Using high-throughput data to imply gene function : assessment of selected genes for roles in mitotic and meiotic DNA processing

The large volume of data generated in high-throughput studies of the 6200 genes of Saccharomyces cerevisiae provides a valuable resource. An in-silico screen, mining existing data-sets including interaction, expression, localisation and phenotype data was developed with the aim of selecting partially uncharacterised genes involved in meiotic DNA processing. Based on the in-silico selection procedure, 81 deletion mutants were constructed and tested for phenotypic abnormalities. During vegetative growth 12 of these mutants showed hypersensitivity to hydroxyurea, five to methyl methanesulfonate and four to ionizing radiation. During meiosis three mutants were identified with reduced gene conversion, and one with increased levels of chromosomal nondisjunction, while six mutants showed very low levels of nuclear divisions. Of those six, five showed a significant delay in the initiation and progression of pre-meiotic DNA replication, while one could not initiate meiosis. One of the five mutants with delayed initiation and progression of pre-meiotic DNA replication, also displayed inefficient synapsis between homologues. Finally, a mutant was identified that produced asci containing more than four inviable spores. The insilico selection procedure used in this study, represents an example of how existing data sets may be used to define gene sets enriched for roles in specified biological processes. Common Abbreviations Common Abbreviations 4 Deletion mutant aa Amino acid(s) ADE Adenine Amp Ampicillin AmpR Ampicillin resistance APC/C Anaphase promoting complex I cyclosome ARG Arginine ARS Autonomously replicating sequence bp Base pair CAN Canavinine CDK Cyclin dependent kinase cm Centimetre CO Crossover COi Crossover interference CY3 N,N -(dipropyl)-tetramethylindocarbocyanine CYGD Comprehensive Yeast Geiome Database dATP Deoxyadenosine triphosphate DAPI 4',6-Diamidino-2-p4enylindole dCTP Deoxycytosine triphosphate dGTP Deoxyguanosine triphosphate dHJ Double Holliday Junction DIP Database of Interacting Proteins DNA Deoxyribonucleic acid DNAP DNA processing DNAPIS DNA processing interactor selection dNTP deoxyribonucleic triphosphate (dATP, dCTP, dGTP and dTTP) DSB Double strand break DSBR Double strand break repair DTT Dithiothreitol dTTP Deoxythymine triphosphate EDTA Ethylene-diamine-tetra-acetic acid Common Abbreviations EUFOFAN European Functional Analysis Network EUROSCARF European Saccharomyces cerevisiae archive functional analysis FACS Fluorescent-activated cell sorting FITC Fluorescein isothiocyanate g Gram(s) GFP Green fluorescent protein GRID General Repository for Interaction Datasets h Hour(s) HA Haemagglutinin HC1 Hydrochloric acid HU Hydroxyurea JR Ionizing radiation Kan Kanamycin (G4 18 sulphate) KanR Kanamycin resistance kb Kilobase(s) L Litre(s) LYS Lysine M Molar MCC Mitotic checkpoint complex mg Milligram(s) min Minute(s) ml Millilitre(s) mM Millimolar mmol Millimole(s) IVIIMS methyl methanesulfonate mRNA Messenger Ribonucleic Acid NCO Non-crossover ng Nanogram(s) nt Nucleotide(s) °C Degrees Celsius 0D600 Optical wavelength.of 600 nm ORC Origin of replication ORF Open Reading Frame Common Abbreviations PBS Phosphate buffered saline PCNA Proliferating cell nuclear antigen PCR Polymerase Chain Reaction pmol Picomole(s) Pre-RC Pre-replication complex PRR Post-replicative repair prom Promoter RNA Ribonucleic Acid RNase Ribonuclease rpm Revolutions per minute RT Room Temperature SAC Spindle assembly checkpoint SC Synaptonemal complex SDS Sodium Dodecyl Sulphate SDSA Synthesis dependent strand annealing SET Single-end invasion SGD Saccharomyces Genome Database ssDNA Single strand DNA TAE Tris/Acetate Buffer tetR Tetracycline repressor tetO Tetracycline operator Tris Tris(hydroxymethyl)aminomethane U Unit(s) UV Ultraviolet radiation V Volt(s) v/v Volume per unit Volume w/v Weight per unit Volume YIVIM Yeast Minimal Medium YPDA Yeast/Peptone/Dextrose/Adenine (medium) Microgram(s) Microlitre(s) pm Micrometer(s) jimol Micromole(s)