Increased Template Activity Research Articles

Identification of regulatory sequences contained in the 5'-flanking region of Drosophila lysine tRNA2 genes.

Transcription of Drosophila tRNA genes is controlled by signals within and outside the region coding for the mature tRNA. Deletion analysis has revealed an oligonucleotide sequence in the 5'-flanking region of a Drosophila tRNA2Lys gene to be responsible for the poor transcriptional activity of this and of other tRNA genes. The low template activity of this gene was maintained even after deletion of the 5'-flanking region up to nucleotide -23. Removal of nine additional nucleotides resulted in complete loss of transcriptional repression. The oligonucleotide responsible for transcriptional repression is GGCAGTTTTTG and is located 13 nucleotides upstream from the mature tRNA coding sequence. Since the sequence of the undecanucleotide is well conserved within the 5'-flanking region of all known Drosophila tRNA2Lys genes, we have investigated why the transcription of all these genes is not similarly repressed. Deletion or insertion of nucleotides between the mature tRNA coding region and this oligonucleotide resulted in tRNA genes with increased template activity. This observation suggests that the position of this oligonucleotide relative to some element downstream influences the extent of transcriptional repression.

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.

Binding of N-hydroxy acetylaminofluorene to eu- and heterochromatin fractions of rat liver in vivo

The in vivo binding of N-hydroxy acetylaminofluorene (N-OH AAF) to rat liver DNA was studied in hetero- and euchromatin fractions prepared by sedimentation through sucrose gradients. The greater transcriptional capacity of the slowly sedimenting (euchromatin) fractions was confirmed by their enhanced incorporation of radioactive precursors into RNA in vivo and their increased template activity for in vitro RNA synthesis by purified RNA polymerase. N-OH AFF was bound in 4- to 5-fold greater amounts to euchromatin DNA than to heterochromatin 2 h after a single injection of the compound. However, the bound carcinogen appeared to be eliminated more rapidly from euchromatin than from heterochromatin by DNA repair processes.

Increased template activity in injured tissue

Nuclei and chromatin, isolated from control and injured (1 hr at 37°C in saline) mouse kidneys, were tested for template activity by measuring RNA synthesis with E. coli RNA polymerase. The template activity of both the nuclei and the chromatin isolated from the ischemic kidneys was increased. Removing protein with 0.7 M NaCl from the chromatin eliminated this difference in template activity. The size of the RNA produced on the control and ischemic templates was the same as measured by polyacrylamide gel electrophoresis. The difference in template activity persisted under conditions in which reinitiation of the RNA polymerase was blocked. These results are consistent with an increased number of E. coli RNA polymerase binding sites in injured cells.

Increased template activity in chromatin from cadmium chloride treated pea tissues

Increases in the synthesis of the isoflavonoid, pisatin, and the activity of phenylalanine ammonia lyase (PAL) are induced in excised pea pods by low concentrations (5×10 −4M) of CdCL 2. The induction of pisatin synthesis and PAL are suppressed if RNA-synthesis-inhibiting concentrations of 6-methyl purine, actinomycin D or ∝-amanitin are applied within 1 h of inducer application. Cycloheximide (0.1 mg/ml) blocks the induction of these responses if applied to tissues within 6 h after inducer application. Within 1 h after CdCl 2 (5×10 −4M) is applied to pods there is an increase in the rate of synthesis of all sizes of RNA as well as an increase in the template activity and dye binding capacity of pea chromatin. The results support the hypothesis that conformational changes in DNA are associated with the induction process.

Protein phosphokinases of chick muscle: Changes during embryonic development

Protein phosphorylation is a widespread phenomenom [l] involving, among others, such distinct cell functions as enzyme conversions [2] , changes ’ in membrane permeability [3], and regulation of RNA transcription [4]. Protein phosphokinases with different substrate specificity have been found [5,6] and histone phosphorylation has been particularly implicated in gene activation through increased template activity [7]. In fact, the increased rate of nucleoprotein phosphorylation is concomitant with an active cell division like that occurring e.g. in regenerating liver [ 81 , in unilaterally nephrectomized kidney and fast growing tumoral ascites cells [9] , and the developing mammary gland [lo]. Protein kinase from rabbit muscle phosphorylates several proteins, including glycogen synthetase [ 5, 1 l] . Since the pattern during embryonic development of chick muscle glycogen synthetase kinase has been described [ 121, it was interesting to know the patterns with the other substrates. In fact, eventual differences would indicate that protein kinases with changing functions might be present during muscle differentiation. It is the purpose of this communication to report

Gene Activation in WI-38 Fibroblasts Stimulated to Proliferate: ROLE OF NON-HISTONE CHROMOSOMAL PROTEINS

Abstract An increase in chromatin template activity occurs within 1 hour after confluent monolayers of WI-38 human diploid fibroblasts are stimulated to proliferate by a change of medium. The present experiments were designed to investigate the role of non-histone chromosomal proteins in the stimulation of chromatin template activity. Chromatin from unstimulated WI-38 cells or from WI-38 cells 1 hour following stimulation was dissociated and then reconstituted with HeLa cell DNA, pooled histones, and non-histone chromosomal proteins from either unstimulated WI-38 cells or WI-38 cells 1 hour after stimulation. The template activity of chromatin reconstituted with non-histone chromosomal proteins from stimulated WI-38 cells was higher than the template activity of chromatin reconstituted with nonhistone chromosomal proteins from unstimulated cells. These results imply that the increased template activity of chromatin from stimulated WI-38 cells is due to changes in the constituent non-histone chromosomal proteins.

In vitro RNA synthesis and nuclear proteins of isolated sea urchin embryo nuclei

Isolated nuclei and/or chromatin from Strongylocentrotus purpuratus gametes and early developmental stages were analyzed for their basic protein content as well as their ability to serve as templates for in vitro RNA synthesis. 1. 1. Sperm chromatin exhibited little or no template activity. Unfertilized egg nuclei directed 1.5 % of the RNA synthesized by pure sperm DNA. This level of template activity increased at 32–64 cell, blastula and gastrula stages reaching a maximum of 6 % during pluteus stage. 2. 2. Trypsin digestion of sperm chromatin and blastula nuclei caused a 6–8-fold increase in template activity. Similarly treated egg nuclei showed no increase in available template. Sonication of egg and blastula nuclei resulted only in a slight increase in template activity. This was less pronounced for egg nuclei. Sonicated and trypsinized blastula nuclei showed an increased template activity over controls. Similar treatment of egg nuclei resulted in no release of genetic restriction. 3. 3. Typical histone patterns could not be electrophoretically demonstrated from extracted nuclear protein prior to blastula stage. 4. 4. The above results indicate a lack of histones during early sea urchin development although there is substantial template restriction.

Studies on in vitro RNA synthesis by chromatin fractions from rat liver and Novikoff hepatoma

Eu- and heterochromatin fractions prepared from normal liver and Novikoff hepatoma were tested for their capacity to direct the synthesis of RNA. Using purified RNA polymerase, template activity was measured by the incorporation of C 14 GTP into RNA. Unlike DNA, chromatin fractions had increased template activity at increased salt concentrations. No difference was observed in the priming capacity of either eu- or heterochromatin fractions derived from normal liver or Novikoff hepatoma.

Regulation of gene activity by hormones.

Regulation of gene expression, i.e. the reading of the genetic message, is of great importance for many vital processes. According to Jacob and Monod, genes are regulated by a “repressor” (a protein) which may interact with an “inducer” (a small molecule). In higher organisms, hormones often function as inducers. Evidence stems from (i) the induction of puffs in giant chromosomes by the insect hormone ecdysone, (ii) the stimulation of nucleic acid synthesis by hormones in vivo and in vitro, (iii) the induction of enzymes by hormones, e.g. cortisol, and (iv) the action of hormones on isolated nuclei where the activity of the DNA-dependent RNA polymerase is stimulated. This is most probably due to an increased template activity of the chromatin. — The implication of these findings for developmental processes are discussed.

Subcellular distribution of testosterone in rat prostate and its possible relationship to nuclear ribonucleic acid synthesis

The template capacity of rat prostatic whole nuclei from testosterone-implanted castrates was higher than that of castrates when assayed using an Escherchia coli RNA polymerase system. Attempts to localize this increased template activity in the “euchromatin” or “heterochromatin” fractions were unsuccessful. The E. coli RNA polymerase enzyme appeared to be saturated by an extremely low concentration of chromatin DNA. Both “heterochromatin” and “euchromatin” fractions are heavily repressed, and can be activated by the removal of histones. Prostatic “heterochromatin,” “euchromatin,” and intact nuclei in vivo and intact nuclei in vitro isolated from castrated rats take up more testosterone- 3H than the corresponding fractions obtained from testosterone-implanted castrated rats. In vivo this increased uptake is observed 2 hours after castration, and in vitro it is inhibited by 2,4-dinitrophenol. Specific uptakes of testosterone- 3H by the “euchromatin” fraction in vivo appeared to be higher than those onto “heterochromatin.”

On the mechanism of hormone action, X. Increased template activity for RNA synthesis of rat liver nuclei incubated with cortisol "in vitro".

On the mechanism of hormone action, X. Increased template activity for RNA synthesis of rat liver nuclei incubated with cortisol "in vitro".

Increased template activity of liver chromatin, a result of hydrocortisone administration.

We have found that the administration of hydrocortisone to adrenalectomized rats increases the template activity of their liver chromatin for RNA synthesis. Such administration is known to cause a two- to threefold increase in rate of nuclear RNA synthesis in the liver (1,2). This increase is followed by an increase in the activities of a series of liver enzymes (3-5). Since the induction of these enzymes by hydrocortisone is abolished by simultaneous treatment with actinomycin D, it is clear that new RNA synthesis is required to support their formation (4,5). The increased rate of liver RNA synthesis caused by administration of hydrocortisone might in principle be due to changes in the template activity of the liver genetic material such as would accompany derepression of genes previously repressed. We shall show below that the administration of hydrocortisone does result in an increased availability of the genetic material for transcription.