Isolation Of Ribonucleic Acid Research Articles

β-Adrenergic receptor subtype gene expression in timed-pregnant rat myometrium

OBJECTIVE: Classic radioligand binding techniques have suggested that β 1- and β 2-adrenergic receptor subtype proteins are expressed in myometrial tissue; however, to date these observations have not been confirmed at the level of the messenger ribonucleic acid for these clinically important membrane receptors. The studies described in this report sought to use quantitative reverse transcriptase–polymerase chain reaction techniques to confirm expression of messenger ribonucleic acid for the β 1- and β 2-adrenergic receptors in myometrial tissue and to determine whether messenger ribonucleic acid expression for these two adrenergic receptors is modulated during pregnancy. STUDY DESIGN: For these studies total cellular ribonucleic acid was isolated from myometrial tissue obtained from timed-pregnant Sprague-Dawley rats by the guanidium thiocyanate–phenol–chloroform extraction technique; formaldehyde-agarose gels then confirmed isolation of intact ribonucleic acid. Random hexamer primers and reverse transcriptase were used to synthesize complementary deoxyribonucleic acid. Subsequently, polymerase chain reaction was performed with subtype specific 20-mer sense and antisense oligonucleotide primers specific for the rat β 1- and β 2-adrenergic receptors. Inclusion of internal standard deoxyribonucleic acid sequences allowed quantification of the reverse transcriptase–polymerase chain reaction results. RESULTS: By use of total cellular ribonucleic acid isolated from myometrial tissue, reverse transcriptase–polymerase chain reaction generated the expected 328 bp product for the β 1-receptor and the expected 559 bp product for the β 2-receptor along with internal standard deoxyribonucleic acid sequences for both. The identity of the β 1- and β 2-adrenergic receptor polymerase chain reaction products was confirmed on the basis of restriction endonuclease digestions producing the expected deoxyribonucleic acid fragments and by Southern blots using β 1- and β 2-adrenergic receptor–specific complementary deoxyribonucleic acid probes. The reverse transcriptase–polymerase chain reaction studies confirmed a gradual decline in β l-receptor messenger ribonucleic acid and stable expression of β 2-receptor messenger ribonucleic acid during the second half of gestation in pregnant rat myometrial tissue. CONCLUSIONS: In summary, these studies have confirmed, at the messenger ribonucleic acid level, expression of the β 1- and β 2-adrenergic receptor subtypes in timed-pregnant rat myometrial tissue.(Am J Obstet Gynecol 1997;176:349-57.)

Isolation of Ribonucleic Acid from the Unactivated Rat Liver Glucocorticoid Receptor

We have reported that the 7-8S form of the rat liver glucocorticoid receptor is associated with RNA. Whether the unactivated 9-10S form of the glucorticoid receptor is also associated with RNA is less clear. Here we provide evidence that the unactivated 9-10S receptor is indeed associated with RNA. Unactivated 9-10S receptor was partially purified by diethylaminoethyl (DEAE)-cellulose chromatography in the presence of molybdate, an activation inhibitor. This preparation was then bound to BuGR-2, a mouse monoclonal antibody of the immunoglobulin G (IgG)-2 class to the rat liver glucocorticoid receptor, or to nonspecific mouse IgG-2. The antibody-antigen complex was then bound to protein A sepharose and washed to remove extraneous RNA. When the receptor was dissociated from the antibody and the RNA extracted and end-labeled, a distinct band of approximately 170 nucleotide (nt) was found that was specific for the BuGR-2 purified receptor. This band could also be found in DEAE-cellulose receptor that had been isolated from sucrose gradients. The DEAE-cellulose receptor was then cross-linked with formaldehyde before mixing with BuGR-2 in order to permit more vigorous washing of the antigen-antibody complex. In addition to the 170 nt RNA band, another distinct band at approximately 400 nt was seen that was specific to the BuGR-2 derived isolate. These results provide evidence that the 9-10S form of the glucocorticoid receptor from rat liver is associated with RNA.

14] Isolation of specific RNAs using DNA covalently linked to diazobenzyloxymethyl cellulose or paper

Publisher Summary This chapter discusses the isolation of specific ribonucleic acids (RNAs) using deoxyribonucleic acid (DNA) covalently linked to diazobenzyloxymethyl (DBM)-cellulose or DBM paper. Substantial amounts of DNA can be linked covalently to the finely divided DBM cellulose or DBM paper. Either form of insoluble DNA can then be used to select specific species of RNA by hybridization. If RNA is radioactive, DNA paper can be used to analyze the fraction of RNA complementary to the insoluble DNA in a procedure very similar to the one originally developed by Gillespie and Spiegelman. Either DNA-cellulose or DNA paper can be used preparatively to select intact, specific RNAs that can be used subsequently for translation in vitro as a template for reverse transcriptase, as a source of pure infectious RNA, or for any other experiment in which pure, intact RNA is required. The chapter describes the purification of intact RNAs and discusses a quantitative analysis of specific labeled RNAs using DNA paper.

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.

Quantitative isolation of ribonucleic acid from the immature rat uterus—Examination of ribonucleic acid synthesis following in vivo 17β-estradiol administration

A CsCl centrifugation procedure was utilized to extract 85%–90% of the total cellular RNA from the immature rat uterus. This total RNA was deaggregated by treatment with sarkosyl and further fractionated by cellulose chromatography and acrylamide gel electrophoresis. Using these techniques, no specific RNA synthesis could be detected following in vitro incorporation of [ 3H]- and [ 14C]-adenosine into rat uterine RNA 15 min, 30 min and 5 h after in vivo 17β-estradiol administration.

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

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.

Isolation of Ribonucleic Acids Having Template Activities from Particulate Components of Soybean Seeds

Ribonucleic acids having template activities were obtained from particulate components prepared from the postribosomal supernatant of soybean seeds. These RNA were 9 S and 18 S in size, and these corresponded to the components (9 S, 18 S) of high molecular weight RNA (H–RNA) prepared from the supernatant of 100,000×g centrifugation. The sizes of the particulate components were 37 S and 59 S, respectively. Larger particles contained 18 S and 9S RNA, and smaller particles contained 9S RNA, but not 18 S RNA. Those particulate components differed in absorption pattern and in the behaviour on sucrose gradient centrifugation depending on the concentration of Mg27+ from the subunits of ribosomes.

Technique for the Isolation of Ribonucleic Acid-Rich Mutants from Escherichia coli

A technique for the isolation of ribonucleic acid (RNA)-rich mutants of Escherichia coli is described. Mutagenized cells were centrifuged to isopycnic equilibrium on potassium tartrate or cesium sulfate gradients. Samples from the region of the gradients slightly denser than the majority of the cells were spread on agar plates, and the resulting clones were tested for increased RNA to protein ratios.

Use of DEAE-cellulose for isolation of ribonucleic acids from bacteriophages Qβ and MS2

Use of DEAE-cellulose for isolation of ribonucleic acids from bacteriophages Qβ and MS2

Isolation and characterization of ribonucleic acid from Musca domestica (L.)

1. 1. Ribonucleic acid isolated from adult female houseflies was fractionated by sucrose gradient centrifugation and by methylated albumin-Kieselguhr columns. 2. 2. Three principal fractions were obtained with approximate sedimentation coefficients of 28 S 18 S and 4 S. 3. 3. Differences in base composition between the three fractions and those of rat livers as well as Drosophila have been found. 4. 4. Only the 29 S fraction was complementary with G = C and A = U.

The isolation of ribonucleic acid from plant, bacterial or animal cells

1. 1. An adaptation of the phenol procedure is described for the isolation of relatively undegraded ribonucleic acid from plant tissue, which is also useful for the isolation of ribonucleic acid from bacterial and animal cells. The novel aspects that make the isolation procedure feasible is the use of a basic buffer (pH 9.5) in the presence of versene. Ribonucleic acid was isolated from white potato tubers, the leaves, stems and roots of etiolated peas, Escherichia coli K-12, rat fat pads, ribosomes from human reticulocytes, rabbit reticulocytes and rabbit liver. 2. 2. The preparations, analyzed by sucrose-gradient centrifugation, revealed that the two ribosomal RNA fractions of pea seedlings were present in equimolar concentrations and had molecular weights of 1.25 · 10 6 and 5.7 · 10 5 estimated from sedimentation coefficients of 24.5 S and 16 S. 3. 3. The molar ratios (cytidine:adenine:guanine:uridine) of the 25 S, 16 S, and 4 S RNA of pea seedlings were 22.7:23.6:32.1:21.5, 20.1:23.7:31.1:25.2, and 25.5:20.8:32.4:21.2, respectively. 4. 4. Pea seedling leaves, which have a higher metabolic activity than stems and roots of the same tissue, also have a higher content of soluble ribonucleic acid (% of the total ribonucleic acid).

Isolation and Characterization of Ribonucleic Acid from Cerebral Cortex of Rat

Abstract Ribonucleic acid isolated from cerebral cortex of mature male and female rats was fractionated by chromatography on methylated albumin Kieselguhr columns or by sucrose gradient centrifugation. Three principal fractions were obtained with analytical ultracentrifuge s20,w0 values of 4, 17, and 28. By comparison with ribonucleic acid prepared from purified rat brain ribosomes, the 17 S and 28 S fractions were shown to be predominantly ribosomal ribonucleic acid. Differences in base analyses between the three fractions were small but significant. Compositions of all fractions were markedly complementary with G ≈ C and A ≈ U.

Isolation of Ribonucleic Acid from Cow Mammary Gland

There is increasing evidence that various forms of ribonucleic acid (RNA) perform essential roles in the transfer of genetic information and protein synthesis (10). Though there have been some studies on the isolation of and the base composition of RNA from rat mammary gland (2, 9), little attention has been paid to the isolation of RNA from cow mammary gland. Some procedures employed successfully to isolate rat liver RNA were examined for the isolation of RNA from cow mammary gland. Results are presented in this paper. Pieces of cow mammary gland were removed from the udder immediately after slaughter and frozen in dry ice. In most cases, the extraction of RNA was done immediately after sampling. In other cases, the mammary gland was stored at −20C; 20–200-g pieces of the mammary gland were minced by a mechanical chopper or scissors into small pieces before extraction. The following three procedures for the isolation of RNA were examined:

Chemical interaction of S35-6-mercaptopurine and ribonucleic acid.

SummaryThese data indicate that 6-mercaptopurine can combine with ribonucleic acid both in vitro and in vivo. Binding is enhanced by added metals and the combination is stable to ribonucleic acid isolation procedures.

On the chromatographic evidence for the occurrence of an infective ribonucleic acid fraction in leaves infected with tobacco mosaic virus

Chromatography on carboxymethyleellulose (CM-cellulose) of tobacco mosaic virus (TMV)-infected tobacco leaf has failed to yield fractions, the infectivity of which could be attributed to the presence of a free ribonucleic acid (RNA). This result is in disagreement with that obtained by Cochran and Chidester (1957). When infective RNA which had been prepared from TMV was ground with tobacco leaf and CM-cellulose in pH 4.5 buffer, it was adsorbed to the insoluble residue and the infectivity could not be recovered. It is, therefore, unlikely that CM-cellulose will be of use for the isolation of free RNA from TMV-infected leaves.

The inactivation of ribonuclease during the isolation of ribonucleic acids and ribonucleoproteins from yeast.

The isolation of ribonucleic acids and ribonucleoproteins in an undegraded state is complicated by the presence of degradative enzymes, particularly ribonuclease. It has been particularly difficult to find an inhibitor for this enzyme that was suitable for use in preparative work on the isolation of ribonucleoproteins. Inhibitors such as sodium cholate (Jones, Marsh & Rizvi, 1957) and sodium dodecyl sulphate (Crestfield, Smith & Allen, 1955), which have been used for the isolation of ribonucleic acids, cannot be used for the isolation of ribonucleoproteins because of their possible denaturing action on the proteins. Polyawidic substances have been used to inhibit pancreatic ribonuclease (Zoliner & Fellig, 1953; Vandendriessche, 1956) and, although these could possibly be used in the isolation of ribonucleoproteins, the removal of the inhibitor from the final product would be difficult. Copper and Zn2+ ions inhibit pancreatic ribonuclease (Zittle, 1946; Davis & Allen, 1955) but, as the present investigations have shown, these have limited use in isolation procedures for ribonucleic acids and ribonuclebproteins. The object of the present work was to find inhibitors suitable for this purpose.

Infectivity of polio virus ribonucleic acid for embryonated eggs and unsusceptible cell lines.

THE isolation of ribonucleic acid from various viruses grown in animals or in tissue culture systems has been reported recently. These preparations have been shown to be infectious for susceptible cell monolayers1–5.

A new method for the isolation of ribonucleic acids from mammalian tissues.

A new method for the isolation of ribonucleic acids from mammalian tissues.

Cytoplasmic localization of highly polymerized liver ribonucleic acid

Highly polymerized ribonucleic acid (RNA) of rat liver cytoplasm was found to be localized almost exclusively in the microsomes. Only traces of RNA were obtained from the supernatant fraction after centrifugation of the microsomes at 100,000 times gravity. The average sedimentation velocity constant, 6.4, of the microsomal RNA was about 30 per cent lower than that of RNA isolated directly from unfractionated cytoplasmic extracts, probably as a result of ribonuclease activity during the fractionation procedure. RNA could not be isolated from mitochondria by the guanidine hydrochloride method used for the isolation of microsomal RNA; a more drastic method yielded mitochondrial RNA with an S20 of 3.7. The yield corresponded to a content in mitochondria of at least 3.3 per cent RNA.