CDNA CLONING AND CHARACTERIZATION OF NONMUSCLE MYOSIN FROM XENOPUS LAEVIS

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Nonmuscle myosins are a distinct group of myosins found in a variety of eukaryotic cells. Recently, in collaboration with Dr. Igor Dawid (LMG, NICHD), I have cloned overlapping cDNAs representing a nonmuscle myosin gene B from Xenopus Laevis. The sequence shows remarkable conservation between chicken, human and Xenopus myosin heavy chain genes. Messenger RNA for the Xenopus gene is expressed throughout development, starting from unfertilized eggs to the swimming tadpole stage. In adult tissues, mRNA is relatively abundant in lung, heart and brain. No message is detected in skeletal muscle. In the past three months, I have extended the characterization of this protein. Using a peptide antibody raised against the B-specific sequence of the carboxy terminus of the chicken B protein (Takahashi et al., J. Biol. Chem., in press 1992), I have performed immunohistochemistry to localize this protein throughout Xenopus development. The peptide sequence against which the antibody is raised is 99% conserved between Xenopus and chicken. There is only one conservative change from leucine to isoleucine between Xenopus and chicken. Preliminary observations suggest that like mRNA, the protein is also ubiquitous and is expressed in the entire embryo throughout embryogenesis. I have also expressed the carboxy terminus portion of Xenopus nonmuscle myosin in E. coli as a glutathione fusion protein. Currently, I am in the process of affinity purifying the antibody against this fusion protein. The affinity purified antibody will be used for the Western blots on various embryonic and tissue extracts. In the head region of the Xenopus cDNA, there is a 16 amino acid insert. The site of this inserted sequence is highly conserved between Xenopus, chicken and human sequences. However, in chicken, the inserted sequence is nervous system-specific (Ibid). In Xenopus, so far, I have not detected any evidence for the noninserted cDNA. Therefore, in order to obtain precise evidence of alternative splicing, I have cloned, by PCR, the genomic DNA surrounding the insert. Two fragments, 1.4 kb and 1.6 kb, respectively, have been subcloned in Bluescript. Preliminary evidence does suggest some splicing around the insert. Sequence analysis is currently underway to confirm whether or not there is a site for alternate splicing in this region. If this site is found, it will suggest the existence of a noninserted form of the myosin heavy chain, similar to chickens.