Proteins associated with heterogeneous nuclear RNA in eukaryotic cells

When HeLa cell nuclei axe mechanically disrupted in either hypotonic or isotonic buffers, heterogeneous nuclear RNA is recovered from the post-nucleolar fraction in the form of EDTA-resistant ribonucleoprotein particles, which sediment between 40 S and 250 S in sucrose gradients containing 0.01 m or 0.15 m-NaCl. That the RNA in these particles is HnRNA² is indicated by its heterodisperse sedimentation (20 to 80 S) and its continued synthesis in concentrations of actinomycin D that selectively inhibit the synthesis of ribosomal RNA. The specificity of the HnRNA-protein complexes is evidenced by the failure of deliberate attempts to generate artificial RNP by the addition of deproteinized HnRNA to intact or disrupted nuclei at low ionic strength.The proteins bound to HnRNA are complex. In HeLa cells, HnRNP particles contain proteins with molecular weights from 39,000 to approximately 180,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric points between 4.9 and 8.3 (analytical isoelectric focusing). They are readily distinguishable from proteins in other cell fractions, including those in chromatin.Exposure of HeLa HnRNP particles to 0.5 m-NaCl reduces their average sedimentation velocity by approximately 30%. CsCl density-gradient analysis reveals that this is accompanied by the loss of a major portion of the proteins. However, a significant fraction of the HnRNP (25 to 30%) is resistant to high salt concentrations and continues to band at the same density as native HnRNP (1.43 g/cm³). This is true even after prolonged exposure (24 h) to high salt. The salt-resistant HnRNP is enriched for proteins above 60,000 molecular weight. In at least these two respects, this sub-class of HnRNP resembles “messenger RNP” prepared from cytoplasmic polyribosomes, which is also salt-stable and contains relatively high molecular weight proteins.HnRNP particles can also be recovered from HeLa cell nuclei lysed in high salt but these contain many extra proteins, notably histones, and sediment much faster in sucrose gradients than particles prepared as above. HnRNP is not liberated by extracting HeLa nuclei in 0.14 m-NaCl, pH 8.0 (Samarina et al., 1967) unless the temperature is 20 °C or higher. In this case the particles are converted to 45 S structures, which contain partially degraded HnRNA. 45 S particles can also be produced by subjecting 40 to 250 S HnRNP to a very limited digestion with pancreatic ribonuclease (1 to 2 hits/molecule).HnRNP particles have similar sedimentation velocities (40 to 300 S) when isolated under physiological ionic conditions from a variety of mammalian cells, including WI38 human diploid fibroblasts, mouse L-cells, monkey kidney cells and rat liver. However, electrophoresis reveals a distinct pattern of HnRNP proteins for each cell type. It is proposed that this cell-specificity reflects a situation in which HnRNA molecules that differ in nucleotide sequence are complexed with different sets of proteins, so that the resulting HnRNP particles are biochemically distinct at each genetic locus. This hypothesis is discussed in relation to the cytology of lampbrush and polytene chromosomes.     

Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17β-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or ε-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0     

Reassembly of flagellar B (alpha beta) tubulin into singlet microtubules: consequences for cytoplasmic microtubule structure and assembly

B(alpha beta) tubulin was obtained from a homogeneous class of microtubules, the incomplete B subfiber of sea urchin sperm flagellar doublet microtubules, by thermal fractionation. The thermally derived soluble B tubulin fraction (100, 000 g-h) repolymerizes in vitro, yielding microtubule-like structures. The microtubule-associated protein (MAP) composition and certain assembly parameters of thermally derived B tubulin are different from those reported for sonication- derived flageller tubulin and purified vertebrate tubulin. The "microtubules" reassembled from thermally prepared B tubulin are composed of 12-15 protofilaments (73% possess 14 protofilaments). A certain number possess a single "adlumenal component" applied to their inside walls, regardless of the number of protofilaments. Following the first cycle of polymerization, 81% of the B tubulin and essentially 100% of the MAPs remain cold insoluble. Evidence suggests that B tubulin assembles faithfully into a B lattice, creating a j seam between two protofilaments that are laterally bonded in a A-lattice configuration. The significance of these seams is discussed in relation to the mechanism of microtubule assembly, the stability of observed ribbons of protofilaments, and the three-dimensional organization of microtubule-associated components.     

Effect of jejunoileal bypass in the rat on the enteroinsular axis

The effect of jejunoileal bypass (JIB) on the enteroinsular axis was studied in vivo and in vitro in the rat. Glucose, insulin and GIP responses to oral glucose were compared in JIB and control rats. The effect of glucose and GIP on insulin release from the isolated perfused pancreas of the same animals was investigated to determine if JIB altered the sensitivity of the beta cell. Immunocytochemical studies of gut and pancreas were also carried out. Glucose, insulin and GIP responses to a glucose load were blunted after JIB, although basal GIP levels were elevated in these animals. The insulin response of the perfused JIB pancreas to GIP was 70% reduced from controls although the insulin response to glucose appeared normal. The size and area of JIB islets were unchanged from controls as was the distribution of insulin, glucagon, somatostatin and pancreatic polypeptide. GIP immunoreactive cells were present in all regions of the intestine including the JIB blind loop. This study confirms the findings of others that a relationship exists between reduced GIP and insulin response to oral glucose after JIB, and indicates that a decrease in sensitivity of the beta cell to GIP occurs following JIB that is not rapidly reversible. GIP secreted from blind loop mucosa may contribute to the high basal GIP found in JIB rats and may be causally connected to the fall in beta cell sensitivity.     

A single lineage of r2 retrotransposable elements is an active, evolutionarily stable component of the Drosophila rDNA locus

R2 elements are non-long-terminal-repeat (non-LTR) retrotransposons that insert specifically in the 28S rRNA genes of many insects. Previous reports concerning this element in the genus Drosophila have suggested that R2 elements are absent from many species of this genus, particularly those species from the subgenus Drosophila. In this report, we present an extensive study of the distribution and evolution of R2 elements in Drosophila. A PCR survey of 59 species from 23 species groups of the two major Drosophila subgenera found that R2 elements are present in all but two species of the melanogaster species subgroup. Phylogenetic analysis based on partial nucleotide sequences of R2 elements from 23 species demonstrates that the relationships of R2 elements are congruent with those of the Drosophila species phylogeny, suggesting that these elements have been vertically inherited since the divergence of this genus some 60 MYA. Sequence variation between different copies of R2 elements within each species was less than 0.16%, indicating that these elements are undergoing concerted evolution similar to that of the 28S genes. Several properties of the R2 sequences suggest that these elements depend on retrotransposition in addition to simple recombination to remain within the rDNA locus: the rates of synonymous substitutions averaged 4.8 times the rate of replacement substitutions, 82 of 83 R2 copies partially sequenced contained intact open reading frames, and, finally, length variation associated with the poly(A) 3' tails indicated that many R2 copies are the direct result of retrotransposition.