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1.
Isolated nucleoli, nucleolar chromatin, and nucleolar DNA were used as templates for DNA synthesis in appropriately supplemented systems in which RNA polymerases other than RNA polymerase I were blocked by alpha-amanitin. With the aid of nucleotide analysis, DNA-RNA hybridization, and homochromatography fingerprinting, it was found that isolated nucleoli and nucleolar chromatin serve primarily as templates for synthesis of rRNA. However, the products formed with purified nucleolar DNA as a template do not contain the specific rRNA oligonucleotides nor are they appreciably hybridized to the rDNA region on cesium chloride gradients. These results indicate that whole nucleoli and nucleolar chromatin contain control mechanisms that restrict readouts by RNA polymerase I of nucleolar DNA to rDNA. 相似文献
2.
A procedure is described to isolate nucleoli from Tetrahymena pyriformis which contain extrachromosomal ribosomal DNA. Macronuclei isolated by the Nonidet procedure were sonicated at a reduced magnesium concentration, and the sonicate was fractionated by isopycnic centrifugation in a metrizamide density gradient. The heaviest band, designated Band IIb, contains exclusively ribosomal DNA, thus constituting the nucleolar fraction. The purity of the nucleolar fraction on a DNA basis, which is defined as the percentage of ribosomal DNA and determined by equilibrium centrifugation in a CsCl density gradient, was around 70%. Electron microscopic examination revealed that the isolated nucleoli retained fairly well the ultrastructure of the in situ nucleoli. Some of the biochemical properties of the isolated nucleoli are also presented. 相似文献
3.
Nucleoli from auxin-treated tissues ( Glycine max L. var Wayne or Kaoshiung No. 3) were isolated and purified by Percoll density gradient centrifugation. There was a 2.1-fold increase in RNA and a 2.8-fold increase in protein after a 24-h auxin treatment per unit nucleolar DNA. More than 150 acid-soluble protein spots were associated with the auxin-treated nucleoli on two dimensional (2-D) gel electropherograms. Nucleoli from auxin-treated tissue were fractionated by suspension in 20 millimolar dithiothreitol at room temperature for 20 minutes into two distinct fractions referred to as the nucleolar chromatin and preribosomal particle fractions. The DNA:RNA:protein ratio of the chromatin fraction was 1:2.5:14. Most of RNA polymerase 1 activity and nucleolar DNA recovered in this fraction. The acid-soluble proteins in the chromatin were resolved into 32 protein spots on 2-D gel electropherogram. The most abundant spots were identified as histones. The nucleolar preribosomal particle fraction had a DNA:RNA:protein ratio of 1:24:102 and contained only trace amounts of RNA polymerase 1 activity and only 10 per cent of the nucleolar DNA. Acid-soluble proteins associated with these particles were resolved into 78 protein spots; 72 of these (acid-soluble) protein spots corresponded in 2-D gel electrophoresis to 80S cytoplasmic ribosomal proteins. Some 15 protein spots found in 80S ribosomal proteins were absent in the preribosomal particles. It seems reasonable, based on these data, that the enlargement of nucleoli after auxin treatment is primarily due to the large increase in ribosomal proteins and rRNA which accumulate and assemble in the nucleoli in the form of preribosomal particles. 相似文献
7.
In maturing oocytes of the newt Triturus viridescens, the nucleoli undergo a series of morphological changes that are very similar to those described by Callan for the axolotl, Ambystoma mexicanum. The nucleoli first assume the form of spheroids which then become extended into ring or necklace shapes that are DNase-sensitive; in mature oocytes the nucleoli revert to a spheroidal form. Short term in vitro incorporation studies with uridine- 3H on both species show that RNA synthesis occurs in a restricted, eccentric portion of the spheroidal nucleoli, thereby producing an asymmetrical pattern of labeling. In the ring forms, however, the localization of the radioactivity suggests that synthesis takes place symmetrically throughout their entire length. The changes in nucleolar morphology apparently reflect the fact that the component DNA has undergone a redistribution from a localized region in the spheroidal nucleoli to an extended circle in the rings; the patterns of uridine- 3H incorporation, therefore, parallel the distribution of DNA in both the spheroidal and the ring nucleoli. Ultrastructurally, the nucleoli contain a fibrillar component that corresponds in position to that of the DNA. The typical spheroidal nucleolus consists of a fibrillar core situated eccentrically and surrounded by a hull of granular, ribonucleoprotein material. The ring nucleoli are composed of a central fibrous region that is ensheathed all around its circumference by a layer of similar granular material. This granular substance is thicker at intervals along the length of the rings, representing the "beads" of the necklaces. 相似文献
11.
A rapid isolation procedure of nucleoli from detergent purified nuclei of some tumor and tissue culture cells is described. The procedure makes use of a non-ionic detergent, Nonidet P40 and sodium deoxycholate to purify nuclei followed by the addition of Ca 2+ or Mg 2+ to strengthen the nucleoli against sonication. Enzymatically active (with respect to nucleolar RNA polymerase) nucleoli containing undegraded nucleolar RNAs may be isolated from a mouse hepatoma MH134, Ehrlich ascites tumor, HeLa cells, L cells and C3H2K cells with this procedure. 相似文献
12.
A large extrachromosomal mass of Feulgen positive material, the DNA body, has been visualized in early prophase oocytes of crickets (Orthoptera: Gryllidae) representative of the closely related subfamilies Gryllinae and Nemobiinae. A similar structure is present in oocytes of representatives of two subfamilies of crickets (subfamilies Oecanthinae and Gryllotalpinae) which taxonomically and phylogenetically are quite separate from those mentioned previously. In situ hybridization demonstrates that the body contains amplified copies of genes coding for ribosomal RNA. Unlike the DNA body in early diplotene oocytes of representatives of the subfamily Gryllinae, which is closely associated with the developing nucleolar apparatus, the DNA body in oocytes of the Oecanthinae and Gryllotalpinae cannot be demonstrated during diplotene. In the Oecanthinae, the nucleolar apparatus of early diplotene stage oocytes is composed of four to seven separate structures, the ribonucleoprotein of which has a characteristically lamellated appearance. During late diplotene, these nucleoli give rise to many smaller structures which are distributed throughout the germinal vesicle. In early diplotene stage oocytes of Scapteriscus acletus (Subfamily: Gryllotalpinae), the nucleolar apparatus consists of a single compact mass of ribonucleoprotein. In contrast to the oocytes of all other crickets that have been studied, the nucleolus of S. acletus remains single throughout diplotene. In situ hybridization analysis indicates that the amplified genes coding for rRNA which are localized in the DNA body of early prophase oocytes become incorporated into this compact nucleolar mass. Differences in nucleolar structure appear to reflect differences in the organization of amplified genes coding for rRNA. 相似文献
13.
The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed. 相似文献
14.
A study has been made of the content and composition of RNA in cytoplasm, nucleoplasm, and nucleoli from growing oocytes of the starfish Asterias rubens. The determinations were carried out, using ultramicrochemical methods, on units isolated by microdissection from fixed sections. Macrochemical and interferometric control experiments show that RNA can be quantitatively evaluated in this way. The results show that the growing oocyte represents a system in which the relations between the quantities of nucleolar, nucleoplasmic, and cytoplasmic RNA undergo great changes. These changes are continuous for nucleolar and cytoplasmic RNA so that their amounts may be predicted from the size of the cell. Nucleoplasmic RNA, on the other hand, shows great variations among different cells, independent of cell size. Purine-pyrimidine analyses show that each cell component contains an RNA which differs significantly from that of the other two. Cytoplasmic and nucleolar RNA are closely related, the only difference being a slightly higher guanine/uracil quotient for the nucleolar RNA. They are both of the usual tissue RNA type, i.e., they show a preponderance of guanine and cytosine over adenine and uracil. Nucleoplasmic RNA deviates grossly from the RNA of the other two components. Here the concentrations of adenine and uracil are higher than those of guanine and cytosine, respectively. This RNA consequently shows some resemblance to the general type of animal DNA although the purine/pyrimidine ratio is far from unity. Our data favor a nucleolar origin for the stable part of the ribosomal RNA and a nucleoplasmic one for the unstable part (the messenger RNA). 相似文献
15.
Amplification of the genes coding for ribosomal RNA oocurs in the oocytes of a wide variety of organisms. In oocytes of various species of crickets (Orthoptera: Gryllidae) the amplified DNA is contained in a large extrachromosomal DNA body. Multiple nucleoli form about the periphery of the DNA body during the diplotene stage of meiosis I. In contrast to the general pattern of orthopteran oocytes, oocytes of the cockroach Blattella germanica demonstrate a single large nucleolus instead of many nucleoli. In order to determine whether the genes coding for rRNA are amplified in the oocytes of B. germanica, the relative amount of rDNA in oocytes was compared with the rDNA content of spermatocytes and somatic cells. An extrachromosomal DNA body similar to that present in crickets is not present in B. germanica. A satellite DNA band which contains nucleotide sequences complementary to rRNA accounts for approximately 3-5% of the total DNA in somatic and in male and female gametogenic tissues. Female cells contain approximately twice as much rDNA as do male cells. An XX-XO sex-determining mechanism is operative in B. germanica. In situ hybridization with rRNA indicates that the nucleolar organizer is located on one end of the X chromosome and that oocytes do not contain more than twice the amount of rDNA found in spermato cytes. The data indicate that rDNA is not amplified in the uninucleolate oocyte of B germanica. 相似文献
16.
A large DNA-containing body is present in addition to the chromosomes in oocytes of the house cricket Acheta domesticus. Large masses of nucleolar material accumulate at the periphery of the DNA body during the diplotene stage of meiotic prophase I. RNA-DNA hybridization analysis demonstrates that the genes which code for 18S and 28S ribosomal RNA are amplified in the ovary. In situ hybridization indicates that the amplified genes are localized within the DNA body of early prophase cells. As the cells proceed through diplotene the DNA which hybridizes with ribosomal RNA is gradually incorporated into the developing nucleolar mass. 相似文献
18.
The pattern of staining for DNA, histone, and nonhistone protein has been studied in whole cells and in nuclei and chromosomes isolated by surface spreading. In whole interphase cells from bovine kidney tissue culture, nuclear staining for DNA and histones reveals numerous small, intensely stained clumps, surrounded by more diffusely stained material. Nuclei in whole cells stained for nonhistone proteins also contain intensely stained regions surrounded by diffuse stain. These intensely stained regions also stain for RNA, indicating that the regions contain nucleolar material. Electron microscopy of kidney cells confirms that multiple nucleoli are present. Kidney nuclei isolated by surface spreading show an even distribution of stain for DNA, histones, and nonhistone proteins, indicating that the surface forces disperse both condensed chromatin and nucleoli. DNA and protein staining was also studied in metaphase chromosomes from testes of the milkweed bug, Oncopeltus fasciatus. Staining for DNA and histones in metaphase chromosomes is essentially the same in sections of fixed and embedded testes as in preparations isolated by surface spreading. However, striking differences are noted in the distribution of nonhistone proteins. In sections, nonhistone stain is concentrated in extrachromosomal areas; metaphase chromosomes do not stain for nonhistone proteins. Chromosomes isolated by surface spreading, however, stain intensely for nonhistone proteins. This suggests that nonhistone proteins are bound to the chromosomes as a contaminant during the isolation procedure. The relationship of these findings to current work with chromosomes isolated for electron microscopy is discussed. 相似文献
19.
The early stages of female and male germ cells have been investigated in Feulgen squash preparations, in unfixed state with phase contrast optics and in the electron microscope. The DNA axes of the ring-shaped multiple nucleoli in the growing oocytes of Gryllus arise from compact DNA bodies which are found in oogonia of young larvae and in oocytes prior to the growth period. The nuclei of the early oogonia contain several little DNA bodies whereas young oocytes at leptotene, zygotene and pachytene have only one body which is bigger than at earlier stages (Pig. 3). At metaphase and anaphase during oogonial mitosis the DNA body has a filamentous shape distinguishable from the compact chromosomes (Fig. 5). In oogonia as well as at leptotene and zygotene stages, nucleoli are produced in the peripheral, uncoiled parts of each DNA body whereas the compact interior is completely free of nucleolar material (Figs. 4, 12). At pachytene, the whole DNA body begins to despiralize, and single DNA strands are released into the nucleoplasm. These strands form hundreds of multiple nucleoli which finally are dispersed in the germinal vesicle (Fig. 11). — Incorporation studies with radio-active thymidine have shown that DNA synthesis in the DNA body is not synchronous with the S-phase of the chromosomes (Fig. 7). — The DNA body is an own formation distinct from the sex chromosomes (in contrast to the opinion of Sotelo and Wettstein, 1964). Although the positive heteropycnotic X-chromosome in the germ cells of the male cricket is very similar to the DNA body of the female (Fig. 8), there is no regular contact between sex chromosome and nucleolus neither in spermatogonia nor in spermatocytes (Figs. 9, 14). In all probability, the site of the nucleolar organizer is autosomal. — It is suggested that the amplification of the nucleolar genes in Gryllus oocytes results in an accumulation of ribosomal RNA for use during the early cleavage stages of the embryo 相似文献
20.
Extrachromosomal nucleoli were isolated from log phase cells of Tetrahymena pyriformis (amicronucleate strain) in a highly purified state. Nucleoli located at the periphery of the macronucleus were detached from the nucleoplasmic mass of isolated macronuclei with agitation and separated from macronuclei by filtration through a Nuclepore membrane filter (pore size 5 m). The filtrate constitutes the crude nucleolar preparation, as judged by electron microscopy and DNA analysis. Further purification of the nucleoli was performed by isopycnic centrifugation of the filtrate in a Metrizamide density gradient. After this step, the purity of the nucleoli, as defined by rDNA content and measured by analytical CsCl centrifugation, was almost 100%. Electron microscopy of the purified nucleoli revealed structures that resemble those of in situ nucleoli. Undergraded 35S pre-rRNA, together with 26S and 17S rRNA, could be isolated from purified nucleoli. In vitro RNA synthetic activity was associated with isolated nucleoli. This activity is insensitive to low and high concentrations of -amanitin, indicating that the form I RNA polymerase is functioning.by M.F. TrendelenburgThis paper is dedicated to the late Dr. Yoshihiro Kato 相似文献
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