The quantitative histochemistry of ribonucleic acid using gallocyanin.

A method for the cytophotometric estimation of ribonucleic acid in tissue sections using gallocyanin-chrome alum is described. The dye obeys Beer's law in gelatin sections. The effect of deoxyribonuclease on the staining of ribonucleic acid is also investigated. The results indicate that this method is of value in the quantitation of ribonucleic acid.     

Cytochemical differentiation of nucleic acids with a Schiff-methylene blue sequence.

A method is described whereby a Feulgen type of hydrolysis of deoxyribonucleic acid is carried out on paraffin sections of routinely fixed tissues by controlled exposure of the sections to Bouin's fluid. Subsequent staining with Schiff reagent followed by methylene blue distinguishes red-to purple-stained deoxyribonucleic acid from blue-stained ribonucleic acid. This Schiff-methylene blue sequence visualizes ribonucleic acid in nucleoli and the chromidial substance of various normal and neoplastic cells and provides an assessment of their protein synthetic activity. The method has proved valuable in demonstrating normal immunocytes and immunoglobulin-forming tumor cells in pathologic specimens.     

Specific demonstration of ribonucleic acid by chemical bromination and immunohistochemistry

In this report we describe a specific staining procedure for detection of ribonucleic acid (RNA), based on bromination of uracil and subsequent immunohistochemical visualization of 5-bromouracil in RNA. This method is applicable for both cryostat and glycol methacrylate (GMA)-embedded sections. Cryostat sections must be fixed in formaldehyde, whereas tissue pieces to be embedded in GMA are fixed in cold acetone. Before bromination, sections must be treated with trypsin. Bromination was performed in a solution of bromine in potassium bromide. After bromination, excess bromine was removed with sodium bisulfite. The monoclonal antibody MoBu-1 specifically bound to brominated RNA. Ribonuclease digestion, in contrast to deoxyribonuclease digestion, abolished staining. This method makes possible precise localization of RNA, especially well demonstrated in plastic-embedded sections.     

The Value of Alcohol for Fixation of Skin

Stained sections of skin fixed in 70% alcohol were compared with others from pieces fixed in 4% formaldehyde-saline. The sections of alcohol-fixed material were much more susceptible to the action of deoxyribonuclease and lipase than those from formalin-fixed, as demonstrated by a standardized hematoxylin staining method and by fluorescence microscopy. After formalin, cytoplasmic basophilia was increased, presumably because formalin fixation caused ribonucleic acid to diffuse from nuclei to cytoplasm. Both types of fixation damaged collagen, as seen in fluorescence induced by 5-anvmo-2-chloro-7-methoxyacridine, but alcohol caused less distortion than formalin. Probably fluorochroming of fresh tissue is the only satisfactory method for studying collagen in pathological conditions.     

The distribution of acid and alkaline ribonuclease activities in preneoplastic and neoplastic rat livers.

Ribonuclease (RNase) activities revealed by the substrate film method were compared with reactions for acid and alkaline RNase obtained by lead precipitation technique in serial sections of preneoplastic livers and hepatomas. The preneoplastic parenchymal tissue giving positive reactions with ribonucleic acid films showed both acid and alkaline RNase activities by lead precipitation technique, and the area of hyperplastic nodules nonreactive against substrate films were deficient in acid and alkaline RNase activities. Preneoplastic hyperbasophilic foci and hepatoma gave weak or negative reactions by either method, but necrotic areas and stromal tissue showed appreciable RNase activities. Thus a good correlation was observed in these tissues between the RNase activities revealed by the film method and those demonstrated by lead precipitation.     

A Methylene Blue-Basic Fuchsin Stain for Mast Cells in Paraffin Sections

In paraffin sections of rat tissue it is possible to stain mast cell granules blue in contrast to red nuclei, pale blue cytoplasmic ribonucleic acid, and colorless collagen. This is done by the following mixture: 1% methylene blue (pure, not polychrome), 9 ml; 0.1% basic fuchsin, 9 ml; glacial acetic acid, 2 ml. Stain formol-fixed, paraffin-processed sections for 5 min, wash in water and pass through acetone, 2 changes, 10 sec total, to xylene and a polystyrene mounting medium.     

The histochemical demonstration of hydrolase activities with films of homopolyribonucleotides.

Films of polycytidylic acid, polyuridylic acid and polyguanylic acid were exposed to tissue sections and the results were compared with those obtained in previous studies on polyadenylic acid and ribonucleic acid. Important variations were observed in the distribution of the hydrolases acting on the different polyribonucleotides, suggesting that a variety of nucleases with marked proclivity for particular nucleotide residues can be demonstrated by the use of films of homopolymers.     

The Isolation and Characterization of Adenosine Monophosphate-rich Polynucleotides Synthesized by Soybean Hypocotyl Cells: Their Relation to Messenger Ribonucleic Acid

Plant ribonucleic acids which have high adenosine monophosphate concentrations were studied. Purified deoxyribonucleic acid-like ribonucleic acid and tenaciously bound ribonucleic acid fractions both contained poly-adenosine monophosphate sequences (those from the latter being longer than those from the former); without these poly-adenosine monophosphate sequences their base compositions were the same. The average poly-adenosine monophosphate sequence from purified tenaciously bound ribonucleic acid was 160 residues long, as measured by gel electrophoresis. However, base hydrolysis and chromatography indicated one 3′-nucleoside (adenosine) per 71 nucleotides, giving a chain length of 72 residues. The dominant species in the cytoplasm, as measured by radioactive precursor incorporation, was tenaciously bound ribonucleic acid, whereas deoxyribonucleic acid-like ribonucleic acid was present in greater amounts in the nucleus. This work provides evidence that deoxyribonucleic acid-like ribonucleic acid and tenaciously bound ribonucleic acid represent forms of messenger ribonucleic acid in soybean, with deoxyribonucleic acid-like ribonucleic acid residing in the nucleus, perhaps as the messenger ribonucleic acid precursor, and tenaciously bound ribonucleic acid residing, as the active messenger ribonucleic acid, in the cytoplasm.     

Improved methods for purification and assay of eukaryotic messenger ribonucleic acids and ribosomes. Quantitative analysis of their interaction in a fractionated reticulocyte cell-free system.

The polyadenylic acid-containing messenger ribonucleic acids of eukaryotic cells are rapidly isolated and deproteinized in a simple and gentle one-step procedure. The polyribosome fraction, dissolved in 0.5 M NaCl/0.5 percent sodium dodecyl sulfate, is passed through an oligo(dT)-cellulose column which is then washed with the solvent until proteins and contaminating ribonucleic acids are fully removed. Deproteinized messenger ribonucleic acid is then eluted by lowering the ionic strength. This method gives highly purified and active messenger ribonucleic acids from all tissues tested. The yield is approximately 1.5 to 2 percent of the polyribosomal ribonucleic acid. Messenger ribonucleic acids are assayed in a rabbit reticulocyte-derived, messenger-dependent, cell-free protein-synthesizing system modified from Crystal et al. (Crystal, R. G., Nienhuis, A. W., Elson, N. A., and Anderson, W.F. (1972) J. Biol. Chem. 247, 5357-5368). This system synthesizes proteins at an almost linear rate for at least 2 hours. During this period, each globin messenger ribonucleic acid directs the synthesis of several globin molecules. Each active ribosome synthesizes a globin molecule every 6 to 7 min, but only a small fraction of the ribosomes or messengers are active at any instant. Translation occurs mainly on di- and monoribosomes although larger sized polysomes also occur. Several lines of evidence suggest that globin messenger ribonucleic acid requires "activation" before it can be utilized and that a messenger activation step of protein synthesis initiation is rate-limiting in this cell-free system.     

Rapid multiparameter analysis of cell stimulation in mixed lymphocyte culture reactions.

A flow-cytofluorometric method, based on the differential stability of deoxyribonucleic acid versus ribonucleic acid with the metachromatic dye, acridine orange, simultaneously measures the following parameters of stimulation in mixed lymphocyte cultures: (a) number of nonstimulated cells; (b) total number of stimulated lymphocytes; (c) number of stimulated lymphocytes in G1, S and G2 + M phases of the cell cycle; (d) number of macrophages; (e) number of dead cells. The progress of lymphocyte stimulation may also be measured by a parameter representing ribonucleic acid accumulation per cell. The method is rapid, avoids cell rinsing, fixation and centrifugation and is applicable to microcultures. Multiparameter analysis of cell stimulation which provides simultaneous measurements of lymphocyte proliferation and accumulation of ribonucleic acid per cell may prove to be a more sensitive assay of histocompatibility than tests based only on cell proliferation (tritiated thymidine incorporation).     

Nature of Ribonucleic Acid Synthesis During Early Sporulation in Saccharomyces cerevisiae

Phosphate uptake in sporulating cultures of Saccharomyces cerevisiae has been found to occur approximately 2 h after the transfer to sporulation medium. Early ribonucleic acid synthesis begins at approximately 4 h and continues to 8 h. Incorporation of phosphate into acid-extractable precursor pools parallels phosphate uptake. In triple-labeling experiments it was observed that the breakdown of vegetatively synthesized ribonucleic acid is not a significant source of precursors for ribonucleic acid synthesis during sporulation. The majority of the ribonucleic acid made in a 10-min period during sporulation does not migrate on gels with precursor or mature ribosomal ribonucleic acid.     

Nuclear Fraction of Bacillus subtilis as a Template for Ribonucleic Acid Synthesis

A "nuclear fraction" prepared from Bacillus subtilis was a more efficient template than purified deoxyribonucleic acid for the synthesis of ribonucleic acid by exogenously added ribonucleic acid polymerase isolated from B. subtilis. The initial rate of synthesis with the nuclear fraction was higher and synthesis continued for several hours, yielding an amount of ribonucleic acid greater than the amount of deoxyribonucleic acid used as the template. The product was heterogenous in size, with a large portion exceeding 23S. When purified deoxyribonucleic acid was the template, a more limited synthesis was observed with a predominantly 7S product. However, the ribonucleic acids produced in vitro from these templates were very similar to each other and to in vivo synthesized ribonucleic acid as determined by the competition of ribonucleic acid from whole cells in the annealing of in vitro synthesized ribonucleic acids to deoxyribonucleic acid. Treatment of the nuclear fraction with heat (60 C for 15 min) or trypsin reduced the capacity of the nuclear fraction to synthesize ribonucleic acid to the level observed with purified deoxyribonucleic acid.     

Cytomegalovirus but not human T lymphotropic virus type III/lymphadenopathy associated virus detected by in situ hybridisation in retinal lesions in patients with the acquired immune deficiency syndrome.

Paraffin sections of retinal tissue from five patients who died from the acquired immune deficiency syndrome (AIDS) and retinopathy were examined by in situ hybridisation experiments with deoxyribonucleic acid (DNA) labelled with sulphur-35 of lentivirus, human T lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV), and cytomegalovirus. HTLV-III/LAV ribonucleic acid (RNA) was not detected in any of the tissue sections. Cytomegalovirus RNA was identified, however, in three of the five patients. Retinopathy induced by cytomegalovirus may thus be one of the many syndromes potentiated by the immunosuppression caused by HTLV-III/LAV.     

Nucleotide sequence of phenylalanine transfer ribonucleic acid from pea (Pisum sativum, Alaska).

Phenylalanine transfer ribonucleic acid from peas (Pisum sativum, Alaska) was completely digested with beef pancreatic ribonuclease (RNase I) and with ribonuclease T1. The resulting oligonucleotides were compared with those from the corresponding hydrolyses of phenylalanine transfer ribonucleic acid from wheat germ. The structures of both ribonucleic acids appeared to be identical. This report is the first to show that identical structures for the same specific acceptor transfer ribonucleic acid are present in two different plant species.     

Potentiation of hemoglobin messenger ribonucleic acid. A step in protein synthesis initiation involving interaction of messenger with 18 S ribosomal ribonucleic acid.

The polyadenylic acid-containing messenger ribonucleic acid from rabbit reticulocyte polyribosomes, isolated by a rapid and very gentle procedure (Krystosek, A., Cawthon, M. L., and Kabat, D. (1975) J. Biol. Chem. 250, 6077-6084), sediments in a sucrose gradient in three sharp peaks, at 9 S, 17 to 18 S, and 28 S. The alpha and beta globin messenger activity follows the absorbance profile in the sucrose gradients and has its major peak at 17 to 18 S. The larger messengers are more active than 9 S messenger by approximately 2-fold per mass unit of ribonucleic acid or by at least 8-fold per molecule. The major 17 to 18 S form of globin messenger was examined further and was shown to be a 1:1 complex of 9 S messenger and 18 S ribosomal ribonucleic acid. The effect of 18 S ribosomal ribonucleic acid on translation of purified 9 S globin messenger was analyzed in a messenger-dependent protein-synthesizing system (Krystosek, A., Cawthon, M. L., and Kabat, D. (1975) J. Biol. Chem. 250, 6077-6084). In the absence of exogenous ribosomal ribonucleic acid, 9 S messenger is inefficiently translated; a large excess of messenger is required to saturate the system; and globin is synthesized mainly on di- and monoribosomes. Exogenous liver or reticulocyte 18 S ribosomal ribonucleic acid potentiates 9 S messenger translation and renders it at least 10 times more efficient. The potentiation reaction can also be accomplished by increasing the concentration of ribosomes in the assay system. However, transfer or messenger ribonucleic acids cannot carry out this reaction. It is proposed that 9 S globin messenger ribonucleic acid is an inactive molecule which is normally potentiated by specific reversible base pairing with an accessible region of ribosomal ribonucleic acid contained in a 40 S ribosomal subunit. The potentiated messenger interacts with initiation factors and with other ribosomal subunits to synthesize protein. Potentiation is the first specific function in protein synthesis demonstrated for the ribosomal ribonucleic acid portion of ribosomes.     

Isolation and identification of yeast messenger ribonucleic acids coding for enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase.

Yeast poly(adenylic acid)-containing messenger ribonucleic acid isolated from two strains of Saccharomyces cerevisiae was fractionated by preparative polyacrylamide gel electrophoresis in the presence of formamide. Three messenger ribonucleic acids, present at high intracellular concentration, were electrophoretically eluted from the polyacrylamide gels and translated in a wheat germ cell-free extract. The in vitro synthesized polypeptides were identified by tryptic peptide analysis. Messenger ribonucleic acids coding for enolase and glyceraldehyde-3-phosphate dehydrogenase were isolated from commercially grown baker's yeast (strain F1), and messenger ribonucleic acid coding for phosphoglycerate kinase was isolated from Saccharomyces cerevisiae (ATCC 24657). Significant differences in the spectrum of abundant messenger ribonucleic acids isolated from commercially grown baker's yeast (strain F1) and strain 24657 were observed. When both strains were grown under identical conditions, however, the spectrum of messenger ribonucleic acid isolated from the cells is indistinguishable.     

Structural and compositional changes associated with chlorine inactivation of polioviruses.

Chlorine inactivation of polioviruses resulted in the loss of viral ribonucleic acid, converting the viruses from 156S particles to 80S particles. However, it was found that virus inactivation occurred before the ribonucleic acid was released from the virions. Extraction of ribonucleic acid from partially inactivated virus suspensions indicated that chlorine inactivation was due to degradation of the ribonucleic acid before release and that ribonucleic acid loss was a secondary event. The empty 80S capsids had the same isoelectric point and ability to attach to host cells as infective virions. Thus, no major capsid conformational changes occurred during chlorine inactivation.     

Structural and compositional changes associated with chlorine inactivation of polioviruses.

Chlorine inactivation of polioviruses resulted in the loss of viral ribonucleic acid, converting the viruses from 156S particles to 80S particles. However, it was found that virus inactivation occurred before the ribonucleic acid was released from the virions. Extraction of ribonucleic acid from partially inactivated virus suspensions indicated that chlorine inactivation was due to degradation of the ribonucleic acid before release and that ribonucleic acid loss was a secondary event. The empty 80S capsids had the same isoelectric point and ability to attach to host cells as infective virions. Thus, no major capsid conformational changes occurred during chlorine inactivation.