Analysis of the in Vitro Translation Product of mRNA Coding for Chick Intestine Apolipoprotein A-I
In all animal species studied so far. apolipoprotein A-I, the main constituent peptide of plasma HDL, is present in plasma as a family of isoforms that have the same molecular weight but different isoelectric points. After in vitro translation of chick intestine mRNA, analysis of the primary translation product of immunoprecipitated apo A-I, by two-dimensional gel electrophoresis, also reveals the presence of at least three isoforms. The origin of these isoforms is still unclear. It may reflect the presence of different mRNAs, each encoding a single apo A-I isoform, or derive from posttranslational modifications of a single translation product. To answer this question we first isolated a “full-size” cDNA representing the coding sequence of chick intestinal mRNA by screening a cDNA library in pEX-1 expression vector. The cDNA insert was then subcloned in p-GEM 1 vector and transcribed with SP 6 polymerase. This in vitro transcribed RNA was then translated in a cell-free system in the presence of [35S]methionine. The primary translation product was immunoprecipitated with anti-apo A-I antiserum and subjected to two-dimensional gel electrophoresis. The isoform pattern of this translation product was found to be identical to that observed after translation of total RNA extracted from chick intestinal mucosa. This finding indicates that the presence of multiple isoforms in the primary translation product of chick intestine apo A-I appears to result from posttranslational modification of a single translation product.
KeywordsTranslation Product cDNA Insert Multiple Isoforms mRNA Code Primary Translation Product
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- Blue, M. L., Ostapchuck, P., Gordon, J. S., and Williams, D. L., 1982, Synthesis of apolipoprotein A-I by peripheral tissues of the rooster, J. Biol. Chem. 257:11151–11159.Google Scholar
- Calandra, S., Tarugi, P., and Ghisellini, M., 1984, Separation of the isoprotein forms of apoprotein A-I of rat, rabbit and human HDL by combined isoelectricfocusing and polyacrylamide gel electrophoresis, Atherosclerosis 50:209–221.Google Scholar
- Chapman, M. J., 1980, Animal Lipoproteins: Chemistry, structure and comparative aspects, J. Lipid Res. 21:789–853.Google Scholar
- Ferrari, S., Tarugi, P., Battini, R., Ghisellini, M., and Calandra, S., 1986b, in: Apolipoprotein Mutants and Atherosclerosis (C. Sirtori and G. Franceschini, eds.) Plenum Press, New York (in press).Google Scholar
- Ghiselli, G., Rohde, M. F., Tanenbaum, S., Krishnan, S., and Gotto, A. M., 1985, Origin of apolipoprotein A-I polymorphism in plasma, J. Biol. Chem. ,260:15662–15668.Google Scholar
- Hermier, D., Forgez, P., and Chapman, M. J., 1985. A density gradient study of the lipoprotein and apolipoprotein distribution in the chicken, Gallus domesticus, Biochim. Biophys. Acta ,836:105–118.Google Scholar
- Shackelford, J. E., and Lebherz, H. G., 1983a. Synthesis and secretion of apolipoprotein A-I by chick breast muscle, J. Biol. Chem. ,258:7175–7180.Google Scholar
- Shackelford, J. E., and Lebherz, H. G., 1983b. Regulation of apolipoprotein A-I synthesis in avian muscles, J. Biol. Chem. ,258:14829–14833.Google Scholar