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G Krohne

Publications and source records attributed to G Krohne.

At least 73 records · Page 4Linked to original sources
PubMed1984

A monoclonal antibody against nuclear lamina proteins reveals cell type-specificity in Xenopus laevis.

Immunofluorescence microscopy shows that the monoclonal murine antibody PKB8 stains the nuclear lamina of various somatic cells from vertebrates as diverse as mammals, birds and amphibia. It also decorates the nuclear periphery of oocytes from rat and chicken but does not react with spermatocytes, spermatids and spermatozoa. Immunoblotting experiments demonstrate reaction with lamina polypeptides A, B and C of rat, with lamina polypeptide A of chicken, and with lamina polypeptides LI and LII of erythrocytes of the frog, Xenopus laevis. Antibody PKB8 does, however, not bind, on blotted polypeptides and on sections through ovaries, to the pore complex-lamina polypeptide of Mr 68000 present in Xenopus oocytes. These results reveal the existence of a common antigenic determinant in all three lamina polypeptides of mammals, in one lamina polypeptide of chicken and in two amphibian lamina polypeptides. The immunological data also indicate that, in Xenopus laevis, pore complex-lamina polypeptides of somatic cells and oocytes are distinct. The Mr 68000 protein of Xenopus oocytes is also different from polypeptides LI and LII of somatic Xenopus cells by tryptic peptide mapping. The results suggest that nuclear pore complex-lamina polypeptides represent a family of related polypeptides containing regions highly conserved during evolution and that these polypeptides can be differentially expressed in cells of at least one species, Xenopus laevis.

Animals↗
PubMed1984

Karyoskeletal proteins and the organization of the amphibian oocyte nucleus.

We have investigated the existence of structural components in the nucleus of the oocyte of Xenopus laevis and other amphibia that are insoluble in non-denaturing detergents and buffers of low and high ionic strength. These cells are particularly suitable for such studies as they have a high frequency of extrachromosomal amplified nucleoli and pore complexes of the nuclear envelope. Using biochemical and immunological techniques, we have shown these structures to contain only two major proteins. These are a polypeptide of Mr 145000, which is located in a meshwork of filaments specific to the nucleolar cortex, and certain nucleoplasmic bodies probably derived therefrom, and a polypeptide of Mr 68000, which is the predominant constituent of the lamina-pore complex structure. We show that the latter protein is related to, but not identical to, lamina proteins ('lamins') of somatic cells, indicating cell type-specificity of the expression of polypeptides of the lamin family. In addition, we describe a protein of Mr 180000, which is the major constituent of the dense fibrillar component of the nucleolus. This can be partially solubilized in buffers of moderately high ionic strength. We interpret proteins of this category as karyoskeletal components involved in the architectural organization of specific functional topology within the nucleus. In contrast to previous reports for other cell types we have found no other prominent high-salt-insoluble structures in the nuclear interior, indicating the absence of an extended internal nuclear matrix in this kind of nucleus.

Animals↗
PubMed1982

Immunological localization of a major karyoskeletal protein in nucleoli of oocytes and somatic cells of Xenopus laevis.

Oocyte nuclei of Xenopus laevis contain two major karyoskeletal proteins characterized by their resistance to extractions in high salt buffers and the detergent Triton X-100, i.e. a polypeptide of 68,000 mol wt which is located in the core complex-lamina structure and a polypeptide of 145,000 mol wt enriched in nucleolar fractions. Both proteins are also different by tryptic peptide maps and immunological determinants. Mouse antibodies were raised against insoluble karyoskeletal proteins from Xenopus oocytes and analyzed by immunoblotting procedures. Affinity purified antibodies were prepared using antigens bound to nitrocellulose paper. In immunofluorescence microscopy of Xenopus oocytes purified antibodies against the polypeptide of 145,000 mol wt showed strong staining of nucleoli, with higher concentration in the nucleolar cortex, and of smaller nucleoplasmic bodies. In various other cells including hepatocytes, Sertoli cells, spermatogonia, and cultured kidney epithelial cells antibody staining was localized in small subnucleolar granules. The results support the conclusion that this "insoluble" protein is a major nucleus-specific protein which is specifically associated with--and characteristic of--nucleoli and certain nucleolus-related nuclear bodies. It represents the first case of a positive localization of a karyoskeletal protein in the nuclear interior, i.e. away from the pore complex-lamina structure of the nuclear cortex.

Animals↗
PubMed1981

A nucleolar skeleton of protein filaments demonstrated in amplified nucleoli of Xenopus laevis.

The amplified, extrachromosomal nucleoli of Xenopus oocytes contain a meshwork of approximately 4-nm-thick filaments, which are densely coiled into higher-order fibrils of diameter 30-40 nm and are resistant to treatment with high- and low-salt concentrations, nucleases (DNase I, pancreatic RNase, micrococcal nuclease), sulfhydryl agents, and various nonionic detergents. This filamentous "skeleton" has been prepared from manually isolated nuclear contents and nucleoli as well as from nucleoli isolated by fluorescence-activated particle sorting. The nucleolar skeletons are observed in light and electron microscopy and are characterized by ravels of filaments that are especially densely packed in the nucleolar cortex. DNA as well as RNA are not constituents of this structure, and precursors to ribosomal RNAs are completely removed from the extraction-resistant filaments by treatment with high-salt buffer or RNase. Fractions of isolated nucleolar skeletons show specific enrichment of an acidic major protein of 145,000 mol wt and an apparent pI value of approximately 6.15, accompanied in some preparations by various amounts of minor proteins. The demonstration of this skeletal structure in "free" extrachromosomal nucleoli excludes the problem of contaminations by nonnucleolar material such as perinucleolar heterochromatin normally encountered in studies of nucleoli from somatic cells. It is suggested that this insoluble protein filament complex forms a skeleton specific to the nucleolus proper that is different from other extraction-resistant components of the nucleus such as matrix and lamina and is involved in the spatial organization of the nucleolar chromatin and its transcriptional products.

Animals↗
PubMed1980

Immunological identification and localization of the predominant nuclear protein of the amphibian oocyte nucleus.

Nuclei of vitellogenic oocytes of the frog, Xenopus laevis, contain a prominent protein, representing about 10% of nuclear protein, which is characterized by a polypeptide of M(r) 30,000. This protein is highly phosphorylated and acidic, displays several isoelectric variants with pI values ranging from 4.7 to 5.3, shows a high thermostability, is not stably complexed with other proteins, and is readily extracted in buffer solutions. Guinea pig antibodies against this protein have allowed its specific immunoprecipitation and localization by immunofluorescence microscopy, using both frozen tissue sections and cells grown in vitro. The protein is located almost exclusively in the nucleus where it appears to be spread throughout the nuclear interior. It is also a major nucleus-specific protein in vitellogenic and previtellogenic oocytes of other amphibian species as well as in other cell types, including hepatocytes, follicle epithelial cells, and cultured Xenopus cells, but is not detected in nuclei of transcriptionally inactive cells such as erythrocytes and spermatids. An immunologically related, if not identical, protein occurs in nuclei of higher vertebrate cells, including mammals. The properties of this abundant nuclear phosphoprotein and its possible relationship to the "nucleosome assembly factor" protein are discussed. It is suggested that this soluble protein serves a general nuclear function.

Amphibians↗
PubMed1978

Introduction of hidden breaks during rRNA maturation and ageing in Tetrahymena pyriformis.

The stability of Tetrahymena pyriformis cytoplasmic rRNAs and nuclear rRNA precursors has been studied by polyacrylamide gel electrophoresis under partly and completely denaturing conditions. Cytoplasmic 17-S rRNA (Mr = 0.66 X 10(6) consists of a continuous polynucleotide chain throughout its lifetime, whereas the bulk of 26-S rRNA (Mr = 1.2m X 10(6) dissociates upon denaturation. Two large fragments (F1, F2) of somewhat different molecular weights (Mr 0.63 X 10(6) and 0.58 X 10(6) and the small 5.8-S rRNA fragment (Mr about 50 000) are regularly observed. Some additional distinct minor fragments (F3-F6) are noted under certain preparative conditions, suggestive of artifactual origin. The following conclusions were made from the data obtained . (a) Newly synthesized 26-S rRNA molecules do not contain the 'central' hidden break (separating F1 and F2) until about 15 min after their appearance in the cytoplasm; however, they release during denaturation the 5.8-S and/or a short-lived 7-S fragment (Mr about 75 000) which might represent a direct precursor to the 5.8-S rRNA. (b) The immediate nuclear precursor to the 26-S rRNA (Mr 1.39 X 10(6) releases a small fragment of similar size (7 S). (c) The largest stable transcription product of the rDNA (pre-rRNA) does not contain any hidden break.

Animals↗
PubMed1978

Localization of a nuclear envelope-associated protein by indirect immunofluorescence microscopy using antibodies against a major polypeptide from rat liver fractions enriched in nuclear envelope-associated material.

The location of a specific major polypeptide present in nuclear pore complex-enriched fractions from rat liver was examined by indirect immunofluorescence microscopy using chicken antibodies against this polypeptide. In both whole cell preparations of cultured cells grown on cover slips (mouse 3 T 3, rat kangaroo PtK2) and in frozen sections through liver and mammary gland tissue a strongly preferential, if not exclusive, binding to the nuclear periphery of interphase cells was observed. The specificity of this localization was demonstrated in these cells by the decoration of chromatin with antibodies against histones and of elements of the endoplasmic reticulum--outer mitochondrial membrane--system with antibodies to cytochrome b5. In addition, the localization was examined by electron microscopy using frozen sections and "immunoperoxidase" techniques. The results suggest that this polypeptide is contained in a protein specific for the nuclear periphery, probably closely associated with the peripheral chromatin.

Animals↗
PubMed1977

Lengths and patterns of transcriptional units in the amplified nucleoli of oocytes of Xenopus laevis.

Transcriptionally active chromatin from peripheral amplified nucleoli of lampbrush-chromosome stage oocytes of Xenopus laevis was dispersed and spread in various solutions of low salt concentrations (including some with additions of detergents) and examined by electron microscopy. Nucleolar material from oocytes of animals with normal (2-nu) and mutant (1-nu) genetical constitution of nucleolus organizers was compared. Histograms showing the distributions of the length of matrix units, apparent spacer intercepts, and the total repeating units of the rDNA containing chromatin axes revealed a significant degree of heterogeity, with indications of subclasses and predominant repeat unit size classes of 3.3 amd 3.8 micron length. The correspondence of matrix unit length to the molecular weight of the first stable product of rDNA transcription was studied using gel electrophoresis of labelled pre-rRNA under non-denaturing and denaturing conditions. Evaluations of individual strands of nucleolar chromatin further demonstrated the existence of both (i) strands with obviously homogeneous repeating units of and (ii) strands with intra-axial heterogeneity of rDNA subunits. "Prelude complexes", i.e. groups of transcriptional complexes in apparent spacer intercepts, were not infrequently noted. The data are compared with the measurements of lengths of repeating units in fragments of rDNA obtained by digestion with EcoRI endonuclease as described by Morrow et al.(1974) and Wellauer et al. (1974,1976a, b). The results are discussed in relation to problems of variations in the modes of arrangement of the pre-rRNA genes, the state of packing of rDNA during transcription, and possible mechanisms of the amplification process.

Animals↗