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P R Cook

Publications and source records attributed to P R Cook.

At least 109 records · Page 6Linked to original sources
PubMed1989

Happy mapping: a proposal for linkage mapping the human genome.

A theoretical approach for linkage mapping the genome of any higher eukaryote is described. It uses the polymerase chain reaction, oligonucleotides of random sequence and single haploid cells. Markers are defined and then the DNA of a single sperm is broken at random (eg by gamma-rays) and physically split into 3 aliquots. Each aliquot is screened for the presence of each marker. Closely-linked markers are more likely to be found in the same aliquot than unlinked markers. The entire process is repeated with further sperm and the frequency that any two markers co-segregate determined. Closely-linked markers co-segregate from most cells; unlinked markers do so rarely. A map can then be constructed from these co-segregation frequencies. A specific application for determining the order and distance between sets of closely-linked and previously-defined markers is also described.

Chromosome Mapping↗
PubMed1989

Blood-brain barrier to pertechnetate following drug-induced hypotension.

The integrity of the blood-brain barrier (BBB) was examined in rabbits, in terms of the partition of 99mTc-pertechnetate (99mTcO4-) between brain and blood, following intracarotid injection of hypertonic arabinose, hypertension (168 mm Hg and 10% inspired carbon dioxide), or hypotension (less than 20 mm Hg for 15 min). Corrections were made for changes in tissue blood contents, using chromium-51 as a red cell marker. In control animals the mean brain:blood ratio was 0.038 (range 0.027-0.052). Following arabinose there was a five-fold increase in mean BBB permeability (mean brain:blood ratio 0.192 (0.070-0.378)). There was no change after hypertension and carbon dioxide (mean ratio 0.034) or after hypotension (mean ratio 0.032), despite an increase in cerebral extracellular potassium. Examination of other tissues showed no change in the 99mTcO4- tissue/blood partition in heart muscle in any study but, following hypotension, ratios in the kidney (mean ratio 1.63) and, to a lesser extent, the liver (mean ratio 1.37) had increased, suggesting an abnormality of active transport under these conditions. We conclude that, while 99mTcO4- tissue/blood partitioning revealed osmotic disruption of the BBB, profound hypotension with evidence of brain cell damage did not change BBB permeability to the same marker. Hypotension may influence active transport of this ion in liver and kidney.

Animals↗
PubMed1988

Visualization of a filamentous nucleoskeleton with a 23 nm axial repeat.

Whether nucleoskeletons seen after extracting cells are preparative artefacts is controversial. Using an extraction method that preserves vital nuclear functions, we have visualized part of a nucleoskeleton by electron microscopy of thick resinless sections. Cells encapsulated in agarose microbeads are lysed using Triton in a physiological buffer; the agarose coat prevents aggregation and protects fragile cell contents. These extracted cells are accessible to small molecules and transcribe and replicate at rates close to those in vivo. After electroeluting most chromatin after treatment with HaeIII, a skeleton is uncovered which ramifies throughout the nucleus. Individual filaments are approximately 10 nm wide with an axial repeat of 23 nm, characteristic of intermediate filaments.

Cell Nucleus↗
PubMed1988

A gentle method for preparing cyto- and nucleo-skeletons and associated chromatin.

We describe a method for permeabilizing and extracting cells that preserves both structure and function whilst allowing the cell derivatives to be handled freely. Cells are encapsulated in microbeads of agarose; the coat of agarose, which is freely permeable to small molecules, forms a protective layer around fragile cell constituents. Cells are then permeabilized by the non-ionic detergent Triton X-100 or antibody and complement in a buffer whose ionic composition mimics that of the cytoplasm. The resulting structures have been characterized morphologically (by immunofluorescence and electron microscopy) and biochemically. Lysis with Triton removes both cell and nuclear membranes, and extracts most of the cytoplasm to leave chromatin surrounded by cytoskeleton; nucleus and cytoplasm then become accessible to triphosphates, enzymes and antibodies. Lysis with complement permeabilizes the cell membrane but leaves the nuclear membrane intact; triphosphates and restriction enzymes, but not antibodies, can then enter both nucleus and cytoplasm. Both types of lysis yield preparations whose chromatin template remains essentially intact, and which is replicated and transcribed at rates close to, or greater than, those found in vivo. Treatment of complement-lysed cells with Triton reduces the very efficient DNA synthesis, implying that the nuclear membrane is involved, directly or indirectly, in replication.

Cell Nucleus↗
PubMed1986

A cell-cycle-dependent DNA polymerase activity that replicates intact DNA in chromatin.

An insoluble DNA polymerase activity that replicates the intact chromatin template at 85% of the rate found in vivo has been partially characterized. HeLa cells, encapsulated in agarose microbeads, are lysed using an isotonic salt concentration: the resulting encapsulated nuclei contain polymerase associated with a nucleoskeleton and the unbroken template. This preparation can be manipulated freely without aggregation or breaking the DNA and yet is accessible to enzymes and other probes. The major activity, which is sensitive to aphidicolin, is found only in S-phase nuclei and replicates DNA semi-conservatively, forming intermediates that are ligated efficiently into larger products.

Autoradiography↗
PubMed1986

Different populations of DNA polymerase alpha in HeLa cells.

Three different populations of HeLa DNA polymerase alpha have been distinguished using a novel preparation of chromatin isolated using an isotonic salt concentration, which contains intact DNA. One synthesizes DNA in vitro at 85% of the rate in vivo, is found only in S-phase nuclei tightly associated with the nucleoskeleton and requires unbroken DNA in the form of chromatin as a template: we assume this is the authentic S-phase activity. On incubation at 37 degrees C, this activity dissociates from the nucleoskeleton to give a soluble activity that prefers broken templates. This soluble activity is in turn heterogeneous, containing active complexes of about 0 X 75 X 10(6) and 3 X 10(6) Mr. The third activity is also soluble and released by lysing cells at any stage of the cell cycle. It, too, prefers broken templates. The authentic activity is obscured by the soluble ones if broken templates are provided.

Antibodies↗
PubMed1986

Part of the human ribosomal RNA locus stabilizes a plasmid in yeast.

Most yeast plasmids--particularly those containing chromosomal replicators (ARS)--are unstable and do not segregate equally to mother and daughter cells unless they contain centromeric sequences. We have screened a fraction of the human genome for sequences that stabilize YRp7, a plasmid containing ARS1. We selected a fraction which we hoped would be enriched in human centromeric sequences--the DNA attached to the nucleoskeleton. We obtained one human sequence that partially stabilized a yeast plasmid and, surprisingly, it contained sequences homologous to those coding for the 3' end of 18s rRNA, the transcribed spacer and 5' end of 28s rRNA. This sequence did not show any ARS activity nor did it increase the copy number of the plasmid and so probably improved partition of the plasmid between mother and daughter cells. It had no homology to yeast centromeres.

Cloning, Molecular↗
PubMed1986

Replication occurs at a nucleoskeleton.

The site of S-phase DNA synthesis has been the subject of recurring controversy. All recent evidence supporting a site fixed to some nuclear sub-structure is derived from studies in which cells or nuclei have been extracted in hypertonic salt concentrations. The controversy centres on whether the resulting nuclear matrices or cages have counterparts in vivo or are simply artefacts. Using isotonic conditions throughout the isolation and analytic procedures we have now reinvestigated the site of replication. Cells are encapsulated in agarose microbeads and lysed to leave encapsulated nuclei which are nevertheless completely accessible to enzymes. After incubation with endonucleases, most chromatin can be electroeluted from beads: however, nascent DNA and active DNA polymerase remain entrapped. Since chromatin particles containing DNA the size of 125 kbp can electroelute, we conclude that the polymerizing complex is attached to a nucleoskeleton which is too large to escape. We have also studied various artefacts induced by departure from isotonic conditions. Perhaps surprisingly, the hypotonic conditions used during isolation of nuclei by conventional procedures are a significant source of artefact.

Cell Division↗
PubMed1985

The chromatin structure of Rous sarcoma proviruses is changed by factors that act in trans in cell hybrids.

In several lines of Rous sarcoma virus (RSV)-transformed rat cells the proviruses are in a configuration typical of active eukaryotic genes. They are sensitive to pancreatic DNase I, with sites hypersensitive to nuclease near the 5' end of the genome, they are close to the nuclear 'cage' and they show a low level of cytosine methylation in CpG doublets. In contrast, in phenotypically untransformed hybrids between these cells and uninfected rat or mouse cells, RSV inactivity is associated with hypermethylation of the provirus, reduced DNase I sensitivity (in two out of three examples) and, where examined, relative remoteness from the nuclear cage. These changes in proviral configuration, which occur rarely in spontaneous reversion of transformed cells, can thus be induced at high frequency and stability in cell hybrids by trans-acting influences of the uninfected parents.

5-Methylcytosine↗
PubMed1985

A general method for preparing chromatin containing intact DNA.

A simple and general method is described for preparing chromatin from eukaryotic cells using isotonic conditions. First, cells are encapsulated in agarose microbeads and then lysed using Triton X-100 in the presence of a chelating agent and a physiological concentration of salt. Most cytoplasmic proteins and RNA diffuse rapidly out through pores in the beads to leave encapsulated chromatin which is nevertheless completely accessible to enzymes and other probes. This chromatin can be manipulated freely without aggregation in a variety of different salt and detergent concentrations. It also contains intact DNA since removal of the histones releases superhelical DNA. Conditions are described for incubating this chromatin at 37 degrees C in the presence of Mg2+ ions without any nicking of the DNA. We illustrate the usefulness of this chromatin in investigations on the attachment of nascent RNA to the nucleoskeleton, the accessibility of the ribosomal locus to EcoRI and the properties of the endogenous RNA polymerase II. This type of chromatin preparation should prove useful for both structural and functional studies.

Cell Nucleus↗
PubMed1985

Transcription occurs at a nucleoskeleton.

Native chromatin aggregates under isotonic conditions so it is generally studied using higher or lower salt concentrations. This has led to different interpretations of how transcription might occur. Studies using hypertonically-isolated preparations suggest that DNA functions in close association with a skeletal nuclear substructure, the matrix or cage, but such a structure is not usually seen under hypotonic conditions (e.g., in 'Miller-spreads'). Using a novel method for preparing chromatin under isotonic conditions we have investigated the site of transcription. We find that all three constituents of the transcription complex, nascent transcripts, active RNA polymerase and genes being transcribed are all closely associated with some structure too large to be electroeluted from the nucleus. Hypotonic treatment partly disrupts this association. We suggest a model for transcription that involves the participation of a nucleoskeleton at the active site and reconcile the contradictory results obtained using different salt concentrations.

Cell Nucleus↗
PubMed1984

Attachment of repeated sequences to the nuclear cage.

Nuclear DNA is probably organized into loops by attachment to a sub-structure in vivo. When HeLa cells are lysed in Triton and 2M NaCl the resulting nucleoids contain naked DNA which is supercoiled so the loops must remain intact. We have attempted to identify sequences responsible for attaching these loops to the nuclear sub-structure by progressively detaching DNA with various nucleases. Fragments at the 5' end of the ribosomal RNA locus, and a variety of transcribed and repeated sequences, are shown to lie relatively close to attachment points. This implies that sequences cannot be arranged randomly. However no "attachment sequence" could be identified.

Base Sequence↗
PubMed1984

The spatial organization of sequences involved in initiation and termination of eukaryotic DNA replication.

Nuclear DNA is looped by attachment to a matrix or cage. As this cage is the site of DNA synthesis, sequences in the loops must attach before they are replicated. We have tested whether sequences which initiate replication are usually out in the loop and attach only during S phase or whether they are attached but quiescent during most of the cell-cycle. Sequences which permit plasmids to replicate autonomously in yeast cells (ARS's) are strong candidates for initiating sequences. Four different human ARS's all map remote from attachment points to the HeLa nuclear cage. In addition a potential terminus of replication is also remote from the cage. We conclude that sequences involved in initiation are usually out in the loop and that DNA synthesis is initiated by their attachment.

Base Sequence↗
PubMed1984

A general method for preparing intact nuclear DNA.

Naked nuclear DNA is easily sheared. Two general methods are described for preparing intact DNA in a stable form that can be pipetted without breaking it. Cells are encapsulated in agarose microbeads and then lysed in a non-ionic detergent (i.e., Triton X-100) and 2 M NaCl or an ionic detergent (e.g., sodium or lithium dodecyl sulphate) in low salt. Most cellular protein and RNA then diffuse out through pores in the beads to leave encapsulated and naked DNA which is nevertheless accessible to enzymes and other probes. Remarkably, considerable structure is preserved since the DNA is supercoiled and chromosomes retain their shape.

Cell Nucleus↗
PubMed1984

Pituitary alcohol injection for cancer pain. Use in a district general hospital.

The use of trans-sphenoidal pituitary alcohol injection was assessed in a district general hospital for the treatment of diffuse bilateral cancer pain in 26 patients. Fourteen patients received a total of 43 injections. Hormonal and non-hormonal dependent cancers were treated, with complete pain relief in ten patients and partial relief in a further seven patients. The duration of relief was variable, and usually less than 6 weeks, although two patients had complete pain relief for 5 and 7 months respectively. No cases of meningitis or cerebrospinal fluid rhinorrhoea occurred. Nasal antiseptics and vasoconstrictors were not used. Temporary pupil dilatation occurred in eight patients, of whom one had prolonged blurred vision for small print. Transient diabetes insipidus was common (53%), although only five patients needed permanent treatment. Death from their disease occurred in 65% of patients within 3 months. Survival was shorter than in series from specialist centres, suggesting later referral for pain relief. The technique is an inexpensive and valid form of treatment for cancer pain in a district hospital, but should not be undertaken lightly in view of the possible complications.

Adult↗