Studies on nuclear deprimerones: isolation, fractionation, characterization and control of gene expression.

A procedure was developed for the isolation of low molecular weight peptides (deprimerones) from calf thymus nuclei and other tissues. These peptides are active in controlling transcription and translation in cell-free systems, and stabilize the double stranded structure of DNA. The procedure involves extraction of nuclei with 80% ethanol at pH 9.5 and fractionation of the extracted peptides on Sephadex G-25. The isolated deprimerones represent heterogenous peptidic fractions which can be separated into four activity peaks on Sephadex G-25 column and into ten peptidic spots on two-dimensional cellulose gel thin layer chromatography. One of the peaks is identical to a chromatin peptidic fraction isolated previously by binding to DNA-cellulose. The deprimerones contain about 8 or 9 different amino acids. It was demonstrated that the cytoplasm contains about 10% of the deprimerones present in nuclei. They are also ubiquitous, since they were found in each of the tissues studied: calf thymus, mouse thymus, mouse spleen and mouse liver.

Poly(A)-mRNA deprimerones in rat liver and Novikoff hepatoma cells.

The poly (A)-mRNA fraction isolated by chloroform deproteinization of liver polysomes and poly(U)-Sepharose chromatography contains a low molecular weights (congruent to 1000) peptidic fraction. The peptides which we suggested to call deprimerones (1) were extracted with 80% ethanol at pH 9.5; after ethanol evaporation, they were purified on Sephadex G-25 column as a fraction of mol. wt. between 1600 and 600, yielding about 9 mg/mg mRNA. If deproteinization is performed with phenol-chloroform the yield is about 2 mg/mg mRNA. In Novikoff hepatoma the yield of the same preparation is only 2.7 mg/mg mRNA (about 70% decrease). The obtained deprimerones are active in inhibiting transcription of thymus DNA with E. coli RNA polymerase and [3H]-GTP by about 90% at a ratio peptide/DNA = 2. For comparison the deprimerones obtained previously (2) by extraction of deproteinized DNA inhibit transcription only by about 50% at the same peptide/DNA ratio. The results demonstrated a decrease of the poly (A)-mRNA deprimerone level during carcinogenesis and further support the previously demonstrated specific occurrence of deprimerones with poly(A)-mRNA. They remain in accordance with and provide further support for the deprimerone theory of carcinogenesis postulated earlier (1).

Nuclear deprimerones in rat liver and Novikoff hepatoma.

Extraction of total low molecular weight peptides controlling transcription (deprimerones) from rat liver and Novikoff hepatoma nuclei was perfromed with 80% ethanol at pH 9.5. The extracted material was fractionated on a Sephadex G-25 column and active peptidic fractions were collected as Sephadex fraction II of mol. wt. between 1600 and 600. The yield of this peptide fraction was 600 micrograms/mg DNA from rat liver nuclei and 200 micrograms/mg DNA from Novikoff hepatoma nuclei. The results confirm a previously found decrease in chromatin deprimerones in tumor cells using different experimental approach and remain in accordance with the postulated deprimerone theory of carcinogenesis.

Inhibition of exogenous mRNA translation in a cell-free system by chromatin peptides from calf thymus.

Low molecular weight chromatin peptides isolated from calf thymus by affinity chromatography on DNA-cellulose inhibit significantly translation of exogenous isolated mRNA in a reticulocyte cell-free system. Translation with endogenous mRNA present in the system is not inhibited by low peptide concentrations. The data obtained combined with the previous findings suggest that chromatin peptides control gene expression at two levels: transcription and translation.

Control of transcription and translation by low molecular weight peptides (deprimerones) from chromatin and poly(A)-messenger RNA. Implication in the mechanism of carcinogenesis.

Poly(A)-mRNA isolated by phenol/chloroform extraction of rat liver polysomes, subtilism digestion, and poly(U)-Sepharose chromatography, contains a low molecular weight (approx. 1000) peptidic fraction. The peptides were extracted from a poly(A)-mRNA fraction by treatment with 80% ethanol; after ethanol evaporation they were purified on a Sephadex G-25 column and high-performance liquid chromatography on muBondapak C18. The isolated peptides were analyzed by cellulose gel thin-layer chromatography, high-pressure liquid chromatography and their amino acid composition was determined. They were compared with a chromatin peptidic fraction isolated from calf thymus nucleic by affinity chromatography on DNA-cellulose or on Sephadex G-25 column. Both groups of peptides from chromatin and from poly(A)-mRNA bind to the purified DNA thereby increasing its melting point; they significantly inhibit DNA transcription and RNA translation in reconstituted cell-free, peptide-free systems. It is suggested that these peptides are endogenous natural regulatory substances controlling gene expression in eucaryotic cells. We propose to name these regulatory peptides 'deprimerones' (from Latin 'deprimere') and describe various fractions of them as chromatin deprimerones, messenger deprimerones, gene deprimerones (for specific genes). Loss or decreased level of these deprimerones during the promotion of carcinogenesis is responsible for uncontrolled gene expression observed in cancer.