The Isolation of Nucleoli from Ungerminated Pea Embryos

Rat liver nuclei were freed of cytoplasmic contamination by washing with Triton-X-100 and subsequent centrifugation through 2.2 M sucrose. Electron microscopic examination showed that the outer membranes of the nuclei had been removed, but that the nuclei otherwise resembled the nuclei of intact liver. Morphological studies, chemical estimations of DNA, RNA, and protein and the estimation of cytoplasmic "marker" enzymes suggested that contamination of nuclei by cytoplasmic components was limited. These nuclei were obtained in yields of about 70% and were suitable for the isolation of nucleoli. Nucleoli were isolated by the breaking of the nuclei by ultrasound and subsequent differential centrifugation. In ultrastructural appearance, the isolated nucleoli resembled nucleoli in intact tissue. However, at high magnifications the "granular" component of isolated nucleoli appeared to consist of tightly twisted fibers. The nucleoli could be obtained in yields of at least 30%, and the values for the chemical composition of the isolated nucleoli agreed with values previously reported.

Section: Introductionmentioning

confidence: 99%

Electron Microscopic and Biochemical Characteristics of Nuclei and Nucleoli Isolated From Rat Liver

Sadowski

Steiner

1968

“…Nuclei and nucleori of "Arthur" peas were isolated as previously described (3). Subcellular fractions were suspended in 2.0 ~ sucrose from which appropriate volumes were withdrawn for analysis.…”

Section: Methodsmentioning

confidence: 99%

Some Chemical Properties of Isolated Pea Nucleoli

Stern

Johnston

Setterfield

1959

Self Cite

Isolated nuclei and nucleoli of ungerminated pea embryos have been analyzed chemically for their content of DNA, RNA, zinc, iron, phosphorus, and protein sulfhydryl groups. The values obtained cannot be considered to represent the whole of the riving nucleolar body as an undetermined amount of material is extracted from nucleoli in the course of their isolation. Only negligible amounts of DNA have been found in the isolated nucleofi; most of the DNA released on disruption of nuclei appears in a fraction showing very few structures under the light microscope. RNA is more concentrated in the nucleolus than in the nucleus or cytoplasm, but since nucleolar protein is 6 per cent of nuclear and less than 1 per cent of cytoplasmic protein, the total amount of nucleolar RNA is comparatively small. None of the other components listed occurs in high concentration in either nucleus or nucleolus.

“…3. NUCLEOTIDE COMPOSITION OF VARIOUS NUCLEAR RNA's RESULTS: Table IV gives the nucleotide composition of the RNA's of the nucleolar and nucleoplasmic fractions and of the salt insoluble and salt extractable RNA's obtained from some of these fractions.…”

Section: Distributionlmentioning

confidence: 99%

“…The first, proposed by Litt et al (1) and by Monty et al (2), relied on sonic vibrations to disrupt liver cell nuclei and disperse their nucleoplasm; it yielded a nucleolar fraction with a high content of DNA. In the second approach (3,4), the "solubilization" of the nucleoplasm was obtained through Ca 2+ removal effected by suspending the nuclei in a citrate-containing medium; the nuclear envelope was subsequently ruptured and the nucleoplasm dispersed by stirring the suspension in a mixer. In a third approach (5), isolated nuclei were homogenized in a high density medium and the liberated nucleoli separated by differential centrifugation upon dilution of this homogenate.…”

Section: Introductionmentioning

confidence: 99%

Studies on Isolated Nuclei

Maggio

Siekevitz

Palade

1963

This paper dcscribes thc snbfractionation of nuclei isolatcd from guinea pig liver by the procedure presented in thc first article of the series (8). Centrifugation in a density gradicnt system of nuclear fractions disruptcd by sonication permits the isolation of the following subfractions: (a) a nucleolar subfraction which consists mainly of nuclcoli surroundcd by a variablc amount of nuclcolus-associated chromatin and contaminated by chromatin blocks derived primarily from von Kupffcr cell nuclei; (b) and (c), two nucleoplasmic subfractions (I and If) which consist mainly of chromatin threads in a coarser (1) or finer (II) degree of fragmentation. The protein, RNA, and DNA content of these subfractions was dctermined, and their RNA's characterized in tcrms of NaCl-solubility, nucleotide composition, and in vivo nucleotidc turnover, using inorganic 32p as a markcr. The results indicate that therc are at Icast threc types of RNA in the nucleus (onc in the nucleolus and two in the nucleoplasm or chromatin), which differ from onc another in NaCl-solubility, nucleotide composition, turnover, and possibly sequcnce. Possible relations among these RNA's and thosc of the cytoplasm arc discussed.