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R S Jack

Publications and source records attributed to R S Jack.

31 records · Page 2Linked to original sources
PubMed1992

The elusive nuclear matrix.

The structure of the interphase nucleus is a major area of current interest in cell biology. It is thought likely that the nucleus is organised around some form of structural matrix and that this matrix will play a role in processes as diverse as chromosome replication and the integration of gene expression. However, the structure of the matrix within the nucleus has remained elusive, largely because attempts to define it have been dogged by technical problems arising from the great complexity of this organelle. This situation is now being changed by the application of in situ analysis and of molecular genetic methodologies which are opening up this hitherto intractable field.

Animals↗
PubMed1991

Restriction enzymes permit quantitative determination of defined chromatin structures within the chromosome.

Open chromatin structures, operationally defined as nuclease-hypersensitive sites, are frequently found spanning the controlling regions of genes and they may ensure that trans-acting factors have ready access to their genomic substrates. The rapidity and extent of induction of a gene may be dependent on the probability that its promoter is folded into an open structure. We show that restriction enzymes can be used to estimate the probability that a given promoter region is contained within a defined structure in the chromosome. In the case of the Drosophila major heat-shock-protein gene, we show that an individual promoter element is folded in an accessible form in at least 75% of embryonic chromosomes. This efficient maintenance of the hypersensitive region may be a necessary precondition for a rapid heat-shock response.

Animals↗
PubMed1991

An ectopic copy of the Drosophila ftz associated SAR neither reorganizes local chromatin structure nor hinders elution of a chromatin fragment from isolated nuclei.

In vitro assays using detergent extracted nuclei allow the operational definition of SARs--specific sequences in the chromosome which are thought to interact with a structural matrix or scaffold. This interaction results in the formation of large stable protein-DNA complexes. We have used P-element transformation to introduce a characterized SAR into the Drosophila genome. The standard assay, which uses detergent extracted nuclei, shows that the ectopic SAR is indeed bound to the scaffold. However, in unextracted nuclei, a chromatin fragment containing the SAR sequence is eluted from the nucleus as readily as a fragment which lacks an SAR. Furthermore, an analysis of the accessibility of the neighbouring chromosomal restriction sites in unextracted nuclei indicates that the introduction of this ectopic SAR does not influence the local structure of chromatin. We conclude that the ectopic SAR site is not attached to a nuclear scaffold or matrix in vivo.

Animals↗
PubMed1990

An unusually stable DNA binding protein can locate its specific binding site in the presence of high concentrations of urea.

A sequence specific DNA binding protein has been demonstrated in extracts of Drosophila melanogaster third instar larval nuclei which binds close to a chromosome-scaffold associated region. This protein has proven difficult to work with because of its strong tendency to aggregate. Here I show that the protein can be readily maintained in solution in the presence of high concentrations of urea. Surprisingly, the protein turns out to be remarkably resistant to denaturation by urea. It is capable of mediating sequence specific DNA binding in the presence of urea at concentrations up to 8M. When incubated in 4M urea the binding activity appears to slowly degrade, but in 1.3M urea the protein is active, soluble and stable over extended periods of time at room temperature. The molecular basis of this unexpected finding must await the purification of the protein. The ability to keep the protein both soluble and active should now permit its isolation.

Animals↗
PubMed1990

Restriction enzymes have limited access to DNA sequences in Drosophila chromosomes.

Sequence specific DNA binding proteins in eukaryotic cells must efficiently locate their binding sites in chromosomes. Restriction enzymes provide a simple model system with which to investigate the factors which influence this process. We have used P element mediated transformation to introduce a DNA fragment containing a set of characterized restriction sites into the Drosophila germline. Embryonic nuclei prepared from these transgenic animals were treated with restriction enzymes to probe the accessibility of the target restriction sites. The results show that the insert is within an accessible region of the chromosome and that restriction sites within the inserted sequence can be cut. However, the rate of cutting is biphasic. At each restriction site, a fraction of the chromosomes is cut rapidly after which the remainder is refractory. Similar levels of incomplete cutting are obtained when the same P element construct is examined at a different chromosomal location, when different sequence elements are introduced into the P element vector or when the experiment is carried out on nuclei from different embryonic stages. These results are discussed in terms of how sequence specific DNA binding proteins may locate their genomic targets in vivo.

Animals↗
PubMed1981

Protein component from Drosophila larval nuclei showing sequence specificity for a short region near a major heat-shock protein gene.

Using a nitrocellulose filter binding assay we have detected a protein component in extracts prepared from the nuclei of D. melanogaster third instar larvae which shows sequence-specific binding to a short region on a cloned D. melanogaster DNA segment containing a copy of the major heat-shock protein gene. The specificity of binding was confirmed by direct visualization of the protein DNA complexes in the electron microscope. Protein blotting experiments demonstrated that three polypeptides in the extract were capable of such sequence-specific binding. The protein binding site detected in this cloned sequence lies 800-1000 bp upstream from the presumptive transcription start.

Animals↗
PubMed1977

Idiotypic analysis of the response of C57BL/6 mice to the (4-hydroxy-3-nitrophenyl)acetyl group.

Strain C57BL/6 mice produce a highly restricted primary response to the (4-hydroxy-3-nitrophenyl)acetyl (NP) group. This response is composed of molecules having mu or gamma1 heavy chains and light chains of the lambda type. A guinea pig anti-idiotype was raised to the purified C57BL/6 primary anti-NP immunoglobulin. After suitable absorption, this anti-idiotype was shown to detect markers present on the primary anti-NP immunoglobulin only of those strains which express the Ig-1b allotype. Breeding experiments demonstrated that the marker segregated with the heavy chain linkage group.

Adjuvants, Immunologic↗
PubMed1976

Metal-replacement studies in Bacillus stearothermophilus aldolase and a comparison of the mechanisms of class I and class II aldolases.

A comparison of the product-inhibition patterns during cleavage of D-fructose 1,6-diphosphate by aldolases from yeast, rabbit muscle and Bacillus stearothermophilus shows an ordered reaction sequence for all three enzymes, with dihydroxyacetone phosphate the last-leaving product. Addition of Zn2+, Co2+, Fe2+, Mn2+ or Cd2+ ions to the inactive apo-(Bacillus stearothermophilus aldolase) restores activity to different extents, whereas Ni2+, Mg2+ or Cu2+ ions have no effect. The cleavage activity of this aldolase is not enhanced by added K+ ion. The effects of metal replacement on thermal stability, Km and Vmax. are given and the possible role of the metal is discussed in the light of these results.

Animals↗
PubMedDate not supplied

Human monocytes lacking the membrane-bound form of the bacterial lipopolysaccharide (LPS) receptor CD14 can mount an LPS-induced oxidative burst response mediated by a soluble form of CD14.

Monocytes and macrophages express a glycosyl phosphatidylinositol (GPI)-anchored lipopolysaccharide (LPS) receptor on the cell surface which enables them to detect minute amounts of LPS released from Gram-negative bacteria. A soluble form of CD14 is also found free in serum, though its physiological function is unknown. the interaction of LPS with CD14 on the monocyte surface leads to an activation of the cells which is manifested in the sudden release of reactive oxygen species, a process referred to as an oxidative burst. In patients suffering from the condition known as paroxysmal nocturnal haemoglobinuria (PNH), the synthesis of GPI anchors is blocked in haematopoietic cells which are therefore unable to express GPI-linked proteins on their surface. In severe cases, over 90% of monocytes lack membrane-bound CD14, though normal levels of the soluble form of the receptor-sCD14-are found in the serum. Despite this lack of membrane-bound CD14, monocytes from PNH patients can respond to low concentrations of LPS. Here we show that the LPS-induced oxidative burst of these PNH monocytes requires a component present in serum. The serum-dependent activation can be inhibited by monoclonal antibodies to CD14, can be removed from the serum by passage over a matrix to which an anti-CD14 antibody has been bound, and the depleted serum can be reconstituted by the addition of either purified natural or purified recombinant soluble CD14. We conclude that an LPS-dependent oxidative burst in PNH monocytes can be mediated by soluble CD14.

Acute-Phase Proteins↗