MCM-GINS and MCM-MCM interactions in vivo visualised by bimolecular fluorescence complementation in fission yeast

Background  

Each of the three individual components of the CMG complex (Cdc45, MCM and GINS) is essential for chromosomal DNA replication in eukaryotic cells, both for the initiation of replication at origins and also for normal replication fork progression. The MCM complex is a DNA helicase that most likely functions as the catalytic core of the replicative helicase, unwinding the parental duplex DNA ahead of the moving replication fork, whereas Cdc45 and the GINS complex are believed to act as accessory factors for MCM.     

Canadians' Representation of Chemical,Biological, Radiological,Nuclear, and Explosive (CBRNE) Terrorism: A Content Analysis

The global threat of terrorism raises questions about preparedness and risk communication in the context of public health and security. Although experts discriminate between chemical, biological, radiological, nuclear, and explosive (CNRNE) terrorist events, little is known about how the Canadian public represents these forms of terrorism. A stratified random sample of 1502 Canadians participated in a telephone survey on CBRNE terrorism. A word association technique was used to assess first words or images that came to mind while thinking about different types of terrorist scenarios. Content analysis of this data revealed a number of potential uncertainties and misconceptions regarding different types of terrorism scenarios. Despite most frequently providing agents in response questions surrounding chemical or biological terrorism, respondents frequently confounded agents of biological and chemical nature. Similarly, different aspects of nuclear events were not consistently distinguished. Most notably, however, a sizable proportion of respondents had difficulty providing word associations to the different types of terrorist events or only provided vague responses that closely mirrored the scenario in question. Finally, images regarding the potential impacts of scenarios were infrequent. Implications for risk communication and preparedness are discussed; in particular, the need to provide the public with more detailed information regarding the nature of different forms of CBRNE terrorism and how to best prepare for a potential event.     

Genetics University of Toronto Thrombophilia Study in Women (GUTTSI): genetic and other risk factors for venous thromboembolism in women

Background  

Women may be at increased risk for venous thromboembolism (VTE) as compared with men. We studied the effects of genetic and biochemical markers of thrombophilia in women, in conjunction with other established risk factors for VTE.     

<i>In vitro</i> evaluation of antifungal activity of Agave (<i>Agave scabra</i>, Salm Dyck) extracts against post-harvest mushrooms

The agricultural sector, and particularly the horticultural production, has a singular importance in agriculture, considering that it ranks second on agricultural products, nationally and worldwide. Fungal diseases are one of the major causes of vegetable loss during storage, reducing their nutritional value, quality and sale price. Vegetables are usually exposed to diverse treatments with chemical products before storage; as a result, fungal populations develop an increased resistance over time becoming more difficult to control. Because of this, research efforts toward finding more suitable chemicals to control fungal diseases are needed. Natural extracts may be an alternative solve this problem. In the present investigation the fungicidal activity of aqueous and ethanol extracts of Agave scabra was evaluated on the growth of Botrytis cinerea, Mucor sp., Aspergillus niger, Fusarium sp. and Penicillium sp., whose strains were isolated from potato and tomato. To assess their effects, the agar-dilution and agar-well techniques were performed. The ethanol extract was more effective against Botrytis cinerea and Mucor sp. when the agar-well method was used. However, when using the agar-dilution method the ethanol extract of Agave scabra inhibited the growth of Botrytis cinerea, Mucor sp. and Penicillium sp.     

Isolation, characterisation and molecular cloning of new mutant alleles of the fission yeast p34cdc2+ protein kinase gene: identification of temperature-sensitive G2-arresting alleles

Summary The protein serine-threonine kinase p34 ^cdc2+ plays a central role in the control of the mitotic cell cycle of the fission yeast Schizosaccharomyces pombe. p34 ^cdc2+ function is required both for the initiation of DNA replication and for entry into mitosis, and is also required for the initiation of the second meiotic nuclear division. Recent extensive analysis of p34 ^cdc2+ homologue proteins in higher eukaryotes has demonstrated that p34 ^cdc2+ function is likely to be conserved in all eukaryotic cells. Here we report the isolation and characterisation of five new temperature-sensitive alleles of the cdc ²⁺ gene. All five have been cloned and sequenced, together with the meiotically defective cdc2-N22 allele, bringing the total of p34 ^cdc2+ mutants cloned in this and previous reports to seventeen. The five temperature-sensitive alleles define four separate mutations within the p34 ^cdc2+ protein sequence, two of which give rise to cell cycle arrest in G₂ only, when shifted to the restrictive temperature. The nature of the mutation in each protein is described and possible implications for the structure and function of p34 ^cdc2+ discussed.     

Molecular cloning and sequence analysis of mutant alleles of the fission yeast cdc2 protein kinase gene: Implications for cdc2+ protein structure and function

Summary The cdc2 ⁺ gene function plays a central role in the control of the mitotic cell cycle of the fission yeast Schizosaccharomyces pombe. Recessive temperature-sensitive mutations in the cdc2 gene cause cell cycle arrest when shifted to the restrictive temperature, while a second class of mutations within the cdc2 gene causes a premature advancement into mitosis. Previously the cdc2 ⁺ gene has been cloned and has been shown to encode a 34 kDa phosphoprotein with in vitro protein kinase activity. Here we describe the cloning of 11 mutant alleles of the cdc2 gene using two simple methods, one of which is presented here for the first time. We have sequenced these alleles and find a variety of single amino acid substitutions mapping throughtout the cdc2 protein. Analysis of these mutations has identified a number of regions within the cdc2 protein that are important for cdc2 ⁺ activity and regulation. These include regions which may be involved in the interaction of the cdc2 ⁺ gene product with the proteins encoded by the wee1 ⁺, cdc13 ⁺ and suc1 ⁺ genes.     

The C-terminal zinc finger of the catalytic subunit of DNA polymerase delta is responsible for direct interaction with the B-subunit

DNA polymerase δ (Pol δ) plays a central role in eukaryotic chromosomal DNA replication, repair and recombination. In fission yeast, Pol δ is a tetrameric enzyme, comprising the catalytic subunit Pol3 and three smaller subunits, Cdc1, Cdc27 and Cdm1. Previous studies have demonstrated a direct interaction between Pol3 and Cdc1, the B-subunit of the complex. Here it is shown that removal of the tandem zinc finger modules located at the C-terminus of Pol3 by targeted proteolysis renders the Pol3 protein non-functional in vivo, and that the C-terminal zinc finger module ZnF2 is both necessary and sufficient for binding to the B-subunit in vivo and in vitro. Extensive mutagenesis of the ZnF2 module identifies important residues for B-subunit binding. In particular, disruption of the ZnF2 module by substitution of the putative metal-coordinating cysteines with alanine abolishes B-subunit binding and in vivo function. Finally, evidence is presented suggesting that the ZnF region is post-translationally modified in fission yeast cells.     

Genome stability: a self-sufficient DNA repair machine

The repair of DNA double-strand breaks often requires the broken ends to be processed prior to religation. New results describe a bacterial enzyme with processing and rejoining activities encoded in a single polypeptide chain.     

Positive selection and sequence rearrangements generate extensive polymorphism in the gamete recognition protein bindin

Bindin is a gamete recognition protein of sea urchins that mediates species-specific attachment of sperm to an egg-surface receptor during fertilization. Sequences of bindin from closely related urchins show fixed species-specific differences. Within species, highly polymorphic bindin alleles result from point substitution, insertion/deletion, and recombination. Since speciation, positive selection favoring allelic variants has generated diversity in bindin polypeptides. Intraspecific bindin variation can be tolerated by the egg receptor, which suggests functional parallels between this system and other flexible recognition systems, including immune recognition. These results show that polymorphism in mate recognition loci required for rapid evolution of sexual isolation can arise within natural populations.      

The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27.

cdc1+ is required for cell cycle progression in Schizosaccharomyces pombe. Cells carrying temperature-sensitive cdc1 mutants undergo cell cycle arrest when shifted to the restrictive temperature, becoming highly elongated. Here we describe the cloning and sequencing of cdc1+, which is shown to encode a 462 residue protein that displays significant sequence similarity to the small subunit of mammalian DNA polymerase delta. cdc1+ interacts genetically with pol3+, which encodes the large subunit of DNA polymerase delta in fission yeast, and the Cdc1 protein binds to Pol3 in vitro, strongly suggesting that Cdc1 is likely to be the small subunit of Pol delta. In addition, we show that cdc1+ overexpression is sufficient to rescue cells carrying temperature-sensitive cdc27 alleles and that the Cdc1 and Cdc27 proteins interact in vivo and in vitro. Deletion of either cdc1+ or cdc27+ results in cell cycle arrest with the arrested cells having a single nucleus with 2C DNA content. No evidence was obtained for a cut phenotype, indicating that neither cdc1+ nor cdc27+ is required for checkpoint function. cdc1 mutant cells are supersensitive to the DNA synthesis inhibitor hydroxyurea and to the DNA damaging agent MMS, display increased frequency of mini-chromosome loss and have an extended S phase.