Identification of the Sea Urchin Egg Myosin Binding Protein Gene, /

Abstract

A novel myosin binding protein designated 53K (p53EMBP) has been identified in unfertilized sea urchin eggs. (Yabkowitz and Burgess, 1987). Antibody against p53EMBP was used to select recombinant cDNAs from a sea urchin oocyte library. An approximately 1,000 base pair sequence was obtained, a very small fragment of a much larger gene. Based on Southern blot analysis it was found that this gene codes for a very large rnRNA. This indicated that the gene was much larger than the approximately 1,000 base pair sequence that had been obtained thus far. In addition, some data suggest that p53EMBP is a fragment of a much larger protein (Yabkowitz and Burgess, 1987). The goal of this study is to obtain the entire EMBP gene from sea urchin genomic DNA. Sea urchin genomic DNA was isolated from sperm of sea urchin species Stongylocentrotus purpuratus. Following the isolation of genomic DNA, a restriction digest was done to create appropriate ends in the DNA obtained. DNA agarose gel electrophoresis was performed and the proper size fragments were obtained by e1ectroelution of the gel. Vector DNA (A DNA) also prepared by a restriction digest was ligated to the insert genomic DNA. Next, the ligated DNA were packaged into phage particles in vitro and then were used to infect a E.Coli culture. The bacteria containing the genomic library were grown on bacterial lawns in the hopes that plaques would be formed. In continuing work on this project p53EMBP cDNA probes will be used to screen the library. This will enabled us to obtain the gene for the sea urchin myosin binding protein designated p53EMBP.