An analysis of results from 305 compounds tested with the yeast RAD54-GFP genotoxicity assay (GreenScreen GC)-including relative predictivity of regulatory tests and rodent carcinogenesis and performance with autofluorescent and coloured compounds.

Data from 305 non-proprietary compounds tested using the yeast RAD54-GFP (Green Fluorescent Protein) assay, GreenScreen GC, are presented, together with a detailed comparison with results from in vitro and in vivo genotoxicity tests and rodent carcinogenesis. In addition, observations on reproducibility and the performance of the test with autofluorescent and coloured compounds are described. Like the Ames test, the GreenScreen assay is shown to exhibit high specificity (82%), meaning that compounds with positive results are very likely to be genotoxic carcinogens. This is in contrast to mammalian cell tests established for use in regulatory testing that provide disappointingly low specificity and the inevitable generation of confounding false positive data. The analysis confirmed the observations of earlier studies, showing that a combination of an Ames test (or surrogate) with the yeast test provides high specificity as well as high sensitivity in the identification of rodent carcinogens.

An assessment of the utility of the yeast GreenScreen assay in pharmaceutical screening.

In this paper we describe an initial reproducibility study of 12 proprietary compounds followed by the assessment of 51 marketed pharmaceuticals and, lastly, a summary of the data so far from 2698 proprietary compounds from the Johnson & Johnson (J&J) compound library, in the yeast GreenScreen assay (GSA). In this assay, a reporter system in the yeast cells employs the DNA damage inducible promoter of the RAD54 gene, fused to the extremely stable green fluorescent protein (GFP). The assay proved to be very robust, the Excel templates provided by Gentronix with the assay interfaced well with in-house J&J systems with little adaptation, the assay was very rapid to perform and used very little compound. The results confirm previous work which suggests that the yeast GSA detects different classes of genotoxic compounds to the Ames assay and as a result can help screen out important genotoxic compounds at the pre-regulatory test phase that are missed by Ames-test-based screens alone. A combination of SAR evaluation of genotoxicity plus an Ames-test-based screen and the GSA provides a powerful pre-regulatory test battery to aid in the selection of successful drug candidates.

Genetic modification and variations in solvent increase the sensitivity of the yeast RAD54-GFP genotoxicity assay.

The yeast (Saccharomyces cerevisiae) RAD54-GFP DNA repair reporter assay (GreenScreen assay, GSA) can be used for early genotoxicity screening in drug discovery. During the initial validation of this preregulatory assay, a subset of known genotoxic compounds that did not give reproducibly clear positive GSA results was identified. Cell permeability, inherent drug resistance mechanisms, metabolic activation and compound solubility were identified as possible barriers to the detection of specific compounds. In this study three types of modification to the existing assay protocol were explored in order to address these possibilities: (i) modification of the reporter host strain by deletion of genes involved in cell wall integrity or with products functioning as efflux pumps (PDR5, ERG6, SNQ2, YOR1); (ii) expression in the host yeast of human phase I metabolic activation genes and (iii) variation in the test solvent system for compounds with poor aqueous solubility. The modifications described and the assay results presented show how the assay may be tailored to suit specific classes of test compound in a more analytical mode. Improvements in assay sensitivity were seen in the detection of some genotoxins using yeast cell wall mutants and those expressing human cytochrome P450 genes.

Results of a technology demonstration project to compare rapid aquatic toxicity screening tests in the analysis of industrial effluents.

The results of a 'BioWise' demonstration project to assess the comparative sensitivity and practicality of seven new assays for the direct assessment of ecotoxicity in industrial effluents are presented. In addition the aim of the project was to validate the results of the new assays against benchmark data generated from non-proprietary, rapid, microplate screening assays using the regulatory species; freshwater crustacean Daphnia magna and green algae Selenastrum capricornutum, chosen in view of their environmental relevance. The new commercial test assays were: Daphnia magna, Selenastrum capricornutum and Thamnocephalus platyurus Toxkits supplied by Vickers Laboratories Ltd, containing dormant, immobilised life stages of the test species; GreenScreen EM, a yeast based assay for genotoxicity and general acute toxicity supplied by Gentronix Ltd; and CellSense a mediated, amperometric whole cell biosensor based on immobilised activated sludge and E. coli. 38 effluent samples supplied by members of SOCSA (Specialised Organic Chemicals Sector Association) were examined over a period of 13 months, in the project co-ordinated by the AstraZeneca Brixham Environmental Laboratory, and part funded by BioWise via the UK Government Department of Trade and Industry.

Unequal sister chromatid exchange in the rDNA array of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae the nucleolar organiser region (NOR) is located on chromosome XII. It contains 100-200 copies of rDNA--a minimum of 20 rDNA genes in tandem--and is termed the RDN locus. Yeast cells may exist in either haploid or diploid form. There are two forms of life cycle: haploid and diploid cells double by mitosis, and diploid cells are reduced to the haploid state by meiosis. Diploid cells have two homologous chromosomes for each of the 16 chromosomes. They are usually of the same size. However, in this study it is shown that homologous chromosomes XII can become different in size due to unequal sister chromatid exchange during mitosis in 'old' cells.

The GreenScreen genotoxicity assay: a screening validation programme.

A yeast (Saccharomyces cerevisiae) DNA repair reporter assay termed the GreenScreen assay (GSA) is described. This is a novel, cost-effective genotoxicity screen, developed to provide a pre-regulatory screening assay for use by the pharmaceutical industry and in other applications where significant numbers of compounds need to be tested. It provides a higher throughput and a lower compound consumption than existing eukaryotic genotoxicity assays and is sensitive to a broad spectrum of mutagens and, importantly, clastogens. We describe a simple, robust assay protocol and a validation study. The end-point of the test reflects the typically eukaryotic chromosomes and DNA metabolizing enzymes of yeast. The capacity for metabolic activation (MA) in yeast is limited compared with the mammalian liver or its extracts, but the assay does detect a subset of compounds that would require MA in existing genotoxicity tests. The GSA detects a different spectrum of compounds to bacterial genotoxicity assays and thus, together with an in silico structure-activity relationship (SAR) screen, and possibly a high throughput bacterial screen, would provide an effective preview of the regulatory battery of genotoxicity tests.

Glycosylation deficiency phenotypes resulting from depletion of GDP-mannose pyrophosphorylase in two yeast species.

The genes encoding GDP-mannose pyrophosphorylase from Saccharomyces cerevisiae (SRB1/PSA1) and Candida albicans (CaSRB1) were expressed under the control of the tightly regulated promoters of MET3 and CaMET3 respectively. Northern analysis showed that the addition of methionine effectively blocks the transcription of pMET3-SRB1/PSA1 and pCaMET3CaSRB1 expression cassettes, which had been integrated into the genomes of appropriate mutants. Methionine-mediated repression of CaSRB1 caused loss of viability in C. albicans, demonstrating that, as in S. cerevisiae, the gene is essential for growth. Depletion of GDP-mannose pyrophosphorylase had a highly pleiotropic effect in the two yeasts. The major phenotypes observed were lysis, failure of cell separation and/or cytokinesis, impaired bud growth and bud's site selection, clumping and flocculation, as well as increased sensitivity to a wide range of antifungal drugs and cell wall inhibitors, and impaired hyphal switching ability. These phenotypes resulted from defects in glycosylation, as demonstrated by reduced affinity for Alcian blue and sensitivity to hygromycin B. Our results provide new information about the roles of protein glycosylation in yeast and, in particular, the steps that require GDP-mannose in the fungal pathogen C. albicans.

Replicative ageing in the fission yeast Schizosaccharomyces pombe.

Saccharomyces cerevisiae has been widely used as a model organism in studies of replicative ageing and senescence. The relevance of these studies to ageing in other organisms has, however, been questioned, since this yeast divides by budding rather than fission, the more common pattern in higher organisms. Here we report that, contrary to popular belief, the fission yeast Schizosaccharomyces pombe also undergoes replicative senescence and in a manner superficially analogous to budding yeast. These experiments provide the first evidence of age asymmetry in cell fission and are consistent with the hypothesis of Jazwinski, that asymmetric division underlies culture immortality. Given their evolutionary divergence, comparison of the ageing determinants in fission and budding yeasts may help identify common mechanisms of the ageing process.

Disruption of six novel ORFs on the left arm of chromosome XII reveals one gene essential for vegetative growth of Saccharomyces cerevisiae.

Deletion via PCR-mediated gene replacement, together with basic functional and bioinformatic analyses, have been performed on six novel open reading-frames (ORFs) on the left arm of chromosome XII of Saccharomyces cerevisiae(YLL033w, YLL032c, YLL031c, YLL030c, YLL029w and YLL028w). ORF deletion was realized using either a short-flanking homology (SFH) or a long-flanking homology (LFH) replacement cassette in the diploid strain FY1679. Sporulation and tetrad analysis showed that YLL031c is the only essential gene of the six. Microscopic examination of the non-growing spores carrying a disrupted copy of the essential gene showed that most of them were blocked after one or two cell divisions with heterogeneous bud size. The standard EUROFAN growth tests failed to reveal any obvious phenotype resulting from the deletion of each the five non-essential ORFs. Bioinformatic analysis revealed that YLL029w is probably an aminopeptidase for mitochondrial or nuclear protein processing and YLL028w may be involved in drug resistance in S. cerevisiae. Replacement cassettes, comprising the promoter and terminator regions of each of the six ORFs, were cloned into pUG7 and demonstrated to efficiently mediate gene replacement in an alternative diploid strain, W303. All the cognate gene clones were constructed, using either PCR products amplified from genomic DNA, or gap-repair. All clones and strains generated have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt).

Development of a green fluorescent protein reporter for a yeast genotoxicity biosensor.

A reporter system, constructed for a laboratory screen for new genes involved in DNA repair in the brewer's yeast Saccharomyces cerevisiae, has been developed for use in a genotoxicity biosensor. The strain produces green fluorescent protein (yEGFP) when DNA damage has occurred. yEGFP is codon optimised for yeasts. The reporter does not respond to chemicals which delay mitosis, and responds appropriately to the genetic regulation of DNA repair. Data is presented which demonstrate strain improvements appropriate to biosensor technology: improved signal to noise ratio, ease of data collection and uncomplicated material handling.

Green fluorescent protein as a reporter for the DNA damage-induced gene RAD54 in Saccharomyces cerevisiae.

The green fluorescent protein (GFP) of Aequorea victoria is now an established marker for gene expression and subcellular localization in budding yeast. Relatively high expression (greater than 2500 copies per cell) of GFP is required for direct microscopic visualization. This report provides a method for studying the expression of less highly expressed genes by the analysis of crude cell extracts--a simple and cheap alternative to the fluorescent activated cell sorter (FACS). The utility of this marker is demonstrated in a study of the expression of the RAD54 gene. It is shown that the induction of the RAD54 promoter leads to the accumulation of Rad54p and of GFP and that the fluorescence induction is correctly regulated. This method should allow the screening of large numbers of novel gene disrupters for their effects on RAD54 expression and so identify trans-acting factors involved in the cellular response to DNA damage.

Partial deletion of the Saccharomyces cerevisiae GDH3 gene results in novel starvation phenotypes.

A small-scale functional analysis screen has revealed several new phenotypes associated with a large deletion of GDH3, one of two Saccharomyces cerevisiae genes known to encode NADP-linked glutamate dehydrogenase. Diploids heterozygous for the deletion are able to sporulate in rich media, while haploid deletants produce dark, wrinkled colonies containing pseudohyphal cells. The haploid cells rapidly lose viability upon starvation.

The oncogenic potential of Candida in the female genital tract.

The possible role of Candida species in carcinogenesis at the uterine cervix was investigated in 226 females attending a colposcopy clinic. Approximately 34% of the 226 subjects harbored Candida species in cervical/vaginal secretions, but there was no association with any particular histologic abnormality. Two independent analytical procedures were used for strain discrimination of the isolates of C. albicans, but again no relationship was found between individual strains and histologic diagnoses. Only three C. glabrata strains were isolated, but they were all in association with cervical intraepithelial neoplasia (CIN) II or III. A total of 18 strains of C. albicans, one C. glabrata and one C. parapsilosis all inhibited the formation of the nitrosamine nitrosodimethylamine (NDMA) from precursors. Furthermore, C. albicans strains did not convert NDMA to carcinogenic metabolites. The results of this study do not suggest that C. albicans has a role in cervical carcinogenesis.

A new, sensitive polynucleotide probe for distinguishing Candida albicans strains and its use with a computer assisted archiving and pattern comparison system.

The repetitive DNA sequence poly[d(GT).d(CA)],(polyGT) can be used to generate DNA fingerprints that distinguish different yeast genera. In this study we demonstrate that the probe can also be used to distinguish individual strains of clinical isolates of Candida albicans. Isolates were fingerprinted by probing Southern blots of restriction enzyme-cleaved DNA samples with radioactively labelled polyGT. The discrimination between strains was clearer than can be achieved by direct visualization of ethidium bromide stained gels and was comparable to that achieved with previous DNA probes. However, the advantage of this probe is that it is not limited to Candida species since polyGT sequences appear to be ubiquitous in eukaryotes. Fingerprints were also generated from Southern blots using a commercial (AMBIS) radioanalytical imaging computer system. A proprietary software package (MICRO PM) was effective in discriminating between the C. albicans strains. These preliminary results indicate the potential value of this probe for discrimination between Candida isolates in epidemiological studies of candidosis.

Vision assisted robotics and tape technology in the life-science laboratory: applications to genome analysis.

Recent proposals to analyze a number of major genomes have created a need for methods of rapidly manipulating and assaying very large numbers of specimens containing DNA fragments. Systems for sampling semi-solid biological material from mixed disordered arrays are required. These samples need to be sorted in an ordered format and stored in minimum space at known locations. It should be possible to recall them quickly from stock, individually or collectively, for duplication or re-ordering into new arrays for appropriate analysis. This article reviews a range of problems associated with high speed multiple specimen handling and assay in the molecular biology laboratory and outlines the solutions currently available to address these difficulties. Designs for automatic systems to manipulate biosamples and analyze DNA are presented.

Yeast telomere length varies in response to changes in the amount of polyC1-3A in the cell.

Yeast chromosomes terminate in a GC-rich tail of DNA. Previous investigations have shown that the length of this tail can change in response to genetic variation. Here we present data that show that the length can also alter in response to changes in the amount of the GC-rich DNA found elsewhere in the nucleus.

Thymidylate synthase-deficient Chinese hamster cells: a selection system for human chromosome 18 and experimental system for the study of thymidylate synthase regulation and fragile X expression.

Chinese hamster lung (CHL) V79 cells already deficient in hypoxanthine phosphoribosyltransferase were exposed to uv light and selected for mutations causing deficiency of thymidylate synthase (TS) by their resistance to aminopterin in the presence of thymidine and limiting amounts of methyl tetrahydrofolate. Three of seven colonies chosen for initial study were shown to be thymidylate synthase deficient (TS-) by enzyme assay, thymidine auxotrophy, and their inability to incorporate labeled deoxyuridine into their DNA in vivo. Complementation analysis of human X TS- hamster hybrids revealed that TS activity segregated with human chromosome 18. Southern analysis of a panel of 14 human X hamster hybrids probed with complementary DNA from mouse TS confirmed the chromosome assignment of TS to human chromosome 18; quantitative Southern blotting using unbalanced human cell lines further localized the gene to 18q21.31----qter. Another hybrid was generated that contained a human X chromosome with the Xq28 folate-dependent fragile site as its only human chromosome in a hamster TS- background. The fragile site could be easily and reproducibly expressed in this hybrid without the use of antimetabolites simply by removing exogenous thymidine from the medium. These TS-deficient cells are useful for: somatic cell genetics as a unique selectable marker for human chromosome 18, studies on regulation of the TS gene, and analysis of the fragile (X) chromosome and other folate-dependent fragile sites.

Genetic control of chromosome length in yeast.

The chromosomes of the yeast Saccharomyces cerevisiae terminate with sequences that have the form poly(C1-3-A). In this paper, we show that within an individual yeast strain all chromosomes end with tracts of poly(C1-3-A) of similar lengths; however, different strains can have tracts that vary in length by a factor of two. By a genetic analysis, we demonstrate that yeast cells have a mechanism that allows them to change rapidly the length of their chromosomes by altering the length of the poly(C1-3-A) tract.

Replicon size of yeast ribosomal DNA.

The ribosomal RNAs of the yeast Saccharomyces cerevisiae are transcribed from a 9K bp stretch of DNA which is reiterated about 120-fold in a continuous array, about 360 microns long, on chromosome XII. Although ARS activity has been detected in the repeat unit, the size and disposition of replicons along this array of identical genes has not hitherto been determined. We have used immobilised rRNA as a probe to examine the size of radioactively labelled rDNA replicons resolved on alkaline sucrose gradients. The replicons were found to be uniformly sized, about 5 repeat units in length, and groups of 4 adjacent replicons may be activated simultaneously. These observations suggest that replicon initiation events are not determined solely by the recognition of specific DNA sequences that function as origins of replication.