Mammalian phosphatidylinositol 4-kinases

Three phosphatidylinositol 4-kinase isoforms, PI4K 230, 92 and 55 have been cloned and sequenced allowing a much wider characterization than the previously employed enzymological typing into type II and III enzymes. PI4K 230 and 92 contain a highly conserved catalytic core, PI4K55 one with a much lower degree of similarity. Candidate kinase motifs, deduced from the protein kinase super family, are absolutely conserved in all isoforms. Kinase activities are described based on their sensitivity and reactivity towards wortmannin, phenylarsine oxide (PAO) and 5'-p-fluorosulfonylbenzoyladenosine (FSBA). Localization of all isoforms in the cell is reported. All enzymes contain nuclear localization and export sequence motifs (NLS and NES) leading to the expectation that they can be transferred to the nucleus. PI4K230 has been found in the nucleolus, PI4K92 in the nucleus, additionally further broadening the function of these enzymes. In the cytoplasm of neuronal cells, PI4K230 is distributed evenly on membranes that are ultra structurally cisterns of the rough endoplasmatic reticulum, outer membranes of mitochondria, multivesicular bodies, and are in close vicinity of synaptic contacts. PI4K92 is functionally characterized as a key enzyme regulating Golgi disintegration/reorganization during mitosis probably via phosphorylation by cyclin-dependent kinases on well-defined sites. PI4K55 is involved in the production of second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3) at the plasma membrane, moreover, in the endocytotic pathway in the cytoplasm.     

Distribution of manganese superoxide dismutase in rat stomach: application of Triton X-100 and suppression of endogenous streptavidin binding activity.

The distribution of rat manganese superoxide dismutase (Mn-SOD) was immunohistochemically investigated in the rat stomach with a specific polyclonal antibody and a labeled streptavidin-biotin immunoglobulin detection system in cryosections. Parietal cells in the stomach were intensely stained, whereas the other epithelial cells in the gastric gland and pit exhibited only slight staining. Rapid-freezing and freeze-substitution immunoelectron microscopy revealed that Mn-SOD in parietal cells was mainly localized in mitochondria. Therefore, the large amount of Mn-SOD in parietal cells is due to the abundant mitochondria, in which Mn-SOD is considered to play important roles in protecting the ion pump and the cell itself from superoxide insult. Application of Triton X-100, cryosectioning, and the streptavidin-biotin system are needed to distinctly visualize Mn-SOD with our antibody. Treatment of the cryosections with Triton X-100 enhanced not only the immunoreactivity but also the false-positive staining, which showed a similar distribution pattern to that of Mn-SOD and thus made it difficult to determine the localization. The most plausible cause of the false-positive staining is thought to be endogenous biotin in the stomach, which survives paraformaldehyde fixation and is revealed by Triton X-100 treatment. Suppression of the endogenous streptavidin binding activity is important when cryosections, the streptavidin-biotin system, and Triton X-100 are employed.     

Location of RNase activity in nuclear residual structures

We report here the partial isolation of an RNase activity which copurifies with the nuclear residual structure prepared from both rat liver and Herpes-infected BHK cells. After the successive treatment of purified nuclei with DNase, low salt and high salt, the RNase activity is found both in the high salt soluble supernatant fraction and in the residual nuclear structure. Triton X-100 treatment of this structure solubilizes the RNase activity. From this we conclude that some of the RNase activity associated with the nuclear residual structure may be located in either the phospholipidic or protein moieties that were extracted with Triton X-100. This RNase cuts rRNA non-randomly into characteristic degradation products. Its molecular weight, on a glycerol gradient, was determined to be 25,000.     

Characterization of a nuclear phosphatidylinositol 4-kinase in carrot suspension culture cells

We have shown previously that a nuclear phosphatidylinositol (PI) 4-kinase activity was present in intact nuclei isolated from carrot suspension culture cells (Daucus carota L.). Here, we further characterized the enzyme activity of the nuclear enzyme. We found that the pH optimum of the nuclear-associated PI kinase varied with assay conditions. The enzyme had a broad pH optimum between 6.5–7.5 in the presence of endogenous substrate. When the substrate was added in the form of phosphatidylinositol/phosphatidylserine (PI/PS) mixed micelles (1 mM PI and 400 μM PS), the enzyme had an optimum of pH 6.5. In comparison, the pH optimum was 7.0 when PI/Triton X-100 mixed micelles (1 mM PI in 0.025 %, v/v final concentration of Triton X-100) were used. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at Triton X-100 concentrations greater than 0.3 % (v/v). Calcium at 10 μM inhibited 100 % of the nuclear-associated enzyme activity. The K_m for ATP was estimated to be between 36 and 40 μM. The nuclear-associated PI kinase activity was inhibited by both 50 μM ADP and 10 μM adenosine. Treatment of intact nuclei with DNase, RNase, phospholipase A₂ and Triton X-100 did not solubilize the enzyme activity. Based on sensitivity to calcium, ADP, detergent, pH optimum and the product analysis, the nuclear-associated PI 4-kinase was compared with previously reported PI kinases from plants, animals and yeast.     

Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230

PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence ⁹¹⁶NFNHIHKRIRRVADKYLSG⁹³⁴ comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin α/β mechanism employing importins α1 and α3 but not α5. This transport was inhibited by wheat germ agglutinin and GTPγS. The sequence ¹⁴¹⁴SKKTNRGSQLHKYYMKRRTL¹⁴³³, a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166–1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin α1/β or α3/β complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.     

Determination of lysosome membrane stability]

Effect of different concentration of non-ionic detergents (Triton X-100, Triton X-305, BRIJ-35 and Triton WR-1339) on total and non-sedimentable activity of 8 rat liver lysosome enzymes (acid phosphatase, acid DNase, acid RNase, arylsulphatases A and B, beta-glucuronidase, beta-galactosidase, beta-glucosidase and beta-acetylglucosaminidase) was studied. Only Triton X-100 at the concentration of 0.1% (and higher) was found to release completely lysosome enzymes. Low concentrations of Triton X-100 (0.025-0.05%) were used to characterize the strength of enzyme binding: the level of releasing acid DNase, beta-galactosidase, beta-glucuronidase and acid phsophatase being considerably higher than that of other lysosome enzymes studied. On the basis of the data obtained a method is worked out, which is suitable for series studies of the stability of lysosome membranes under different physiological and pathological conditions. The essence of the method is the treatment of membrane particles with increasing concentrations of Triton X-100 (0.025; 0.05 AND 0.1%) AND THE SUCCESSIVE ESTIMATION OF NON-Sedimentable activity of marker enzymes. The method detected troubles in the stability of rat liver lysosome membranes under starvation, protein deficiency and aging.     

p63cdc13, a B-type cyclin, is associated with both the nucleolar and chromatin domains of the fission yeast nucleus.

The cellular distribution of the fission yeast mitotic cyclin B, p63cdc13, was investigated by a combination of indirect immunofluorescence light microscopy, immunogold electron microscopy, and nuclear isolation and fractionation. Immunofluorescence microscopy of wild-type cells and the cold-sensitive mutant dis2.11 with a monospecific anti-p63cdc13 antiserum was consistent with the association of a major subpopulation of fission yeast M-phase protein kinase with the nucleolus. Immunogold electron microscopy of freeze-substituted wild-type cells identified two nuclear populations of p63cdc13, one associated with the nucleolus, the other with the chromatin domain. To investigate the cell cycle regulation of nuclear labeling, the mutant cdc25.22 was synchronized through mitosis by temperature arrest and release. Immunogold labeling of cells arrested at G2M revealed gold particles present abundantly over the nucleolus and less densely over the chromatin region of the nucleus. Small vesicles around the nucleus were also labeled by anti-p63cdc13, but few gold particles were detected over the cytoplasm. Labeling of all cell compartments declined to zero through mitosis. Cell fractionation confirmed that p63cdc13 was substantially enriched in both isolated nuclei and in a fraction containing small vesicles and organelles. p63cdc13 was not extracted from nuclei by treatment with RNase A, Nonidet P40 (NP-40), Triton X-100, and 0.1 M NaCl, although partial solubilization was observed with DNase I and 1 M NaCl. A known nucleolar protein NOP1, partitioned in a similar manner to p63cdc13, as did p34cdc2, the other subunit of the M-phase protein kinase. We conclude that a major subpopulation of the fission yeast mitotic cyclin B is targeted to structural elements of the nucleus and nucleolus.     

Considerations in the isolation of rat liver nuclear matrix,nuclear envelope,and pore complex lamina

A number of recent studies have demonstrated a salt-, nuclease, and detergent-resistant subnuclear structure termed the nuclear protein matrix which consists of a fibrogranular intranuclear network, residual components of the nucleolus, and a peripheral lamina. Other workers, however, have shown that somewhat similar methods result in the isolation of the peripheral lamina devoid of the intranuclear components. In this report we demonstrate that seemingly slight changes in the isolation procedure cause major changes in the morphology of the residual structures obtained. When freshly purified rat liver nuclei were digested with DNase I and RNase A and then extracted with buffers of low magnesium ion concentration (LS buffer) and high ionic strength (HS buffer), the resulting structures isolated prior to or after Triton X-100 extraction lacked the extensive intranuclear network and the easily identifiable residual nucleoli present in the nuclear protein matrix. Systematic modification of this extraction procedure revealed that morphologically identifiable residual nucleoli were present when digestion with RNase A followed extraction with HS buffer but were absent when the order of these steps was reversed. The removal of the nucleolus by RNase A and HS buffer correlated with the removal of nuclear RNA by the same treatments. These coordinate events could not be prevented by treatment with protease inhibitors but were prevented by treatment of the RNase A with diethylpyrocarbonate, an RNase inhibitor. The extensive intranuclear network seen in the nuclear protein matrix was sparse or absent when residual structures were prepared from DNase- and RNase-treated nuclei under conditions which minimized the oxidation of protein sulfhydryl groups. In contrast, an extensive non-chromatin intranuclear network was seen if the formation of intermolecular protein disulfide bonds was promoted by extraction of nuclei with cationic detergents, by overnight incubation, or by treatment with oxidizing agents like sodium tetrathionate prior to nuclease digestion and subsequent extraction. By varying the order of extraction steps and the extent of disulfide cross-linking, it is possible to isolate from a single batch of nuclei residual structures with a wide range of morphologies and compositions.     

Fibrillarin: a new protein of the nucleolus identified by autoimmune sera

Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.     

ErbB-4 and TNF-alpha converting enzyme localization to membrane microdomains

Sequential proteolytic processing of ErbB-4 occurs in response to ligand addition. Here, we assess the localization of cleavable and non-cleavable ErbB-4 isoforms to membrane microdomains using three methodologies: (1) Triton X-100-insolubility, (2) Brij98-insolubility, and (3) detergent-free density gradient centrifugation. Whereas ErbB-4 translocated to a Triton X-100-insoluble fraction upon treatment of T47D cells with heregulin, it constitutively associated with a Brij98-insoluble fraction and a lipid raft fraction isolated using detergent-free methodology. Comparison of cleavable and non-cleavable isoforms of ErbB-4 revealed that both ErbB-4 isoforms are constitutively localized to either a Triton X-100-soluble or Brij98-insoluble fraction. In contrast, addition of heregulin resulted in translocation of the cleavable isoform to a detergent-free lipid raft. Tumor necrosis factor-alpha converting enzyme (TACE), the ectodomain secretase for ErbB-4, was present predominantly in its mature active form in most microdomains analyzed. These data suggest the assembly of ErbB-4 ectodomain cleavage apparatus in a membrane microdomain.     

Isolation and preliminary characterization of the nucleus and the nucleomorph of a cryptomonad, Pyrenomonas salina

The cryptomonad cell has presumably arisen by a secondary symbiotic event involving two eukaryotes, and thus is composed of four different DNA-containing compartments (nucleus, nucleomorph, plastid, and mitochondrion). In the present paper, the isolation and quantitative DNA estimation of the host cell nucleus and the nucleomorph, a vestigial eukaryotic nucleus, is presented. In the presence of CaCl2, the host nucleus could be isolated from cells lysed by Triton X-100. For isolation of the nucleomorph, cells were slightly fixed with glutardialdehyde and thereafter, lysed by treatment with proteinase K and Triton X-100, leaving an intact nucleomorph-pyrenoid complex. Nuclei were further purified by isopycnic Percoll density gradient centrifugation. Purity and quality of the two nuclear fractions were checked by means of DAPI-epifluorescence microscopy and electron microscopy. The DNA content of the host nucleus and nucleomorph, determined by the diphenylamine method and by means of quantitative microspectrofluorometry, respectively, was found to be more than 700 times higher in the host nucleus than in the nucleomorph.     

Ultrastructure and enzyme digestion of nucleoli and associated structures in hypothalamic nerve cells viewed in resinless sections

Estrogen has been shown to affect ventromedial hypothalamic (VMH) nerve cell nucleoli in ovariectomized rats, by causing an increase in the number of electron-dense aggregates associated with nucleoli. In order to characterize these nucleolus-associated structures and other nuclear components, we examined the ultrastructure of ventromedial hypothalamic nucleoli and nuclei revealed by enzyme digestions (pepsin, RNase and DNase) in resinless thin sections. Digestion by pepsin did not cause obvious alterations in the morphology of the nucleolus or its related structures. Pepsin treatment followed by RNase, however, reduced the density of the nucleolus, while that of the nucleolus-associated structure and other related structures remained unchanged. Conversely pepsin treatment followed by DNase, reduced the density of nucleolus-associated and other chromatin structures, but had no effect on the density of the nucleolus. Pepsin treatment followed by RNase and then DNase treatment, reduced the density of the nucleolus and nucleolus-associated structures. A residual nucleolus and nucleolus-associated structure remained after this treatment. Stereo viewing of resinless sections shows that the nucleolus, its associated structures, and other related structures, are associated with fine filaments that may comprise the nuclear matrix. The nucleolus-associated structure containing DNA may direct RNA synthesis at an increased rate in estrogen-treated hypothalamic cells.     

Nonhistone protein BA is a glutathione S-transferase localized to interchromatinic regions of the cell nucleus

A DNA-binding nonhistone protein, protein BA, was previously demonstrated to co-localize with U-snRNPs within discrete nuclear domains (Bennett, F. C., and L. C. Yeoman, 1985, Exp. Cell Res., 157:379-386). To further define the association of protein BA and U-snRNPs within these discrete nuclear domains, cells were fractionated in situ and the localization of the antigens determined by double-labeled immunofluorescence. Protein BA was extracted from the nucleus with the 2.0 M NaCl soluble chromatin fraction, while U-snRNPs were only partially extracted from the 2.0 M NaCl-resistant nuclear structures. U-snRNPs were extracted from the residual nuclear material by combined DNase I/RNase A digestions. Using an indirect immunoperoxidase technique and electron microscopy, protein BA was localized to interchromatinic regions of the cell nucleus. Protein BA was noted to share a number of chemical and physical properties with a family of cytoplasmic enzymes, the glutathione S-transferases. Comparison of the published amino acid composition of protein BA and glutathione S-transferases showed marked similarities. Nonhistone protein BA isolated from saline-EDTA nuclear extracts exhibited glutathione S-transferase activity with a variety of substrates. Substrate specificity and subunit analysis by SDS polyacrylamide gel electrophoresis revealed that it was a mixture of several glutathione S-transferase isoenzymes. Protein BA isolated from rat liver chromatin was shown by immunoblotting and peptide mapping techniques to be two glutathione S-transferase isoenzymes composed of the Yb and Yb' subunits. Glutathione S-transferase Yb subunits were demonstrated to be both nuclear and cytoplasmic proteins by indirect immunolocalization on rat liver cryosections. The identification of protein BA as glutathione S-transferase suggests that this family of multifunctional enzymes may play an important role in those nuclear domains containing U-snRNPs.     

Role of the FYVE finger and the RUN domain for the subcellular localization of Rabip4

Rabip4 is a Rab4 effector, which possesses a RUN domain, two coiled-coil domains, and a FYVE finger. It is associated with the early endosomes and leads, in concert with Rab4, to the enlargement of endosomes, resulting in the fusion of sorting and recycling endosomes. Our goal was to characterize the role of these various domains in Rabip4 subcellular localization and their function in Chinese hamster ovary cells. Although the FYVE finger domain specifically bound phosphatidylinositol 3-phosphate and was necessary for the function of Rabip4, it was not sufficient for the protein association with membranes. Indeed a protein containing the FYVE finger and the Rab4-binding site was cytosolic, whereas the total protein was mostly associated to the membrane fraction, whether or not cells were pretreated with wortmannin. By contrast, a construct corresponding to the N-terminal end, Rabip4-(1-212), and containing the RUN domain was membrane-associated. The complete protein partitioned between the Triton X-100-insoluble and -soluble fractions and a wortmannin treatment increased the amount of the protein in the Triton X-100 fraction. Rabip4-(1-212) was totally Triton X-100-insoluble, and confocal microscopic examination showed that it labeled not only the endosomes, positive for Rabip4, but also a filamentous network with a honeycomb appearance. The Triton X-100-insoluble fraction that contains Rabip4 did not correspond to the caveolin or glycosylphosphatidylinositol-enriched lipid rafts. Rabip4 did not appear directly linked to actin but seemed associated to the actin network. We propose that the subcellular localization of the protein is primarily driven by the RUN domain to endosomal microdomains characterized by Triton X-100 insolubility and that the FYVE domain and the Rab4-binding domain then allow for the recruitment of the protein to lipophilic microdomains enriched in phosphatidylinositol 3-phosphate.     

Monoclonal antibody against microtubule associated protein-1 produces immunofluorescent spots in the nucleus and centrosome of cultured mammalian cells

A monoclonal antibody was raised against the highest molecular weight protein associated with microtubules (MAP-1). Its specific binding to MAP-1 was determined by immunoblotting of the gel electrophoretogram of microtubule proteins prepared from porcine brain. The antibody reacted only with MAP-1, not with MAP-2, tau or tubulin. Indirect immunofluorescent staining by this antibody showed bright intranuclear spots, the centrosome and the faint meshwork of the cytoplasm in several types of cultured mammalian cells; HeLa, PtK2, human skin fibroblasts, mouse melanoma cells, Chinese hamster ovary cells. The nuclear spots in the interphase cells, were replaced by diffuse enhanced fluorescence throughout the cell except for chromosomes during mitosis. They reappeared in late telophase, first in the cytoplasm, late in the nucleus. The punctate pattern of nuclear immunofluorescence was not affected by microtubule-depolymerizing agents. The result that it persisted on residual cell structures after extraction with a high salt concentration buffer containing Triton X-100 followed by digestion with DNase I and RNase A suggests that the antigen is associated with the nuclear skeleton.     

Basic Domains Target Protein Subunits of the RNase MRP Complex to the Nucleolus Independently of Complex Association

The RNase MRP and RNase P ribonucleoprotein particles both function as endoribonucleases, have a similar RNA component, and share several protein subunits. RNase MRP has been implicated in pre-rRNA processing and mitochondrial DNA replication, whereas RNase P functions in pre-tRNA processing. Both RNase MRP and RNase P accumulate in the nucleolus of eukaryotic cells. In this report we show that for three protein subunits of the RNase MRP complex (hPop1, hPop4, and Rpp38) basic domains are responsible for their nucleolar accumulation and that they are able to accumulate in the nucleolus independently of their association with the RNase MRP and RNase P complexes. We also show that certain mutants of hPop4 accumulate in the Cajal bodies, suggesting that hPop4 traverses through these bodies to the nucleolus. Furthermore, we characterized a deletion mutant of Rpp38 that preferentially associates with the RNase MRP complex, giving a first clue about the difference in protein composition of the human RNase MRP and RNase P complexes. On the basis of all available data on nucleolar localization sequences, we hypothesize that nucleolar accumulation of proteins containing basic domains proceeds by diffusion and retention rather than by an active transport process. The existence of nucleolar localization sequences is discussed.     

Detection of S-100b protein in Triton cytoskeletons: an immunocytochemical study on cultured Schwann cells

We investigated the subcellular distribution of S-100b protein in primary cultures of Schwann cells. The subcellular localization of the protein in cells fixed and then permeabilized is similar, if not identical, to that seen in Schwann cells in peripheral nerves, i.e., S-100b protein is found in the cytoplasm and associated with membranes and filamentous structures. In cells either fixed in the presence of Triton X-100 or exposed to Triton X-100 for a short time before fixation (Triton cytoskeletons), the immune reaction product is considerably less intense, and the protein is associated with filaments running parallel to the long axis of the cell as well as in a submembranous position. Including CaCl2 in the buffer during fixation in the presence of Triton X-100 does not result in any increase in the intensity of the immune reaction product in Triton cytoskeletons, suggesting that, within the limits of the technique employed, no binding of additional S-100b protein to the Triton X-100-resistant material can be induced. On the other hand, including EGTA results in a substantial decrease in the intensity of the immune reaction product in Triton cytoskeletons. Altogether, these findings suggest that a remarkable fraction of S-100b protein in cultured Schwann cells is associated with elements of the cytoskeleton and that Ca2+ exerts some regulatory role in the association of S-100b protein with the cytoskeleton.     

Dynamic localization of RNase MRP RNA in the nucleolus observed by fluorescent RNA cytochemistry in living cells

The dynamic intra-nuclear localization of MRP RNA, the RNA component of the ribonucleoprotein enzyme RNase MRP, was examined in living cells by the method of fluorescent RNA cytochemistry (Wang, J., L.-G. Cao, Y.-L. Wang, and T. Pederson. 1991. Proc. Natl. Acad. Sci. USA. 88:7391-7395). MRP RNA very rapidly accumulated in nucleoli after nuclear microinjection of normal rat kidney (NRK) epithelial cells. Localization was specifically in the dense fibrillar component of the nucleolus, as revealed by immunocytochemistry with a monoclonal antibody against fibrillarin, a known dense fibrillar component protein, as well as by digital optical sectioning microscopy and 3-D stereo reconstruction. When MRP RNA was injected into the cytoplasm it was not imported into the nucleus. Nuclear microinjection of mutant MRP RNAs revealed that nucleolar localization requires a sequence element (nucleotides 23-62) previously implicated as a binding site for a nucleolar protein, the To antigen. These results demonstrate the dynamic localization of MRP RNA in the nucleus and provide important insights into the nucleolar targeting of MRP RNA.     

A possible skeletal substructure of the macronucleus of Tetrahymena

Upon removal of chromatin from isolated macronuclei of tetrahymena, residual structures are obtained, the organization of which faithfully reflects the distinctive architecture of the macronucleus. Macronuclei are isolated by a new procedure in which cells are lysed by immersion in citric acid and Triton X-100. This method is rapid and efficient and leaves the nuclear structures stripped of nuclear envelope and nucleoli. The remaining interconnected chromatin bodies are structurally differentiated into a dense outer shell and a fibrillar inner core. The fibrillar component is identified as chromatin because it is removed upon digestion with DNase and extraction with 2 M NaCl. The dense shell of the chromatin body is unaffected by the digestion procedure, which leaves a skeletal structure comprised of hollow spherical bodies. Analysis of the protein composition by SDS acrylamide gel electrophoresis before and after digestion with DNase and RNase and high-salt extraction shows that histones are diminished, whereas the nonhistone protein composition remains unchanged. It was found the DNase not only extracts chromatin but also protects the nonchromatin structure from the otherwise disruptive effects of high-salt extraction. The method used for isolating the nuclei also affects the structure remaining after the digestion procedure the citric acid/Triton X-100 method enhances the stability of the interconnected spherical bodies. The results indicate that the method for isolating nuclei and the procedure by which chromatin is extracted are both major factors contributing to the detection of a possible nonchromatin nuclear skeleton.