Jesse F. Scott

Studies in histochemistry. I. Determination of nucleic acids in microgram amounts of tissue.

A method for the analysis of nucleic acids in microgram amounts of tissue has been devised. Results obtained by this method in rat liver, kidney, and spleen have been compared with results obtained on the same tissues by other methods of analysis for tissue nucleic acid. This method is not designed to replace the more conventional methods of analysis when milligram quantities of tissue are available, although it has been used successfully for that purpose.

The interaction of steroid hormones and coenzyme components

Abstract 1. 1. The distribution-equilibrium method has been refined to permit the measurement of equilibrium constants for complex formation between several steroids and coenzyme components in aqueous solution. 2. 2. From the relation of the structure of the steroid and the coenzyme component to the equilibrium constant, it is concluded that complexes result from interactions between the purine nucleus of coenzyme components which contain purines, and the α-side of rings, C, D, and part of ring B of the steroid. The flatness of this region of the steroid is of primary importance for complex formation. Interactions found to occur with riboflavine phosphate are presumably of a similar type. 3. 3. The nature of the forces leading to complex formation are discussed. The interaction appears to be of a non-polawr character, most of the energy being derived from the recombination of water molecules which are displaced from the interacting surfaces.

SOME NUCLEOTIDE SEQUENCES FROM PARTIALLY PURIFIED TRANSFER RIBONUCLEIC ACIDS.

1. 1. Transfer RNA from bakers yeast and from Escherichia coli have been fractionated by partition and ion-exchange chromatography. 2. 2. Partially purified samples of transfer RNA from yeast specific for serine and valine and from E. coli specific for phenylalanine and lysine, as well as unfractionated transfer RNA from both sources have been subjected to digestion with alkali, pancreatic RNAase (EC 2.7.7.16), and takadiastase RNAase, T1 (EC 3.1.4.8). The products of the digestions were separated by paper ionophoresis and chromatography in a two-dimensional system and identified. 3. 3. It was found that: (a) The nucleotide sequences of unfractionated transfer RNAs are non-random. (b) It appears unlikely that methylated purines occur in the serine transfer RNA reported on, and none have been identified in the partially purified samples from E. coli. (c) In transfer RNA from E. coli no runs of adenylic acid longer than 3 residues were found. (d) The following provision list of nucleotide sequences occurring in pancreatic ribonuclease digest of serine transfer RNA from yeast has been deduced: AC, 1; AU, 2; GC, 4; GU, 2; A2C, 1; A2U, 1; G2C, 2; G2U, 1; G2ψ, 1; [AG]C, 1; [AG]U, 2; [AG2]U, 2; C, 11; U, 10; and ψ, 1.

Conservation of nucleic acids by Ehrlich ascites-tumor cells II. Conservation of ribonucleic acid purines

Abstract Ehrlich mouse ascites-tumor cells have been labelled in vivo by the intraperitoneal injection of [8-14C]adenine. 1 to 2 days after labelling the cells are transplanted into new host animals. The radioactivity of the RNA-purines of the transplanted cells is completely retained over a period of 9-days growth. Determination of the radioactivity of the separated purines of RNA shows that from the 3rd to the 9th day of growth there is no change in the ratio of either specific or total activities of the RNA-purines. The total radioactivity contained in the acid-soluble purine nucleotides during this period is of the order of 1% of the radioactivity in the RNA. Between day 0 and day 3 of growth there is a consistent fall in the ratio of specific activities of RNA-purines which is not found after a second transplantation of labelled cells. If cells are allowed to remain in animals into which the labelled precursor has been injected there is a steady decline in the ratio of specific activities which ceases when the cells are transferred into hosts which have received no isotope. These findings suggest that: i, the RNA which is labelled on day 3 is stable; ii, there is some fraction of RNA still labelled 2 days after exposure to the precursor which is depleted of label within 3 days after transplantation; iii, there is a measurable effect of contribution of purine precursors by the host to the nucleic acids of the tumor.

Evidence that the polyadenylic acid segment of “35S” RNA of avian myeloblastosis virus is located at the 3′-OH terminus

Summary By means of a periodate oxidation-[3H]borohydride labeling technique, evidence has been obtained which indicates that most of the 3′-OH ends of the high molecular weight RNA of avian myeloblastosis virus are occupied by polyadenylic acid segments, approximately 30 residues long. Of the polyadenylic acid sequences released by endonucleolytic digestion of this high molecular weight RNA, at least 90 per cent have a 3′-OH terminus, and are thus terminally, and not internally, located.

Isolation of ribonucleic acid from animal tissue by use of chaotropic agents.

Abstract A method is presented for solubilizing and isolating pulse-labeled RNA from isolated nuclei, whole cells, and the insoluble material which collects at the interface between the phenol and aqueous layers during phenol extraction procedures. For this we have used solutions of the chaotropic salt, lithium trichloroacetate. It appears that the method may be useful for extracting the more stable RNA species as well. The RNA extracted from whole cells and phenol interface precipitate is undegraded, as determined by acrylamide gel electrophoresis after exposure to dimethyl sulfoxide. Pulse-labeled RNA extracted from isolated nuclei shows a marked tendency to aggregate and this fraction is being investigated further.