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R W Parish

Publications and source records attributed to R W Parish.

At least 37 records · Page 2Linked to original sources
PubMed1988

A mycoplasma high-affinity transport system and the in vitro invasiveness of mouse sarcoma cells.

FS9 mouse sarcoma cells were previously shown to be highly invasive when confronted with chicken heart fibroblasts using Abercrombie's confronted explant technique. This invasion could be inhibited by addition to the assay of Fab fragments of a monoclonal antibody directed against p37, a protein associated with the surface of FS9 cells. We have cloned and sequenced the gene for p37. We show that it originates from Mycoplasma hyorhinis and that UGA is a tryptophan codon in this organism. We present evidence that the p37 gene is part of an operon encoding two additional proteins which are highly similar to components of the periplasmic binding-protein-dependent transport systems of Gram-negative bacteria, and we suggest that p37 is part of a homologous, high-affinity transport system in M. hyorhinis, a Gram-positive bacterium. We discuss the influence of p37 and M. hyorhinis on contact inhibition of locomotion of mammalian cells.

Amino Acid Sequence↗
PubMed1987

Mechanisms of tumour cell metastasis.

Abercrombie and his colleagues have accumulated evidence that changes in the heterotypic contact-inhibition response are largely responsible for the invasiveness of cells, at least in culture. We have identified a 37,000 Mr protein on the surface of mouse fibrosarcoma cells that is involved in their in vitro invasion. Blocking this protein with specific antibodies inhibits the invasion of chicken heart fibroblasts by the tumour cells and normal heterotypic contact inhibition is restored. These results are presented in the general framework of metastatic mechanisms and we review a selection of more recent studies aimed at describing the metastatic phenotype in molecular terms.

Animals↗
PubMed1986

Hypersensitive sites in the 5' and 3' flanking regions of the cysteine proteinase I gene of Dictyostelium discoideum.

The cysteine proteinase I gene of Dictyostelium discoideum is a developmentally regulated single copy gene. Specific sites in the 5' and the 3' flanking regions of the gene were cleaved by an endogenous nuclease when the gene was being transcribed. The majority of these sites were not cut when the gene was inactive. A dramatic change in the pattern of micrococcal nuclease and DNase I hypersensitive sites occurred in the 5' flanking region when transcription commenced at the 8 h stage of development. The major sites, doublets at -220/-300 bp and -670/-770 bp upstream of the transcription start site, corresponded to those cut by the endogenous nuclease. When transcription subsequently ceased the hypersensitive sites did not significantly change, indicating the gene remained in an activated state. The micrococcal nuclease hypersensitive sites in the 3' flanking region did not change significantly during development.

Base Sequence↗
PubMed1986

Mapping of endogenous nuclease-sensitive regions and of putative topoisomerase sites of action along the chromatin of Dictyostelium ribosomal RNA genes.

Indirect end-labelling and the digestion patterns of endogenous and exogenous nucleases were used to analyse chromatin organization along the ribosomal RNA genes of Dictyostelium discoideum cells. A zone just upstream from the 5' end of the coding region was particularly sensitive to endogenous nucleases. In exponentially growing cells, this hypersensitive zone extended from -350 to -1600 bp relative to the transcription start. In sharp contrast, the DNA between 0 and -350 bp was strongly protected. In differentiating cells, in which the ribosomal RNA transcription rate is low, the 5' hypersensitive zone was more diffuse than in exponentially growing cells, and the protected region at the 5' end of the transcribed region was less pronounced. It is known that where DNA topoisomerase is acting on DNA, the addition of sodium dodecyl sulphate will result in cleavage of the DNA and covalent attachment of the enzyme to the cut DNA end. Treatment of nuclei from both exponentially growing cells and differentiating cells with SDS caused double-stranded cleavages at -200 (i.e. within the protected region), at -2200, and at two sites at about -17 kb. A fraction of the cleavage products appeared to be strongly associated with protein. Novobiocin, a DNA topoisomerase II inhibitor, did not inhibit the SDS-induced cleavages in vegetative cells. However, it significantly reduced the extent of nuclease cleavage within the -350 to -1600 bp hypersensitive zone. The possibility is discussed that there are two DNA topoisomerase-like activities on the ribosomal genes. One is site-specific and novobiocin-insensitive. We speculate that the other is responsible for maintaining DNA at the 5' end of the gene in a torsionally strained, nuclease-hypersensitive state.

Chromatin↗
PubMed1986

Methidiumpropyl-EDTA-iron(II) cleavage of ribosomal DNA chromatin from Dictyostelium discoideum.

We have used methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)] in parallel with micrococcal nuclease to investigate the chromatin structure of the extrachromosomal palindrome ribosomal RNA genes of Dictyostelium. Confirming our earlier results with micrococcal nuclease (1,2), MPE.Fe(II) digested the coding region of rapidly transcribing rRNA genes as a smear, indicating the absence or severe disruption of nucleosomes, whereas in slowly transcribing rRNA genes, a nucleosomal ladder was produced. In the central non-transcribed spacer region of the palindrome, MPE.Fe(II) digestion resulted in a normal nucleosomal repeat, whereas micrococcal nuclease gave a complex banding pattern. The difference is attributed to the lower sequence specificity of MPE.Fe(II) compared to micrococcal nuclease. In the terminal region of the palindrome, however, both substances gave a complex chromatin digestion pattern. In this region the DNA appears to be packaged in structures strongly positioned with respect to the underlying DNA sequence.

Cell Nucleolus↗
PubMed1986

Indole-3-acetic acid and fusicoccin cause cytosolic acidification of corn coleoptile cells.

Microelectrodes were used to measure simultaneously the effects of indole-3-acetic acid (IAA) on membrane potential and cytosolic pH of corn coleoptile cells. IAA caused an initial depolarization followed by hyperpolarization, the latter displaying rhythmic oscillations. The extent of the changes in membrane potential was dependent on IAA concentration, and hyperpolarization, but not depolarization, could be detected with concentrations of IAA as low as 10 nM. Membrane hyperpolarization was preceded by a decrease in cytosolic pH. The decrease commenced approximately 5 min after adding IAA and continued for 15-20 min before reaching a new steady state approximately 0.1 pH unit lower than the original pH. The decrease in pH was readily detectable after treatment with 0.1 muM IAA. Fusicoccin and acetate, which, like IAA, induce elongation growth, caused a similar drop in cytosolic pH and subsequent membrane hyperpolarization, the decrease in pH commencing within seconds. The addition of fusicoccin to IAA-treated cells resulted in a further cytosolic acidification and membrane hyperpolarization. The two substances probably change cytosolic pH via different mechanisms. The results imply that one of the primary effects of auxins in coleoptiles is to lower cytosolic pH.

Journal Article↗
PubMed1985

A lysine-rich protein functions as an H1 histone in Dictyostelium discoideum chromatin.

Mononucleosomes released from Dictyostelium discoideum chromatin by micrococcal nuclease contained two distinctive DNA sizes (166-180 and 146 bp). Two dimensional gel electrophoresis suggested a lysine-rich protein protected the larger mononucleosomes from nuclease digestion. This was confirmed by stripping the protein from chromatin with Dowex resin. Subsequently, only the 146 bp mononucleosome was produced by nuclease digestion. Reconstitution of the stripped chromatin with the purified lysine-rich protein resulted in the reappearance of the larger mononucleosomes. Two-dimensional gel electrophoresis showed the protein was associated with mononucleosomes. Hence, the protein functions as an H1 histone in bringing the two DNA strands together at their exit point from the nucleosome. Trypsin digestion of the lysine-rich protein in nuclei resulted in a limiting peptide of approx. 10 kilodaltons. Trypsin concentrations which degraded the protein to peptides of 12-14 kilodaltons and partially degraded the core histones did not change the DNA digestion patterns obtained with micrococcal nuclease. Thus, the trypsin-resistant domain of the lysine-rich protein is able to maintain chromatosome structure.

Animals↗
PubMed1984

Psoralen-crosslinking of DNA as a probe for the structure of active nucleolar chromatin.

Trimethylpsoralen was used to crosslink the extrachromosomal ribosomal DNA in nucleoli or nuclei of growing Dictyostelium discoideum cells. The DNA was extracted and was examined by spreading under denaturing conditions for electron microscopy. Intact 95,000 base ribosomal DNA molecules were seen, showing regularly spaced, single-stranded bubbles of about 200 to 400 bases in size, interrupted twice by 11,000 base heavily crosslinked stretches, which correspond to the known positions of the coding regions. The bubbles on the nontranscribed regions indicate the presence of nucleosomes during crosslinking. The DNA was digested with restriction enzymes and analysed by gel electrophoresis in parallel with DNA not treated with psoralen. Fragments from the non-coding region had the same mobility as untreated DNA, while those from the coding region had a markedly lower mobility, though not as low as that of crosslinked pure DNA. This shifting of the bands, specific to the coding region, was also seen when whole cells were treated with psoralen. Treatment of nucleoli with 2 m-NaCl (which is known to dissociate histones) before addition of psoralen led to strong crosslinking all along the ribosomal DNA, resulting in a decreased electrophoretic mobility of bands from the non-coding region, but no further retardation of those from the coding region. In differentiating Dictyostelium cells, slugs, where ribosomal RNA synthesis is very much reduced, the extent of psoralen-crosslinking in the coding region was reduced, but not completely to the level of that of the non-transcribed spacer. In order to test whether psoralen itself alters chromatin structure, crosslinked and non-crosslinked nucleoli from growing cells were lysed with heparin and spread for electron microscopy. There was no difference in the appearance or the frequency of the transcription units seen. Digestion of crosslinked nuclei with micrococcal nuclease indicated an undisturbed structure for bulk chromatin, as well as for the chromatin in the non-transcribed spacer of the ribosomal DNA. Thus psoralen-crosslinking does not lead to extensive disruption or distortion of the structure of either inactive or active chromatin. We conclude, taking the results presented in the Appendix into account, that the extent of psoralen-crosslinking in chromatin DNA is diagnostic for the structure of undistorted chromatin.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Nucleolus↗
PubMed1984

Studies of the invasiveness of the chemically induced mouse sarcoma FS9. I. Monoclonal antibodies to a 37,000 dalton membrane glycoprotein inhibit invasion of fibroblasts in vitro.

Monoclonal antibodies against the mouse sarcoma FS9 were prepared. Antibodies recognizing a 37,000 dalton glycoprotein on FS9 tumor cells inhibit invasion by FS9 sarcoma cells of chicken heart fibroblasts in vitro as assessed by Abercrombie's confronted explant assay. Antibodies to other membrane proteins of FS9 tumor cells failed to inhibit invasiveness of FS9 sarcoma cells. The 37,000 dalton glycoprotein, which is neither a histocompatibility antigen nor a gp37 glycoprotein of Rous sarcoma virus nor the MEP described by Gottesman, is present on the surface and in the cytoplasm of FS9 sarcoma cells. The plasma membrane of the non-invasive mouse cell line L929 contains only low concentrations of the 37,000 dalton antigen. Hence, a relationship apparently exists between the increased concentration of this protein on the surface of FS9 cells and their invasiveness.

Animals↗
PubMed1984

A structural concept for nucleoli of Dictyostelium discoideum deduced from dissociation studies.

We aimed to establish whether there is a matrix structure in the nucleolus to which the ribosomal DNA (rDNA) is strongly attached. To detect artifacts that might occur during the harsh histone extraction procedures frequently used for matrix preparation, we dissociated nucleoli of Dictyostelium discoideum with a range of NaCl or heparin concentrations. With heparin treatment significant amounts of rDNA were solubilized into the dissociating solution. When the residual nucleoli were digested with Eco RI, none of the Eco RI fragments of the rDNA remained preferentially bound to the residual nucleoli, indicating that there is no matrix attached to a specific site on the rDNA. When residual nucleoli were examined by electron microscopy, a correlation was found between the extent of solubilization of rDNA, the loss of nucleosomes, and, in heparin-treated nucleoli, the loss of ribonucleoprotein-bound components. These results suggest that the rDNA is released from the nucleoli as soon as nucleosomes have been dissociated and transcription complexes disrupted. Electron microscopy also showed that the NaCl concentration required for dissociation of nucleosomes was higher when divalent cations (Ca2+, Mg2+, Cu2+) were used during the isolation or the treatment of the nucleoli prior to dissociation in high salt. Furthermore, the residual, high-salt-resistant structures were much larger when nucleoli were pretreated with divalent cations or when they were purified in the presence of Ca2+ than when they were purified in its absence. Hence divalent cations, which induce chromatin condensation, prevented nucleolar dissociation whereas treatment with chelating agents, which loosen chromatin compaction, led to much smaller residual matrixlike structures. Nucleoli could be dissociated with heparin to a larger extent than with NaCl so that in Ca2+-free preparations no residual nucleolar matrixlike structures could be detected. Our results suggest that the nucleolar "matrix" seen in the electron microscope is due to incomplete dissociation of the nucleolar material. We propose that in nucleoli of Dictyostelium the rDNA is not attached to a tightly binding matrix structure, but that nucleoli are stabilized by side-to-side contacts between chromatin fibers and transcription complexes.

Cell Fractionation↗
PubMed1984

Invasive behavior of mouse sarcoma cells is inhibited by blocking a 37,000-dalton plasma membrane glycoprotein with Fab fragments.

Abercrombie 's confronted explant technique was used to study the role of tumor surface antigens in malignant invasion. Plasma membranes were isolated from mouse sarcoma cells ( FS9 ) and a mouse cell line (L929) of the same H-2 haplotype. FS9 cells are highly invasive when confronted with chicken heart fibroblasts, whereas the L929 cells are not [ Abercrombie , M. (1979) Nature (London) 281, 259-262]. The FS9 plasma membranes contained significantly higher concentrations of a 37,000-dalton glycoprotein. When antiserum directed against FS9 plasma membranes was preabsorbed with L929 cells, the antibodies remaining reacted predominantly with the 37,000-dalton antigen. Fab fragment prepared from the preabsorbed antiserum inhibited the invasion of chicken heart fibroblasts by FS9 cells. Fab prepared from a monoclonal antibody directed against the 37,000-dalton antigen also inhibited invasivity , whereas monoclonal antibodies reacting with two other FS9 cell surface antigens did not. The results imply a relationship between the increased concentration of the 37,000-dalton glycoprotein on the surface of the FS9 cells and their invasivity .

Animals↗
PubMed1983

Chromatin structure along the ribosomal DNA of Dictyostelium. Regional differences and changes accompanying cell differentiation.

The ribosomal genes of Dictyostelium discoideum are extrachromosomal palindromic DNA molecules situated in the nucleolus. Each molecule comprises ribosomal RNA coding regions and non-transcribed spacer regions. We used both biochemical and electron microscopic approaches to investigate the structure of transcribing and non-transcribing chromatin. Nucleoli from exponentially growing cells were digested with micrococcal nuclease, and the resulting DNA fragments were separated by gel electrophoresis and transferred to DBM paper. They were hybridized with cloned EcoRI fragments derived from different parts of the ribosomal gene. Probes of the coding region showed a smear, while probes of the non-transcribed regions gave pronounced banding patterns more complex than typical nucleosome repeats, but not due solely to sequence-specific cutting by micrococcal nuclease. The DNA of the coding region was digested more quickly than that of the non-transcribed ones. When nucleoli were digested with restriction enzymes, sites within the coding region were accessible and sites in the non-transcribed region were protected. The structure of ribosomal chromatin in differentiating cells, in which the rate of ribosomal RNA synthesis is reduced, was examined using essentially the same methods. The coding region, probed by hybridization to micrococcal digests, then showed a typical DNA repeat pattern indicating that this region had become condensed into nucleosomes, and its accessibility to restriction enzymes was very much reduced. On electron micrographs of lysed nucleoli from exponentially growing cells, two types of chromatin were observed, one with a beaded nucleosomal appearance, the other with putative RNA polymerase molecules attached to fibres indistinguishable from free DNA adsorbed to the same grid. The combined results suggest that whereas regions that are not transcribed are packaged with proteins that protect them from nuclease digestion, actively transcribing ribosomal genes are associated with few macromolecular constituents apart from those required for transcription and its regulation.

Cell Nucleolus↗
PubMed1980

Evidence that a developmentally regulated glycoprotein is target of adhesion-blocking Fab in reaggregating Dictyostelium.

The target site of adhesion-blocking Fab in aggregating Dictyostelium cells is a plasma membrane glycoprotein of approximate molecular weight 82,000 (refs 1--3), which is no longer synthesized after aggregation, and disappears from the plasma membrane. However, since cell adhesion remains important during later stages of differentiation, presumably important during later stages of differentiation, presumably another proteins(s) takes over its function. One candidate is a glycoprotein (molecular weight 95,000) whose synthesis commences at the tip stage and continues until the completion of development. Both proteins are strongly antigenic. When pseudoplasmodia ('slugs', a late developmental stage) are disaggregated and replated, the cells recapitulate the previous sequence of morphological changes much more quickly than before, although the glycoprotein of molecular weight 82,000 is not resynthesized during reaggregation. We report here that monovalent antibody (Fab) directed against slug plasma membranes inhibits reaggregation by binding to the glycoprotein of molecular weight 95,000 which thus seems to be involved in cell adhesion.

Animals↗