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. 1980 Feb;77(2):765–769. doi: 10.1073/pnas.77.2.765

Cloning of sea urchin actin gene sequences for use in studying the regulation of actin gene transcription

Glenn T Merlino *, Robert D Water *, John P Chamberlain *, David A Jackson †, M Raafat El-Gewely *, Lewis J Kleinsmith *,‡
PMCID: PMC348361  PMID: 6928677

Abstract

In order to investigate the regulation of actin gene transcription during early sea urchin development, a specific hybridization probe for actin sequences is required. Such a probe was produced by cloning cDNA transcribed from a sea urchin poly(A)-containing mRNA preparation enriched for actin message. Double-stranded DNA was ligated into the BamHI restriction site of plasmid pBR322, and the resulting hybrid molecules were used to transform the Escherichia coli strain ML100. After preliminary screening of bacterial colonies by antibiotic sensitivity and hybridization back to the original cDNA, clones containing sea urchin DNA were further characterized by a positive translation assay in which total sea urchin mRNA was hybridized to plasmid, and the hybridized message then was eluted and translated in a reticulocyte cell-free protein-synthesizing system. In this way, one clone (pSA38) was found to hybridize selectively to sea urchin mRNA coding for a protein of 43,000 daltons. This protein was identified as actin by three criteria: electrophoretic migration in two-dimensional polyacrylamide gels, affinity for DNase I, and peptide mapping. Restriction endonuclease and heteroduplex mapping of pSA38 indicate that it contains a 1.5-kilobase-pair insert and is therefore likely to contain a large portion of the actin coding sequence. By using pSA38 as a hybridization probe, it has been found that the level of actin-specific RNA sequences increases dramatically during early sea urchin development.

Keywords: recombinant plasmid, positive hybridization-translation, restriction mapping, Southern hybridization, heteroduplex analysis

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