Open AccessCloning of cDNA for the contact site A glycoprotein of Dictyostelium discoideum
Author(s) -
Lu Min Wong,
ChiHung Siu
Publication year - 1986
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.83.12.4248
Subject(s) - dictyostelium discoideum , complementary dna , biology , microbiology and biotechnology , rna , cdna library , coding region , messenger rna , molecular cloning , dna , expression cloning , gene , biochemistry
A cell surface glycoprotein ofDictyostelium discoideum withM 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 inEscherichia 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 λgt11. 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, λDdgp80c-19, contained the coding sequence for the amino terminus of gp80. DNA-RNA hybridization showed that the insert of λDdgp80c-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 inD. discoideum cells, suggesting that gp80 expression is regulated at the transcriptional level.