CDNA CLONING AND CHARACTERIZATION OF NONMUSCLE MYOSIN FROM XENOPUS LAEVIS

含有叉头结构域的蛋白质是一类不同的DNA结合 存在于从果蝇到人类的各种生物体中的蛋白质。在… 到目前为止，叉头家族中至少有10个不同的成员是已知的。 这些蛋白质包括一些肺和/或肝脏特异的蛋白质以及 普遍表达的叉头蛋白。在非洲爪哇，有两项发展-- 特定的叉头基因已经被克隆出来。最近，在协作中 与FDA的米兰·贾姆里奇博士合作，我们克隆了一种新型叉头 利用简并引物聚合酶链式反应分析非洲爪哇基因。最初，一种聚合酶链式反应 从DG文库中克隆了该片段。使用该片段作为探针，一个 从Stage 13文库中分离到全长CDNA克隆F3。CDNA 在两条链上使用Sequenase Version 2.0进行测序。该序列 分析显示，一个483个氨基酸的蛋白质可能与DNA结合 77个氨基酸组成的结构域。这77个氨基酸高度保守 在不同的叉头蛋白之间。接下来是DNA结合域 由一些碱性残基组成。全长CDNA用于探针对Northern 从不同发育阶段制备的RNA的斑点。A 2.3 KB 转录首先在阶段10中期检测到，也就是在发病之后 原肠形成。成绩单保持不变，直到阶段40。整体 安装原位杂交使用全长的cDNA作为 探测器。在第13阶段左右，转录本定位于早熟 中胚层，在后期，只出现在新形成的体节中。 由于在阶段10中期检测到较强的条带，因此动物帽诱导 进行化验。从第8期(囊胚期)解剖动物头盖骨 胚胎，在含有或不含有激活素的缓冲液中孵化。核糖核酸是 两组都在四个小时后准备好，用于Northern印迹。 用含有c-末端编码的片段进行杂交检测 F3克隆的区域。仅在处理过的动物帽子中检测到F3RNA 用激活素。进一步实验鉴定F3的表达 蛋白质及其与其他肌肉特异性转录因子的关系 目前正在进行中。

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.