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S McCready

Publications and source records attributed to S McCready.

16 recordsLinked to original sources
PubMed1998

Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe.

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G2 phase of the cell cycle. However, in this study we show that cells held in G1 of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G2 following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G2 DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in response to inhibition of DNA synthesis by hydroxyurea.

Caffeine↗
PubMed1998

Immune-mediated congenital heart block (CHB): identifying and counseling patients at risk for having children with CHB.

OBJECTIVE: To identify patterns of maternal antibodies associated with an increased risk of having a child with congenital heart block (CHB) and to provide a basis for counseling women with a previously affected child. METHODS: This retrospective clinical study of the obstetric histories of 46 Finnish women with a CHB child compared the strength and specificity of the immune response to SS-A/Ro and SS-B/La, as determined by immunoblot and ELISA, in 44 affected women with 85 women with systemic lupus erythematosus (SLE) and 32 women with primary Sjögren's syndrome (SS) with healthy children. RESULTS: High levels of anti-SS-A/Ro and anti-SS-B/La by practically all assays were associated with a significantly increased risk of having a CHB child. The best single test to identify high-risk mothers was anti-52 kd SS-A/Ro by immunoblot (OR 18.9), and it was the only assay to detect mothers at increased risk of CHB as compared with controls with primary SS. Low risk of CHB was indicated by undetectable or low levels of antibodies in the ELISA assays and no reactivity on immunoblot. Mothers with a previous child with CHB had a history of fetal loss (mostly spontaneous abortions) or a history of recurrent fetal losses (> or = 3) slightly more often than controls. Late-trimester obstetric complications in non-CHB pregnancies were insignificant. The relative risk for a female child compared with a male child to have CHB was 1.9 (1.2-2.9, P = .009), and the risk of the mother having another child with CHB was 12% (4 of 34). CONCLUSION: Although there is no unique antibody profile specific for CHB, mothers with a high or low risk of having a child with CHB can be identified. Female children appear to have an increased risk of CHB, but the risk of the mother having another child with CHB is low.

Abortion, Spontaneous↗
PubMed1998

HSP 12 is a LEA-like protein in Saccharomyces cerevisiae.

LEA group I, II and III antibodies all recognised soluble proteins present in an extract of yeast (Saccharomyces cerevisiae). The smaller protein of the two recognised by the group I antibody displayed identical migration on SDS-PAGE to the pea seed LEA group I protein against which the antibody was raised. However, the antibody failed to recognise the predominant protein present after heating the extract at 80 degrees C for 10 min. This predominant protein, which also displayed identical migration on SDS-PAGE, was purified from the supernatant of the extract heated at 80 degrees C for 10 min. Peptide sequencing after CNBr cleavage identified the isolated protein as the heat shock protein HSP 12. Despite a previous report that HSP 12 is a heat shock protein, HSP 12 was found to increase in yeast grown at 37 degrees C compared with growth at 30 degrees C. However, increased amounts of HSP 12 were present in yeast after entry into stationary phase; this was enhanced by growth in the osmolytes NaCl and mannitol.

Amino Acid Sequence↗
PubMed1996

The repair of ultraviolet light-induced DNA damage in the halophilic archaebacteria, Halobacterium cutirubrum, Halobacterium halobium and Haloferax volcanii.

Extremely halophilic archaebacteria have been reported to have no capacity for dark repair (excision repair) of ultraviolet damage and to rely on very efficient photoreactivation for recovery after UVC irradiation. Post-UV incubation in the light restores 100% survival in these organisms. This has been taken to indicate that cyclobutane dimers are the only significant UV-induced lesions and that they are completely repaired by photoreactivation. However, in all organisms studied to date, pyrimidine (6-4) pyrimidone photoproducts are a significant cytotoxic and mutagenic lesion and constitute 10-30% of UV photoproducts. The question arises, therefore--are 6-4 photoproducts induced in the halophilic archaebacteria and, if they are, how are they repaired? This paper shows that both cyclobutane dimers and 6-4 photoproducts are induced in the extremely halophilic archaebacteria, Halobacterium cutirubrum, Halobacterium halobium and Haloferax volcanii, at similar levels as in other organisms. Furthermore, contrary to previous reports, there is dark repair of both lesions. As in other organisms, 6-4 photoproducts are removed more efficiently than cyclobutane dimers in the dark. In the light, cyclobutane dimers are repaired very rapidly and there is also photoenhanced repair of 6-4 photoproducts. This work confirms that organisms such as Halobacterium and Haloferax which live in conditions of high exposure to sunlight have very efficient rates of repair of UV lesions in the light.

DNA Damage↗
PubMed1996

The levels of repair of endonuclease III-sensitive sites, 6-4 photoproducts and cyclobutane pyrimidine dimers differ in a point mutant for RAD14, the Saccharomyces cerevisiae homologue of the human gene defective in XPA patients.

In the accompanying paper we demonstrated that endonuclease III-sensitive sites in the MAT alpha and HML alpha loci of Saccharomyces cerevisiae are repaired by the Nucleotide Excision Repair (NER) pathway. In the current report we investigated the repair of endonuclease III sites, 6-4 photoproducts and cyclobutane pyrimidine dimers (CPDs) in a rad14-2 point mutant and in a rad14 deletion mutant. The RAD14 gene is the yeast homologue of the human gene that complements the defect in cells from xeroderma pigmentosum (XP) patients belonging to complementation group A. In the point mutant we observed normal repair of endonuclease III site (i.e. as wild type), but no removal of CPDs at the MAT alpha and HML alpha loci. Similar experiments were undertaken using the recently created rad14 deletion mutant. Here, neither endonuclease III sites nor CPDs were repaired in MATa or HMRa. Thus the point mutant appears to produce a gene product that permits the repair of endonuclease III sites, but prevents the repair of CPDs. Previously it was found that in the genome overall, repair of 6-4 photoproducts was less impaired that repair of CPDs in the point mutant. The deletion mutant repairs neither CPDs nor 6-4 photoproducts in the genome overall. This finding is consistent with the RAD14 protein being involved in lesion recognition in yeast. A logical interpretation is that the rad14-2 point mutant produces a modified protein that enables the cell to repair endonuclease III sites and 6-4 photoproducts much more efficiently than CPDs. This modified protein may aid studies designed to elucidate the role of the RAD14 protein in lesion recognition.

DNA Repair↗
PubMed1996

Recombination associated with replication of malarial mitochondrial DNA.

Mitochondrial DNA of the malarial parasite Plasmodium falciparum comprises approximately 20 copies per cell of a 6 kb genome, arranged mainly as polydisperse linear concatemers. In synchronous blood cultures, initiation of mtDNA replication coincides with the start of the 4-5 doublings in nuclear DNA that mark the reproductive phase of the erythrocytic cycle. We show that mtDNA replication coincides with a recombination process reminiscent of the replication mechanism used by certain bacteriophages and plasmids. The few circular forms of mtDNA which are also present do not replicate by a theta mechanism, but are themselves the product of recombination, and we propose they undergo rolling circle activity to generate the linear concatemers.

Animals↗
PubMed1995

Fission yeast rad17: a homologue of budding yeast RAD24 that shares regions of sequence similarity with DNA polymerase accessory proteins.

Following DNA damage or a block to DNA synthesis, checkpoint pathways act to arrest mitosis and prevent the attempted segregation of damaged or unreplicated DNA. The rad17 locus of Schizosaccharomyces pombe is one of seven known radiation-sensitive (rad) loci which are absolutely required to prevent mitosis following DNA damage in fission yeast. Six of these (rad1, rad3, rad9, rad17, rad26 and hus1) are also required for the checkpoint which prevents mitosis from occurring before DNA replication is complete. We report here that the predicted rad17 gene product is a basic hydrophilic protein of 606 amino acids which contains five domains with sequence homology to replication factor C (RF-C)/activator 1 subunits. Western analysis and fusion with Green Fluorescent Protein indicate that the abundance and electrophoretic mobility of Rad17 is not significantly modified following a block to DNA synthesis or following DNA damage, and that Rad17 is localized in the nucleus. Rad17 function is not essential for growth, but is required for the function of the DNA structure-dependent checkpoints. Site-directed mutagenesis has been used to demonstrate the biological significance of the RF-C/activator 1-related domains. These studies have also defined an element of the radiation sensitivity caused by loss of Rad17 function which is not associated with the radiation-induced G2 arrest defect seen in the rad17.d null mutant cells.

Base Sequence↗
PubMed1995

Cloning of a pea cDNA encoding a polypeptide of the light-harvesting complex associated with photosystem I using a monoclonal antibody.

A monoclonal antibody (MAb UB42) is described that binds to thylakoids in pea chloroplasts, as shown by EM-immunogold labelling. The antibody recognised proteins of ca. 23-29 kDa in western blots of a pea leaf homogenate. A cDNA library was prepared from pea epidermal cells in the vector lambda ZAP II, and immunoscreening of the library with UB42 led to the isolation of a clone, pUB42. This was sequenced and had an open reading frame of 269 codons encoding a predicted polypeptide of 28.9 kDa. The sequence showed extensive homology with three closely related polypeptides belonging to a family of chlorophyll a/b-binding proteins from the light harvesting complex of photosystem I (LHCI). Collectively, the results suggest that MAb UB42 recognises an epitope on the type II chlorophyll a/b-binding protein from LHCI and that clone pUB42 encodes this protein.

Amino Acid Sequence↗
PubMed1995

The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair.

The rad18 mutant of Schizosaccharomyces pombe is very sensitive to killing by both UV and gamma radiation. We have cloned and sequenced the rad18 gene and isolated and sequenced its homolog from Saccharomyces cerevisiae, designated RHC18. The predicted Rad18 protein has all the structural properties characteristic of the SMC family of proteins, suggesting a motor function--the first implicated in DNA repair. Gene deletion shows that both rad18 and RHC18 are essential for proliferation. Genetic and biochemical analyses suggest that the product of the rad18 gene acts in a DNA repair pathway for removal of UV-induced DNA damage that is distinct from classical nucleotide excision repair. This second repair pathway involves the products of the rhp51 gene (the homolog of the RAD51 gene of S. cerevisiae) and the rad2 gene.

Amino Acid Sequence↗
PubMed1994

Repair of 6-4 photoproducts and cyclobutane pyrimidine dimers in rad mutants of Saccharomyces cerevisiae.

Repair rates of both pyrimidine-pyrimidone (6-4) photoproducts and cyclobutane pyrimidine dimers have been measured in the UV-sensitive mutants of Saccharomyces cerevisiae: rad1 to rad12 and rad14 to rad24. A dot blot immunoassay for UV photoproducts was used which measures lesions in the genome as a whole and which distinguishes 6-4 photoproducts from cyclobutane dimers. The principal findings are: (1) Wild-type yeast cells, like normal mammalian cells, repair 6-4 photoproducts more rapidly than cyclobutane dimers. (2) All mutants that are defective in repair are defective in repair of both lesions. (3) The most sensitive alleles of rad1, rad2, rad3, rad4 and rad10 show no repair of either lesion. (4) Leaky alleles of rad1, rad3 and rad14 show a very marked difference in repair rates of the two lesions, rather like the human XPA revertant cell line XP129 and the Chinese hamster mutants UV61 and V-H1. (5) No mutant repairs cyclobutane dimers more rapidly than 6-4 photoproducts.

DNA Damage↗
PubMed1993

Repair of 6-4 photoproducts in Saccharomyces cerevisiae.

We have developed a dot blot immunoassay for UV photoproducts which distinguishes 6-4 photoproducts from cyclobutane dimers. The assay uses a polyclonal antiserum that is specific for UV-irradiated DNA. Cyclobutane dimers are measured in DNA samples which have been treated with hot alkali to destroy 6-4 photoproducts. 6-4 Photoproducts are measured using blots that have been incubated in photoreactivating enzyme to eliminate cyclobutane dimers. A combination of the two treatments leaves no detectable antigenic lesions. Wild-type S. cerevisiae repairs 6-4 photoproducts, in the genome overall, more rapidly than cyclobutane dimers. The most sensitive alleles of rad1, rad2, rad3 and rad4 are completely unable to repair either kind of photoproduct. We conclude that 6-4 photoproducts are repaired by essentially the same mechanism as are cyclobutane dimers.

Animals↗
PubMed1993

Repair of cyclobutane pyrimidine dimers and 6-4 photoproducts in the fission yeast Schizosaccharomyces pombe.

We have measured repair of both of the major lesions induced by ultraviolet irradiation (cyclobutane pyrimidine dimers and 6-4 photoproducts) in wild-type Schizosaccharomyces pombe and in selected rad mutants, including mutants with deletions in genes from the main phenotypic groups. We find that rad13 delta, rad15 and rad16 delta, which are the S. pombe homologues of the excision-defective Saccharomyces cerevisiae rad2, rad3 and rad1, respectively, repair lesions somewhat more slowly than the wild type, but still have considerable repair capacity. rad2 delta, also a presumed excision-defective mutant, behaves similarly. rad8 and rad9 delta, which belong to different phenotypic groups, repair lesions at the same rate as wild-type cells. These findings provide new evidence that S. pombe has a second repair system for removing ultraviolet damage, which is absent in S. cerevisiae. Surprisingly, this second mechanism repairs lesions very efficiently; its possible nature is discussed.

Cyclobutanes↗
PubMed1988

Mitochondrial DNA of the human malarial parasite Plasmodium falciparum.

Covalently closed circular DNA molecules were isolated from Plasmodium falciparum total DNA by isopycnic centrifugation in CsCl gradients containing either ethidium bromide or 2',6-diamidino-2-phenylindole. The circular molecules had an average contour length of 11.1 +/- 0.5 micron, similar to the analogous molecules previously isolated from the simian malaria parasite P. knowlesi. Both circular molecules shared considerable sequence homology and conserved restriction sites. The nucleotide sequence of one 936 bp fragment of the P. falciparum molecule was determined and identified, by a data base homology search, as part of a mitochondrial small rRNA subunit, thus confirming the mitochondrial origin of the circular DNAs of both malarial species.

Animals↗
PubMed1985

Nuclear and mitochondrial DNA of the primate malarial parasite Plasmodium knowlesi.

Restriction analyses and DNA/DNA hybridisation of parasite DNA isolated from monkeys infected with the malarial parasite Plasmodium knowlesi has permitted unambiguous identification of the nuclear DNA of this species. Its (G+C) content, as determined by estimations of buoyant density as well as by direct analysis, is about 38%, essentially indistinguishable from that of its primate laboratory host, and grossly different from that of the major human malaria parasite, P. falciparum, which has a (G+C) content of approx. 19%. In addition, gradient fractionation of total P. knowlesi DNA revealed a minor DNA component (approx. 1% of the total) with a (G+C) content of about 19%. This DNA comprises covalently closed circular molecules which have a contour length about 11.6 microns, carry a small cruciform structure, and are thought to originate in the parasite's mitochondria.

Animals↗
PubMedDate not supplied

Reverse transcriptase activity and Ty RNA are associated with virus-like particles in yeast.

The Ty element of yeast represents a class of eukaryotic transposons that show remarkable structural similarity to retroviral proviruses. Recently, these comparisons have been strengthened by a series of observations on the yeast Ty element: Ty transposes via an RNA intermediate; it contains a sequence (Fig. 1) which, when translated, is homologous to a conserved region found in all reverse transcriptases; a fusion protein encoded by Ty is produced by a frameshift event that is directly analogous to the production of Pr180gag-pol in a retrovirus such as Rous sarcoma virus. Here we identify the reverse transcriptase activity that, until now, has been presumed to mediate Ty transposition and show that it is sequestered in virus-like particles that also contain Ty RNA.

DNA Transposable Elements↗