Mary L. Stephenson

The preparation and some properties of a low molecular weight ribonucleic acid from baker's yeast.

Abstract A low molecular weight RNA has been extracted from whole bakers yeast cells with phenol and compared with yeast S-RNA. Several properties which have been investigated are very similar for both products. Among these properties are the sedimentation constants, the chromatographic behavior on DEAE-cellulose columns, the formation of dinitrophenylhydrazones, the base composition, and the valine and adenylate accepting activity. The extraction procedure is adaptable to large scale preparations.

Mechanisms of matrix degradation in rheumatoid arthritis

In the inflammatory synovium production of collagenase is probably responsible for the degradation of collagen in the extracellular matrix and distortion of the architecture and function of the joints. Major collagenase-producing cells are mesenchymal cells such as fibroblasts and chondrocytes, which synthesize and secrete the enzyme influenced by the action of cytokines produced by adjacent mononuclear cells. The cytokines act primarily through cell-surface receptors, whose signal is probably then mediated by complexes of nuclear oncoproteins, to activate transcription of the procollagenase gene. The increased production of collagenase ultimately is the result of a cascade of cellular effects involving complex interactions of different ligands in a system characterized by amplification and feedback loops.

Stimulation of procollagenase synthesis parallels increases in cellular procollagenase mRNA in human articular chondrocytes exposed to recombinant interleukin 1β or phorbol ester

Interleukin 1, a product predominantly of monocytes, increases the synthesis and release of procollagenase and prostaglandin E2 by mesenchymal target cells such as synovial fibroblasts and articular chondrocytes, an effect mimicked by some phorbol esters. In order to determine the mechanisms underlying these responses primary cultures of human articular chondrocytes were preincubated with recombinant human interleukin 1 beta or the phorbol ester, phorbol 12-myristate 13-acetate, in the presence or absence of the cyclooxygenase inhibitor, indomethacin. Interleukin 1 beta or phorbol ester increased the levels of procollagenase (assayed after trypsin activation) and the labeling of several medium proteins by cells incubated with [35S]methionine, independent of prostaglandin synthesis. The labeling of a 55 kD protein immunocomplexed with antibodies to procollagenase was also increased. The increased synthesis of procollagenase was paralleled by increased cellular levels of procollagenase mRNA, determined with a cDNA probe coding for human procollagenase. Thus the increased synthesis of procollagenase in response to the inflammatory mediator, interleukin 1, is controlled at a pretranslational level, possibly at the level of transcription.

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.

Partial purification of transfer RNA.

Nineteen years ago F. Lipmann suggested’ that the peptide-bond forming step in protein synthesis might proceed by means of a phosphoanhydride of the carboxyl-activated amino acid. Findings of the past five years have richly borne out this prophecy. The problem of how amino acid sequence is arranged with such unerring accuracy is, however, still an unsolved one. It is patent that there is great specificity in the activating enzyme^,**^^^-^ and it seems probable that a separate activating enzyme is present for each natural amino acid. I t has also become clear that the same enzyme that activates a given amino acid is also responsible for its transfer to ester linkage on a soluble RNA molecule.“.’ The acceptor site for the amino acid on the cytoplasmic S-RNA molecule is a terminal adenyl group,Bn9 and immediately inside this are located either one or two cytidyl units.’OJl J ~ , ~ ~ The presence of this terminal nucleotide constellation is essential to the amino acid acceptor function of the S-RNA.8*14s6-9 Among S-RNA’s obtained from mammalian, yeast, and microbial origins and also in S-RNA’s from the same source16 there is variation in the ability of the RNA sample to accept amino acid. This may reflect a difference in the degree to which the -pCpCpA-end group is present on the RNA as isolated. There has been some discussion in this and other publications of the possibility of the presence of “junk RNA” in the S-RNA fraction, and the following data are related to this question. It has been shown that RNA prepared by direct phenol extraction of whole yeast cells is very similar to the S-RNA prepared by the usual homogenization and centrifugation techniques.I6 Our evidence suggests that direct phenol extraction of yeast cells results in better preservation of these end groups than does preparation of the S-RNA fraction by grinding or homogenization techniques.I6 These latter procedures expose the S-RNA fraction to attack by various nucleotidases as well as to the reversible removal of the terminal mononucleotides by the action of the enzyme(s) that attaches them to the S-RNA molecule. This degradative possibility has a bearing on the question of whether there exists in the S-RNA fraction an appreciable proportion of RNA chains incapable of serving as amino acid acceptors. Following grinding of yeast in aqueous media as illustrated in TABLE 1, the S-RNA fraction obtained has a lower amino acid and terminal AMP-accepting ability (as measured by the use of ascites cell enzymes) than does the RNA obtained * Publication No. 992 of the Harvard University, Cambridge, Mass., Cancer Commission. The work reported in this paper was supported in part by grants from the United States Atomic Energy Commission, Washington, D.C., the American Cancer Society, New York, N.Y., and the National Cancer Institute, United States Public Health Service, Bethesda, Md. Paul M. Doty has kindly carried out experiments that indicate that the sedimentation and diffusion constants of the dye RNA are very similar to those of the RNA. It is thus unlikely that a polymer of dye RNA actually occurs.