Localization of a highly divergent mammalian testicular alpha tubulin that is not detectable in brain.

Abstract

Sequence analysis of a mouse testicular a-tubulin partial cDNA, pRDaTTl, reveals an isotype that differs from both the somatic and the predominant testicular a tubulins at approximately 30% of the 212 amino acid residues determined. Although this mouse testicular cDNA retains the highly conserved sequence, Glu-GlyGlu-Glu, found in the carboxyl termini of many a tubulins, the protein extends substantially beyond this sequence and does not terminate with a C-terminal tyrosine. Using rabbit antiserum prepared to a novel synthetic peptide predicted from this mouse testis a-tubulin cDNA, we have detected by immunoblot and indirect immunofluorescence an antigenic epitope present in testicular a tubulin that is not detectable in brain a tubulins. We find that the antiserum specifically binds to the manchettes and meiotic spindles of the mouse testis but not with neural fibers or tubulin extracts of the adult mouse brain. These results demonstrate that at least one of the multiple a-tubulin isotypes of the mammalian testis is expressed and used in male germ cells but not in the brain.Microtubules, formed from heterodimers of a and ,3 tubulin, constitute the primary structural components of mitotic and meiotic spindles, eucaryotic cilia and flagella, and elongated neuronal processes (3). The differential expression of specific tubulins during development in higher eucaryotes (3), the identification of a testis-specific i tubulin in Drosophila melanogaster (6), testis-specific a tubulins in mouse (4, 16) and chicken (13) testes, and a male-specific Drosophila a tubulin (14) suggest that tissue-or cell-type-specific microtubules could exist in organs such as the testis.Microtubules play an important role in the dramatic morphological changes in cell structure and shape that occur during the differentiation events of spermatogenesis. Of the microtubular structures peculiar to spermatogenesis, the meiotic spindle and the manchette are highly notable. The manchette forms during the haploid phase of spermatogenesis, when round spermatids first begin to lose their spherical shape, and disappears as the male germ cell elongates during late spermatid differentiation (2). The manchette structure consists of a parallel arrangement of up to 1,100 microtubules attached to a circumnuclear, dense protein plate. Although the function of the manchette is not known, it effectively partitions the cytoplasm of the spermatid, may play a role in shaping the nucleus of the spermatozoon, and is likely involved with transport of cytoplasmic components from the anterior to the posterior portion of the developing male gamete.Identification of a divergent testicular a tubulin. A 1,650-nucleotide cDNA insert that contains coding and 3'-untranslated sequences of the rat brain a tubulin, pILaT1 (10), was used to isolate a novel a-tubulin cDNA from a mouse testis cDNA library (4). Approximately 20,000 colonies were screened by colony hybridization. 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.1% sodium dodecyl sulfate), and 100 ,ug of soni...

Identification of α-tubulin as a granzyme B substrate during CTL-mediated apoptosis

“…6) (for review, see Luduena, 1993). Moreover, examination of tubulin isoforms in other model systems has revealed different developmental and tissuespecific expression patterns, suggesting that the variable Cterminus imparts unique activities (Hecht et al, 1988;Pratt and Cleveland, 1988;Villasante et al, 1986). We have also noted an isoform of human ␣-tubulin with a truncated C-terminus that appears very similar to the grB-cleaved form of tubulin (see Fig.…”

A novel Ca2+/calmodulin-dependent protein kinase and a male germ cell-specific calmodulin-binding protein are derived from the same gene.

“…More than 30 other examples exist for expression of germ cell-specific mRNAs (24). Molecular mechanisms responsible include differential transcriptional initiation of a gene expressed in somatic cells (13), expression of testis-specific genes encoding an isoform of a somatic cell counterpart (17), alternative choice of polyadenylation sites (3), change in the extent of mRNA polyadenylation (1,33) and translational control of mRNA expression (5,33). More rare are instances in which a germ cell-specific transcript is generated from a somatic cell transcription unit by transcriptional initiation within the usual amino acid-coding region of the gene.…”

Expression of glycylated tubulin during the differentiation of spermatozoa in mammals

“…In the mammalian testis, germ cell microtubules are made of three major tubulin isotypes: m␤ 3 and m␣ 3/7 [Lewis and Cowan, 1988] and pRD␣TT1 [Hecht et al, 1988;Simerly et al, 1993]. These isotypes are either expressed as unmodified polypeptides or as posttranslationally modified isoforms.…”