CDNA CLONING AND CHARACTERIZATION OF NONMUSCLE MYOSIN FROM XENOPUS LAEVIS

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Forkhead domain containing proteins are a distinct class of DNA binding proteins found in a variety of organisms from Drosophila to humans. At least 10 different members of the forkhead family are known to date. These include some lung- and/or liver-specific proteins as well as ubiquitously expressed forkhead proteins. In Xenopus, two development- specific forkhead genes have been cloned. Recently, in collaboration with Dr. Milan Jamrich of FDA, we have cloned a novel class of forkhead genes from Xenopus using degenerate primer PCR. Initially, a PCR fragment was cloned from DG library. Using this fragment as probe, a full-length CDNA clone, F3, was isolated from stage 13 library. The CDNA was sequenced on both strands using Sequenase version 2.0. The sequence analysis reveals a 483 amino acid protein with a putative DNA binding domain of 77 amino acids. These 77 amino acids are highly conserved between different forkhead proteins. The DNA binding domain is followed by some basic residues. The full-length CDNA was used to probe Northern blots of RNA prepared from various developmental stages. A 2.3 kb transcript is first detected at mid-stage 10, that is after onset of gastrulation. The transcript remains constant until stage 40. Whole mount in situ hybridization was performed using full-length CDNA as a probe. Around stage 13, the transcript is localized in presomitic mesoderm and, in later stages, it appears only in newly formed somites. Since a strong band is detected at mid-stage 10, an animal cap induction assay was performed. Animal caps were dissected from stage 8 (blastula) embryos and incubated in buffer with or without activin. RNA was prepared after four hours from both sets and used for Northern blots. The blot was probed with the fragment containing the c-terminus coding region of the F3 clone. F3 RNA is detected only in animal caps treated with activin. Further experiments to characterize the expression of F3 protein and its relation to other muscle-specific transcription factors are presently underway.