Large-scale purification of viroid RNA using Cs2SO4 gradient centrifugation and high-performance liquid chromatography

A procedure for the purification of viroid RNA from tomato plants is described which yields up to a milligram of viroid RNA of gel electrophoretic homogeneity within 2 days. This technique is at least three times as fast as previous methods and is generally applicable to other RNA species. Plant material was homogenized and phenol extracted. In a Cs₂SO₄ density gradient, viroid RNA together with low-molecular-weight RNA, was separated from large single-stranded RNA, DNA, polysaccharides, polyphenols, and other compounds. The separation is based on the differences in the buoyant density and on the selective precipitation of large single-stranded RNA in Cs₂SO₄. Further purification of viroid RNA was achieved by HPLC over a weak anion exchanger linked to silica gel of optimized pore size. The elution was carried out by a salt gradient with complete exclusion of divalent metal ions. The procedures were applied to whole plants, leaves, stems, roots, cells, and protoplasts. The yields of nucleic acids at the different steps of purification are given for leaves, stems, and roots.     

Purification of bovine neurosecretory granule membranes by density gradient centrifugation

A procedure for the subfractionation of neurosecretory granules into membrane and content components is described. The procedure involves the hypotonic lysis of the secretory granule fraction and further purification of the membranes by centrifugation through a discontinuous sucrose gradient. The neurosecretory granule membranes represented 5.2% of the total proteins of the neurosecretory granule fraction and were highly enriched in cytochrome b561. Electron microscopic analysis of the purified membranes showed vesicles devoid of electrodense content.     

Isolation of intramitochondrial bodies in bovine adrenocortical cells by density gradient centrifugation

Summary Two methods for isolating the intramitochondrial bodies from bovine adrenocortical cells are proposed. Electron microscopic examination shows that discontinuous sucrose density gradient centrifugation can separate the fraction rich in intramitochondrial bodies, but some indistinguishable fragments remain among them. Continuous sucrose density gradient centrifugation is probably superior to the former method in obtaining a highly purified fraction of the bodies. The amido black positive granules, presumed to be intramitochondrial bodies, are collected in the fractions of the sucrose density of around 1.27 (1.23–1.30), which lack cytochrome c oxidase activity.This work was supported by a Scientific Research Grant, No. 144017, from the Ministry of Education of Japan to Professor M. Yasuda     

Isolation of intramitochondrial bodies in bovine adrenocortical cells by density gradient centrifugation.

Two method for isolating the intramitochondrial bodies from bovine adrenocortical cells are proposed. Electron microscopic examination shows that discontinuous sucrose density gradient centrifugation can separate the fraction rich in intramitochondrial bodies, but some indistinguishable fragments remain among them. Continuous sucrose density gradient centrifugation is probably superior to the former method in obtaining a highly purified fraction of the bodies. The amido black positive granules, presumed to be intramitochondrial bodies, are collected in the fractions of the sucrose density of around 1.27 (1.23--1.30), which lack cytochrome c oxidase activity.     

Enucleation of cells by density gradient centrifugation

HEp-2 cells can be enucleated by ultracentrifugation in a colloidal silica (PTL) density gradient, containing cytochalasin B. Under optimal conditions, more than 70% of the cells are enucleated. Purification up to 97% is carried out by centrifugation at low speed through a second, preformed PTL density gradient. The enucleated cells show a high viability, as tested by [³H]leucine incorporation. The method described was developed for enucleation of high quantities of cells and has the advantage that it can be used for cell types which do not adhere firmly enough to a carrier to be centrifuged as a monolayer.     

Separation of hormone-sensitive yeast cells by density gradient centrifugation

Cells in cultures of haploid strains of Saccharomyces cerevisiaein stationary phase were separated into interface fraction andpellet fraction by density gradient centrifugation. Cells inpellet fraction expanded in response to yeast sexual hormoneand animal sex hormones, whereas cells in interface fractiondid not. ¹Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka, Japan (Received July 16, 1970; )     

Purification of Rickettsia tsutsugamushi by Percoll density gradient centrifugation

Purification of Rickettsia tsutsugamushi has been achieved by Percoll density gradient centrifugation. The microorganisms purified showed good retention of infectivity and intracellular morphology. Budding rickettsiae in the egressing stage and intracellular rickettsiae in the multiplying process were harvested separately and purified by this technique. In electron microscopic observations, the intracellular rickettsiae obtained were surrounded with double membrane-layers of cell wall and cell membrane, and the budding rickettsiae were enveloped with an additional outermost membrane which may have originated from host cell membrane obtained in the budding process.     

Assessing the utility of partitioning primary productivity by density gradient centrifugation

We assess here a method for determining what contribution variouscomponents of natural phytoplankton assemblages make to measuredcommunity primary productivity. Following incubation with ¹⁴C-bicarbonate-labelledplankton and unlabelled controls are physically separated intosets of parallel subpopulations by centrifugation in densitygradients. The relative activity of the former subaliquots isdetermined by scintillation counting while the latter subaliquotsare enumerated by a modified Utermohl inverted microscope method.Field and laboratory data are presented illustrating partitioningsof coastal and oceanic plankton population carbon uptake. Thedescribed procedure could be used to partition the uptake ofother isotopes or biomass parameters such as chlorophyll orATP concentration. The advantages and disadvantages of the densitygradient approach are discussed relative to available alternatives.     

Separation of keratinocytes by density gradient centrifugation for DNA cytofluorometry

A method for the separation of guinea pig epidermal keratinocytes, in which the Feulgen-stainable material suffers minimal damage, has been investigated. The principal stage involves trypsin treatment of the epidermal sheet, stripped from the dermis with ethylenediamine tetraacetic acid. The epidermal cells thus isolated are separated into three groups by centrifugation on a continuous colloidal silica (Percoll) density gradient. The resulting arrangement of the keratinocytes in the centrifuge tube corresponds to their arrangement in situ, with basal cells at the bottom and the more differentiated cells above. By morphological examination, it can be shown that relatively pure fractions of basal cells, spinous cells, and granular cells are obtained by this method. With respect to DNA distribution pattern, there was good agreement between that of keratinocytes separated by the microdissection-ultrasonic irradiation method, or by the chymotrypsin method as reported previously by us, and that obtained by the present method.     

Fractionation of plant protoplast types by iso-osmotic density gradient centrifugation

Summary A simple effective technique for the fractionation of protoplast populations is described. Protoplasts are separated by low-speed centrifugation in an iso-osmotic, discontinuous density gradient system on the basis of differences in their buoyant densities. At a constant osmolality of 660±20 mOs/kg H₂O, the gradients provide a density range from 1.017 to 1.069 g/cm³ at 20 °C which corresponds to the buoyant densities of most protoplast types studied. Characteristics of the KMC/S-density gradient system and factors affecting the fractionation were investigated. Protoplasts were isolated from various tissues and cultivars of tobacco, barley, wheat, rye, oat and maize. Their density-dependent distribution profiles in KMC/S-gradients and their average buoyant densities were determined under standardized conditions. Great differences in the buoyant densities were found between protoplasts of different tissues. Mixed populations of two types of protoplasts, differing in buoyant density by about 15–20 mg/cm³, were separated to give highly purified fractions. Factors affecting the buoyant densities of protoplasts have been investigated. Ploidy level and species differences did not significantly affect the fractionation profiles. However, an age-dependent variation in the average buoyant density of tobacco mesophyll protoplasts was observed. Fractionation of tobacco mesophyll protoplasts and their subsequent regeneration to plants demonstrates the practicability and physiological compatibility of the KMC/S-density gradient system under sterile conditions. The morphogenetic potential of protoplasts was not affected by the separation procedure or the gradient components.     

Separation of keratinocytes by density gradient centrifugation for DNA cytofluorometry

Summary A method for the separation of guinea pig epidermal keratinocytes, in which the Feulgen-stainable material suffers minimal damage, has been investigated. The principal stage involves trypsin treatment of the epidermal sheet, stripped from the dermis with ethylenediamine tetraacetic acid. The epidermal cells thus isolated are separated into three groups by centrifugation on a continuous colloidal silica (Percoll) density gradient. The resulting arrangement of the keratinocytes in the centrifuge tube corresponds to their arrangement in situ, with basal cells at the bottom and the more differentiated cells above. By morphological examination, it can be shown that relatively pure fractions of basal cells, spinous cells, and granular cells are obtained by this method. With respect to DNA distribution pattern, there was good agreement between that of keratinocytes separated by the microdissection-ultrasonic irradiation method, or by the chymotrypsin method as reported previously by us, and that obtained by the present method.     

Plasmodium falciparum merozoites: isolation by density gradient centrifugation using Percoll and antigenic analysis

Merozoites of Plasmodium falciparum were isolated and immunocytochemically analyzed. Mature parasites from knobby (K+) and knobless (K-) strains were incubated for 4 to 5 hr in RPMI 1640 with 10% serum and 10% RBC extract. About 12 to 14% of the merozoites released were recovered by density gradient centrifugation using Percoll. From 1 to 3 X 10(9) merozoites were obtained per collection. The merozoite preparations were contaminated with 10% residual bodies, about 0.1% infected and uninfected erythrocytes, about 0.1% RBC-free trophozoites and schizonts, and numerous small (less than 0.5 microns) membrane vesicles. Merozoites from the K+ and K- strains were morphologically and, by an indirect, ferritin-labeled antibody assay using serum from immune Aotus, antigenically indistinguishable. Although the residual body coats reacted with the immune Aotus serum, the membrane vesicles, some of which were seen to be blebbing from merozoites, did not react with this serum or a serum against erythrocytes. This paper describes a procedure that can be used to obtain large numbers of merozoites with little contamination by host erythrocytes.