Bindin is a major protein of the sea urchin sperm acrosome granule which mediates the species-specific adhesion and binding of sperm to the egg. Bindin protein has been purified from the sperm of the sea urchin Strongylocentrotus purpuratus (S. purpuratus) and the protein has been partially sequenced. The work presented in this thesis is the isolation and the sequence analysis of bindin cDNA and gene, and the study of the expression of the bindin gene.
A λgt10 cDNA library was constructed from S. purpuratus testes poly(A)+ RNA. The library which was screened with a synthetic DNA probe prepared according to the known protein sequence yielded clones representing bindin cDNA. One of these clones containing an 1873 base pair (bp) insert was sequenced and found to code for a bindin precursor (prebindin) which is twice as large as the mature bindin protein. Upon immunoprecipitation with bindin antibody, the testes poly(A)+ RNA in vitro translation product yields a larger precursor. The bind in cDNA was used to study the tissue specificity of expression. The results show that bindin is a sperm specific protein -- its messenger RNA is not detected in the eggs, ovaries, early embryos, coelomocytes, tubefeet, lantern tissue or intestine that we tested.
Sperm DNA isolated from several individuals was probed with bindin cDNA and reveals that there is one bindin gene per haploid genome. Haploid female coelomocyte DNA also possesses one bindin gene.
Bindin cDNA was used to screen an EcoRI partially digested sea urchin S. purpuratus DNA charon 4A library. A clone containing a 14 kilobase (Kb) insert hybridized to the 3' end of the bindin cDNA. It also has overlapping restriction enzyme recognition sites with the 3' end of the bindin cDNA. There is an intron in the genomic clone upstream of this overlapping region since it did not hybridize with bindin cDNA. A λgt10 mini-genomic library was made and a 1.3 Kb genomic DNA clone which hybridized with bindin cDNA has been characterized and partially sequenced. It contains a 219 bp exon in which the 5' end lies 2 bp upstream of the AUG translation initiation codon. This exon is flanked by introns on either side. Thus, there are at least three introns in the bindin gene.
