Developmental regulation and identification of an isotype encoded by altB, an alpha-tubulin locus in Physarum polycephalum.

A subcloned portion of the 5' nontranslated sequence from a Physarum alpha-tubulin cDNA is specific for a single alpha-tubulin locus, altB, of Physarum polycephalum. We find that this locus is expressed only in the plasmodium and encodes at least an alpha 1-tubulin isotype, which we have designated alpha 1B. Hybridization patterns of other subclones of this cDNA reveal two sequences for alpha-tubulin at the altB locus.     

Recognition of specific Physarum alpha-tubulin isotypes by a monoclonal antibody. Sequence heterogeneity around the acetylation site at lysine 40

The monoclonal antibody 6-11B-1 recognises specifically the acetylated form of alpha-tubulin. The acetylation event occurs on a unique lysine residue, lysine 40. Using 6-11B-1, acetylated alpha-tubulin was detected in myxamoebae but not plasmodia of Physarum polycephalum. Following chemical acetylation plasmodial alpha-tubulin was detected by 6-11B-1. The monoclonal antibody KMP-1 recognises certain Physarum alpha-tubulin isotypes but only in non-acetylated form. Whilst recognising all the non-acetylated fraction of myxamoebal alpha-tubulin only a proportion of plasmodial alpha-tubulin was recognised by KMP-1. Peptides were synthesised corresponding to the acetylation domains (containing lysine 40) of myxamoebal alpha-tubulin and the inferred acetylation domains of two plasmodial-specific alpha-tubulin isotypes. The only difference between the two peptides was at a single residue corresponding to amino acid 44 in the polypeptide. Tyrosine was present in myxamoebal alpha-tubulin and glycine was present in the plasmodial specific peptides; the peptides are referred to as the Tyr44 and Gly44 peptides respectively. Both peptides in acetylated form blocked 6-11B-1 reactivity towards acetylated myxamoebal alpha-tubulin. The Tyr44 but not the Gly44 peptide blocked KMP-1 reactivity towards non-acetylated myxamoebal alpha-tubulin. Tyrosine at position 44 is not found in any other known alpha-tubulin. Thus a unique antigenic determinant exists in certain Physarum alpha-tubulin isotypes, close to the acetylation site at lysine 40. This antigenic determinant forms part of the KMP-1 recognition epitope and explains the unique isotype selectivity of this monoclonal antibody.     

Two alleles of a developmentally regulated alpha-tubulin locus in Physarum polycephalum replicate on different schedules.

The replication timing of a pair of natural alleles was compared at two alpha-tubulin loci of the Physarum plasmodium. Taking advantage of the naturally synchronous cell cycle of nuclei within the syncytial plasmodium, we analyzed the replication schedule of specific DNA fragments to a resolution of 10-min intervals within a 3-h S phase. At this level of resolution, differences in replication timing between polymorphic alleles at the same locus can be detected in a heterozygote. Specifically, the 3' region of the altA1 allele completes replication at between 20 and 40 min of S phase. The same region of the altA2 allele completes replication at between 40 and 80 min of S phase. In contrast, both alleles at the altB locus replicate concurrently within the first 10 to 15 min of S phase. Previous studies showed that both altA and altB are expressed in the plasmodium, their message levels peaking at mitosis, just minutes before the onset of S phase. However, altB message is detected at substantially higher levels than altA message on Northern (RNA) blots. The temporal windows over which the altA alleles each replicate are very broad in comparison with the levels of mitotic synchrony and altB replication synchrony in a single plasmodium. The allele-specific replication schedule of the altA locus demonstrates that the temporal organization of replicons is not strictly conserved between homologous chromosomes.     

Amino acid sequence data of alpha-tubulin from myxamoebae of Physarum polycephalum

About 96% of the amino acid sequence of an alpha-tubulin from the slime mould Physarum polycephalum has been determined. Of 430 sequenced amino acids, 30 differ from the deduced amino acid sequence of a recently published alpha-tubulin complementary DNA from the plasmodial form of P. polycephalum. The myxamoebal alpha-tubulin differs from all other known alpha-tubulins in one of the last three C-terminal amino acids that are Gly-Glu-Tyr instead of the usual Glu-Glu-Tyr. These last three amino acids are preceded by 11 residues that appear to be particularly susceptible to mutation. No heterogeneity was found whilst sequencing the myxamoebal alpha-tubulin, indicating that only one type of alpha-tubulin is present in myxamoebae. This alpha-tubulin appears to be less conserved than the previously described plasmodial alpha-tubulin, supporting the hypothesis that the structural constraints on tubulin in axonemes have a significant effect on its rate of mutation.     

Use of monoclonal antibodies to analyse the expression of a multi-tubulin family

We have used a panel of monoclonal antibodies in a study of the expression of multiple tubulins in Physarum polycephalum. Three anti-beta-tubulin monoclonal antibodies, DM1B, DM3B3 and KMX-1 all reacted with the beta 1-tubulin isotypes expressed in both myxamoebae and plasmodia. However, these antibodies showed a spectrum of reduced reactivity with the plasmodial beta 2-tubulin isotype - the competence of recognition of this isotype was graded DM1B greater than KMX-1 greater than DM3B3. The anti-alpha-tubulin monoclonal antibody, YOL 1/34 defined the full complement of Physarum alpha-tubulin isotypes, whilst the anti-alpha-tubulin monoclonal antibody, KMP-1 showed a remarkably high degree of isotype specificity. KMP-1 recognises all of the myxamoebal alpha 1-tubulin isotypes but only recognises 3 out of the 4 alpha 1-tubulin isotypes expressed in the plasmodium (which normally focus in the same 2D gel spot). KMP-1 does not recognise the plasmodial specific alpha 2-tubulin isotype. This monoclonal antibody reveals a new level of complexity amongst the tubulin isotypes expressed in Physarum and suggests that monoclonal antibodies are valuable probes for individual members of multi-tubulin families.     

Genetics of the Tubulin Gene Families of Physarum

The organization of the alpha- and beta-tubulin gene families in Physarum was investigated by Mendelian analysis. Restriction endonuclease-generated DNA fragments homologous to alpha- and beta-tubulin show length polymorphisms that can be used as markers for genetic mapping. Analysis of meiotic assortment among progeny of heterozygotes allowed alpha- and beta-tubulin sequence loci to be defined. There are four unlinked alpha-tubulin sequence loci (altA, altB, altC and altD) and at least three unlinked beta-tubulin sequence loci (betA, betB and betC). The alpha-tubulin loci are not linked to the beta-tubulin loci. --Segregation of tubulin sequence loci with respect to ben mutations that confer resistance to antitubulin benzimidazole drugs was used to investigate whether any members of the alpha- or beta-tubulin gene families are allelic to ben loci. The beta-tubulin sequence locus betB is allelic to the resistance locus benD, the betA locus is probably allelic to benA and the alpha-tubulin sequence locus altC may be allelic to benC. The molecular implications of benzimidazole resistance phenotypes when only one of the expressed beta-tubulin gene family members mutates to drug resistance are discussed in relation to tubulin function.     

Genetic evidence for a dystrophin-glycoprotein complex (DGC) in Caenorhabditis elegans

A novel alpha-tubulin gene (alpha6) was cloned from a genomic library of Naegleria gruberi strain NB-1 and characterized. The open reading frame of alpha6 contained 1359 nucleotides encoding a protein of 452 amino acids (aa) with a calculated molecular weight of 50.5 kDa. The nucleotide sequence of the open reading frame of alpha6 showed considerable divergence (68.4% identity) when compared with previously cloned N. gruberi alpha-tubulin genes, which share about 97% identity in DNA sequences. The deduced aa sequence of alpha6-tubulin was 61.9% identical to that of alpha13-tubulin, which was cloned from the same strain, and showed similar identities to those of alpha-tubulins from other species (54 approximately 62%). These data showed that alpha6-tubulin is one of the most divergent alpha-tubulins so far known. Alpha6-tubulin was found to be expressed in actively growing cells and repressed quickly when these cells were induced to differentiate. Immunostaining with an antibody against alpha6-tubulin showed that alpha6-tubulin is present in the nuclei and mitotic spindle-fibers but absent in flagellar axonemes or cytoskeletal microtubules. These data finally established the presence of an alpha-tubulin that is specifically utilized for spindle-fiber microtubules and distinct from the flagellar axonemal alpha-tubulins in N. gruberi, hence confirmed the multi-tubulin hypothesis in this organism.     

Distribution of acetylated alpha-tubulin in Physarum polycephalum

The expression and cytological distribution of acetylated alpha-tubulin was investigated in Physarum polycephalum. A monoclonal antibody specific for acetylated alpha-tubulin, 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094), was used to screen for this protein during three different stages of the Physarum life cycle--the amoeba, the flagellate, and the plasmodium. Western blots of two-dimensional gels of amoebal and flagellate proteins reveal that this antibody recognizes the alpha 3 tubulin isotype, which was previously shown to be formed by posttranslational modification (Green, L. L., and W. F. Dove, 1984, Mol. Cell. Biol., 4:1706-1711). Double-label immunofluorescence demonstrates that, in the flagellate, acetylated alpha-tubulin is localized in the flagella and flagellar cone. Similar experiments with amoebae interestingly reveal that only within the microtubule organizing center (MTOC) are there detectable amounts of acetylated alpha-tubulin. In contrast, the plasmodial stage gives no evidence for acetylated alpha-tubulin by Western blotting or by immunofluorescence.     

alpha-Tubulin limits its own synthesis: evidence for a mechanism involving translational repression

A Chinese hamster alpha-tubulin cDNA was modified to encode an 11-amino acid carboxyl-terminal extension containing the immunodominant epitope from influenza hemagglutinin antigen (to create HA alpha 1-tubulin) and was cloned into a vector for expression in mammalian cells. 12 stable CHO cell lines expressing this HA alpha 1-tubulin were isolated and characterized. HA alpha 1-tubulin incorporated into all classes of microtubules, assembled to the same extent as the endogenous tubulin, and did not perturb the growth of the cells in which it was expressed. However, overexpression of HA alpha 1-tubulin strongly repressed the synthesis of endogenous alpha-tubulin while having little or no effect on the synthesis of beta-tubulin. Treatment of transfected cells with sodium butyrate to induce even greater expression of HA alpha 1-tubulin led to a further decrease in synthesis of endogenous alpha-tubulin that was fully reversible upon removal of the inducer. Decreased synthesis of alpha-tubulin in transfected cells did not result from decreased levels of alpha-tubulin mRNA, as demonstrated by ribonuclease protection assays. On the other hand, colchicine, a drug previously shown to destabilize the tubulin message, caused a clear reduction in both protein synthesis and mRNA levels for transfected HA alpha 1- tubulin and endogenous alpha-tubulin, thus indicating that the decreased alpha-tubulin synthesis observed as a result of HA alpha 1- tubulin overexpression is distinct from the previously described autoregulation of tubulin. The results are consistent with a mechanism in which free alpha-tubulin inhibits the translation of its own message as a way of ensuring stoichiometric synthesis of alpha- and beta- tubulin.     

alpha-Tubulin genes of Drosophila

Four Drosophila alpha-tubulin genes have been isolated on recombinant DNA molecules. The identity of two of these genes (T alpha 1 and T alpha 2) was established by isolating complementary mRNAs and then examining the in vitro translation products of the mRNAs. The one- and two-dimensional gel patterns and the peptide maps of the in vitro products were indistinguishable from those of embryonic alpha-tubulin. In turn, the embryonic tubulin was identified by determining its amino-terminal sequence. We identified two other cloned alpha-tubulin genes (T alpha 3 and T alpha 4) by their complementarity to T alpha 1 and T alpha 2. Maps of restriction endonuclease sites indicate that the four genes are different. DNA hybridization studies demonstrated, however, that three of them have extensive sequence homology with each other and slight homology with the fourth, T alpha 4. Hybridization to genomic DNA fragments indicated that the four clones genes account for all of the different alpha-tubulin genes of Drosophila melanogaster. Three of them are present only once in the haploid genome; the other, T alpha 1, is present in either one or two copies. Each of the four genes hybridizes in situ to a different site on the third chromosome.     

Increased expression of T alpha 1 alpha-tubulin mRNA during collateral and NGF-induced sprouting of sympathetic neurons

We have examined expression of T alpha 1 alpha-tubulin mRNA in the rat superior cervical ganglion (SCG) to determine whether changes in gene expression accompany neuronal sprouting and to investigate factors that regulate growth-associated genes in intact neurons. Northern blot analysis demonstrates that levels of T alpha 1 alpha-tubulin mRNA increase in the uninjured SCG following transection of contralateral neurons that project to bilaterally innervated, but not unilaterally innervated target organs. The observed increase in uninjured neurons is associated with collateral sprouting, as measured by increased tyrosine hydroxylase immunoreactivity within the pineal gland. These data suggest that target-derived factors may regulate T alpha 1 mRNA in sprouting neurons. Consistent with this hypothesis, systemic NGF treatment of neonatal animals over a developmental interval when T alpha 1 alpha-tubulin mRNA normally decreases led to a 5- to 10-fold increase in T alpha 1 mRNA levels in developing sympathetic neurons. In addition, deafferentation of the SCG, which promotes neuronal sprouting in the ganglion, increases T alpha 1 mRNA in ganglia on the ipsilateral and contralateral sides. Together, these data demonstrate that T alpha 1 alpha-tubulin mRNA elevates as a function of neuronal sprouting, and that T alpha 1 mRNA expression in intact neurons can be regulated by extrinsic cues, including NGF and changes in connectivity.     

Structural features and restricted expression of a human alpha-tubulin gene.

The nucleotide sequence of a human alpha-tubulin gene (b alpha 1) is described. This gene is extensively homologous to a rat alpha-tubulin gene in its coding regions, 3'-untranslated region and, indeed, in segments of its largest intron. However, with the exception of three short conserved blocks of homology, the 5' flanking regions of the rat and human genes are unrelated. Hence, these genes each encoding an identical protein are transcribed under the influence of divergent promoters. Blot analyses using RNA from a variety of transformed cells derived from different tissues indicate that expression of the human alpha-tubulin gene is restricted to cells of neurological origin. Among neurological cell types b alpha 1 expression is further restricted to adherent cells that are morphologically differentiated. The data presented suggest that the b alpha 1 gene encodes a prominent neuronal and glial alpha-tubulin and that b alpha 1 expression is a function of the differentiated state of these cells.     

Accelerated poly(A) loss on alpha-tubulin mRNAs during protein synthesis inhibition in Chlamydomonas

Detachment of flagella in Chlamydomonas reinhardii stimulates a rapid accumulation of tubulin mRNAs. The induced tubulin mRNAs are normally rapidly degraded following flagellar regeneration, but inhibition of protein synthesis with cycloheximide prevents their degradation. alpha-Tubulin poly(A) tail lengths were measured during normal accumulation and degradation, and in cycloheximide-treated cells. To measure alpha-tubulin mRNA poly(A) chain lengths with high resolution, specific 3' fragments of alpha 1- and alpha 2-tubulin mRNAs, generated by RNase H digestion of mRNA-oligonucleotide hybrids, were sized by Northern analysis. Both alpha-tubulin mRNAs have a newly synthesized poly(A) chain of about 110 adenylate residues. The poly(A) tails shorten with time, and show an average length of 40 to 60 adenylate residues by 90 minutes after deflagellation, at which time induced alpha-tubulin mRNA is being rapidly degraded. Poly(A) loss is significantly accelerated in cycloheximide-treated cells, and this loss is not attributible simply to the longer time the stabilized molecules spend in the cytoplasm. A large fraction of alpha-tubulin mRNA accumulates as mRNA with very short poly(A) tails (less than 10 residues) in the presence of cycloheximide, indicating that deadenylated alpha-tubulin mRNAs can be stable in vivo, at least in the absence of protein synthesis. The rate and extent of poly(A) loss in cycloheximide are greater for alpha 2-tubulin mRNA than for alpha 1-tubulin mRNA. This difference cannot be attributed to differential ribosome loading. This finding is interesting in that the two mRNAs are very similar in sequence with the exception of their 3' untranslated regions.     

A combination of posttranslational modifications is responsible for the production of neuronal alpha-tubulin heterogeneity.

We describe the presence of alpha-tubulin and MAP2 acetyltransferase activities in mouse brain. The enzyme(s) copurified with microtubules through two cycles of assembly-disassembly. Incubation of microtubule proteins with [3H]acetyl CoA resulted in a strong labeling of both alpha-tubulin and MAP2. To determine the site of the modification, tubulin was purified and digested with Glu-C endoproteinase. A unique radioactive peptide was detected and purified by HPLC. Edman degradation sequencing showed that this peptide contained epsilon N-acetyllysine at position 40 of the alpha-tubulin molecule. This result demonstrates that mouse brain alpha-tubulin was acetylated at the same site as in Chlamydomonas. Isoelectric focusing analysis showed that acetylated alpha-tubulin was resolved into five isoelectric variants, denoted alpha 3 and alpha 5 to alpha 8. This heterogeneity is not due to acetylation of other sites but results from a single acetylation of Lys40 of an heterogeneous population of alpha-tubulin isoforms. These isoforms are produced by posttranslational addition of one to five glutamyl units. Thus, neuronal alpha-tubulin is extensively modified by a combination of modifications including acetylation, glutamylation, tyrosylation, and other yet unknown modifications.     

Generation of antisera that discriminate among mammalian alpha- tubulins: introduction of specialized isotypes into cultured cells results in their coassembly without disruption of normal microtubule function

To assay the functional significance of the multiple but closely related alpha-tubulin polypeptides that are expressed in mammalian cells, we generated three specific immune sera, each of which uniquely recognizes a distinct alpha-tubulin isotype. All three isotypes are expressed in a tissue-restricted manner: one (M alpha 3/7) only in mature testis, one (M alpha 4) mainly in muscle and brain, and the third (M alpha 6) in several tissues at a very low level. A fourth specific antiserum was also generated that distinguishes between the tyrosinated and nontyrosinated form of a single alpha-tubulin isotype. Because individual tubulin isotypes cannot be purified biochemically, these sera were raised using cloned fusion proteins purified from host Escherichia coli cells. To suppress the immune response to shared epitopes, animals were first rendered tolerant to fusion proteins encoding all but one of the known mammalian alpha-tubulin isotypes. Subsequent challenge with the remaining fusion protein then resulted in the elicitation of an immune response to unique epitopes. Three criteria were used to establish the specificity of the resulting sera: (a) their ability to discriminate among cloned fusion proteins representing all the known mammalian alpha-tubulin isotypes; (b) their ability to uniquely detect alpha-tubulin in whole extracts of tissues; and (c) their capacity to stain microtubules in fixed preparations of cells transfected with sequences encoding the corresponding isotype. The transfection experiments served to demonstrate (a) the coassembly of M alpha 3/7, M alpha 4, and M alpha 6 into both interphase and spindle microtubules in HeLa cells and NIH 3T3 cells, and (b) that the M alpha 4 isotype, which is unique among mammalian alpha-tubulins in that it lacks an encoded carboxy-terminal tyrosine residue, behaves like other alpha-tubulin isotypes with respect to the cycle of tyrosination/detyrosination that occurs in most cultured cells.     

Isotypes of alpha-tubulin are differentially regulated during neuronal maturation

The mRNAs for two isotypes of alpha-tubulin, termed T alpha 1 and T26, are known to be expressed in the rat nervous system. We have compared the expression of these two alpha-tubulin mRNAs during neural development, using RNA blotting and in situ hybridization techniques with probes directed against unique sequences of each mRNA. T alpha 1 mRNA is highly enriched in the embryonic nervous system but is markedly less abundant in the adult brain; T26 mRNA is expressed in many embryonic tissues with little change in abundance during development. Within the nervous system, T alpha 1 mRNA is enriched in regions with neurons actively undergoing neurite extension, such as the cortical plate, whereas T26 mRNA is relatively homogeneous in distribution, with some enrichment in proliferative zones. Expression of T alpha 1 mRNA is also increased in PC12 cells induced to differentiate and extend neurite processes by nerve growth factor. Taken together, the data indicate that T alpha 1-tubulin mRNA is expressed at high levels during the extension of neuronal processes. The abundant expression of T alpha 1-tubulin mRNA may therefore reflect either a means to increase the available pool of alpha-tubulin or a specific requirement for the T alpha 1 isotype for neurite extension.