Comparative Studies of Rat Liver and Sea Urchin Embryo Nuclear Matrices (original) (raw)
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Kinetics of Labeling of the «Cap» of the Nuclear and Cytoplasmic RNA in Sea-urchin Embryos
Bolletino di zoologia, 1978
plasmic giazf RNA in sea rirchin rinbryos. 11. Physicochet~rical rharacferizafion. Cell Differentiation, 3: 297-304. Sconzo G., ROCCHERI hi. C., DI LIBERTO hi. and GIUDICE G., 1977-Studies OIJ !he strrictrire and possible frrtrclion of the RNA "CAP" irr developing sea rirchins. Cell Differentiation, hllREZ F., BAVISTER B. D. and GlUDICE G.,
Experimental Cell Research, 1972
Normal and Li+-treated embryos of Paracentrotus Iividus at different stages of development were pulse-labelled for 15-30 min with 14C-uridine. Clarified homogenates in 0.1 % deoxycholate were analyzed by gradient centrifugation. The sedimentation pattern of the ribosomes was recorded at 260 nm. Consecutive fractions of the gradients were collected on membrane filters, and the isotope content of the adsorbed material was determined. In control embryos the isotope content showed a marked rise from 5 h and reached a maximum at 15 h (early gastrula stage). A significant part of this activity was observed in a subribosomal fraction. In Lit-treated embryos the early increase in labelling was delayed. However, this lag was later followed by an overcompensation, and at the gastrula stage the total amount of labelled material was significantly higher than in the controls. The sedimentation pattern was also modified. The results are discussed from the assumption that the rapidly labelled material represents newly synthesized messenger RNA, which appears in the cytoplasm either as free ribonucleoproteins or associated with ribosomes.
Embryonic stage-related properties of sea urchin embryo chromatin
Biochimica et biophysica acta, 1976
Two chromatin components, obtained by buoyant density centrifugation of the unsheared blastula and gastrula chromatin of a sucrose/glucose gradient, have been comparatively characterized. When compared to the heavy fraction the light fraction (i) represents a far smaller part of chromatin, (ii) contains a higher RNA/DNA mass ratio and a higher proportion of newly synthesized nonhistone proteins and (iii) possesses greater template activity for RNA synthesis. Gastrulation of the embryos was found to render the dense chromatin fraction less compact and both chromatin subpopulations more transcriptable and enriched with newly synthesized non-histone proteins.
Two-dimensinal gel electrophoresis of rat liver nuclear washes, nuclear matrix, and hnRNA proteins
Journal of Cell Biology, 1980
The proteins of rat liver cytoplasm, nuclear washes, matrix, membrane, heterogeneous nuclear (hn)RNA proteins and chromatin were examined by two-dimensional gel electrophoresis . The inclusion in the gels of six common protein standards of carefully selected molecular weight and isoelectric point allowed us to clearly follow the distribution of specific proteins during nuclear extraction . In the nuclear washes and chromatin, we observed five classes of proteins : (a) Exclusively cytoplasmic proteins, present in the first saline-EDTA wash but rapidly disappearing from subsequent washes ; (b) ubiquitous proteins of 75,000, 68,000, 57,000, and 43,000 mol wt, the latter being actin, found in the cytoplasm, all nuclear washes and the final chromatin pellet ; (c) proteins of 94,000, 25,000, 22,500, and 20,500 mol wt specific to the nuclear washes ; (d) proteins present in the nuclear washes and final chromatin, represented by species at 62,000, 55,000, 54,000, and 48,000 mol wt, primarily derived from the nuclear matrix ; and (e) two proteins of 68,000 mol wt present only in the final chromatin . The major 65,000-75,000-mol wt proteins seen by one-dimensional gel electrophoresis of nuclear matrix were very heterogeneous and contained a major acidic, an intermediate, and a basic group . A single 68,000-mol wt polypeptide constituted the majority of the membrane-lamina fraction, consistent with immunological studies indicating that a distinct subset of matrix proteins occurs, associated with heterochromatin, at the periphery of the nucleus . Actin was the second major nuclear membrane-lamina protein . Two polypeptides at 36,000 and 34,000 mol wt constituted 60% of the hnRNP. Approximately 80% of the mass of the nonhistone chromosomal proteins (NHP) from unwashed nuclei is contributed by nuclear matrix and hnRNPs, and essentially the same patterns were seen with chromatin NHP . The concept of NHP being a distinct set of DNA-bound proteins is unnecessarily limiting . Many are derived from the nuclear matrix or hnRNP particles and vary in the degree to which they share different intracellular compartments .
Stabilities of Nuclear and Messenger Rna Molecules in Sea Urchin Embryos
Journal of Cell Biology, 1972
The kinetics of accumulation of radioactive adenosine in adenosine triphosphate and in RNA of nuclear, cytoplasmic, and polysomal fractions of sea urchin embryos have been analyzed. 85% of the RNA synthesized decays in the nucleus with an apparently uniform half-life of about 7 min. The remaining 15% goes to the cytoplasm, mostly entering polysomes, and decays with a quite uniform half-life of about 75 min. The nuclear RNA accounts for one-third and the cytoplasmic RNA accounts for two-thirds of the total unstable RNA which accumulates at steady state in the embryo. The size distribution of shortlabeled nuclear RNA is very similar to that of long-labeled messenger RNA, when both are extracted directly from the cells without a previous cell fractionation .
The Journal of Cell Biology, 1980
Isolated rat liver nuclear matrices have been partially separated by means of mild sonication into a matrix protein (matricin) fraction and a residual ribonucleoprotein (RNP) fraction . The initial matricin fraction is composed largely of protein (91 .1%) but also contains significant amounts of DNA (8.4%) . Reconstruction experiments indicate that this DNA is not the result of the artifactual binding of DNA to the matrix during the extraction procedures. Subsequent treatment with DNase I results in purified matricin composed of >99 .5% protein. SDS acrylamide gel electrophoresis of the matrix protein fibrils reveals only three bands: the primary matrix polypeptides of 62,000, 66,000, and 70,000 daltons. Electron microscopy demonstrates a diffuse reticulum with fibrils as thin as 30-50 A and the presence of 80-100-A globular structures . The residual RNP fraction is composed largely of protein (80 .1%) and RNA (19 .5%), with only traces of DNA (1 .I%) . Over 98% of the total matrix-associated RNA is recovered in this fraction . SDS acrylamide gel electrophoresis indicates an enrichment in both low and high molecular weight secondary matrix polypeptides, although the 60,000-70,000dalton polypeptides are present in significant amounts as well . Ultrastructural analysis of the residual RNP fraction reveals distinct electron-dense-staining matrix particles (150-350 A) attached to a fibrous matricin network.
Cytological identification and characterization of the nuclear matrix
Experimental Cell Research, 1978
The presence of a matrix system in the nucleus has been cytologically demonstrated in intact untreated and cu-amanitin-treated HeLa and rat kangaroo cells. The matrix is Feulgen negative and is stained very faintly with toluidine blue, whereas it can be brightly stained with haemalum or light green. It is highly susce tible to pepsin digestion but resistant to RNase. Ultrastructurally the matrix consists of 50-60 d: tluck fib&. Some granules, which may be ribonuclear protein granules are also found associated with the fibrils. The matrix is essentially a network of acidic protein.
Cellular and molecular bases of biomineralization in sea urchin embryos
Sea urchin embryos construct their skeleton following a precise gene-regulated time-and space-dependent programme, in concert with factors promoting cell adhesion and differentiation. The biomineral is deposited in a privileged extracellular space produced by the fused filopodia processes of the primary mesenchyme cells, the only cells producing a set of necessary matrix proteins. More than ten years ago we showed for the first time that signals from ectoderm cells promoted the expression of one of the major skeleton matrix genes by the primary mesenchyme cells. Since then, many of the crucial steps of this complex activation cascade, from ectoderm cells to embryonic spicules, have been elucidated. The experimental production of skeleton malformations, induced by the exposure to toxic metals or ionizing radiations, served as model to dissect the molecular mechanisms leading to biomineralization. With the aim of understanding the sea urchin skeleton physiology, we analysed the expression of well-known and newly-identified biomineral-related genes, including those coding for growth and transcription factors as well as for skeleton matrix proteins. This review summarizes our recent findings on sea urchin embryo skeletogenesis, with a particular attention to the role played by cellular and molecular signaling, approached by the use of experimentally induced skeleton malformations.