Genomic disruption of six budding yeast genes gives one drastic example of phenotype strain-dependence

Using PCR to construct disruption cassettes, null alleles of six genes have been created in Saccharomyces cerevisiae. In a FY1679 background, no defects were detected in any of the haploid deletion mutants with respect to growth, gross morphology, or mating. A diploid FY1679-derived Δygl194c/Δygl194c homozygous disruptant displayed reduced sporulation. In contrast to the lack of phenotypic consequences of Δyol100w disruptions in the FY1679 background, in the CEN.PK2 strain even a heterozygous disruption of the same gene caused striking effects, very slow vegetative growth and highly impaired sporulation. Tetrad analysis showed YOL100w to be an essential gene in this strain. A copy of the YGL194c or the YOL100w wild-type gene borne on a centromeric episomal plasmid was introduced into a corresponding disruption mutant strain, and in both cases was found to partially complement the defects. © 1998 John Wiley & Sons, Ltd.     

Disruption of 12 ORFs located on chromosomes IV,VII and XIV of Saccharomyces cerevisiae reveals two essential genes

We describe the generation of null-mutants of 12 open reading frames (ORFs), discovered during the systematic sequencing of the Saccharomyces cerevisiae genome. These ORFs are located on chromosome IV (YDL183c), on chromosome VII (YGL139w, YGL140c, YGL141w, YGR280c and YGR284c) or on chromosome XIV (YNL006w, YNR004w, YNR007c, YNR008w, YNR009w and YNR013c). Disruptants were generated using the PCR-based short flanking homology (SFH) strategy in yeast strain FY1679. Tetrad analysis, following sporulation of the heterozygous disruptants, revealed that YGR280c and YNL006w are essential genes for vegetative yeast growth in rich medium. The lethality of the two genes was confirmed by gene complementation analysis. The protein encoded by YNL006w (LST8) is now known to be involved in transport of permeases from the Golgi to the plasma membrane. Basic phenotypic analyses were performed on haploid disruptants from both mating types of 10 non-essential genes. One disruptant (YNR004w) revealed a slow growth rate on glucose-minimal medium at 15 degrees C. For each of the individual ORFs, a disruption cassette and the corresponding cognate gene were cloned into appropriate plasmids.     

Disruption and phenotypic analysis of six open reading frames from chromosome VII of Saccharomyces cerevisiae reveals one essential gene.

Six open reading frames (ORFs) located on chromosome VII of Saccharomyces cerevisiae (YGR205w, YGR210c, YGR211w, YGR241c, YGR243w and YGR244c) were disrupted in two different genetic backgrounds using short-flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YGR211w, recently identified as the yeast ZPR1 gene, is an essential gene. The other five genes are non-essential, and no phenotypes could be associated to their inactivation. Two of these genes have recently been further characterized: YGR241c (YAP1802) encodes a yeast adaptor protein and YGR244c (LSC2) encodes the beta-subunit of the succinyl-CoA ligase. For each ORF, a replacement cassette with long flanking regions homologous to the target locus was cloned in pUG7, and the cognate wild-type gene was cloned in pRS416.     

Generation of null alleles for the functional analysis of six genes from the right arm of Saccharomyces cerevisiae chromosome II

Using PCR‐ligated long flanking homology cassettes, null alleles of six open reading frames (ORFs) from chromosome II have been created in Saccharomyces cerevisiae. Deletants were constructed in three genetic backgrounds: FY1679, W303 and CEN.PK2. Tetrad analysis of heterozygous deletants revealed that none of the ORFs is essential for vegetative growth. Basic phenotypic analysis of haploid deletants showed that deletion of the YBR283c ORF causes a slight growth defect at 30°C and 37°C on glycerol‐complete, glucose‐complete, and glucose‐minimal media only in the FY1679 and W303 backgrounds. Transformation of these deletants with the corresponding cognate gene in a centromeric plasmid complements the defects. Deletion of the YBR287w ORF leads to poor growth on glucose‐minimal medium at 15°C in the FY1679 background. None of the six ORFs seems to be involved in mating or sporulation. Copyright © 1999 John Wiley & Sons, Ltd.     

Disruption of six Saccharomyces cerevisiae genes from chromosome IV and basic phenotypic analysis of deletion mutants

We report here the construction of six deletion mutants and the analysis of their basic phenotype. Deletion cassettes containing the KanMX4 marker module and long flanking regions homologous to the target locus were constructed for each of the six open reading-frames (ORFs YDL088c, YDL087c, YDL086w, YDL085w, YDL084w and YDL082w) located on chromosome IV. Sporulation and tetrad analysis of heterozygous deletant strains revealed that, in the FY1679 genetic background, ORFs YDL088c, YDL087c and YDL084w are essential genes for vegetative growth whereas YDL086w, YDL085w and YDL082w are non-essential. ydl088cΔ and ydl084wΔ haploid strains are viable in the CEN. PK2 genetic background although ydl084wΔ grows at a slower rate than the wild type. Complementation tests by corresponding cognate genes confirmed that gene inactivation was responsible for these growth defects. © 1998 John Wiley & Sons, Ltd.     

Disruption of Six Novel Yeast Genes Reveals Three Genes Essential for Vegetative Growth and One Required for Growth at Low Temperature

We describe here the construction of six deletion mutants and their basic phenotypic analysis. Six open reading frames (ORFs) from chromosome X, YJR039w, YJR041c, YJR043c, YJR046w, YJR053w and YJR065c, were disrupted by deletion cassettes with long (LFH) or short (SFH) flanking regions homologous to the target locus. The LFH deletion cassette was made by introducing into the kanMX4 marker module two polymerase chain reaction (PCR) fragments several hundred base pairs (bp) in size homologous to the promoter and terminator regions of a given ORF. The SFH gene disruption construct was obtained by PCR amplification of the kanMX4 marker with primers providing homology to the target gene. The region of homology to mediate homologous recombination was about 70 bp. Sporulation and tetrad analysis revealed that ORFs YJR041c, YJR046w and YJR065c are essential genes. Complementation tests by corresponding cognate gene clones confirmed this observation. The non-growing haploid segregants were observed under the microscope. The yjr041cΔ haploid cells gave rise to microcolonies comprising about 20 to 50 cells. Most yjr046wΔ cells were blocked after one or two cell cycles with heterogeneous bud sizes. The yjr065cΔ cells displayed an unbudded spore or were arrested before completion of the first cell division cycle with a bud of variable size. The deduced protein of ORF YJR065c, that we named Act4, belongs to the Arp3 family of actin-related proteins. Three other ORFs, YJR039w, YJR043c and YJR053w are non-essential genes. The yjr043cΔ cells hardly grew at 15°C, indicating that this gene is required for growth at low temperature. Complementation tests confirmed that the disruption of YJR043c is responsible for this growth defect. In addition, the mating efficiency of yjr043cΔ and yjr053wΔ cells appear to be moderately a ffected. © 1997 John Wiley & Sons, Ltd.     

Microbial quality of “doubles” sold in Trinidad

A cross-sectional study was conducted to determine the prevalence and characteristics of Escherichia coli, Staphylococcus aureus, Bacillus spp. and Salmonella spp. in “bara”, “channa”, condiments/spices and ready-to-eat “doubles” sold by vendors in the St. George and Caroni counties of Trinidad. Of 196 samples of each of “bara”, “channa”, condiments/spices and ready-to-eat “doubles” examined, E. coli was detected in 0 (0.0%), 14 (7.1%), 96 (49.0%) and 67 (34.2%), respectively; Staphylococci were isolated from 104 (53.1%), 71 (36.2%), 129 (65.8%) and 123 (62.8%) samples, respectively; and Bacillus spp. were recovered from 22 (11.2%), 85 (43.4%), 100 (51.0%) and 88 (44.9%) samples, respectively. Salmonella spp. were not isolated from any sample. Of the 177 isolates of E. coli recovered from all sources, 9 (5.1%), 7 (4.0%) and 47 (26.6%) were mucoid, haemolytic and non-sorbitol fermenters (NSF), respectively, but none agglutinated with O157 antiserum. Of 427 staphylococcal isolates, 130 (30.4%) were confirmed as S. aureus of which 20 (15.4%) were haemolytic and 84 (64.6%) pigmented, while 17 (20.7%) of 82 strains of S. aureus tested produced enterotoxins. Ready-to-eat “doubles”, a popular food in Trinidad, therefore pose a potential health risk to consumers due to the high level of contamination with bacteria.     

Disruption of six novel ORFs from Saccharomyces cerevisiae chromosome VII and phenotypic analysis of the deletants

We describe the disruption and basic phenotypic analysis of six open reading frames (ORFs) of unknown function located in the left arm of Saccharomyces cerevisiae chromosome VII, namely YGL133w, YGL134w, YGL136c, YGL138c, YGL142c and YGL144c. Disruptions were made using the short flanking homology PCR replacement strategy in the FY1679 and CEN.PK2 diploid strains. Sporulation and tetrad analysis of the heterozygous deletants was performed, as well as phenotypic analysis of the corresponding deleted haploid strains. No obvious phenotypes could be attributed to the strains deleted in any of the genes YGL134w, YGL138c and YGL144c under the conditions tested. YGL142c was shown to be an essential gene. Segregants bearing a deletion in YGL136c grew slowly in complete glycerol medium at 37 degrees C. Cells deleted in YGL133w showed abnormal morphology and reduced mating efficiency, but these phenotypes were observed only when the YGL133w disruption was in a MATalpha background. Ygl133 protein was found to localize to the nucleus.     

Annotation of unknown yeast ORFs by correlation analysis of microarray data and extensive literature searches

Changes in the expression of genes were used to elucidate the metabolic pathways and regulatory mechanisms that respond to environmental or genetic modifications. Results from previously published chemostat datasets were merged with novel data generated in the present study. ORFs displaying significant changes in expression that correlated with those of other ORFs were analysed using GO mapping tools and supplemented by literature information. The strategy developed was used to propose annotations for ORFs of unknown function. The following ORFs were assigned functions as a result of this study: YMR090w, YGL157w, YGR243w, YLR327c, YER121w, YFR017c, YGR067c, YKL187c, YGR236c (SPG1), YMR107w (SPG4), YMR206w, YER067w, YJL103c, YNL175C (NOP13) YJL200C, YDL070C (FMP16) and YGR173W.     

Effects of heat-processing regime, pH, water activity and their interactions on the behaviour of Listeria monocytogenes in ground pork. Modelling the boundary of the growth/no-growth areas as a function of pH, water activity and temperature

The influence of four heat‐processing regimes and a storage phase on the behaviour of Listeria monocytogenes in ground pork was studied. The effects of pH and water activity (a_w) were also tested. During the heat process phase, a_w, the heat‐processing regime and its interactions with pH or a_w, had a significant effect on the behaviour of L. monocytogenes. During the storage phase, all parameters tested and their interactions had significant effects. Nevertheless, the area in which the growth of L. monocytogenes was observed at the end of the experiment was not influenced by the heat‐processing regime tested. On the contrary, pH, a_w and their interactions had significant effects on Listeria behaviour. The boundary of the growth area delimited by environmental conditions where growth was higher than 1.0 Log CFU g^?1 from those where growth was lower than this limit was correctly predicted by Augustin's model.     

Immunoglobulin E epitope mapping by microarray immunoassay reveals differences in immune response to genetic variants of caseins from different ruminant species

The allergenicity of the caseins (CN), one of the major allergens in cow milk, is well characterized and their immunoglobulin E (IgE)-binding epitopes have been identified. However, investigations about the allergenic potential of the genetic variants occurring in the caseins are lacking. Therefore, this study determined the influence of the genetic polymorphism on IgE binding to epitopes of bovine casein variants. Furthermore, differences in IgE binding between epitopes of goats and water buffaloes were analyzed. A set of 187 peptides, covering the previously identified sequential IgE-binding epitopes of α_S1-, α_S2-, β-, and κ-CN variants from cows and the corresponding homologous peptides of water buffaloes and goats, were synthesized and tested by means of peptide microarray for IgE binding, using sera from 16 cow milk-sensitized individuals. Seven of the 16 sera samples showed positive signals on microarrays and were included in this study. In 5 α_S1-CN variants (A, B, C, E, and I), the AA substitution or deletion affected the immunoreactivity of epitopes AA 4 to 23, AA 17 to 36, AA 83 to 102, AA 173 to 192, and AA 175 to 194, as well as of the variant-specific peptides AA 184 to 196, AA 187 to 199, AA 174 to 193, and AA 179 to 198, which were found to resist gastrointestinal digestion. Variation in IgE binding was further detected for peptides AA 103 to 123 and AA 108 to 129 of 3 β-CN variants (A¹, A², and B). The majority of sera showed IgE binding to α_S1-CN peptides of cows and the homologous counterpart of goats and water buffaloes. However, α_S1- and β-CN epitopes from goats and water buffaloes had lower immunoreactivity than those of cows, but, in some cases, higher or exclusive IgE binding was observed. The results of this study indicate that genetic variants of the caseins differ in their allergenicity. This might be useful in the search for a suitable protein source for cow milk-allergic patients. In addition, milk from water buffaloes and goats harbor an allergenic potential due to cross-reactivity of IgE antibodies with cow milk caseins and are, therefore, not an acceptable alternative in the nutrition of cow milk-allergic patients.     

Identification of the gene encoding scHelI,a DNA helicase from Saccharomyces cerevisiae

The gene encoding scHelI, a previously characterized DNA helicase from Saccharomyces cerevisiae, has been identified as YER176w, an open reading frame on chromosome V. The gene has been named HEL1 to indicate the DNA helicase activity of the gene product. HEL1 was identified by screening a |glgt11 yeast protein expression library with antiserum to purified scHelI. Several independent immunopositive clones were isolated and shown to contain portions of HEL1 either by sequencing or by hybridization to a probe containing HEL1 sequences. The HEL1 open reading frame includes the seven conserved helicase motifs, consistent with the DNA helicase activity of scHelI, and the predicted size of the protein is in agreement with the size of purified scHelI. Partially purified cellular extracts from a hel1 deletion mutant strain did not contain scHelI activity. Homology searches revealed protein sequence homology between HEL1 and two previously identified and biochemically characterized yeast helicases, encoded by the DNA2 and UPF1 genes. Haploid hel1 deletion strains were constructed and shown to be viable with growth rates equivalent to those of parental strains. These strains did not differ from the parental strains in ultraviolet light sensitivity or the generation of petite colonies. Furthermore, these haploid deletion strains were capable of mating, the resultant diploid homozygous mutants were viable, capable of sporulation, and the spores displayed no reduction in viability. © 1997 John Wiley & Sons, Ltd.     

QCR9, the nuclear gene encoding a small subunit of the mitochondrial cytochrome bc1 complex,maps to the right arm of chromosome VII in Saccharomyces cerevisiae

We present here mapping data for QCR9, a nuclear gene encoding a subunit of the ubiquinol-cytochrome c oxidoreductase complex. Deletion of QCR9 results in the inability of cells to grow on non-fermentable carbon sources at 37°C. Thus, qcr9 mutants can be scored by growing cells on YPE/G at 37°C, or followed by the URA3 marker, which was inserted when making the qcr9 deletion strain, JDP1. The location of QCR9 on the right arm of chromosome VII with respect to the previously mapped genes ADE3, SER2 and PET54 is given.     

Phylogenetic Relationships of Fungal Cytochromes c

The CYC1 gene encoding cytochrome c in the yeast Candida albicans was cloned by complementation of a cytochrome c-deficient mutant of Saccharomyces cerevisiae, and its DNA sequence was determined. The analysis of the amino acid sequences of cytochrome c from 14 fungal species and two isoforms from S. cerevisiae revealed sequences unique to fungi, and revealed a phylogenetic relationship with a pronounced divergence between Schizosaccharomyces pombe and other ascomycetous budding yeast. The C. albicans CYC1 cytochrome c sequence has been assigned Accession Number U57896 in the GenBank/EMBL database. © 1997 John Wiley & Sons, Ltd.