Cloning of cDNA for the contact site A glycoprotein of Dictyostelium discoideum

Abstract

A cell surface glycoprotein of Dictyostelium discoideum with M(r) 80,000 (gp80) has been shown to mediate the formation of the developmentally acquired EDTA-resistant cell-cell binding sites termed contact sites A. We have isolated cDNA clones encoding gp80 by immunological screening of an expression library prepared in Escherichia coli. Double-stranded cDNA was prepared from poly(A)(+) RNA isolated from cells at 8 hr of development and cloned into the bacteriophage expression vector lambdagt11. Two recombinant phages containing cDNA inserts of 1.2 and 0.8 kilobases were isolated and shown to contain sequences coding for gp80 by the immunoselect assay. Partial DNA sequence analysis also confirmed that one of these cDNA clones, lambdaDdgp80c-19, contained the coding sequence for the amino terminus of gp80. DNA-RNA hybridization showed that the insert of lambdaDdgp80c-19 hybridized to a single mRNA transcript of approximately 2.0 kilobases. gp80 mRNA became detectable after 6 hr of development, reached its maximum level at 9 hr, and dropped to a negligible level by 15 hr. This pattern of mRNA accumulation corresponded closely to that of gp80 synthesis in D. discoideum cells, suggesting that gp80 expression is regulated at the transcriptional level.