Isolation and Characterization of Full-Length cDNA Clones for Human α-, β-, and γ-Actin mRNAs: Skeletal but Not Cytoplasmic Actins Have an Amino-Terminal Cysteine that Is Subsequently Removed

作者：Peter William Gunning, Phyllis A. Ponte, Hiroto Okayama, Joanne N. Engel, Helen M. Blau, Larry Kedes · 发表于：Molecular and Cellular Biology · 年份：1983 · DOI：10.1128/mcb.3.5.787-795.1983 · 被引用次数：1211 · 研究领域：Viral Infectious Diseases and Gene Expression in Insects、Virus-based gene therapy research、RNA and protein synthesis mechanisms

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis...