Makabe Kazuhiro W, Satoh Noriyuki
Department of Zoology, Kyoto University, Kyoto 606, Japan and Asamushi Marine Biological Station, Tohoku University, Aomori 039-34, Japan.
Dev Growth Differ. 1989 Feb;31(1):71-77. doi: 10.1111/j.1440-169X.1989.00071.x.
A muscle-specic monoclonal antibody, termed Mu-2, recognizes a single 220-kd polypeptide which first appears in early tailbud-stage embryos of the ascidian Halocynthia roretzi (17). In the present investigation, a cDNA library was prepared from poly(A) RNA isolated from tailbud embryos. The resulting cDNAs were inserted into the expression vector γgt11, and the library was screened with the Mu-2. One of positive clones, containing an insert about 1.6-kb in length, was selected and subcloned. Partial sequence analyses at the 5' terminus and at the 3' terminus of the 1.6-kb cDNA allowed the amino acid sequence. The deduced sequence showed extensive homology to the rodent myosin heavy chain protein. Using an antisense RNA probe generated from the cDNA, Northern blot hybridization was carried out to measure the level of the myosin heavy chain gene transcripts during normal embryogenesis. These transcripts were not detected at pregastruia stages, after which they accumulated rapidly.
一种名为Mu-2的肌肉特异性单克隆抗体可识别一条单一的220-kd多肽,该多肽最早出现在海鞘(Halocynthia roretzi)尾芽期早期胚胎中(17)。在本研究中,从尾芽胚胎中分离的聚腺苷酸RNA制备了一个cDNA文库。将得到的cDNA插入表达载体γgt11中,并用Mu-2筛选该文库。选择了一个阳性克隆,其插入片段长度约为1.6 kb,并进行了亚克隆。对1.6 kb cDNA的5'末端和3'末端进行部分序列分析,得到了氨基酸序列。推导的序列与啮齿动物肌球蛋白重链蛋白具有广泛的同源性。使用从该cDNA产生的反义RNA探针,进行Northern印迹杂交以测量正常胚胎发育过程中肌球蛋白重链基因转录本的水平。在原肠胚形成阶段之前未检测到这些转录本,之后它们迅速积累。