Abstract
alpha- and beta-Tubulin are encoded in vertebrate genomes by a family of approximately 6-7 functional genes whose polypeptide products differ in amino acid sequence. In the chicken, one beta-tubulin isotype (c beta 6) has previously been found to be expressed only in thrombocytes and erythroid cells, where it is assembled into a circumferential ring of marginal band microtubules. In light of its unique in vivo utilization and its divergent assembly properties in vitro, we used DNA transfection to test whether this isotype could be assembled in vivo into microtubules of divergent functions. Using an antibody specific to c beta 6, we have found that upon transfection this polypeptide is freely coassembled into an extensive array of interphase cytoplasmic microtubules and into astral and pole-to-chromosome or pole-to-pole microtubules during mitosis. Further, examination of developing chicken erythrocytes reveals that both beta-tubulins that are expressed in these cells (c beta 6 and c beta 3) are found as co-polymers of the two isoforms. These results, in conjunction with efforts that have localized various other beta-tubulin isotypes, demonstrate that to the resolution limit afforded by light microscopy in vivo microtubules in vertebrates are random copolymers of available isotypes. Although these findings are consistent with functional interchangeability of beta-tubulin isotypes, we have also found that in vivo microtubules enriched in c beta 3 polypeptides are more sensitive to cold depolymerization than those enriched in c beta 6. This differential quantitative utilization of the two endogenous isotypes documents that some in vivo functional differences between isotypes do exist.
Highlights
Abstract. a- and 13-Tubulin are encoded in vertebrate genomes by a family of,x,6-7 functional genes whose polypeptide products differ in amino acid sequence
The principal subunit of each of these different classes of microtubules is a heterodimer of one ~t- and one 13-tubulinpolypeptide, each subunit is encoded in vertebrates by a family of,o6--7functional genes (Lopata et al, 1983; Hall et al, 1983; Lewis et al, 1985a, b; Wanget al., 1986; Sullivan et al, 1986a, b; Elliott et al, 1986; Villasante et al, 1986; Pratt et al, 1987)
We cloned and sequenced the major 13-tubulinsubunit that is assembled into marginal band microtubules in nucleatederythrocytes and thrombocytesof chickens (Murphy et al, 1987)
Summary
Abstract. a- and 13-Tubulin are encoded in vertebrate genomes by a family of ,x,6-7 functional genes whose polypeptide products differ in amino acid sequence. Examination of developing chicken erythrocytes reveals that both 13-tubulins that are expressed in these cells (cl and eli3) are found as co-polymers of the two isoforms These results, in conjunction with efforts that have localized various other 13-tubulin isotypes, demonstrate that to the resolution limit afforded by light microscopy in vivo mierotubules in vertebrates are random copolymers of available isotypes. The existence within a single organism of a group of distinct isotypes of a-tubulin and 13-tubulin has encouraged speculation that divergent functional classes of microtubules are specified, at least in part, by their assembly from different isoforms (Fulton and Simpson, 1976;Stephens, 1975; see Cleveland, 1987for review) This hypothesis has been supported by the identification of evolutionarily conserved isotypes of 13-tubulinwhose primary sequence and patterns of expression have been very highly conserved (Sullivan and Cleveland, 1986). Solomon and co-workers have shown that a chimeric 13-tubulincomposed of an amino terminus of chicken 13~-tuhulinlinked to a divergent carboxy terminus from a yeast 13-tubulinis incorporated into all
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