CsCl Cushion Research Articles

Isolation of Functional RNA From Grapevine Tissues Poor in Nucleic Acid Content

A modified procedure for efficient isolation of intact and biologically active RNA from leaves, berries and cell cultures of grapevine (<i>Vitis vinifera</i> L.) is described. RNA is extracted in the presence of mild denaturants followed by sequential precipitations with potassium acetate and isopropanol. RNA is recovered free of contaminants after centrifugation through a CsCl cushion. Alternatively, RNA from grapevine callus and berry cell suspensions can be purified after the isopropanol step by high-salt precipitations, without a CsCl centrifugation step. The resulting RNA is of high-quality, as judged by agarose gel electrophoresis and northern blot analysis. Furthermore, RNA isolated by this method is biologically active as was evidenced by <i>in vitro</i> translation and analysis of the products by polyacrylamide gel electrophoresis and fluorography. This procedure can also be applied to other perennial woody plant species.

Isolation of RNA from apple skin

A method has been developed for the isolation of RNA from apple skin. The method involves an adaptation of the Manning (1991) method and includes a high-salt extraction step and a final purification step through a CsCl cushion. The RNA isolated was of high quality and produced good hybridization signals in northern blot analyses.

The preparation of poly(A)+mRNA from the hagfish slime gland.

The isolation of translatable poly(A)+mRNA from the slime glands of the Pacific hagfish, Eptatretus stouti, is not possible by the commonly used procedures because of the viscous slime that is formed when the contents of the glands are hydrated. This paper reports on a procedure developed to overcome this problem. Briefly, the tissue was powdered in liquid nitrogen, mixed with sodium lauroylsarcosine and proteinase K and lyophilized. The lyophilized powder was then mixed with 0.3 mm diameter glass beads, thoroughly ground and wetted with buffer and digested at 37 degrees C. The RNA from the digest was recovered by ultracentrifugation through a CsCl cushion. Further purification of the RNA was accomplished by the usual methods with slight modifications.

Ontogenesis of TRH mRNA in the rat pancreas

The rapid changes in TRH levels in the rat pancreas during the neonatal period make this organ an interesting model for the study of the regulation of TRH biosynthesis. Pancreatic RNAs were isolated by the guanidinium thiocyanate method and layered onto CsCl cushion. Northern blot preparations were hybridized with 32P labeled TRH cDNA probe. Pancreatic TRH mRNA was first detected in 19-day old fetuses and reached the highest level on day O, then decreased, being barely detectable 14 days after birth. The neonatal injection of streptozotocin induced a dramatic drop of TRH mRNA levels 24 hours later. This result suggests that the peculiar evolution of TRH level in pancreas is partly due to the evolution of the expression of the TRH gene.

Decline in infectivity of simian virus 40 by sodium deoxycholate and its restoration with the extract of monkey kidney cells

Plaque-forming activity and T-antigen-synthesizing activity in the crude preparation of simian virus 40 (SV40) decreased to 1/20–27 after treatment with 0.5% sodium deoxycholate (DOC) for 30 min at 37°C. A full restoration of the activity occurred after incubation of DOC-treated virions with the extract of monkey CV-1 cells, host cells for productive infection with SV40. Analysis by sedimentation through 15% sucrose to CsCl cushion (ϱ = 1.327 g/cm 3) revealed that virions in the [ 35S]methionine-labeled crude virus preparation sedimented to the interface between CsCl and sucrose, and that treatment with DOC resulted in the loss of infectivity and the appearance of virions sedimentable into CsCl cushion. The [ 35S]methionine-labeled purified virions (prepared after treatment with DOC and sedimentable into CsCl cushion) sedimented to the CsCl-sucrose interface after incubation with the cell extract, with restoration of infectivity. The infectivity-restoring activity of the cell extract was sensitive to ethyl ether, partially sensitive to heating at 75°–97° for 30 min, but resistant to treatment with DNase (50 μg/ml), RNase (40 μg/ml), or trypsin (0.05%) for 30 min at 37°. These results suggest that lipid-related cellular components bind stably to virions of SV40 and facilitate an efficient infection.

Preparation and characterization of mRNAs from rat heart muscle

RNA was prepared from rat heart muscle by a procedure using guanidium thiocyanate and centrifugation on a CsCl cushion. Analysis of the RNA by sucrose gradient centrifugation and electrophoresis shows that major part of it was long sized. Poly (A+) RNA was isolated with a yield of 35 micrograms/g. The average size of the poly (A) tail was 120 nucleotides. RNA was translated in a reticulocyte lysate and the efficiencies were compared with total liver and skeletal muscle RNA. The translational products of poly (A+) RNA were analyzed in a two dimensional gel. They show that many muscle specific proteins have been synthesized in vitro, which indicates that at least, part of the RNAs has remained intact.

Preparation and in vitro translation of mRNA from Fasciola hepatica

Total RNA was extracted from mature and juvenile Fasciola hepatica by homogenizing in 5.0 M guanidine thiocyanate and centrifugation through a 5.7 M CsCl cushion. Yields of 2 mg/g and 1 mg/g wet weight starting material were obtained, respectively. Messenger RNA was separated from the bulk extracted RNA by binding to oligo(dT)-cellulose. About 25% of the extracted RNA bound in both adult and juvenile cases. This material was subsequently tested in a rabbit reticulocyte cell-free translation system. Up to a 12-fold stimulation of incorporation of [ 35S]methionine into trichloroacetic acid-precipitable material was observed over that where no message was added. When the in vitro translation products were analysed by autoradiography of SDS-polyacrylamide gradient gels, polypeptides ranging in apparent molecular weight from about 10 000 to 100 000 were observed. Several minor differences in the electrophoretic patterns obtained from juvenile and adult mRNA were observed.

Synthesis in vitro of keratin polypeptides directed by mRNA isolated from newborn and adult mouse epidermis.

A method is described which allows the isolation of undegraded total cellular RNA from living epidermis in a one-step procedure. Epidermal tissue is homogenized in 4 M guanidinium thiocyanate/2% sarkosyl buffer and centrifuged through a 5.7 M CsCl cushion. Under appropriate density conditions in the homogenate, large amounts of RNA are sedimented free of DNA and protein to the bottom of the tubes. A prerequisite for the applicability of this method is the preparation of pure epidermis under conditions which efficiently inactivate endogenous RNases. Oligo(dT) affinity chromatography of total RNA yields 3-4% of polyadenylated RNA. Translation of poly(A)-rich fractions from both newborn and adult mouse epidermis in a cell-free reticulocyte system leads to the synthesis of proteins with electrophoretic mobilities identical to those of native keratin polypeptides of the corresponding tissue. Similar to native keratin polypeptides, these proteins are highly insoluble and can selectively be enriched by high-salt extraction of the translation assays. The CNBr cleavage pattern of these purified proteins is identical to that of native keratins. There is no evidence for the synthesis of precursors of these proteins.

Size, conformation and purity of chloroplast DNA of some higher plants

1. Chloroplast DNA of Antirrhinum majus, Oenothera hookeri, Beta vulgaris and Spinacia oleracea band at the same buoyant density of 1.697 g · cm −3 in neutral CsCl equilibrium gradients. The corresponding nuclear DNAs band at 1.691, 1.703, 1.695 and 1.695 g · cm −3, respectively. The purity of chloroplast and nuclear DNA can be assessed objectively only in the cases of Antirrhinum and Oenothera. 2. Electron microscopic analysis of chloroplast DNA, purified in CsCl or CsCl ethidium bromide gradients, revealed up to 80% circular molecules. Of these about 15% were of supertwisted conformation. Best yields of circular molecules were recovered when populations of unbroken chloroplasts were subjected to DNAase and phosphodiesterase treatment, and when the DNA was purified from viscous lysates by centrifugation into a CsCl cushion. Treatment of plastids with DNAase alone did not guarantee complete degradation of nuclear DNA. 3. The average contour length of the open circular chloroplast DNA molecules was basically similar for all four plants. They were 45.9 ± 2.1 μm for Antirrhinum, 45.7 ± 1.9 μm for Spinacia, 44.9 ± 1.7 μm for Beta and 45.2 μm for Oenothera. This is comparable to the size derived for the coding capacity of chloroplast DNA from reassociation experiments. As much as 15% of the total population of circles in chloroplast DNA of Spinacia were circular dimers.

Concentration and purification of poliovirus by ultrafiltration and isopycnic centrifugation.

A method is described by which poliovirus can be rapidly and simply concentrated by the use of a Diaflo XM-50 ultrafilter membrane. Freon-extracted ultrafilter concentrates banded in CsCl provided a 1,724-fold volumetric concentration of poliovirus. During concentration, trypsin-digested cellular material can pass through the ultrafilter membrane, thus providing a versatile means of degrading and eliminating extraneous contaminating proteins. The ultrafilter concentration system is compared with the CsCl cushion system of poliovirus concentration, and both systems are further compared by banding virus and virus capsid material in CsCl by use of isopycnic centrifugation.

Synthesis and assembly of simian virus 40. I. Differential synthesis of intact virions and empty shells.

Intact virions and empty shells of simian virus 40 may be rapidly separated from each other and from cell contaminants by a procedure employing a CsCl cushion. This approach permits quantitation of their respective syntheses in infected cells labeled with radioactive amino acids. As much as 5 to 10% of the total acid-precipitable radioactive lysine in infected cell extracts was incorporated into viral particles in a two-hour pulse late in infection. Evidence for multiple origins of empty shells is presented. Some of the empty shells result from breakdown of intact virions. However, empty shells can also form independently of intact virions. First, labeling for periods of 15 min to 2 hr late in the course of infection results in preferential incorporation of (3)H-lysine into empty shells. Secondly, treatment with the deoxyribonucleic acid inhibitor cytosine-beta-d-arabinofuranoside late in infection results in a 50% inhibition in the rate of formation of intact virions with minimal reduction in the rate of appearance of empty shells.

Concentration and Purification of Poliovirus by Ultrafiltration and Isopycnic Centrifugation1

A method is described by which poliovirus can be rapidly and simply concentrated by the use of a Diaflo XM-50 ultrafilter membrane. Freon-extracted ultrafilter concentrates banded in CsCl provided a 1,724-fold volumetric concentration of poliovirus. During concentration, trypsin-digested cellular material can pass through the ultrafilter membrane, thus providing a versatile means of degrading and eliminating extraneous contaminating proteins. The ultrafilter concentration system is compared with the CsCl cushion system of poliovirus concentration, and both systems are further compared by banding virus and virus capsid material in CsCl by use of isopycnic centrifugation.

Simian adenovirus SA7 DNA: chemical, physical, and biological studies.

Experimental Procedures.-M11aterials: A 13SC-1 cell line was obtained from lDr. RI. N. Hull; CV-1 was provided by Dr. Leroy McLaren. Simian adenovirus type SA7 was obtained from Dr. R. N. Hull. Snake venom phosphodiesterase, papaini (2X crvstallized), and electrophoretically purified pancreatic DNase I were purchased from Worthington Biochemical Corporation. RNase, sniake venom (Crotalus adamanteus), subtilisin, and alpha-chymotrypsin were obtained from Sigma Chemical Company. P'2-orthophosphate was obtained from the New England Nuclear Corporation. Cesium chloride was purchased from the American Potash Corporation and the Gallard Schlesinger Company. SV40 tumor antiserum was provided by Dr. W. Rowe; and SV40 tumor antigen was obtained from Flow Laboratories (Rockville, Maryland). Virus and cell culture: All cells were routinely propagated in Eagle's (MEM) medium with 10% fetal calf serum. The virus pools were produced by infecting monolayer cultures at a multiplicity of infection of 1-10 in MEM with 5% fetal calf serum and harvesting the infected cells at 36-48 hr postinfection. Virus purification: The virus was concentrated and purified by a procedure to be described in detail elsewhere.5 Briefly, the infected cells were disrupted by sonication; and the resulting virus suspension was treated sequentially with a combination of RNase, DNase, and snake venom followed by subtilisin anid alpha-chymotrypsin. The virus was then concentrated by centrifugation onto a CsCl cushion in a Spinco SW25.1 rotor at 25,000 rpm for 2 hr. The collected virus ban-d was centrifuged at least two times to equilibrium in a CsCl gradient. The final viruts bands were stored at 4?C in 35% CsCl or dialyzed against 0.15 411 NaCl containinig 0.01 Ml sodium phosphate, p1,1 7, and 0.001 M1 ethylenediaminietetraacetate (ED)TA) and stored frozen at 90C. Extraction of viral DNA: 1B3eta-mereaptoethanol (20 ,l) and papain (I mg) were added to each milliliter of dialyzed virus containing approximately 10 mg of virus. The mixture was incubated for 10-12 mini at 37?C or until visible clearing was obtained. Twenlty mg/ml of solid sodium dodecyl sulfate (SDS) was then added and incubation continued for 7 mimi at 37?C. This mixture was extracted at room temperature with an equal volume of phenol containing 0.2% 8-hydroxyquinoline. The aqueous phase was reextracted two times with fresh phenol. Traces of phenol were removed either by dialysis against 0.1 X SSC (SSC = 0.15 Ml NaCl + 0.015 M sodium citrate), or by precipitation and spooling of the DNA witb ethanol. The final preparation was stored in SSC with a drop of chloroform at 4?C. Density of DNA: The buoyant densities of native and alkali-treated viral DNA were determined in an equilibrium density gradient of CsCl containing 0.01 M tris(hydroxymethyl)aminomethane (tris) buffer, pH 7.4. Alkali-denatured DNA was prepared by exposing DNA, in 0.1 X SSC, to 0.2 N NaOH for 10 min at room temperature followed by neutralization with HCl. In each experiment, Micrococcus lysodeikticus or E. coli DNA was added as a standard. Centrifugations were performed at 25?C and 44,770 rpm in the Spinco model E ultracentrifuge equipped with ultraviolet absorptionl optics. Photographs were analyzed with a Spinco Analytrol densitometer.