Regulation of muscle differentiation: cloning of sequences from alpha-actin messenger ribonucleic acid.
Regulation of muscle differentiation: cloning of sequences from alpha-actin messenger ribonucleic acid.
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肌肉分化的调节：α-肌动蛋白信使核糖核酸序列的克隆。
DOI：
10.1021/bi00566a034
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发表时间：
1980
期刊：
影响因子：
2.9
通讯作者：
Dugaiczyk,A
中科院分区：
文献类型：
--
作者：
Schwartz,RJ;Haron,JA;Rothblum,KN;Dugaiczyk,A
Robert J. Schwartz、* Jay A. Haron、Katrina N. Rothblum 和 Achilles Dugaiczyk 摘要：α-肌动蛋白仅存在于骨骼肌组织中，并且似乎在肌生成过程中被诱导。可以使用与α-肌动蛋白信使核糖核酸（mRNA）序列互补的脱氧核糖核酸（DNA）探针来研究诱导肌肉基因转录的机制。这种探针是通过克隆从富含肌动蛋白 mRNA 的胸肌 RNA 制剂转录的互补 DNA (cDNA) 来产生的。将双链DNA插入质粒pBR322的PstI限制性位点，所得杂合DNA分子用于转化大肠杆菌RR1。通过与两种不同的[32P]cDNA制剂杂交来初步筛选细菌菌落，即其中一种含有α-肌动蛋白mRNA及其主要污染物的序列，而另一种仅含有主要污染物序列。通过翻译测定，在质粒 pAC269 中记录了肌动蛋白特异性基因插入的存在。将总肌肉 mRNA 与 pAC269 DNA-纤维素杂交，然后洗脱杂交信息并在 mRNA 依赖性网织红细胞裂解液中进行翻译。与 pAC269 杂交的 mRNA 指导 42 000 Mr 的蛋白质翻译，随后通过电泳迁移率（一维和二维）、脱氧核糖核酸酶 I (DNase I) 亲和力和氰基化肽图谱将其鉴定为肌动蛋白。 pAC269 的限制性核酸内切酶和异源双链体作图检测到 1.4 KB 插入片段，约为之前测量的肌动蛋白 mRNA 长度的 95%。当使用pAC269作为杂交探针时，发现肌肉特异性α-肌动蛋白序列与非肌肉/3-和y-肌动蛋白序列仅具有70％的同源性。在与 pAC269 杂交的肌肉和非肌肉肌动蛋白 mRNA 之间的热熔解中还观察到 13 C 的差异。这些同源性和热熔解的差异将允许在肌发生过程中对肌动蛋白 mRNA 含量进行特异性定量，并且还有助于鉴定α-肌动蛋白天然基因。 e 肌动蛋白是在所有真核细胞中发现的收缩蛋白家族（Garrels & Gibson，1976；Storti 等，1976；
Robert J. Schwartz,* Jay A. Haron, Katrina N. Rothblum, and Achilles Dugaiczyk abstract: a-Actin is found exclusively in skeletal muscle tissue and appears to be induced during myogenesis. The mechanism underlying the induction of muscle gene tran-scription can be studied with a deoxyribonucleic acid (DNA) probe complementary to the a-actin messenger ribonucleic acid (mRNA) sequence. Such a probe was produced by cloning complementary DNA (cDNA) transcribed from a breast muscle RNA preparation enriched for actin mRNA. Double-stranded DNA was inserted into the Pstl restriction site of plasmid pBR322, and the resulting hybrid DNA molecules were used to transform Escherichia coli RR1. Bacterial colonies were preliminarily screened by hybridization to two different [32P] cDNA preparations, ie, one of which contained sequences of a-actin mRNA and its major contaminant, while the other contained only the major contaminant sequence. The presence of an actin-specific gene insert was documented in plasmid pAC269 with translation assays. Total muscle mRNA was hybridized to pAC269 DNA-cellulose, and the hybridized message was then eluted and translated in a mRNA-dependent reticulocyte lysate. The mRNA which hybridized to pAC269 directed the translation of a protein of 42 000 Mr which was subsequently identified as actin by electrophoretic mobility (one and two dimensions), deoxyribonuclease I (DNase I) affinity, and cyanylation peptide mapping. Restriction en-donuclease and heteroduplex mapping of pAC269 detected a 1.4-kilobase insert which is~ 95% of the previously measured length of the actin mRNA. When pAC269 was used as a hybridization probe, it was found that the muscle-specific a-actin sequence hadonly a 70% homology with thenonmuscle/3-and y-actin sequences. Also a difference of 13 C was observed in thermal melts between muscle and nonmuscle actin mRNAs hybridizedto pAC269. These differences in hom-ology and thermal melting will allow the specific quantitation of actin mRNA content during myogenesis and should also aid in the identification of the a-actin natural gene. e actins are a family of contractileproteins found in all eukaryotic cells (Garrels & Gibson, 1976; Storti et al., 1976;
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发表时间：
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期刊：
Journal of immunology (Baltimore, Md. : 1950)
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