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Biomedical subjects

S J McCready

Publications and source records attributed to S J McCready.

14 recordsLinked to original sources
PubMed1989

The Saccharomyces cerevisiae RAD2 gene complements a Schizosaccharomyces pombe repair mutation.

Two Saccharomyces cerevisiae genes necessary for excision repair of UV damage in DNA, RAD1 and RAD2, were introduced individually, on a yeast shuttle vector, into seven Schizosaccharomyces pombe mutants - rads 1, 2, 5, 13, 15, 16 and 17. The presence of the cloned RAD1 gene did not affect survival of any of the S. pombe mutants. The RAD2 gene increased survival of S. pombe rad13 to near the wild-type level after UV irradiation and had no effect on any of the other mutants tested. S. pombe rad13 mutants are somewhat defective in removal of pyrimidine dimers so complementation by the S. cerevisiae RAD2 gene suggests that the genes may code for equivalent proteins in the two yeasts.

DNA Damage↗
PubMed1987

Excision repair in the yeast, Saccharomyces cerevisiae.

cdc9 mutants of yeast lack detectable DNA ligase activity at restrictive temperatures. They also appear to be more sensitive than wild-type cells to ultraviolet (u.v.) radiation and it has been assumed that this is because the CDC9 ligase is needed for the final ligation step in excision repair. The fact that single-strand breaks have been demonstrated in u.v.-irradiated cdc9 mutants has been regarded as evidence for this interpretation. However, the kinetics of appearance of nicks in the DNA do not support this since maximal levels of strand breaks appear almost immediately after exposure to u.v. light and not progressively as repair events are initiated. We believe, therefore, that these strand breaks are connected with a u.v.-dependent preincision event, possibly connected with reorganization of chromatin.

DNA Repair↗
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↗
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↗
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↗
PubMed1977

A comparison of small circular DNA molecules in psi+ and psi- strains of Saccharomyces cerevisiae.

The psi+ and psi- phenotypes, which affect the efficiency of ochre suppression in yeast, are inherited in a non-Mendelian fashion. There is no apparent difference in length or in length distribution of 2 micronm circular DNA molecules between psi+ and psi- strains. It seems that the psi genetic determinant is probably not connected with the presence or absence of these small circular DNA molecules.

DNA, Circular↗
PubMed1977

The extrachromosomal control of nonsense suppression in yeast: an analysis of the elimination of [psi+] in the presence of a nuclear gene PNM.

When a [psi-] strain of yeast mutates to [psi+], the efficiency of suppression by certain ochre suppressors is increased. The [psi+] phenotype is inherited extrachromosomally. There is a nuclear gene, PNM, which, when mutant, causes loss of the [psi+] phenotype. PNM- is dominant to PNM+ and a heterozygous diploid gradually loses the ability over successive generations, to produce PNM+ [psi+] spores. This paper describes the kinetics of this elimination and the data obtained are discussed in relation to two models of the molecular nature of the [psi] genetic determinant--one considering the [psi] determinant as an autonomous nucleic acid, the other treating the possibility that the [psi] nucleic acid is that which codes for rRNA in the nuclear genome.

Cell Division↗