Jay S. Roth

Chromosomal RNA: An artifact of preparation?

Abstract Total nuclear RNA, chromatin-associated RNA § and chromosomal RNA have been examined in various chicken tissues. The quantity of chromosomal RNA varied from zero to almost 100% of the total chromatin-associated RNA, depending on the conditions of isolation and preparation of chromatin and chromosomal RNA. Sucrose gradient sedimentation analysis of chromosonal RNA isolated under conditions minimizing RNA degradation showed that it was comprised of all of the RNA species found in the nucleus. No evidence was found for the presence of a special class of RNA which is RNase resistant and covalently bound to chromosomal proteins.

Assay of proteolytic enzyme activity using a 14C-labeled hemoglobin

Abstract The measurement of total tissue protcolytic activity with a hemoglobin substrate labeled with K 14 CNO is described. The labeled hemoglobin is stable and its use offers several advantages, particularly in specificity and sensitivity over methods commonly in use.

Ribonuclease. IX. Further studies on ribonuclease inhibitor.

Abstract Ribonuclease inhibitor was found in the livers of five mammalian species, but could not be detected in the liver of chicken or frog. Detailed examination of the ribonuclease activity of chicken-liver homogenate and various subcellular fractions under different conditions failed to provide convincing evidence for the presence of an inhibitor in this tissue. Some factors affecting ribonuclease-inhibitor activity were examined. It was demonstrated that ribonuclease inhibitor slows the hydrolysis of cyclic cytidylic acid by crystalline pancreatic ribonuclease, but this reaction is not suitable for an assay of the inhibitor, due to the interference of tissue enzymes that hydrolyse the cyclic nucleotide.

Some factors affecting the interaction between actin in leukemic L1210 cells and DNASE I.

Summary Total actin content and F/G actin ratios were determined in L1210 leukemic cell cytosol based on inhibition of DNase I by actin. DMSO, when present in the assay mixture, inhibited the interaction between actin and DNase I. Ca ++ and pCMB were also inhibitory with maximum effect at 1.25 μM and 2.5 μM, respectively. For expression of maximum inhibition of DNase I by L1210 cytosol, 0.25 μM ATP is necessary in addition to proper dilution. Preincubation of purified chicken muscle actin or actin in L1210 cytosol with double stranded DNA inhibited the subsequent interaction between DNase I and the actin.

METAL IONS IN RIBONUCLEIC ACID: THEIR NATURE AND INTERFERENCE WITH THE ASSAY FOR RIBONUCLEASE AND RIBONUCLEASE INHIBITOR.

Abstract RNA from some commercial sources has been shown to contain significant quantities of copper and zinc and the amounts these metals present has been determined spectroscopically. Studies of the effects of copper and zinc ions, as well as other metal ions, on RNAase (polyribonucleotide 2-oligonucleotidotransferase (cyclizing), EC 2.7.7.16) and RNAase inhibitor have been made. Of several metal ions studied, only copper and zinc inactivated RNAase inhibitor at low metal-ion concentrations. It was demonstrated that the inactivating effects of different RNA samples RNAase inhibitor are consistent with their metal-ion content. The significance of metal-ion contaminants to biochemical studies involving RNA was discussed.

Evidence for the presence of DNase—actin complex in L1210 leukemia cells

L1210 leukemia cell cytosol was analysed for the presence of DNase I activity. No free activity was determined in crude cytosol. DNase I enzyme was found to occur in a latent form bound to cytoplasmic actin. DNase‐actin complex was partially isolated by Sephadex filtration and DNase I‐like activity was demonstrated after SDS gel electrophoresis of the complex and enzyme renaturation. The results were compared with those for synthetic complex of pancreatic bovine DNase I and chicken muscle actin.

An assay for and some properties of deoxyribonuclease inhibitor in rat liver

Abstract A simple, convenient assay for DNase I protein inhibitor has been devised. Some of the conditions affecting the assay such as Mg++ concentration, pH and ionic strength, have been investigated. Some properties of DNase inhibitor have been studied. The inhibitor is relatively labile to storage, is not affected by sulfhydryl reagents, and is destroyed by heating at 60 °C. Evidence is presented that suggests the mechanism of inhibitor action is through combination with a Mg++-DNA complex. The inhibitor-DNA-Mg++ combination is not wholly resistant to the action of DNase I.

Studies on thymidylate phosphatase, thymidine kinase and thymidylate kinase activities in some transplantable rat hepatomas

Summary Thymidylate phosphatase distribution and specific activity was measured in a series of transplantable hepatomas, the host livers and normal liver. Approximately 90 per cent of the enzyme was recovered in three particulate fractions with nearly equal distribution among them. Activity in the Novikoff hepatoma was only one-third that of normal liver but in the Dunning, another rapidly growing hepatoma, it was higher than in normal liver. In all cases, activity was concentrated in the microsomal fraction. Studies of thymidine kinase and thymidylate kinase activity of the hepa- tomas were carried out. Thymidine kinase activity, in general, correlated well with the proliferative rate of the tumor but thymidylate kinase activity did not. Some implications of the results were discussed.

INHIBITION OF GROWTH OF CHICK EMBRYO BY INHIBITION OF DEOXYCYTIDYLATE DEAMINASE.

Deoxyguanylate when added to chick embryos grown in explant inhibited growth and development. Deoxycytidylate deaminase activity was inhibited both in the explants and in vitro; since the effect was quite specific, it is suggested that this may represent another control mechanism for deoxynucleotide synthesis.

Some aspects of deoxynucleotide metabolism in the developing chick embryo

Abstract The deamination of deoxycytidylate, deoxyguanylate, deoxyadenylate and the corresponding nucleosides was measured in whole chick embryo, embryo liver, and in the hatched chick using a new simplified assay procedure. Deoxycytidylate deaminase activity was high in the early chick whole embryo (3 days) and declined slowly to about the 15th day. The activity in embryo liver also decreased from the 15th to 19th day. Deoxyadenylate deaminase activity could not be detected before the sixth to seventh day. After this, it attained a relatively constant value in whole embryo with about the same activity being measured in embryo liver. No deamination of deoxyguanosine or deoxyguanylate was detected until just after hatching. No deamination of deoxycytidine was observed until the 17th or 18th day, after which the activity increased to moderate levels in liver. Deoxyguanylate phosphatase activity was relatively high and deoxycytidylate phosphatase activity low in the chick embryo (4–8 days). Both increased after this time, but dGMP phosphatase activity was always considerably greater than deoxycytidylate phosphatase. Some implications of these findings with respect to development are discussed.