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Biomedical subjects

P R Cook

Publications and source records attributed to P R Cook.

At least 127 records · Page 7Linked to original sources
PubMed1984

Replication and transcription depend on attachment of DNA to the nuclear cage.

When living cells are lysed in a non-ionic detergent and 2 M-NaCl, structures are released that resemble nuclei. They contain naked nuclear DNA packaged within a flexible cage of RNA and protein. Since the DNA is supercoiled, it must be intact and looped by attachment to the cage. It is argued that this cage is the active site of the key nuclear functions, transcription and replication: outlying sequences are activated by attachment to polymerases at the cage. This thesis is supported by the close and specific association of nascent RNA with cages, the attachment of active viral sequences (in transformed and productively infected cells) and the attachment of nascent DNA during both normal and repair synthesis.

Cell Nucleus↗
PubMed1984

Lesions induced in DNA by ultraviolet light are repaired at the nuclear cage.

In mammalian cells, S-phase DNA synthesis occurs at sites fixed to a sub-nuclear structure, the nuclear matrix or cage. This is an ordered network of non-histone proteins, which maintains its essential morphology even in the absence of DNA. We show here that unscheduled DNA synthesis following exposure of HeLa cells to ultraviolet light also takes place at this sub-structure. We also show that ultraviolet irradiation grossly reorganizes nuclear DNA, arresting S-phase synthesis at the cage and leaving the residual synthesis highly localized.

Autoradiography↗
PubMed1983

The DNA-protein cross: a method for detecting specific DNA-protein complexes in crude mixtures.

A method using crude cellular mixtures is described which permits identification of polypeptides and DNA fragments forming specific complexes. Our procedure incorporates elements of both 'Southern' and 'protein' blotting and combines, in two dimensions, the resolving power of a denaturing protein gel with that of an agarose DNA gel. Conditions for 'crossing' have been established using the lambda repressor-operator system: the specific complex can be detected by crossing total protein from bacteria overproducing the repressor with a mixture of total genomic fragments from a lysogen.

Bacteriophage lambda↗
PubMed1982

Active viral genes in transformed cells lie close to the nuclear cage.

Nuclear DNA is looped by attachment to a matrix or cage. Using nine different lines transformed by polyoma or avian sarcoma virus, we have mapped viral sequences integrated within these loops. In all lines that contain high concentrations of viral transcripts and express the transformed phenotype, the integrated viral genes lie close to the points of attachment to the cage. Integration of polyoma DNA induces outlying cellular sequences to become closely associated with the cage. The strength of this correlation between gene activity and proximity to the cage was examined using sub-clones of one avian sarcoma virus transformant. Proviral sequences are closely associated with the cage in this transformant, much less so in two untransformed 'flat revertants' which contain no detectable viral transcripts but regain their close association with the cage in two retransformed derivatives.

Animals↗
PubMed1981

Conformational changes induced by salt in complexes of histones and superhelical nuclear DNA.

When HeLa cells are lysed in solutions containing a non-ionic detergent and 0.75 M-NaCl, structures are released that retain many of the morphological features of nuclei. These nucleoids contain all the nuclear DNA, RNA and the 'core' histones, but few other proteins characteristic of chromatin. Their DNA is intact. The core histones dissociate on raising the salt concentration. We have probed the structure of nucleoid-histone complexes using the intercalating dye, ethidium, or the RNA polymerase of Escherichia coli. Both have a higher affinity for superhelical DNA than they do for relaxed DNA. The binding of ethidium is measured fluorometrically, and using this probe we find that the DNA of nucleoids containing all the core histones behaves as if it were supercoiled slightly positively. As the salt concentration is increased, free energy characteristic of negative supercoiling appears between 0.92 M and 0.95 M-NaCl. This transition, which is reversible in the presence of the arginine-rich histones, occurs without dissociation of these histones from the DNA and so must reflect a conformational change in the complex. In contrast to the results with ethidium, we find that RNA polymerase can detect the presence of some negative free energy of supercoiling in nucleoids containing the core histones. The transformations of the free energy that can assist the binding of ethidium and RNA polymerase are discussed.

Binding Sites↗
PubMed1981

Reconstruction of complexes of histone and superhelical nuclear DNA.

When HeLa cells are lysed in solutions containing a non-ionic detergent and 2 M-NaCl, structures are released that retain many of the morphological features of nuclei. These nucleoids contain all the nuclear RNA and DNA but few of the proteins characteristic of chromatin. Their DNA is supercoiled and so intact. Using a simple and rapid procedure we have reconstructed nucleohistone complexes from nucleoids and the 'core' histones without breaking the DNA. We have probed the integrity and structure of the reconstructed complexes using a non-destructive fluorometric approach, which provides a general method for detecting agents that bind to DNA and alter its supercoiling. The superhelical status of the DNA in the reconstructed complexes is indistinguishable from that found in control nucleoids containing core histones. Experiments with micrococcal nuclease confirm that the DNA in the reconstructed complexes is organized into nucleosome-like structures. These, however, are spaced 145 base-pairs apart and not 200 base-pairs apart as is found in native chromatin.

DNA, Superhelical↗
PubMed1980

Mapping sequences in loops of nuclear DNA by their progressive detachment from the nuclear cage.

Nuclear DNA is organised into loops, probably by attachment to a supramolecular structure. We describe a method which enables us to map the position of sequences within a loop relative to the point of attachment. Nuclear DNA is isolated unbroken by lysing HeLa cells in 2M NaCl to release structures which retain many of the morphological features of nuclei. Their DNA is supercoiled and so must remain unbroken and looped during lysis. Nucleoids are digested to various degrees with a restriction endonuclease and the cages - and any associated DNA - sedimented free from unattached DNA. The cage-associated DNA is purified and completely fragmented using the same restriction endonuclease. Equal weights of fragmented DNA are separated by gel electrophoresis, transferred to a filter and the relative amounts of the alpha, beta and gamma globin genes on the filter determined by hybridisation to the appropriate probes. The alpha genes, unlike the beta and gamma genes, resist detachment from the cage and so must lie close to the point of attachment to the cage. Our ability to map these genes implies that sequences cannot be attached at random to the cage; rather, specific sequences must be attached, so looping the DNA.

Base Sequence↗
PubMed1980

DNA gyrase stimulates transcription.

The nuclear DNA of HeLa cells can now be isolated unbroken and supercoiled. Using DNA gyrase and the untwisting enzyme, we have prepared an allomorphic series of templates derived from this nuclear DNA, and also from the circular DNA of the bacterial virus, PM2. We have then transcribed these templates using 2 different RNA polymerases--from wheat germ and Escherichia coli. Relaxed DNA is transcribed slowly by both polymerases. Supertwisting the naturally-supercoiled templates with gyrase slightly inhibits transcription by the bacterial polymerase but stimulates dramatically transcription by RNA polymerase II from wheat germ.

Animals↗
PubMed1980

DNA is replicated at the nuclear cage.

Structures resembling nuclei are released when HeLa cells are lysed in a detergent and 2 M salt. These nucleoids, which lack any organized membrane structure, contain all the nuclear DNA packaged within a cage of RNA and protein. Their DNA is supercoiled so that the linear DNA must remain unbroken and looped during lysis. Following digestion with the restriction endonuclease, EcoRI, cages and associated DNA were filtered free of unattached DNA. Pulse-labelled (i.e. newly synthesized) DNA remains preferentially associated with the cages. This association has been confirmed by autoradiography. When nucleoids are prepared for electron microscopy by the Kleinschmidt procedure the DNA spills out to form a skirt around the flattened cage. Labelling, which is restricted to the region of the cage after short pulses, extends out into the skirt as the labelling time increases. A model, based on the premise that replication takes place at the nuclear cage, is presented in the Appendix. The results of the biochemical experiments and electron microscopy both indicate that the average size of the unit of replication is approximately 2 micrometer. This is about one-quarter the size of the average structural unit - the loop. Therefore sequences in the loop must become attached to the nuclear cage prior to the initiation of DNA synthesis.

Autoradiography↗
PubMed1979

Electron-microscopy of intact nuclear DNA from human cells.

Structures retaining many of the morphological features of nuclei may be released by lysing human cells in a non-ionic detergent and 2 M NaCl. Such nucleoids contain all the nuclear DNA packaged within a flexible cage of RNA and protein. HeLa nucleoids have been spread at an air-water interface and viewed in the electron microscope. A tangled network of superhelical fibres surrounds the collapsed cage. Irradiation with gamma-rays abolishes supercoiling and treatment with the untwisting enzyme or a low concentration of ethidium reduces it. A high concentration of ethidium induces supertwisting. The nuclear DNA of higher cells can be isolated naked, supercoiled and intact.

Cell Nucleus↗
PubMed1978

Spectrofluorometric measurement of the binding of ethidium to superhelical DNA from cell nuclei.

Structures retaining many of the morphological features of nuclei may be released by lysing HeLa cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain few chromatin proteins. We have shown that the DNA of nucleoids is quasicircular and supercoiled by measure spectrofluorometrically the amount of the intercalating dye, ethidium, bound to unirradiated and gamma-irradiated nucleoids. Ethidium binds to nucleoids in the manner characteristic of the binding to superhelical DNA: at low concentrations more ethidium binds to unirradiated nucleoids than to their gamma-irradiated counterparts with broken DNA, and at higher concentrations less ethidium binds to the unirradiated nucleoids. The quasi-circles in nucleoids are 22 times less sensitive to gamma-irradiation than are circles of pure PM2 DNA: they must contain about 2.2 X 10(5) base pairs. The constraints that maintain the quasi-circularity of nucleoid DNA are very resistant to extremes of temperature and alkali; some remain under conditions in which the duplex is denatured. The constraints are destabilised by ethidium suggesting that they are stabilised by free energy of supercoiling. Proteolytic enzymes, but not ribonucleases, remove the constraints. Possible structures for the constraining mechanism are discussed.

Binding Sites↗
PubMed1978

The dissociation of nuclear proteins from superhelical DNA.

Structures retaining many of the morphological features of nuclei may be released by gently lysing human cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain superhelical DNA. Using a double-labelling procedure we have compared, at different salt concentrations, the amounts and types of protein associated with human nucleoides containings superhelical or relaxed DNA. We find that the slightly lysine-rich histones (H2A and H2B) but not the arginine-rich histones (H3 and H4) dissociate more slowly from nucleoids containing superhelical DNA than from those containing relaxed DNA. A protein of apparent molecular weight of 22000 also binds more tightly to superhelical DNA. We conclude that this protein and the slightly lysine-rich histones transmute free energy of supercoiling into binding energy when they bind to superhelical DNA.

Arginine↗
PubMed1978

Supercoiling of DNA and nuclear conformation during the cell-cycle.

When cells are lysed in solutions containing high concentrations of salt and a non-ionic detergent, structures are released which retain many of the morphological features of nuclei. These nucleoids contain superhelical DNA but are depleted of nuclear protein. We have analysed DNA conformation in nucleoids derived from HeLa cells synchronized at different stages in the cell cycle. The gross differences in nuclear morphology seen during the cell cycle are reflected in the morphology of the nucleoids; for example, the individual chromosomes of mitotic cells remain identifiable and aggregated within the mitotic nucleoid. The sedimentation rate of nucleoids in sucrose gradients reflects the gross nuclear morphology; the small S-phase nucleoids sediment 9 times faster than the large mitotic nucleoids. Despite these large differences at the gross level of organization, both the degree of supercoiling and the size of the units in which supercoiling is maintained are roughly similar in the nucleoids derived from cells in the different phases. The protein content of the various nucleoids is also very similar. Like the nucleoids made from randomly growing cultures of cells, mitotic nucleoids are excellent templates for the RNA polymerase of Escherichia coli.

Cell Cycle↗