Abstract
We constructed and characterized recombinant cDNA clones containing α- and β-tubulin DNA sequences. The inserted DNA was determined to code for α- and β-tubulin by positive hybridization-selection. The selected mRNA was translated in vitro, and the translation products were shown to be α- or β-tubulin by comigration with flagellar α- and β-tubulin on one- and two-dimensional gels and by immunoprecipitation with antibodies specific for α- and β-tubulin. Hybridization of the cloned tubulin probes with Chlamydomonas DNA indicated that there are at least two genes each for α- and β-tubulin in this organism. No evidence of cross-hybridization between α- and β-tubulin DNA sequences was found. Because previous experiments had shown that tubulin synthesis was stimulated in response to flagellar amputation, the tubulin clones were used to analyze the levels of tubulin sequences in RNA from cells before and after deflagellation. Hybridization of the tubulin cDNA probes with total or polyadenylated RNA indicated that tubulin sequences in RNA increased within 8 min following deflagellation, reached maximal levels by 50 min and began to decrease by 80 min after deflagellation. One hybridization band was detected with use of the β-tubulin probe, but RNA in two size classes hybridized to the α-tubulin probe.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 81-88 |
| Number of pages | 8 |
| Journal | Cell |
| Volume | 24 |
| Issue number | 1 |
| DOIs | |
| State | Published - Apr 1981 |
Bibliographical note
Funding Information:We wish to thank Drs. Paul Lefebvre and Jeff Schlossfor many helpful discussions and assistance in preparation of the manuscript. We also thank Dr. Tim McKeithan for help in designing the flagellar tubulin purification method, and Ms. Ditti Holub for excellent technical assistance. We are grateful to Drs. Joseph Gall, Bambi Batts-Young. Rob Steele. Brian Kay, David Baulcombe and Ed Stephenson for helpful discussions. This work was supported by a research grant from the National Institutes of Health. C. S. was supported by a postdoctoral fellowship from the National Institutes of tiealth. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.