CDNA CLONING AND CHARACTERIZATION OF NONMUSCLE MYOSIN FROM XENOPUS LAEVIS

非肌肉肌球蛋白是在多种肌球蛋白中发现的一组独特的肌球蛋白。 真核细胞 最近，与Igor Dawid博士（LMG， NICHD），我已经克隆了代表非肌肉肌球蛋白的重叠cDNA 非洲爪蟾B基因。 序列显示出显著的保守性 鸡、人和非洲爪蟾肌球蛋白重链基因之间的差异。 信使 非洲爪蟾基因的RNA在整个发育过程中表达， 从未受精的卵到游泳的蝌蚪阶段。 在成人组织中， mRNA在肺、心脏和脑中相对丰富。 没有消息被 在骨骼肌中检测到。 在过去的三个月里， 这种蛋白质的特性。 使用一种肽抗体 针对鸡B羧基端的B特异性序列 蛋白质（Takahashi等，J. Biol. Chem.，1992年出版），我 进行免疫组织化学定位这种蛋白质在整个 非洲爪蟾的发展。 抗体所针对的肽序列是 在非洲爪蟾和鸡之间有99%的保守性。 只有一 从亮氨酸到异亮氨酸的保守变化在爪蟾和 鸡 初步观察表明，与mRNA一样， 也是普遍存在的，在整个胚胎中表达， 胚胎发生 我还表达了 非洲爪蟾非肌肉肌球蛋白E.大肠杆菌作为谷胱甘肽融合蛋白。 目前，我正在进行抗体的亲和纯化 对抗这种融合蛋白 亲和纯化的抗体将是 用于各种胚胎和组织提取物的蛋白质印迹。 在 在非洲爪蟾cDNA的头部区域，有一个16个氨基酸的插入片段。 该插入序列的位点在非洲爪蟾， 鸡和人的序列 然而，在鸡肉中， 是神经系统特异性的（同上）。 到目前为止，在爪蟾中， 检测到任何未插入cDNA的证据。 因此为了 为了获得选择性剪接的精确证据，我通过PCR克隆了， 插入物周围的基因组DNA。 两个片段，1.4 kb和1.6 kb kb，已分别在Bluescript中亚克隆。 初步 有证据表明插入物周围有拼接 序列 目前正在进行分析，以确认是否有一个网站， 选择性剪接的基因 如果这个网站被发现，它将 表明存在非插入形式的肌球蛋白重链， 就像鸡一样。

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.