Abstract
Drosophila alpha-spectrin cDNA sequences were isolated from a lambda gt11 expression library. These cDNA clones encode fusion proteins that include portions of the Drosophila alpha-spectrin polypeptide as shown by a number of structural and functional criteria. The fusion proteins elicited antibodies that reacted strongly with Drosophila and vertebrate alpha-spectrins and a comparison of cyanogen bromide peptide maps demonstrated a clear structural correspondence between one fusion protein and purified Drosophila alpha-spectrin. Alpha-spectrin fusion protein also displayed calcium-dependent calmodulin-binding activity in blot overlay experiments and one fusion protein bound specifically to both Drosophila and bovine brain beta-spectrin subunits on protein blots. A region of the Drosophila cDNA cross-hybridized at lowered stringency with an avian alpha-spectrin cDNA. Together these data show that the composition, structure, and binding properties of the spectrin family of proteins have been remarkably well conserved between arthropods and vertebrates. Drosophila cDNA hybridized to an mRNA of greater than or equal to 9 kb on blots of total Drosophila poly A+ RNA; and hybridized in situ to a single site in polytene region 62B, 1-7. This result and Southern blot analysis of genomic DNA indicate that the sequences are likely to be single copy in the Drosophila genome.
Full Text
The Full Text of this article is available as a PDF (1.9 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bennett V. The membrane skeleton of human erythrocytes and its implications for more complex cells. Annu Rev Biochem. 1985;54:273–304. doi: 10.1146/annurev.bi.54.070185.001421. [DOI] [PubMed] [Google Scholar]
- Birkenmeier C. S., Bodine D. M., Repasky E. A., Helfman D. M., Hughes S. H., Barker J. E. Remarkable homology among the internal repeats of erythroid and nonerythroid spectrin. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5671–5675. doi: 10.1073/pnas.82.17.5671. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
- Calvert R., Bennett P., Gratzer W. Properties and structural role of the subunits of human spectrin. Eur J Biochem. 1980 Jun;107(2):355–361. doi: 10.1111/j.1432-1033.1980.tb06036.x. [DOI] [PubMed] [Google Scholar]
- Coleman T. R., Harris A. S., Mische S. M., Mooseker M. S., Morrow J. S. Beta spectrin bestows protein 4.1 sensitivity on spectrin-actin interactions. J Cell Biol. 1987 Mar;104(3):519–526. doi: 10.1083/jcb.104.3.519. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Crowl R., Seamans C., Lomedico P., McAndrew S. Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli. Gene. 1985;38(1-3):31–38. doi: 10.1016/0378-1119(85)90200-8. [DOI] [PubMed] [Google Scholar]
- DODGE J. T., MITCHELL C., HANAHAN D. J. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys. 1963 Jan;100:119–130. doi: 10.1016/0003-9861(63)90042-0. [DOI] [PubMed] [Google Scholar]
- Davis J., Bennett V. Brain spectrin. Isolation of subunits and formation of hybrids with erythrocyte spectrin subunits. J Biol Chem. 1983 Jun 25;258(12):7757–7766. [PubMed] [Google Scholar]
- Dieckmann C. L., Tzagoloff A. Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. J Biol Chem. 1985 Feb 10;260(3):1513–1520. [PubMed] [Google Scholar]
- Dubreuil R., Byers T. J., Branton D., Goldstein L. S., Kiehart D. P. Drosophilia spectrin. I. Characterization of the purified protein. J Cell Biol. 1987 Nov;105(5):2095–2102. doi: 10.1083/jcb.105.5.2095. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Elgsaeter A., Stokke B. T., Mikkelsen A., Branton D. The molecular basis of erythrocyte shape. Science. 1986 Dec 5;234(4781):1217–1223. doi: 10.1126/science.3775380. [DOI] [PubMed] [Google Scholar]
- Forster A. C., McInnes J. L., Skingle D. C., Symons R. H. Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acids Res. 1985 Feb 11;13(3):745–761. doi: 10.1093/nar/13.3.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldstein L. S., Laymon R. A., McIntosh J. R. A microtubule-associated protein in Drosophila melanogaster: identification, characterization, and isolation of coding sequences. J Cell Biol. 1986 Jun;102(6):2076–2087. doi: 10.1083/jcb.102.6.2076. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Higgins R. C., Dahmus M. E. Rapid visualization of protein bands in preparative SDS-polyacrylamide gels. Anal Biochem. 1979 Mar;93(2):257–260. doi: 10.1016/s0003-2697(79)80148-7. [DOI] [PubMed] [Google Scholar]
- Itoh N., Slemmon J. R., Hawke D. H., Williamson R., Morita E., Itakura K., Roberts E., Shively J. E., Crawford G. D., Salvaterra P. M. Cloning of Drosophila choline acetyltransferase cDNA. Proc Natl Acad Sci U S A. 1986 Jun;83(11):4081–4085. doi: 10.1073/pnas.83.11.4081. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson D. A., Elder J. H. Antibody directed to determinants of a Moloney virus derived MCF GP70 recognizes a thymic differentiation antigen. J Exp Med. 1983 Nov 1;158(5):1751–1756. doi: 10.1084/jem.158.5.1751. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Litman D., Hsu D. J., Marchesi V. T. Evidence that spectrin binds to macromolecular complexes on the inner surface of the red cell membrane. J Cell Sci. 1980 Apr;42:1–22. doi: 10.1242/jcs.42.1.1. [DOI] [PubMed] [Google Scholar]
- Marchesi V. T. Stabilizing infrastructure of cell membranes. Annu Rev Cell Biol. 1985;1:531–561. doi: 10.1146/annurev.cb.01.110185.002531. [DOI] [PubMed] [Google Scholar]
- Olmsted J. B. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem. 1981 Dec 10;256(23):11955–11957. [PubMed] [Google Scholar]
- Pepinsky R. B. Localization of lipid-protein and protein-protein interactions within the murine retrovirus gag precursor by a novel peptide-mapping technique. J Biol Chem. 1983 Sep 25;258(18):11229–11235. [PubMed] [Google Scholar]
- Sigel M. B., Sinha Y. N., VanderLaan W. P. Production of antibodies by inoculation into lymph nodes. Methods Enzymol. 1983;93:3–12. doi: 10.1016/s0076-6879(83)93031-8. [DOI] [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Speicher D. W., Morrow J. S., Knowles W. J., Marchesi V. T. Identification of proteolytically resistant domains of human erythrocyte spectrin. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5673–5677. doi: 10.1073/pnas.77.10.5673. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
- Yoshino H., Marchesi V. T. Isolation of spectrin subunits and reassociation in vitro. Analysis by fluorescence polarization. J Biol Chem. 1984 Apr 10;259(7):4496–4500. [PubMed] [Google Scholar]
- Young R. A., Davis R. W. Yeast RNA polymerase II genes: isolation with antibody probes. Science. 1983 Nov 18;222(4625):778–782. doi: 10.1126/science.6356359. [DOI] [PubMed] [Google Scholar]