The third and fourth tropomyosin isoforms of Caenorhabditis elegans are expressed in the pharynx and intestines and are essential for development and morphology
The third and fourth tropomyosin isoforms of Caenorhabditis elegans are expressed in the pharynx and intestines and are essential for development and morphology
复制标题
DOI：
10.1006/jmbi.2001.5052
复制
发表时间：
2001-10-26
影响因子：
5.6
通讯作者：
Kagawa, H
中科院分区：
文献类型：
--
作者：
Anyanful, A;Sakube, Y;Kagawa, H
秀丽隐杆线虫原肌球蛋白基因tmy-1/lev-11全长14.5kb，通过选择性剪接编码3种异构体。为了鉴定、表征和比较第四种亚型的基因组和组织表达，采用快速扩增cDNA末端和显微注射lacZ和gfp融合质粒的技术。我们阐明了CeT-MIV，tmy-1的第四种亚型，其编码256个残基的多肽。CeTMIV同种型具有与CeTMIII同种型相似的启动子区，但被选择性剪接以产生在两个外显子中不同的cDNA。tmy-1：：lacZ和tiny-1：：gfp融合基因在咽和肠细胞中表达，其中tmy-1：：lacZ和tiny-1：：gfp融合基因具有3.2kb启动子序列和1.1kb CeTMIV亚型特异性外显子。序列的进一步单向缺失位于起始密码子上游的初级启动子区853。我们发现在上游区域，存在B和C亚元件样序列的myo-2，这可能是用来刺激咽部的表达。尽管存在一个ges-1样序列，我们无法定位肠表达所需的两个加塔位点。用新构建的融合质粒重新评估CeTMIII同种型的组织表达，我们显示除了咽表达之外，在种系组织和肠细胞中进一步表达。最后，为了证明原肌球蛋白对发育是必不可少的，我们通过RNA介导的干扰使体壁和咽特异性亚型失活。除了50- 75%的胚胎致死率外，在体壁干扰中存活的蠕虫具有异常的身体形态和不协调的运动，而在咽干扰中存活的蠕虫具有变形的咽和肠道区域。这些结果显示了原肌球蛋白在正常肌丝组装和胚胎发育中的功能，并阐明了原肌球蛋白异构体在C.优美的(C)北京：科学出版社.
The tropomyosin gene tmy-1/lev-11 of Caenorhabditis elegans spans 14.5 kb Science and Technology and encodes three isoforms by alternative splicing. To identify, characterize and compare the genome and tissue expression of a fourth isoform, the technique of rapid amplification of cDNA ends and microinjection with lacZ and gfp fusion plasmids were employed. We elucidated CeT-MIV, a fourth isoform of tmy-1, which encoded a 256 residue polypep tide. CeTMIV isoform had a similar promoter region to CeTMIII isoform, but was alternatively spliced to generate a cDNA that differed in two, exons. The tmy-1::lacZ and tiny-1::gfp fusion genes, with 3.2 kb promoter sequence and 1.1 kb of CeTMIV isoform specific exons, were expressed in the pharyngeal and intestinal cells. Further unidirectional deletion of the sequence located the primary promoter region 853 by upstream from the initial codon. We show within the upstream region, the presence of B and C subelement-like sequences of myo-2, which may be used to stimulate pharyngeal expression. Despite the presence of a ges-1 like sequence, we were unable to locate the two GATA sites required for intestinal expression. Reassessing tissue expression for CeTMIII isoform with newly constructed fusion plasmids, we showed further expression in germ-line tissue and intestinal cells in addition to pharyngeal expression. Finally, to demonstrate that tropomyosin is essential for development, we inactivated the body wall and pharynx-specific isoforms by RNA-mediated interference. In addition to 50-75 % embryonic lethality in both cases, the worms that survived body wall interference had abnormal body morphology and uncoordinated movements, and those that survived pharynx interference had deformed pharynges and gut regions. These results show the function of tropomyosin in normal muscle filament assembly and embryonic development, and illustrate the different expression patterns characteristic of tropomyosin isoforms in C. elegans. (C) 2001 Academic Press.