CDNA CLONING AND CHARACTERIZATION OF NONMUSCLE MYOSIN FROM XENOPUS LAEVIS

叉头结构域蛋白是一类独特的DNA结合蛋白， 在从果蝇到人类的各种生物中发现的蛋白质。 在 迄今为止已知叉头家族的至少10种不同成员。 这些包括一些肺和/或肝特异性蛋白以及 广泛表达的叉头蛋白。 在非洲爪蟾中，有两个发展- 已经克隆了特定的叉头基因。 最近，在合作 与FDA的Milan Jamrich博士合作，我们克隆了一类新的叉头 用简并引物PCR法从非洲爪蟾中扩增出10个目的基因。 首先，PCR 从DG文库中克隆了该片段。 使用此片段作为探针， 从第13阶段文库中分离全长cDNA克隆F3。 的cdna 使用Sequenase 2.0版对两条链进行测序。 序列 分析揭示了一个483个氨基酸的蛋白质，具有推定的DNA结合 77个氨基酸的结构域。 这77个氨基酸高度保守 不同的叉头蛋白之间的差异。 DNA结合结构域如下： 一些碱性残留物。 全长cDNA用于探针北方 从不同发育阶段制备的RNA印迹。 A 2.3 kb 转录本首先在中期10检测到，即在转录起始后， 原肠胚形成 转录本保持不变，直到阶段40。 整个 使用全长cDNA作为探针进行原位杂交。 探针 在第13期左右，转录本定位于体前区， 中胚层，在后期，它只出现在新形成的体节。 由于在第10阶段中期检测到强条带， 进行测定。 从第8阶段（囊胚）解剖动物帽 胚胎并在含有或不含激活素的缓冲液中孵育。 将rna 在4小时后从两组制备并用于北方印迹。 用含有C-末端编码的片段探测印迹。 F3克隆区。 F3 RNA仅在处理的动物帽中检出 激活素。 表征F3表达的进一步实验 蛋白质及其与其他肌肉特异性转录因子的关系 正在进行中

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.