Nuclear peptides from calf liver: large scale isolation and fractionation; control of gene expression in cell-free systems, and inhibition of growth of cells in culture.

DNA and nuclear RNA fractions contain small peptides (mol. wt. 600-1500) attached noncovalently. A large scale isolation procedure was developed for the extraction of such peptides directly from the lysed nuclei. Further purification and fractionation was performed with the chromatography on Sephadex, silica gel and H.P.L.C. C18 reverse phase columns. H.P.L.C. fractionation yielded eleven peaks. The peptides are rich in serine, glycine, alanine and acidic amino acids. They do not contain sulfur-containing amino acids. Only occasionally tyrosine, phenyalanine, histidine, arginine, and very moderate amount of lysine are found. These peptides are active in inhibiting gene expression in cell-free systems and incorporation of labeled thymidine in L 1210 murine leukemic cell culture. Thorough and exhaustive analysis demonstrated that the isolated peptides are not degradative products of histone or nonhistone chromosomal proteins.

Identification of messenger RNA for glutamate dehydrogenase using a spectrophotometric probe.

Specific messenger RNA for glutamate dehydrogenase was partially purified from a calf liver polysomal poly(A)-mRNA fraction by sucrose density gradient centrifugation. Enzyme activity in the translational incubation mixture was detected by measuring NADH oxidation in the presence of alpha-ketoglutarate and ammonia as a decrease in absorbency 340-442 nm in a dual wavelength Aminco DW-2 spectrophotometer.

Small peptides bound to polysomal RNA inhibit gene expression in cell-free systems, replication of stimulated lymphocytes and DNA repair in isolated chromatin.

Polysomal poly(A)+-RNA prepared from isolated calf liver polysomes by deproteinization and affinity chromatography on oligo(dT)-Sepharose at pH 6 contains low molecular weight peptides (between 600-1500 daltons) bound noncovalently. These peptides were extracted from the poly(A)+-RNA-peptides complex by precipitation of the nucleic acids with 80% (v/v) ethanol at alkaline pH (9.5) and purified on Sephadex G-25 and G-15 columns. Further fractionation was performed by silica gel chromatography and high performance liquid chromatography (h.p.l.c.). The amino acid composition of the isolated peptidic fraction was compared with similar peptides obtained from rat liver, rabbit reticulocyte and calf thymus polysomes. Effluent (ribosomal) RNA contains only negligible amount of peptides. Isolated polysomal RNA peptides were named "deprimerones" (from Latin "deprimere"), since they have a general depressing effect on gene expression in vitro (Hillar & Przyjemski, 1979). Isolated deprimerones not only inhibit DNA transcription, RNA translation in reconstituted cell-free systems, but also DNA replication by DNA polymerase beta with single- and double-stranded DNA template and synthetic deoxyribonucleotide polymers. The inhibitory effect on replication was correlated with the inhibition of [3H]-deoxyribonucleotide incorporation in isolated chromatin and in stimulated lymphocyte cell cultures. The isolated deprimerones are characterized by similar amino acid compositions in various species.

Nuclear peptides from calf liver: large scale isolation and fractionation; control of gene expression in cell-free systems, and inhibition of growth of cells in culture.

DNA and nuclear RNA fractions contain small peptides (mol. wt. 600 - 1500) attached noncovalently. A large scale isolation procedure was developed for the extraction of such peptides directly from the lysed nuclei. Further purification and fractionation was performed with the chromatography on Sephadex, silica gel and h.p.l.c. C18 reverse phase columns. H.p.l.c. fractionation yielded eleven peaks. The peptides are rich in serine, glycine, alanine and acidic amino acids. They do not contain sulphur-containing amino acids. Only occasionally tyrosine, phenylalanine, histidine, arginine, and very moderate amount of lysine are found. These peptides are active in inhibiting gene expression in cell-free systems and incorporation of labeled thymidine in L 1210 murine leukemic cell culture. Thorough and exhaustive analysis demonstrated that the isolated peptides are not degradative products of histone or nonhistone chromosomal proteins.

Nuclear deprimerones (low molecular weight peptides controlling gene expression) are associated with DNA and nuclear RNA.

Isolated, deproteinized nucleic acids from calf thymus and rat liver nuclei were fractionated on Bio-Gel A-5m into DNA and RNA fractions. Extraction of deproteinized DNA and nuclear RNA with 80% ethanol at pH 9.5 yielded low molecular weight peptides (deprimerones) capable of inhibiting DNA transcription in a cell-free system. The extracted peptidic fractions were further fractionated on a Sephadex G-25 column and collected as a fraction of mol. wt. between 1 500 and 600. The yield of DNA deprimerones was about 30 microgram/mg DNA and of nuclear RNA deprimerones 150-200 microgram/mg RNA (measured by amino acid composition). The nucleoplasm contained a negligible amount of free, soluble peptides (about 18 microgram/g tissue). The DNA and RNA deprimerones were characterized by their amino acid composition and by two-dimensional thin layer chromatography on cellulose gel plates which yielded two major and two minor fractions. DNA deprimerones isolated in this way are very similar, if not identical, to the affinity DNA deprimerones isolated from a nuclear extract on DNA-cellulose but differ from RNA deprimerones. DNA and RNA deprimerones were also compared with the nuclear deprimerones extracted directly from the lysed nuclei. Their amino acid composition is different from both DNA and nuclear RNA deprimerones. Two-dimensional thin layer chromatography on cellulose plates yielded about ten fractions (spots) with various amino acid composition and inhibitory activity for cell-free transcription.

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.

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.

Low molecular weight peptidic fraction in the chromatin from normal and cancer cells: control of transcription.

DNA isolated from cell nuclei by intensive deproteinization with chloroform/isoamyl alcohol and phenol extractons contains a low molecular weight peptidic fraction in a quantity of about 20 micrograms/mg DNA. These peptides were characterized by chromatography on CM-Sephadex, Sephadex G-25, high performance liquid chromatography on microBondapak C18 and amino acid composition. The peptides control transcription in a reconstituted cell-free system with prokaryotic and eukaryotic RNA polymerase and stabilize the structure of double stranded DNA, while increasing its melting point. Their level is markedly decreased (by about 40%) in DNA prepared from tumor cells as compared to normal cell DNA. Transcriptional studies showed only a slightly increased template activity of DNA extracted at pH 9.5 versus DNA extracted at pH 6.0 for DNA preparations from tumor cells. However, there was a marked increase in template activity for DNA preparations treated at pH 9.5 from normal cells--232%, 124%, 97% and 78% for rat liver, mouse liver, mouse thymus and fibroblast L-929 cells, respectively. Also there was no difference in the melting point between these two preparations of DNA from tumor cells; normal cell DNA preparations showed increased melting point of preparations treated at pH 6.5. The data obtained indicate that the loss of low molecular weight peptides from tumor DNA during carcinogenesis is responsible for uncontrolled gene expression observed in cancer.

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.

Glutamate dehydrogenase activity in subcellular fractions of mouse fibrosarcoma.

Glutamate dehydrogenase activity was found to be present at a high level in neoplastic cytosol and microsomes, 3.45 and 9.90 nmoles NADH/min mg protein, respectively. This remains in accordance with the high rate of RNA and protein synthesis in the neoplastic process.

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.

Membrane-bound mitochondrial DNA: isolation, transcription and protein composition.

Mitochondrial membrane-bound DNA complex from bovine heart mitochondria lysed in the presence of Triton X-100 was isolated by differential centrifugation. The yield of "nucleoid" is about 30 microgram protein/mg mitochondrial protein. It contains about 3-5 microgram DNA/mg protein and varying amounts of RNA. The heart mitochondrial nucleoid actively synthesizes RNA. The nucleoid fraction contains about sixteen different proteins as evidenced by urea-SDS gel electrophoresis and about twenty-one proteins as evidenced by acid-urea gel electrophoresis. It appears that the nucleoid is attached to the inner membrane since it does contain cytochromes.

Translation of glutamate dehydrogenase mRNA in a reticulocyte cell-free system.

Messenger RNA template activity for glutamate dehydrogenase was detected in poly(A)-rich RNA extracted from rat liver polysomes. Enzyme synthesized in cell-free reticulocyte system was detected by measuring enzyme activity in the translation incubation mixture using dual wavelength spectrophotometric technique. The translation product was also identified by a partial purification of the labeled synthesized enzyme and by coelectrophoresis with the carrier enzyme preparation from mitochondrial matrix.

Histone inhibition of mitochondrial proton transport.

Histone blocks proton uptake by mitochondria incubated in the presence of valinomycin or DNP. In the presence of DNP valinomycin-induced H+ uptake is not affected by histone. H+ uptake induced by nigericin is not affected by histone as well. Postulated mechanism of histone action involves the immobilization of proton translocation in mitochondrial membrane and induction of local change in H+ concentration, the prevention of the interaction between H+ and natural K+-carrier and Mg2+ transport system or valinomycin.

Histone-produced magnesium extrusion from mitochondria and magnesium binding to histone.

Histone (60 microgram/mg mit. protein) extrudes Mg2+ from mitochondria by 30% with the utilization of endogenous substrates; in the presence of rotenone extrusion drops to about 18%. Dinitrophenol and ADP prevent this effect of histone. Mg2+ extrusion produced by histone depends on histone concentration being at a maximum (100% extrusion) at 107 microgram histone/mit. protein. It was found also that histone alone binds Mg2+ (1.6 nmol Mg2+/microgram histone).

Spectra of glutamate dehydrogenase with diethylstilbestrol.

Glutamate dehydrogenase displays hyperchromicity at 256 nm and at 276 nm upon binding of diethylstilbestrol. Increase in absorbancy is linear at both regions up to 250 micrometer DES, and becomes parabolic at higher concentration of DES. ADP in the presence of DES causes decrease in absorbancy at 256 nm; absorbancy at 276 nm increased by DES is not affected by ADP. DES prevents spectral effects produced by GTP (decrease in absorbancy at 254 nm and at 276 nm). ADP still decreases absorbancy at 254 nm, leaving the 276 nm region unchanged. ADP enhances spectral effects produced by GTP. GTP, however, prevents changes produced by ADP.

Translation of mRNA for glutamate dehydrogenase and spectrophotometric procedure to follow the enzyme biosynthesis.

Heterogeneous poly (A)-mRNA fraction was isolated from rat liver microsomes using phenol-chloroform extraction, millipore filtration and poly (U)-agarose affinity chromatography. Obtained fractions were characterized with respect to their secondary structure and poly (A) content. Isolated poly (A)-mRNA fraction contained high template activity for glutamate dehydrogenase in cell-free systems with microsomes or polysomes. A spectrophotometric procedure to follow enzyme biosynthesis was also developed.

Studies on a rat liver subcellular poly A-rich RNA fraction with glutamate dehydrogenase template activity.

A heterogeneous poly A-mRNA fraction was isolated from rat liver microsomes by phenol:chloroform extraction, millipore filtration, and poly U-agarose affinity chromatography. The fractions were characterized by their secondary structures and poly A contents. From translational studies, the isolated fraction was found to have high glutamate dehydrogenase template activity in cell-free systems containing microsomes or polysomes. A spectrophotometric procedure for following enzyme biosynthesis was also developed.

Hormonal induction of glutamate dehydrogenase in rat liver.

Glutamate dehydrogenase rapidly increases in microsomes and appears in the cytoplasm after administration of cortisone, cAMP, hydrocortisone-acetate. Prolonged administration of ACTH maintains high level of enzyme in the mitochondria and microsomes. Hydrocortisone-acetate, insulin and cortisone decrease drastically enzyme in mitochondria.