Microtubule architecture specified by a β-tubulin isoform

Science

Volumn 275, Issue 5296, 1997, Pages 70-73

  Raff, Elizabeth C ^a   Fackenthal, James D ^a,b   Hutchens, Jeffrey A ^a   Hoyle, Henry D ^a   Turner, F Rudolf ^a  

^a INDIANA UNIVERSITY   (United States)

^b UNIVERSITY OF CHICAGO   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BETA TUBULIN; CYTOSKELETON PROTEIN;

AMINO ACID SEQUENCE; ANIMAL CELL; ANIMAL EXPERIMENT; ANIMAL TISSUE; ARTICLE; AXONEME; CONTROLLED STUDY; CYTOSKELETON; DROSOPHILA MELANOGASTER; LEPIDOPTERA; MALE; MICROTUBULE; NONHUMAN; PRIORITY JOURNAL; SEQUENCE HOMOLOGY; SPERMATOGENESIS; TESTIS;

AMINO ACID SEQUENCE; ANIMALS; ANIMALS, GENETICALLY MODIFIED; DROSOPHILA MELANOGASTER; HUMANS; MALE; MICROTUBULES; MOLECULAR SEQUENCE DATA; MOTHS; SPERMATIDS; SPERMATOGENESIS; TUBULIN;

EID: 0031022787     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5296.70     Document Type: Article

References (36)

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20. Even when the synthesis rate was the same, less Hvβt protein accumulated than β2; the moth protein is thus less stable than the endogenous isoform. However, testes of sterile males carrying multiple inserts of the Hvβt transgene accumulated Hvβt in amounts equivalent to those of β2 that we have shown to be sufficient to support microtubule function. Thus, the functional deficit of the moth isoform is not attributable simply to its relative instability in Drosophila cells. The total failure of Hvβt to support microtubule function contrasts with ectopic expression of Drosophila β3 in the male germ cells; β3 can support wild-type function of one class of cytoplasmic microtubules, even though it cannot assemble axonemes, spindles, or other microtubule arrays (3).
21. In a given spermatid, the threshold may be lower; we did not detect Hvβt in motile sperm in males in which the amount of Hvβt was sufficiently low as to allow fertility.
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26. Vertebrate isoforms are designated by the organism, followed by the isotype class to which the sequence shown belongs (1): class II, major neuronal; class III, minor neuronal; class IVb, predominant testis; class VI, hematopoetic. COOH-termini of isoforms of the same class in other species are identical or very similar to those shown. Widely expressed class I and V isotypes also lack the axoneme motif. β-Tubulins in other Drosophila species appear to be identical to those in D. melanogaster [(4); F. Michiels et al., Chromosoma 95, 387 (1987)].
27. Aspergillus nidulans has two β-tubulin genes; benA also lacks the axoneme motif.
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31. To generate the Hvβt transgene, we generated cloning sites 29 base pairs (bp) 5′ and 27 bp 3′ of the Hvβt coding sequence, and the resulting fragment was inserted between 2.1 kb of the 5′ β2 genomic sequences and 1.5 kb of the 3′ β2 genomic sequences previously shown to be sufficient to drive expression of heterologous proteins in the postmitotic male germ cells with correct developmental specificity and at the same level of expression as wild-type β2 (2, 3) (J. Hutchens, H. Hoyle, F. R. Turner, E. C. Raff, Mol. Biol. Cell, in press), Intronless and intron-containing versions were constructed; in the latter, an oligonucleotide matching the 59-bp β2 intron sequence was inserted between Hvβt codons 73 and 74. Transgenes were inserted into the CaSpeR vector [ V. Pirrotta, Biotechnology 10, 437 (1988)] and introduced into the Drosophila genome by P element-mediated transformation (2, 3). Multiple transgenic lines were obtained and testis tubulins analyzed on two-dimensional gels as described previously (2, 3); the level of Hvβt expression depended on the site of insertion and presence of the intron. We obtained wild-type β2-like levels of expression (as in Fig. 2) only with an intron-containing insert, suggesting that splicing may be important in normal β2 expression. All transgenic lines exhibited the same suite of defects in spermatogenesis; thus, the phenotype is attributable solely to expression of the moth β-tubulin.
32. To generate the Hvβt transgene, we generated cloning sites 29 base pairs (bp) 5′ and 27 bp 3′ of the Hvβt coding sequence, and the resulting fragment was inserted between 2.1 kb of the 5′ β2 genomic sequences and 1.5 kb of the 3′ β2 genomic sequences previously shown to be sufficient to drive expression of heterologous proteins in the postmitotic male germ cells with correct developmental specificity and at the same level of expression as wild-type β2 (2, 3) (J. Hutchens, H. Hoyle, F. R. Turner, E. C. Raff, Mol. Biol. Cell, in press), Intronless and intron-containing versions were constructed; in the latter, an oligonucleotide matching the 59-bp β2 intron sequence was inserted between Hvβt codons 73 and 74. Transgenes were inserted into the CaSpeR vector [ V. Pirrotta, Biotechnology 10, 437 (1988)] and introduced into the Drosophila genome by P element-mediated transformation (2, 3). Multiple transgenic lines were obtained and testis tubulins analyzed on two-dimensional gels as described previously (2, 3); the level of Hvβt expression depended on the site of insertion and presence of the intron. We obtained wild-type β2-like levels of expression (as in Fig. 2) only with an intron-containing insert, suggesting that splicing may be important in normal β2 expression. All transgenic lines exhibited the same suite of defects in spermatogenesis; thus, the phenotype is attributable solely to expression of the moth β-tubulin.
33. Electron microscopy and tannic acid staining were done as previously described (2, 3).
34. The morphology of doublet microtubules and the central pair is the same in moths, flies, and transgenic flies. Doublets have a 13-pf A-tubule and a 10-pf shared-wall B-tubule; central pair microtubules are 13-pf. Accessory microtubules in fly axonemes are 13-pf, but 16-pf in moth. Most accessory microtubules in transgenic males are 13-pf, but the abnormal large-diameter accessory microtubules are 16-pf.
35. Accessory microtubules begin as a projection of a protofilament sheet from the B-tubule of each doublet, but completed accessory microtubules are no longer physically associated with the doublet. Completed accessory microtubules in immature axonemes of flies and moths were of a slightly larger diameter than in mature axonemes; thus, adjacent protofilaments in the walls of the accessory microtubules appear to "tighten up" as they form.
36. We thank M.-T. Davis and S. Miller for providing us with the H. virescens testis-specific β-tubulin cDNA clone; C.-S. Hong and M. Martin for their enthusiastic participation and contributions as undergraduate research students in the early parts of this study; and W. Saxton and R. Raff for critical reading of the manuscript. This work was supported by a grant from National Institute of Child Health and Human Development (of NIH) to E.C.R.