Isolation and characterization of prolactin-copy DNA

Abstract

The novel properties of rat pituitary tumor cells (GH-cells) in culture have been utilized to isolate prolactin-copy DNA (cDNA PRL). The cDNA PRL has been characterized by i) polyacrylamide gel electrophoresis ii) by sedimentation on alkaline sucrose gradients and by iii) the kinetic study of the reassociation of “cDNA PRL” with polysomal RNA and with polysomal poly(A) RNA isolated from cells which contain mRNA PRL in abundance and from cells which do not contain any translatable mRNA PRL sequences. The cDNA PRL fraction reassociated with polysomal RNA and polysomal poly(A) RNA isolated from PRL producing cells (PRL +) with pseudo first order kinetics, whereas no significant reassociation was observed when the cDNA PRL was hybridized with the same two RNA fractions isolated from PRL-nonproducing cells (PRL −). The [ 3H] cDNA PRL moved as a sharp band of radioactivity when analyzed by polyacrylamide gel electrophoresis and sedimented in alkaline sucrose gradients in the size range of 6.7s. A mRNA fraction which is highly enriched for PRL-specific mRNA (mRNA PRL) has been isolated from PRL + cells. The cDNA PRL hydridized with mRNA PRL with an eRot 1 2 value of 0.007–0.008. From these results it is calculated that more than 75% of the mRNA PRL enriched fraction contains mRNA PRL sequences.