tubulins encoded in the genomes of thaumarchaeota of the genus Nitrosoarchaeum that we denote artubulins Phylogenetic analysis results are compatible with the origin of eukaryotic tubulins from artubulins. These findings expand the emerging picture of the origin of key components of eukaryotic functional systems from ancestral forms that are scattered among the extant archaea.

Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the ß-tubulin isotype 1 encoding gene in Haemonchus contortus.

Resistance to benzimidazole anthelmintics in the nematode Haemonchus contortus has been correlated with single nucleotide polymorphisms (SNPs) on the ß-tubulin isotype 1 gene. Three mutations can be used as markers for the detection of resistance, namely SNPs at position 200 and 167 (both TTC to TAC) or at position 198 (GAA to GCA). Harbouring a resistance genotype at any one of these codons can lead to a resistant phenotype. Our objective in this study was to analyse the frequencies of the three mutations when the albendazole dose rate and selection pressure were increased. We used adult H. contortus (males and females) collected directly from the abomasum of untreated lambs, or lambs treated with the manufacturer's recommended dose rate (5mg/kg), three times the recommended dose rate (15 mg/kg), or nine times the recommended dose rate (45 mg/kg). Anthelmintic efficacy was determined by worm and egg count reductions. For the surviving worms of the four treatment groups, the frequencies of each resistance SNP at codons 167, 200 and 198 were measured using pyrosequencing. Our results showed a strong relationship between an increasing dose rate and an increase in the frequency of the (TAC)(200) SNP and a decrease in the (TAC)(167) SNP. All worms genotyped were GAA at codon 198.

Expression profiling of tubulin isotypes and microtubule-interacting proteins using real-time polymerase chain reaction.

Real-time polymerase chain reaction (PCR) has been used for quantification of intracellular mRNA levels in cell culture and tissue samples. It is an important tool for studying antimitotic drug effects on tubulin isotype and microtubule-interacting protein levels and for measuring differences in normal and tumor tissue samples that could have predictive or prognostic applications. Both quantitative and comparative methods are valuable approaches; however, the selection of either approach requires an understanding of their benefits and challenges. In this chapter, we provide detailed protocols for real-time PCR experiments, discuss issues to consider in selecting real-time PCR methodologies, and give examples utilizing either quantitative or comparative approaches.

Molecular basis for class V beta-tubulin effects on microtubule assembly and paclitaxel resistance.

Vertebrates produce at least seven distinct beta-tubulin isotypes that coassemble into all cellular microtubules. The functional differences among these tubulin isoforms are largely unknown, but recent studies indicate that tubulin composition can affect microtubule properties and cellular microtubule-dependent behavior. One of the isotypes whose incorporation causes the largest change in microtubule assembly is beta5-tubulin. Overexpression of this isotype can almost completely destroy the microtubule network, yet it appears to be required in smaller amounts for normal mitotic progression. Moderate levels of overexpression can also confer paclitaxel resistance. Experiments using chimeric constructs and site-directed mutagenesis now indicate that the hypervariable C-terminal region of beta5 plays no role in these phenotypes. Instead, we demonstrate that two residues found in beta5 (Ser-239 and Ser-365) are each sufficient to inhibit microtubule assembly and confer paclitaxel resistance when introduced into beta1-tubulin; yet the single mutation of residue Ser-239 in beta5 eliminates its ability to confer these phenotypes. Despite the high degree of conservation among beta-tubulin isotypes, mutations affecting residue 365 demonstrate that amino acid substitutions can be context sensitive; i.e. an amino acid change in one isotype will not necessarily produce the same phenotype when introduced into a different isotype. Modeling studies indicate that residue Cys-239 of beta1-tubulin is close to a highly conserved Cys-354 residue suggesting the possibility that disulfide formation could play a significant role in the stability of microtubules formed with beta1- but not with beta5-tubulin.

Ixabepilone: targeting betaIII-tubulin expression in taxane-resistant malignancies.

Microtubule-targeting agents, such as taxanes and epothilones, block mitosis and cell proliferation by targeting the dynamics of the cytoskeleton. The taxanes are widely used for treatment of various malignancies, but primary and acquired resistance to chemotherapy remains a significant clinical concern. Class I, II, III, IV, and V beta-tubulin isotypes are expressed in human tumors. Overexpression of the betaIII-tubulin isotype is one mechanism that can render tumor cells resistant to taxanes. The relative expression of betaIII-tubulin correlates with clinical outcomes in several tumor types, including breast cancer, non-small cell lung cancer, and ovarian cancer. A novel analogue of epothilone B, ixabepilone, has recently been approved in combination with capecitabine for the treatment of patients with anthracycline- and taxane-resistant locally advanced or metastatic breast cancer and as monotherapy in patients whose tumors are resistant or refractory to an anthracycline, a taxane, and capecitabine. The significant antitumor activity of ixabepilone in taxane-resistant tumors may be related to its preferential suppression of the dynamic instability of alpha/betaIII-microtubules in cells expressing high levels of betaIII-tubulin.

Identification of tubulin drug binding sites and prediction of relative differences in binding affinities to tubulin isotypes using digital signal processing.

Microtubules are involved in numerous cellular processes including chromosome segregation during mitosis and, as a result, their constituent protein, tubulin, has become a successful target of several chemotherapeutic drugs. In general, these drugs bind indiscriminately to tubulin within both cancerous and healthy cells, resulting in unwanted side effects. However, differences between beta-tubulin isotypes expressed in a wide range of cell types may aid in the development of anti-tubulin drugs having increased specificity for only certain types of cells. Here, we describe a digital signal processing (DSP) method that is capable of predicting hot spots for the tubulin family of proteins as well as determining relative differences in binding affinities to these hot spots based only on the primary sequence of 10 human tubulin isotypes. Due to the fact that several drug binding sites have already been characterized within beta-tubulin, we are able to correlate hot spots with the binding sites for known chemotherapy drugs. We have also verified the accuracy of this method using the correlation between the binding affinities of characterized drugs and the tubulin isotypes. Additionally, the DSP method enables the rapid estimation of relative differences in binding affinities within the binding sites of tubulin isotypes that are yet to be experimentally determined.

De novo production of K-alpha1 tubulin-specific antibodies: role in chronic lung allograft rejection.

Lung transplantation is the treatment option for a variety of end-stage pulmonary diseases. Posttransplant development of Abs against donor HLA and non-HLA Ags have been associated with acute and chronic rejection of transplanted organs. Development of bronchiolitis obliterans syndrome (BOS) following lung transplantation has been correlated with de novo production of anti-donor-HLA Abs. However, only a portion of the patients with BOS demonstrate detectable anti-donor-HLA Abs. Airway epithelium is considered as a major target for lung allograft rejection. In this study we demonstrate that many BOS(+) patients (12 of 36) develop Abs reactive to epithelial cell Ag that are distinct from HLA. Furthermore, de novo production of antiepithelial cell Ab precedes clinical onset of BOS. N-terminal sequencing and blastx analysis as well as blocking with K-alpha1 tubulin-specific Ab identified the epithelial Ag as K-alpha1 tubulin. Binding of the de novo-produced anti-K-alpha1 tubulin Abs to the airway epithelial cells resulted in the increased expression of transcription factors (TCF5 and c-Myc), leading to increased expression of fibrogenic growth factors, activation of cell cycle signaling, and fibroproliferation, the central events in immunopathogenesis of BOS following human lung transplantation.

Is class III beta-tubulin a predictive factor in patients receiving tubulin-binding agents?

On the basis of preclinical studies that show overexpression of class III beta-tubulin is associated with resistance to tubulin-binding agents, several investigators have addressed the relation between class III beta-tubulin and outcome in patients treated with such agents. High expression of class III beta-tubulin has been found to be correlated either with low response rates in patients treated with regimens containing taxanes or vinorelbine or with reduced survival in patients with non-small-cell lung cancer, in breast, ovarian, and gastric cancers, and in cancers of unknown primary site. Two studies have shown patients with advanced non-small-cell lung cancer receiving paclitaxel whose tumours expressed high levels of class III beta-tubulin had a lower response to paclitaxel and shorter survival, whereas this variable was not found to be predictive in patients receiving regimens without tubulin-binding agents. Conversely, analysis of samples from patients in the JBR-10 trial, which compared adjuvant chemotherapy to no further therapy in operable non-small-cell lung cancer, showed that chemotherapy seemed to overcome the negative prognostic effect of high levels of expression of class III beta-tubulin and the greatest benefit from cisplatin/vinorelbine was seen in patients with high levels of expression of class III beta-tubulin. Further analyses in operable and advanced non-small-cell lung cancer showed a relation between high expression of class III beta-tubulin and baseline factors such as age under 60 years, adenocarcinoma and large-cell carcinoma histologies, and advanced stage of disease. These results suggest that class III beta-tubulin could be both a prognostic and a predictive factor. Large randomised studies are warranted to determine the prognostic or predictive value of class III beta-tubulin in different settings and tumours.

The Princeton Protein Orthology Database (P-POD): a comparative genomics analysis tool for biologists.

Many biological databases that provide comparative genomics information and tools are now available on the internet. While certainly quite useful, to our knowledge none of the existing databases combine results from multiple comparative genomics methods with manually curated information from the literature. Here we describe the Princeton Protein Orthology Database (P-POD, http://ortholog.princeton.edu), a user-friendly database system that allows users to find and visualize the phylogenetic relationships among predicted orthologs (based on the OrthoMCL method) to a query gene from any of eight eukaryotic organisms, and to see the orthologs in a wider evolutionary context (based on the Jaccard clustering method). In addition to the phylogenetic information, the database contains experimental results manually collected from the literature that can be compared to the computational analyses, as well as links to relevant human disease and gene information via the OMIM, model organism, and sequence databases. Our aim is for the P-POD resource to be extremely useful to typical experimental biologists wanting to learn more about the evolutionary context of their favorite genes. P-POD is based on the commonly used Generic Model Organism Database (GMOD) schema and can be downloaded in its entirety for installation on one's own system. Thus, bioinformaticians and software developers may also find P-POD useful because they can use the P-POD database infrastructure when developing their own comparative genomics resources and database tools.

Heightened sensitivity to paclitaxel in Class IVa beta-tubulin-transfected cells is lost as expression increases.

Stably transfected Chinese hamster ovary cell lines expressing increasing levels of beta4a, a class IV neuronal-specific beta-tubulin, were compared for effects on microtubule organization, assembly, and sensitivity to antimitotic drugs. It was found that beta4a reduced microtubule assembly in proportion to its abundance and thereby caused supersensitivity to microtubule disruptive drugs such as colcemid, vinblastine, and nocodazole. However, the response to paclitaxel was more complex. Low expression of beta4a caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that beta4a may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of beta4a expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by paclitaxel binding, and drug supersensitivity is lost. beta4a-Tubulin differs from the more ubiquitous beta4b isotype at relatively few amino acid residues, yet beta4b expression has little effect on microtubule assembly or drug response. To determine which amino acids mediate the effects of beta4a expression, beta4a and beta4b were altered by site-directed mutagenesis and expressed in Chinese hamster ovary cells. The introduction of N332S or N335S mutations into beta4b-tubulin was sufficient to confer microtubule disruption and increased colcemid sensitivity. On the other hand, mutation of Ala(115) to serine in beta4a-tubulin almost completely reversed heightened sensitivity to paclitaxel, but introduction of an S115A mutation into beta4b had no effect, suggesting that a complex interaction of multiple amino acids are necessary to produce this phenotype.

Bcl-2 down-regulation and tubulin subtype composition are involved in resistance of ovarian cancer cells to vinflunine.

Vinflunine, a new microtubule-targeting drug, has a marked antitumor activity in vitro and in vivo. Here, we studied the mechanisms mediating resistance to vinflunine. We investigated the response to vinflunine of ovarian cancer cells initially selected as paclitaxel-resistant cells (A2780-TC1 cells). By comparison with A2780-wild-type (wt) cells, we showed that A2780-TC1 cells were highly resistant to vinflunine, with resistance factors reaching 800 and 1,830 for IC(50) and IC(70), respectively. We showed that P-glycoprotein minimally participated in this cell resistance. The examination of tubulin composition revealed increased levels of acetylated alpha-tubulin, betaII-tubulin, and betaIII-tubulin in A2780-TC1 cells before vinflunine treatment. As a consequence, vinflunine unequally affected microtubule network organization and function in A2780-wt and A2780-TC1 cells. Whereas the drug depolymerized microtubules and induced a mitotic block in A2780-wt cells, it did not depolymerize microtubules and induced a G(2) block in A2780-TC1 cells. Elsewhere, the mitochondrial protein Bcl-2 was down-regulated in A2780-TC1 cells. This down-regulation was related to resistance, as A2780-TC1 cells stably transfected with a Bcl-2 construct recovered a partial sensitivity to vinflunine. Lastly, we confirmed the role played by Bcl-2 by showing that the mitochondrial membrane potential was only disrupted by vinflunine in cells expressing Bcl-2. Altogether, our results indicate that modifications acquired during treatment (i.e., paclitaxel) have significant consequences on cell response to the following drug (i.e., vinflunine). Especially, this study shows that a specific pool of tubulin subtypes and a down-regulation of Bcl-2 are associated with resistance of ovarian cancer cells to vinflunine.

Telonemia, a new protist phylum with affinity to chromist lineages.

Recent molecular investigations of marine samples taken from different environments, including tropical, temperate and polar areas, as well as deep thermal vents, have revealed an unexpectedly high diversity of protists, some of them forming deep-branching clades within important lineages, such as the alveolates and heterokonts. Using the same approach on coastal samples, we have identified a novel group of protist small subunit (SSU) rDNA sequences that do not correspond to any phylogenetic group previously identified. Comparison with other sequences obtained from cultures of heterotrophic protists showed that the environmental sequences grouped together with Telonema, a genus known since 1913 but of uncertain taxonomic affinity. Phylogenetic analyses using four genes (SSU, Hsp90, alpha-tubulin and beta-tubulin), and accounting for gamma- and covarion-distributed substitution rates, revealed Telonema as a distinct group of species branching off close to chromist lineages. Consistent with these gene trees, Telonema possesses ultrastructures revealing both the distinctness of the group and the evolutionary affinity to chromist groups. Altogether, the data suggest that Telonema constitutes a new eukaryotic phylum, here defined as Telonemia, possibly representing a key clade for the understanding of the early evolution of bikont protist groups, such as the proposed chromalveolate supergroup.