AI Article Synopsis
- Poly(A)-RNAs from rat livers treated with hydrocortisone and cycloheximide were refined and used to create double-stranded cDNAs, which were then cloned into E. coli.
- Seven cloned cDNAs were linked to mRNAs in liver that respond to steroid treatment, with two showing rapid induction by insulin, cAMP, and hydrocortisone.
- The regulated mRNA encodes a 53 kDa protein, undergoes quick degradation in cells, and is derived from a single copy gene that shows increased transcription in response to hormones.
Article Abstract
Poly(A)-RNAs were prepared from livers of rats treated with hydrocortisone and cycloheximide, then enriched for large mRNAs by successive sucrose gradients and gel electrophoresis. The size-selected RNAs were used as templates for synthesis of double-stranded cDNAs that were cloned in Escherichia coli using the pBR322 plasmid vector. Recombinant plasmids characterized as carrying inserts of potential interest were further analyzed by differential hybridization to mRNAs from untreated and hydrocortisone-treated rats. Seven of the cloned cDNAs were identified as complementary to mRNAs whose content in liver is sensitive to modulation by the steroid. Further screening for hormonal responsiveness revealed that two of the cloned cDNAs hybridize to a 3.4-kilobase mRNA that is rapidly induced in liver by treatment with insulin or cAMP as well as by hydrocortisone; restriction enzyme analysis demonstrated that the cDNAs from these two clones are derived from the same mRNA. In isolated nuclei, the rate of transcription of this mRNA is increased by each of the inducing hormones. This unusually regulated mRNA codes for a protein of 53 kDa on denaturing gels, undergoes rapid intracellular degradation that is prevented by cycloheximide, and appears to be the product of a single copy gene.
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