Novy R E, Lin J L, Lin C S, Lin J J
Department of Biological Sciences, University of Iowa, Iowa City 52242-1324.
Cell Motil Cytoskeleton. 1993;25(3):267-81. doi: 10.1002/cm.970250307.
A tropomyosin-specific oligonucleotide probe (REN29) designed to hybridize to all known human tropomyosin isoforms was used to study tropomyosin mRNA levels in normal and transformed human cells. At least four different sizes of RNAs were detected in normal human fibroblast KD cells by Northern blot analysis. The major bands of 1.1 kb RNA for hTM1 and 3.0 kb RNA for hTM4 were decreased substantially in various transformed cell lines. One of the minor RNA bands (2.0 kb for hTM2 and hTM3) appeared to be absent in a human pancreatic carcinoma cell line. The level of the other minor RNA band (2.5 kb for hTM5) was found to be unchanged or slightly decreased in transformed cells. This differential expression of tropomyosin isoforms at the RNA level was not totally in agreement with the difference in the protein amounts found in normal and transformed cells, suggesting that translational control may also play an important role in the expression of some tropomyosin isoforms. The REN29 probe was further used to screen lambda gt10 and lambda gt11 cDNA libraries, which were constructed from poly(A)+ RNAs of human fibroblast cell lines HuT-14 and WI-38, respectively. In addition to cDNA clones encoding known isoforms, we obtained three classes of new cDNA clones that encode two low M(r) isoforms (hTM5a and hTM5b), and a high M(r) isoform (hTMsm alpha). Sequence comparison revealed that hTM5a and hTM5b are alternatively spliced products derived from the same gene that encodes hTM2 and hTM3. Northern blot analysis and amino acid sequence comparison suggested that the hTMsm alpha represents a smooth muscle tropomyosin which is also expressed in human fibroblasts. The exon specific for, and common to, hTM5a and hTM5b was found to be highly expressed in small intestine. However, there was no detectable expression of this exon in stomach and skeletal muscle. The difference in tissue-specific expression suggests that different isoforms may perform distinct functions in different tissues.
一种旨在与所有已知人类原肌球蛋白异构体杂交的原肌球蛋白特异性寡核苷酸探针（REN29）被用于研究正常和转化的人类细胞中原肌球蛋白mRNA水平。通过Northern印迹分析在正常人成纤维细胞KD细胞中检测到至少四种不同大小的RNA。在各种转化细胞系中，hTM1的1.1 kb RNA主带和hTM4的3.0 kb RNA主带大幅减少。其中一条较小的RNA带（hTM2和hTM3为2.0 kb）在一种人胰腺癌细胞系中似乎缺失。发现另一条较小的RNA带（hTM5为2.5 kb）在转化细胞中的水平未变或略有下降。原肌球蛋白异构体在RNA水平上的这种差异表达与正常细胞和转化细胞中蛋白质含量的差异并不完全一致，这表明翻译控制在某些原肌球蛋白异构体的表达中可能也起着重要作用。REN29探针进一步用于筛选分别由人成纤维细胞系HuT-14和WI-38的poly(A)+ RNA构建的λgt10和λgt11 cDNA文库。除了编码已知异构体的cDNA克隆外，我们还获得了三类新的cDNA克隆，它们编码两种低分子量异构体（hTM5a和hTM5b）以及一种高分子量异构体（hTMsmα）。序列比较显示，hTM5a和hTM5b是源自编码hTM2和hTM3的同一基因的可变剪接产物。Northern印迹分析和氨基酸序列比较表明，hTMsmα代表一种平滑肌原肌球蛋白，它也在人成纤维细胞中表达。发现hTM5a和hTM5b特有的且共有的外显子在小肠中高表达。然而，在胃和骨骼肌中未检测到该外显子的表达。组织特异性表达的差异表明不同的异构体可能在不同组织中发挥不同的功能。
