Molecular cloning and characterization of a developmentally regulated sea urchin gene expressed in the embryonic ectoderm

Abstract

A developmentally regulated gene expressed in the embryonic ectoderm of the Hawaiian sea urchin, Tripneustes gratilla, was characterized by molecular cloning and analyzing representative cDNA and genomic DNA sequences. A previously identified 0.5 kb cDNA was used to isolate a 2.0 kb and 3.1 kb cDNA. Nonoverlapping probes from the three cDNAs were hybridized to Northern transfers of pluteus embryo poly-A RNA to detect messages of 5.8 kb, 2.1 kb, and 1.6 kb that have relative abundance ratios of one, fifty, and five, respectively. Northern transfers in conjunction with sequence analysis of the cDNAs and genomic clones indicated that the three mRNAs overlap and are most likely transcribed from the same gene. The three messages have long 3' untranslated sequences. The two longer mRNAs, and most likely the smaller mRNA, contain repeats in the 3' untranslated sequences which are present in the genome but not in other mRNAs. The most abundant 2.1 kb mRNA was present in prism stage embryo polysomes and released from polysomes by EDTA which suggest that this mRNA is translated. Sequence analysis of cDNAs complementary to the 2.1 kb mRNA and of genomic clones containing its transcription unit reveals an open reading frame encoding an 87 amino acid peptide. This hypothetical translation product contains a 16 amino acid hydrophobic leader followed by a peptide cleavage site and a conspicuous stretch of 6 consecutive arginines. A comparative search of the protein database with this peptide identified no extensive homology to known proteins. The hypothetical peptide minus the hydrophobic leader was expressed as a fusion protein to the carboxyl terminus of tryptophan synthetase in ,g. coli. Three genomic clones spanning 34 kb of the gene region were isolated, restriction mapped, and mRNA encoding regions were partially sequenced. This gene, named i21 after its previously identified cDNA, is a complex transcriptional unit coding for at least three different mRNAs. The transcription initiation site for the 2.1 kb mRNA was determined by primer extension of prism embryo poly-A RNA. The transcription unit for the 2.1 kb mRNA extended 12 kb and consisted of three exons of 146 bp, 69 bp, and 1836 bp separated by two introns of 6.5 kb and 3.0 kb. Sequence analysis located CAAT and TATA boxes in the 5' promoter region, confirmed exons and introns, and identified polyadenylation signals and sites. The transcription unit for the 1.6 kb mRNA most likely mimics that of the 2.1 kb mRNA but utilizes a slightly further 5' initiation site and an earlier polyadenylation signal. The 5.8 kb mRNA terminated further 3' than the 2.1 kb mRNA, and it is suspected to initiate at a different upstream promoter and/or be spliced differently.