Expression Of Beta-tubulin Isotypes Research Articles

Abstract A079: SB-216 inhibits oncogenic beta-tubulin subtypes in PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies, with only 12% of patients living longer than five years after diagnosis. Recent studies have shown that inhibiting microtubule dynamics may be a promising therapeutic avenue for treating patients with this cancer. Microtubules are critical in cancer cell growth and division, as well as intracellular cytoskeletal support and internal trafficking. In PDAC, βIII- and βIVb-tubulin are highly upregulated in contrast to normal pancreas cells. Decreased expression of βIII- and βIVb-tubulin, through silencing or inhibition, correlates with reduced PDAC cell growth, tumorigenic potential, metastases in xenograft models, and chemoresistance. We hypothesized that a novel colchicine binding site beta-tubulin inhibitor, SB-216, will decrease cell growth while also decreasing the expression of oncogenic βIII- and βIVb-tubulin. To examine the anti-proliferative effects, PDCL110, a PDAC patient-derived cell line, cells were seeded (6,000 cells/well), and after 24 hours, treated with 0.1% DMSO (control), 2 nM, 5 nM, 10 nM, or 15 nM SB-216. Plates were analyzed on the IncuCyte Live-Cell Analysis System for six days, where phase contrast images of confluency were captured every four hours. To characterize the mRNA expression of βIII- and βIVb-tubulin, Panc-1 and PDCL110 cells were treated with 0.1% DMSO and 5 nM SB-216 for 48 hours. Next, total RNA was extracted using a TRIzol reagent. cDNAs were prepared using the SYBR Green RNA Reverse Transcription kit, and mRNA expression of beta-tubulin isotypes was quantified using the QuantStudio Real-Time PCR system. The expression of tubulin isotypes was normalized with the internal control ACTIN. Cell growth curves illustrated a significant (p<0.006) decrease in proliferation after 48 hours of treatment at all tested concentrations of SB-216. After 72 hours, cell growth inhibition became more significant (p<0.0001) in a dose-dependent manner. SB-216 treatment of a commercially available PDAC cell line (Panc-1) led to decreased mRNA expression of TUBB3 (class βIII, p<0.05) and TUBB4B (class βIVb, p<0.05) compared to DMSO control. Similarly, TUBB4B expression was significantly (p<0.05) decreased in the PDCL110 cells upon SB-216 treatment. These results enhance our confidence in utilizing SB-216 as a therapeutic agent against PDAC. Future studies are directed at elucidating the effects of SB-216 on metastases, chemoresistance, and mitochondrial function. Through an improved understanding of the mechanisms behind microtubule inhibition, we can meet the urgent clinical need of finding targeted treatments for this complex cancer. Citation Format: Lauren C Gattie, Chun Cai, Rui Wang, Wei Li, Evan Glazer. SB-216 inhibits oncogenic beta-tubulin subtypes in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A079.

Cancer Cells Acquire Mitotic Drug Resistance Properties Through Beta I-Tubulin Mutations and Alterations in the Expression of Beta-Tubulin Isotypes

BackgroundAnti-mitotic compounds (microtubule de-stabilizers) such as vincristine and vinblastine have been shown clinically successful in treating various cancers. However, development of drug-resistance cells limits their efficacies in clinical situations. Therefore, experiments were performed to determine possible drug resistance mechanisms related to the application of anti-mitotic cancer therapy.Principal FindingsA KB-derived microtubule de-stabilizer-resistant KB-L30 cancer cell line was generated for this study. KB-L30 cells showed cross-resistance to various microtubule de-stabilizers including BPR0L075, vincristine and colchicine through multiple-drug resistant (MDR)-independent mechanisms. Surprisingly, KB-L30 cells showed hyper-sensitivity to the microtubule-stabilizer, paclitaxel. Results of the RT-PCR analysis revealed that expression of both class II and III β-tubulin was down-regulated in KB-L30 cells as compared to its parental KB cancer cells. In addition, DNA sequencing analysis revealed six novel mutation sites present in exon four of the βI-tubulin gene. Computational modeling indicated that a direct relationship exists between βI-tubulin mutations and alteration in the microtubule assembly and dynamic instability in KB-L30 cells and this predicted model was supported by an increased microtubule assembly and reduced microtubule dynamic instability in KB-L30 cells, as shown by Western blot analysis.Conclusions and SignificanceOur study demonstrated that these novel mutations in exon four of the βI-tubulin induced resistance to microtubule de-stabilizers and hyper-sensitivity to microtubule stabilizer through an alteration in the microtubule assembly and dynamics in cancer cells. Importantly, the current study reveals that cancer cells may acquire drug resistance ability to anti-mitotic compounds through multiple changes in the microtubule networks. This study further provided molecular information in drug selection for patients with specific tubulin mutations.

Impact, mechanisms, and novel chemotherapy strategies for overcoming resistance to anthracyclines and taxanes in metastatic breast cancer

Despite advances in treatment of patients with metastatic breast cancer (MBC), prognosis remains poor and median survival is 2-3 years. Resistance to antineoplastics mediated by many factors, potentially including overexpression of drug efflux proteins or altered beta-tubulin isotype expression limits the effectiveness of MBC chemotherapy. Capecitabine, approved for anthracycline- and taxane-resistant MBC, has produced modest responses, highlighting the need for more effective treatments for MBC resistant to multiple chemotherapeutic agents. Agents with potential to reverse drug resistance have not proven effective. Albumin-bound paclitaxel is a formulation that may enhance delivery to tumor tissues. Conversely, ixabepilone, an epothilone analog, has been reported to have lower susceptibility to at least some mechanisms of tumor resistance and clinical activity in resistant/refractory MBC. The topoisomerase-I inhibitor irinotecan also has low cross-resistance with other antineoplastics, and has shown some activity against refractory MBC. Development of new agents and identification of genetic biomarkers in translational studies promise to improve management of patients with resistant/refractory breast cancer.

Preclinical efficacy spectrum and pharmacokinetics of ixabepilone

Ixabepilone, a semisynthetic analog of natural epothilone B, was developed for use in cancer treatment. This study extends previous findings regarding the efficacy of ixabepilone and its low susceptibility to tumor resistance mechanisms and describes the pharmacokinetics of this new antineoplastic agent. The cytotoxicity of ixabepilone was assessed in vitro in breast, lung, and colon tumor cell lines and in vivo in human xenografts in mice. Antitumor activities of ixabepilone and taxanes were compared in multidrug-resistant models in vivo. Differential drug uptake of ixabepilone and paclitaxel was assessed in a P-glycoprotein (P-gp)-resistant colon cancer model in vitro. The pharmacokinetic profile of ixabepilone was established in mice and humans. Ixabepilone demonstrated potent cytotoxicity in a broad range of human cancer cell lines in vitro and in a wide range of xenografts in vivo. Ixabepilone was *3-fold more potent than docetaxel in the paclitaxel-resistant Pat-21 xenograft model (resistant due to overexpression of betaIII-tubulin and a lack of betaII-tubulin). Ixabepilone activity against P-gp-overexpressing breast and colon cancer was confirmed in in vivo models. Cellular uptake of ixabepilone, but not paclitaxel, was established in a P-gp-overexpressing model. The pharmacokinetics of ixabepilone was characterized by rapid tissue distribution and extensive tissue binding. Cytotoxicity studies against a range of tumor types in vitro and in vivo demonstrate that ixabepilone has potent and broad-spectrum antineoplastic activity. This is accompanied by favorable pharmacokinetics. Ixabepilone has reduced susceptibility to resistance due to P-gp overexpression, tubulin mutations, and alterations in beta-tubulin isotype expression.

Expression of beta-tubulin isotypes in human primary ovarian carcinoma

Objective. Selective expression of beta-tubulin isotypes has been reported to be one of the important mechanisms of taxane resistance. The purpose of this study was to evaluate the immunohistochemical expression of beta-tubulin isotypes using clinical samples of ovarian carcinoma treated by taxanes and to examine whether the protein levels of each of the beta-tubulin isotypes were correlated with the clinical features. Experimental design. We examined tumor samples taken from 77 ovarian carcinoma patients (54 patients treated with a taxane-based regimen and 23 treated with a taxane-free regimen), for the intrinsic protein level of beta-tubulin isotype (classes I, II, III and IV) expression using immunohistochemistry, and we evaluated the correlation of this protein level with the clinical features. The expression levels were scored by the proportion and intensity of the immunoreactive tumor cells. Results. High protein levels of classes I and IV beta-tubulin, and very low protein levels of class II beta-tubulin, and intermediate protein levels of class III beta-tubulin expression were demonstrated in a total of 77 ovarian carcinomas. As for the samples taken from the 54 patients treated with the taxane-based regimen, 40 samples demonstrated undetectable levels of class II beta-tubulin protein. The class II beta-tubulin expression-absent group was significantly correlated with advanced stage ( p = 0.024) and with a short period of progression-free survival (log-rank test, p = 0.022). Multivariate analyses demonstrated that the only significant independent prognostic indicator of a short period of progression-free survival was advanced stage, although a high expression of class III beta-tubulin was also prone to be associated with a short period of progression-free survival, but not significantly so ( p = 0.081). No such correlations or propensities were demonstrated in the 23 patients treated with the taxane-free regimen. Conclusions. In cases of ovarian carcinoma treated by taxanes, high expression of class III beta-tubulin seems to be associated with earlier recurrence, which is believed likely to be resistant relapse. In addition, loss of class II beta-tubulin expression is correlated with advanced stage, which may represent aggressive tumor progression.

Alterations of β‐tubulin isotypes in breast cancer cells resistant to docetaxel

Docetaxel is one of the most active drugs used to treat breast cancer. The cellular target of docetaxel is the microtubule, specifically the beta-tubulin subunit, that comprises a series of isotypes and that can modulate function. This study has examined the role of alteration in beta-tubulin isotypes in vitro and has sequenced the beta-tubulin gene to determine if there were mutations, both of which may represent important mechanisms of acquired resistance to docetaxel. Breast cancer cells, MCF-7 (oestrogen-receptor positive) and MDA-MB-231, (oestrogen-receptor negative) were made resistant to docetaxel in vitro. Expression of beta-tubulin isotypes (class I, II, III, IVa, IVb, and VI) was determined at the RNA and protein level using RT-PCR and western analysis, respectively. DNA sequencing evaluated the beta-tubulin gene. At the mRNA level, class I, II, III, and IVa beta-tubulin mRNA isotypes were over-expressed in docetaxel-resistant MCF-7 cells when compared with the docetaxel-sensitive parental cells. However, class VI beta-tubulin mRNA isotype expression was decreased in resistant cells. In MDA-MB-231 cells, there was a decrease in expression of the class I and class IVa beta-tubulin mRNA. However, there were increased expressions in class II, IVb, and VI beta-tubulin mRNA isotypes in resistant cells. Western analysis has confirmed corresponding increases in beta-tubulin protein levels in MCF-7 cells. However, in MDA-MB-231 cells, there were decreased protein levels for class II and class III beta-tubulin. This study demonstrates that altered expression of mRNA beta-tubulin isotypes and modulation of beta-tubulin protein levels are associated with acquired docetaxel resistance in breast cancer cells. This allows further understanding and elucidation of mechanisms involved in resistance to docetaxel.

Requirement for the betaI and betaIV tubulin isotypes in mammalian cilia.

Nielsen et al., [2001: Curr Biol 11:529-533], based on studies in Drosophila, have proposed that beta tubulin in axonemal microtubules must contain a specific acidic seven amino acid sequence in its carboxyl terminus. In mammals, the two betaIV isotypes (betaIVa and betaIVb) contain that sequence. In order to test the application of this hypothesis to mammals, we have examined the expression of beta tubulin isotypes in four different ciliated tissues (trachea, ependyma, uterine tube, and testis) using isotype-specific antibodies and indirect immunofluorescence. We find that betaIV tubulin is present in all ciliated cell types examined, but so is betaI tubulin. Taken together with recent studies that show that betaI and betaIV tubulin are both present in the cilia of vestibular hair cells, olfactory neurons, and nasal respiratory epithelial cells, we propose that both betaI tubulin and betaIV tubulin may be required for axonemal structures in mammals.

Characterization of isotype-specific regions of five classes of canine beta-tubulin and their expression in several tissues and cell culture.

The structure of isotype-specific regions of classes 1, II, III, IVa and IVb of canine beta-tubulin was characterized by 3'-RACE and the expression of these isotypes in canine tissues was examined by ribonuclease protection assay (RPA). Furthermore, a malignant mammary tumor-derived osteosarcoma-like cell line was established and the altered expression of beta-tubulin isotypes in taxol-resistant sublines was analyzed. The deduced amino acid sequences in isotype-specific regions corresponding to classes I, II and IVb were identical to those of humans and mice, but those in classes III and IVa showed slight differences among species. RPA revealed that classes I and IVb were widely distributed, but classes II, III and IVa were restricted to the brain. Because RPA could clearly distinguish the expression of class IVa from that of class IVb, it was thought to be more useful than northern blot for analysis of beta-tubulin isotype expression. In vitro, taxol-resistant sublines displayed a significant increase in class IVa as compared with taxol-sensitive cells, suggesting that altered expression of class IVa was associated with taxol resistance in these cell lines.

Expression of Oncogenic Epidermal Growth Factor Receptor Family Kinases Induces Paclitaxel Resistance and Alters β-Tubulin Isotype Expression

Oncogenic transformation confers resistance to chemotherapy through a variety of mechanisms, including suppression of apoptosis, increased drug metabolism, and modification of target proteins. Oncogenic epidermal growth factor receptor family members, including EGFRvIII and HER2, are expressed in a broad spectrum of human malignancies. Cell lines transfected with EGFRvIII and HER2 are more resistant to paclitaxel-mediated cytotoxicity, and tubulin polymerization induced by paclitaxel is suppressed compared with cells expressing wild type epidermal growth factor receptor. Because differential expression of beta-tubulin isotypes has been proposed to modulate paclitaxel resistance, we analyzed beta-tubulin isotypes expressed in cell lines transfected with different oncogenes. EGFRvIII- and HER2-expressing cells demonstrated equivalent total beta-tubulin protein compared with cells transfected with wild type receptor or untransfected controls. EGFRvIII-expressing cells demonstrated increases in class IVa (2.5-fold) and IVb (3.1-fold) mRNA, and HER2-expressing cells showed increases in class IVa (2. 95-fold) mRNA. Expression of oncogenic Ha-Ras did not change class IV RNA levels significantly. Inhibition of EGFRvIII kinase activity using a mutant allele with an inactivating mutation in the kinase domain decreased expression of class IVa by 50% and partially reversed resistance to paclitaxel. Expression of oncogenic epidermal growth factor receptor family members is associated with modulation of both beta-tubulin isotype expression and paclitaxel resistance in cells transformed by expression of the receptor. This effect on tubulin expression may modulate drug resistance in human malignancies that express these oncogenes.

Differentiation of human colon cancer cells changes the expression of beta-tubulin isotypes and MAPs.

The human colon adenocarcinoma HT29-D4 cell line is an interesting model for studies on epithelial cell differentiation. Undifferentiated cells are malignant proliferating cells, whereas differentiated cells act like epithelial polarized cells. In the present study, we first characterized the action of taxoids on the microtubular network of HT29-D4 cells according to the state of differentiation. Microtubular bundles were found in undifferentiated cells but not in differentiated cells, even with 500-fold higher taxoid concentrations for 96 h. This finding led us to study changes in microtubules according to the polarity status of the cell. E-MAP-115 was expressed only in differentiated cells; expression of β-tubulin isotypes was altered in them relative to undifferentiated cells. Classes I, II, III, IVa and IVb isotypes were expressed in both phenotypes; however, differentiated epithelial cells displayed a specific increase in class III β-tubulin. Thus, the increase in expression of this β-tubulin isotype in differentiated cells is not restricted to neuronal cells. Moreover, these expression changes may reflect a higher stability of microtubular network in differentiated cells, which may explain the lower activity of anti-microtubule agents, independently of the mitotic process. These results indicate that the composition of microtubules should be considered as one of the criteria involved in the response of tumour cells to chemotherapy with anti-microtubule agents. © 1999 Cancer Research Campaign

Paclitaxel-resistant human ovarian cancer cells have mutant beta-tubulins that exhibit impaired paclitaxel-driven polymerization.

Acquired resistance to paclitaxel can be mediated by P-glycoprotein or by alterations involving tubulin. We report two paclitaxel-resistant sublines derived from 1A9 human ovarian carcinoma cells. Single-step paclitaxel selection with verapamil yielded two clones that are resistant to paclitaxel and collaterally sensitive to vinblastine. The resistant sublines are not paclitaxel-dependent, and resistance remained stable after 3 years of drug-free culture. All cell lines accumulate [3H]paclitaxel equally, and no MDR-1 mRNA was detected by polymerase chain reaction following reverse transcription. Total tubulin content is similar, but the polymerized fraction increased in parental but not in resistant cells following the paclitaxel addition. Purified tubulin from parental cells demonstrated paclitaxel-driven increased polymerization, in contrast to resistant cell tubulin, which did not polymerize under identical conditions. In contrast, epothilone B, an agent to which the resistant cells retained sensitivity, increased assembly. Comparable expression of beta-tubulin isotypes was found in parental and resistant cells, with predominant expression of the M40 and beta2 isotypes. Sequence analysis demonstrated acquired mutations in the M40 isotype at nucleotide 810 (T --> G; Phe270 --> Val) in 1A9PTX10 cells and nucleotide 1092 (G --> A; Ala364 --> Thr) in 1A9PTX22 cells. These results identify residues beta270 and beta364 as important modulators of paclitaxel's interaction with tubulin.

Taxol differentially modulates the dynamics of microtubules assembled from unfractionated and purified beta-tubulin isotypes.

Substoichiometric binding of taxol to tubulin in microtubules potently suppresses microtubule dynamics, which appears to be the most sensitive antiproliferative mechanism of taxol. To determine whether the beta-tubulin isotype composition of a microtubule can modulate sensitivity to taxol, we measured the effects of substoichiometric ratios of taxol bound to tubulin in microtubules on the dynamics of microtubules composed of purified alphabeta(II)-, alphabeta(III)-, or alphabeta(IV)-tubulin isotypes and compared the results with the effects of taxol on microtubules assembled from unfractionated tubulin. Substoichiometric ratios of bound taxol in microtubules assembled from purified beta-tubulin isotypes or unfractionated tubulin potently suppressed the shortening rates and the lengths shortened per shortening event. Correlation of the suppression of the shortening rate with the stoichiometry of bound taxol revealed that microtubules composed of purified alphabeta(II)-, alphabeta(III)-, and alphabeta(IV)-tubulin were, respectively, 1.6-, 7.4-, and 7.2-fold less sensitive to the effects of bound taxol than microtubules assembled from unfractionated tubulin. These results indicate that taxol differentially modulates microtubule dynamics depending upon the beta-tubulin isotype composition. The results are consistent with recent studies correlating taxol resistance in tumor cells with increased levels of beta(III0- and beta(IV)-tubulin expression and suggest that altered cellular expression of beta-tubulin isotypes can be an important mechanism by which tumor cells develop resistance to taxol.

Altered Expression of Mβ2, the Class II β-Tubulin Isotype, in a Murine J774.2 Cell Line with a High Level of Taxol Resistance

A series of taxol- and taxotere-resistant J774.2 cell lines has been characterized with respect to altered expression of beta-tubulin, the cellular target for these drugs. Vertebrates have six classes of beta-tubulin isotypes, each displaying a distinct pattern of expression. Although the functional significance of multiple beta-tubulins has not been fully defined, there is evidence that the individual isotypes contribute to differences in microtubule dynamics and drug binding. To determine if alterations in the expression of beta-tubulin isotypes play a role in taxol resistance, a PCR-based methodology was developed that permits highly specific amplification of each of the six known murine beta-tubulin isotypes. Two isotypes, M beta 5 and M beta 3, were expressed abundantly in the drug-sensitive parental J774.2 cells. Although expressed at an extremely low level in the parental cells, expression of the M beta 2 isotype was increased 21-fold (< 0.005) in the cell line most resistant to taxol. These findings suggest that a cell can alter its relative tubulin isotype composition in response to an external stress and specifically imply that altered expression of M beta 2, the class II beta-tubulin isotype, may contribute to the development of high resistance to taxol.

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.

Expression and function of β-tubulin isotypes in Chinese hamster ovary cells

The availability of isotype-specific antisera for beta-tubulin, coupled with genetic and biochemical analysis, has allowed the determination of beta-tubulin isotype expression and distribution in Chinese hamster ovary (CHO) cells. Using genetic manipulations involving selection for colcemid resistance followed by reversion and reselection for drug resistance, we have succeeded in isolating cell lines that exhibit three major and one minor beta-tubulin spots by two-dimensional gel electrophoresis. In concert with isotype-specific antibodies, analysis of these mutants demonstrates that CHO cells express two copies of isotype I, at least one copy of isotype IV, and very small amounts of isotype V. All three isotypes assemble into both cytoplasmic and spindle microtubules and are similar in their responses to cold, colcemid, and calcium-induced depolymerization. They have comparable turnover rates and are equally sensitive to depression of synthesis upon colchicine treatment. These results suggest that beta-tubulin isotypes are used interchangeably to assemble microtubule structures in CHO cells. However, of 18 colcemid-resistant mutants with a demonstrable alteration in beta-tubulin, all were found to have the alteration in isotype I, thus leaving open the possibility that subtle differences in isotype properties may exist.

Differential distribution of beta-tubulin isotypes in cerebellum.

We describe the structure and expression of a mammalian beta-tubulin isotype (M beta 6) that is weakly expressed in testis but is abundant in developing brain, with transcripts declining to lower levels in the adult brain. The expression of M beta 6 was undetectable in any other mouse tissue examined. A serum specific for this isotype was prepared using a cloned fusion protein as immunogen. M beta 6 is one of five known beta-tubulin isotypes expressed in brain, and using the anti-M beta 6 serum along with sera, anti-M beta 2, anti-M beta 3/4 and anti-M beta 5, previously characterized, we have examined the pattern of expression of beta-tubulin isotypes in rat cerebellum. The isotypes each have characteristic cell-type specific patterns of localization in cerebellum. M beta 2, M beta 3/4 and M beta 5 are present in both neuronal and non-neuronal cells, but in contrast M beta 6 was only detectable in neurons in tissue sections and in dissociated cerebellar cell culture. The majority of sequence differences among the beta-tubulin isotypes lie at the carboxy terminus, the region of beta-tubulin involved in MAP binding. In the case of M beta 2 and M beta 6, the patterns of expression are similar or identical to the patterns of expression of MAP3 and MAP1A respectively. These results suggest that beta-tubulin isotypes may contribute to the determination of the specific association of MAPs with microtubules of diverse function. However, the strict subcellular segregation of other MAPs in brain may be determined by other factors.

Differential and developmental expression of beta-tubulins in a higher plant.

By using two-dimensional gel electrophoresis and immunoblotting, we have analyzed the expression of beta-tubulin isotypes in the higher plant, carrot. We report a complex expression of beta-tubulins that is dependent on the developmental stage of the tissues analyzed. Consequently, each tissue examined can be identified by its unique composition of beta-tubulins. In total, there are six electrophoretically separable beta-tubulins. In no tissue, however, is there less than two or more than five beta-tubulins. Within this framework we have detected a beta-tubulin specific to seedling tissue beta 6, and a beta-tubulin, beta 5, that is found only in the vegetative tissues of the mature plant. Traced from stem to midrib to leaf lamina, the beta 5 isotype becomes progressively dominant relative to beta 1. Another beta-tubulin isotype, beta 4, appears in marked abundance in immature and mature stamens. In isolated mature pollen the beta 4-tubulin overwhelmingly predominates the ubiquitously expressed beta 2-tubulin isotype. The remaining beta-tubulin isotypes also have specific expression programs with beta 1 present in all tissues except pollen and beta 3 absent only from pollen and leafy tissues.

The sequence and expression of the divergent beta-tubulin in chicken erythrocytes.

We report here the complete sequence of a highly divergent chicken erythrocyte beta-tubulin, c beta 6, which appears to represent a major exception to the observation that the primary sequences and sites of expression of beta-tubulin isotypes are conserved within vertebrates. The amino acid sequence was deduced from overlapping cloned cDNAs identified in a chicken erythroblast cDNA library contained in the expression vector, lambda gt11. Compared with other chicken beta-tubulins, among which the maximum sequence divergence is only 8%, c beta 6-tubulin is more hydrophobic, contains seven fewer net negative charges, and exhibits a surprising 17% overall divergence in its amino acid sequence. DNA and RNA blot analyses show that c beta 6-tubulin is present as a single gene copy in the chicken genome and is specifically expressed in the bone marrow. Comparisons of RNA blots and immunoblots of various cells and tissues confirm that this beta-tubulin isotype is contained specifically in erythrocytes and thrombocytes and accounts for 75% of the beta-tubulin mRNA species contained in developing erythroblasts. Interestingly, c beta 6-tubulin exhibits 18% amino acid sequence divergence relative to MB1, the analogous hematopoietic beta-tubulin contained in mouse.