Haploid Colonies Research Articles

BEST: Barcode Enabled Sequencing of Tetrads

Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. Barcode Enabled Sequencing of Tetrads (BEST)1 replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by virtue of a sporulation-specific GFP fusion protein that permits fluorescence-activated cell sorting of tetrads directly onto agar plates, where the ascus is enzymatically digested and the spores are disrupted and randomly arrayed by glass bead plating. The haploid colonies are then assigned sister spore relationships, i.e. information about which spores originated from the same tetrad, using molecular barcodes read during genotyping. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. Here we present a detailed description of the experimental procedures required to perform BEST in the yeast Saccharomyces cerevisiae, starting with a heterozygous diploid strain through the isolation of colonies derived from the haploid meiotic progeny.

BEST: barcode enabled sequencing of tetrads.

Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. Barcode Enabled Sequencing of Tetrads (BEST)1 replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by virtue of a sporulation-specific GFP fusion protein that permits fluorescence-activated cell sorting of tetrads directly onto agar plates, where the ascus is enzymatically digested and the spores are disrupted and randomly arrayed by glass bead plating. The haploid colonies are then assigned sister spore relationships, i.e. information about which spores originated from the same tetrad, using molecular barcodes read during genotyping. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. Here we present a detailed description of the experimental procedures required to perform BEST in the yeast Saccharomyces cerevisiae, starting with a heterozygous diploid strain through the isolation of colonies derived from the haploid meiotic progeny.

Mutational Analysis of the Ras Converting Enzyme Reveals a Requirement for Glutamate and Histidine Residues

The Ras converting enzyme (RCE) promotes a proteolytic activity that is required for the maturation of Ras, the yeast a-factor mating pheromone, and certain other proteins whose precursors bear a C-terminal CAAX tetrapeptide motif. Despite the physiological importance of RCE, the enzymatic mechanism of this protease remains undefined. In this study, we have evaluated the substrate specificity of RCE orthologs from yeast (Rce1p), worm, plant, and human and have determined the importance of conserved residues toward enzymatic activity. Our findings indicate that RCE orthologs have conserved substrate specificity, cleaving CVIA, CTLM, and certain other CAAX motifs, but not the CASQ motif, when these motifs are placed in the context of the yeast a-factor precursor. Our mutational studies of residues conserved between the orthologs indicate that an alanine substitution at His194 completely inactivates yeast Rce1p enzymatic activity, whereas a substitution at Glu156 or His248 results in marginal activity. We have also determined that residues Glu157, Tyr160, Phe190, and Asn252 impact the substrate selectivity of Rce1p. Computational methods predict that residues influencing Rce1p function are all near or within hydrophobic segments. Combined, our data indicate that yeast Rce1p function requires residues that are invariably conserved among an extended family of prokaryotic and eukaryotic enzymes and that these residues are likely to lie within or immediately adjacent to the transmembrane segments of this membrane-localized enzyme.

Human tryptophanyl-tRNA synthetase can efficiently complement the Saccharoymces cerevisiae homologue, Wrs1P

Aminoacyl-tRNA synthetases corresponding to each of the 20 amino acids are essential proteins for nearly all cells. The tryptophan-specific enzyme in the cytoplasm of Saccharomyces cerevisiae (ScWRS) is a 49-kDa protein encoded by the WRS1 gene and required for survival. The human enzyme (HsWRS) is a 54-kDa protein with 46% sequence identity to ScWRS. HsWRS has a kinase domain in the N-terminal region and a serine phosphorylation site near the C-terminus not present in the yeast enzyme. To determine if the gene encoding the human protein could functionally complement the WRS1 gene, HsWRS cDNA was cloned in the expression vectors pVT100U and pYES then transformed into a diploid yeast where one copy of WRS1 had been replaced with a G418 R gene. Tetrads derived from these transformants were dissected and spores germinated on media selecting for the presence of the plasmids. Haploid colonies were then tested for resistance to G418. G418 R cells were unable to grow in the presence of 5-fluoroorotic acid, indicating their dependence on the plasmids for viability. Polymerase chain reaction analysis of these cells confirmed the presence of the HsWRS gene and the absence of WRS1. Growth rates of cells expressing HsWRS are essentially identical to the parent. This demonstrates that the human enzyme can function in yeast and effectively replace the ScWRS protein in spite of the presence of two unique functions and a >50% overall difference in amino acid sequence.

Cytochrome c is not essential for viability of the fungus Aspergillus nidulans.

The filamentous fungus Aspergillus nidulans is an obligate aerobe, which is capable of anaerobic survival, but not anaerobic growth. Since cytochrome c forms an essential part of the oxidative respiratory pathway it was expected that mutants lacking this component would be non-viable. Gene replacement of one homologue of the cycA (cytochrome c) gene was carried out in a diploid strain. Benomyl-induced haploidisation of this diploid yielded all cycA+ haploid colonies, initially suggesting that loss of cycA was indeed lethal. However, use of an alternative unbiased method to recover haploids yielded viable, but slow-growing, cycA- mutants. Replacement of the cycA locus in the cycA- mutants was verified by Southern blotting. Spectral analysis confirmed the absence of detectable levels of cytochrome c, and respiratory insensitivity to cyanide suggested the absence of cytochrome c-dependent respiration. Growth parameters were consistent with those expected of a CycA- mutant. Compared to the wild type, the mutants grew slowly on fermentable carbon sources, did not grow on non-fermentable carbon sources, and produced higher levels of ethanol. To our knowledge, this is the first report of a filamentous fungus that remains viable after complete elimination of a functional cytochrome c gene. We propose that the mutants are viable due to their ability to ferment and to use alternative respiratory pathways.

A novel SMC protein complex in Schizosaccharomyces pombe contains the Rad18 DNA repair protein.

In Schizosaccharomyces pombe, rad18 is an essential gene involved in the repair of DNA damage produced by ionizing radiation and in tolerance of UV-induced DNA damage. The Rad18 protein is a member of the SMC (structural maintenance of chromosomes) superfamily, and we show that, like the other SMC proteins in condensin and cohesin, Rad18 is a component of a high-molecular-weight complex. This complex contains at least six other proteins, the largest of which is Spr18, a novel SMC family member closely related to Rad18, and likely to be its heterodimeric partner. SMC proteins have ATP-binding domains at the N- and C-termini, and two extended coiled-coil domains separated by a hinge in the middle. We show that the N-terminal ATP-binding domain of Rad18 is essential for all functions, and overexpression of an N-terminal mutant has a dominant-negative effect. We have identified an important mutation (S1045A) near the C-terminus of Rad18 that separates its repair and essential roles. Potential models for the role of the Rad18-Spr18 complex during DNA repair are discussed.

Nuclear Migration in Diploid-Haploid Pairings ofArmillaria Ostoyae

Diploid-haploid pairings to identify field isolates of Armillaria to species generally rely on morphological differences between diploid and haploid colonies (flat and fluffy colonies, respectively) and morphological changes in haploid tester colonies. Presumably, the change in morphology of the haploid tester is due to diploidization. Isozyme markers, aconitase and glucosephosphate isomerase, were used to follow nuclear migration from putatively diploid isolates into a haploid tester strain of A. ostoyae. After pairing diploid field isolates with a haploid tester for 3 or 4 weeks, subcultures were taken from the haploid tester at 2 and 10 mm from the confrontation zone. Subcultures from 2 mm were mostly flat; subcultures from 10 mm were fluffy, flat, or fluffy with flat sectors. Hyphal tip cultures from the subcultures were analyzed for the isozyme markers. All fluffy hyphal tip cultures had the isozyme phenotype of the haploid tester. Flat hyphal tip cultures either had the isozyme phenotype of the diploid parent or had a hybrid isozyme phenotype with markers of both the diploid parent and the haploid tester. The data suggest that in conspecific diploid-haploid pairings, the diploid nucleus usually migrates into the haploid mycelium and eventually displaces the haploid nucleus.

Nuclear Migration in Diploid-Haploid Pairings of Armillaria ostoyae

Diploid-haploid pairings to identify field isolates of Armillaria to species generally rely on morphological differences between diploid and haploid colonies (flat and flufly colonies, respectively) and morphological changes in haploid tester colonies. Presumably, the change in morphology of the haploid tester is due to diploidization. Isozyme markers, aconitase and glucosephosphate isomerase, were used to follow nuclear migration from putatively diploid isolates into a haploid tester strain of A. ostoyae. After pairing diploid field isolates with a haploid tester for 3 or 4 weeks, subcultures were taken from the hap!oid tester at 2 and 10 mm from the confrontation zone. Subcultures from 2 mm were mostly flat; subcultures from 10 mm were flufly, flat, or flufly with flat sectors. Hyphal tip cultures from the subcultures were analyzed for the isozyme markers. All flufly hyphal tip cultures had the isozyme phenotype of the haploid tester. Flat hyphal tip cultures either had the isozyme phenotype of the diploid parent or had a hybrid isozyme phenotype with markers of both the diploid parent and the haploid tester. The data suggest that in conspecific diploid-haploid pairings, the diploid nucleus usually migrates into the haploid mycelium and eventually displaces the haploid nucleus.

Nuclear fusion-defective phenocopies in Chlamydomonas reinhardtii: mating-type functions for meiosis can act through the cytoplasm.

Nuclear fusion in newly formed Chlamydomonas reinhardtii zygotes can be inhibited by drugs that affect microtubule stability, which include colchicine, amiprophosmethyl, oryzalin, and taxol. This inhibition can be monitored genetically by the production of haploid meiotic products from conjugations between haploid and diploid parents. Such zygotes would normally produce aneuploid progeny. Inhibition of nuclear fusion by colchicine requires treatment of gametic cells both before conjugation and after formation of the zygotes. These results suggest that nuclear fusion requires dynamic microtubules. Treated zygotes formed from a haploid-diploid mating can produce six spores, but only four spores germinate to form viable haploid colonies. No contribution from the nuclear genome of the haploid parent is recovered, whereas all loci from the diploid parent are recovered. The four viable products from the diploid parent of inhibited zygotes show normal segregation of loci located on linkage groups segregating according to Mendelian laws. Levels of meiotic recombination were tested for pairs of loci on linkage groups XVIII and XIX and found to be unchanged by inhibition of nuclear fusion. Thus, similar to Saccharomyces cerevisiae, C. reinhardtii mating-type functions necessary for nuclear fusion are not nuclear limited and can act through the cytoplasm. Inhibition of nuclear fusion can be used to analyze diploid Chlamydomonas that cannot enter meiosis. This technique permits direct analysis of dominant mutations, dominant suppressors and enhancers, and new alleles of identified loci that have been isolated in diploid strains.

Effect of mutagens, chemotherapeutic agents and defects in DNA repair genes on recombination in F′ part6ial diploid Escherichia coli

The ability of mutagenic agents, nonmutagenic substances and defects in DNA repair to alter the genotype of F′ partial diploid (F30) Escherichia coli was determined. The frequency of auxotrophic mutants and histidine requiring (His −) haploid colonies was increased by mutagen treatment but Hfr colonies were not detected in F30 E. coli even with specific selection techniques. Genotype changes due to nonreciprocal recombiantion were determined by measuring the frequency of His − homogenotes, eg. F′ hisC780, hisI +/ hisC780, hisI +, arising from a His + heterogenote, F′ hisC780 hisI +/ hisC +, his1903. At least 75% of the recombinants were homozygous for histidine alleles which were present on the F′ plasmid (exogenote) of the parental heterogenote rather than for histidine alleles on the chromosome. Mutagens, chemotherapeutic agents which block DNA synthesis and a defective DNA polymerase I gene, polA1, were found to increase th frequency of nonreciprocal recombination. A defect in the ability to exercise thymine dimers, uvrC34, did not increase spontaneous nonreciprocal recombination. However, UV irradiation but not methyl methanesulfonate (MMS) induced greater recombination in this excision-repair defective mutant than in DNA-repair-proficient strains. Mutagenic agents, with the exception of ethy methanesulfonate (EMS), induced greater increases in recombination than the chemotherapeutic agents or the polA1 mutation. EMS, which causes relatively little degradation of DNA, was more mutagenic but less recombinogenic than MMS, a homologous compound that induces rapid degradation of DNA. One explanation for these results is that inhibition of DNA synthesis increases recombination by prolonging the lifetime of naturally occurring single-stranded regions in replicative intermediates of the DNA. Mutagens which cause the rapid breakdown of DNA may, in addition, intorduce lesions into the genome that increase the number of single-stranded regions thus inducing even higher frequencies of recombination.

Evidence for Parthenogenesis in Aspergillus nidulans

Colonies from ascospores of perithecia from a diploid strain of Aspergillus nidulans, synthesized from haploid strains 182 and 183, were either diploid or were recombinants and presumably haploid; the former constituted approximately 94 per cent of the colonies. Although perithecia from diploid colonies contained a low number of germinating ascospores, perithecia from recombinants had either a high or a low number of such ascospores and four presumably haploid colonies did not have any detectable perithecia. It was possible to obtain only diploid, only haploid, or both diploid and haploid ascospores from a perithecium.

Visible changes in the vegetative mycelium of Coprinus lagopus Fr. at the time of fruiting

Until 2 days old, diploid colonies of Coprinus lagopus in plate cultures are entirely composed of uniformly cylindrical, slender hyphae. After this, many hyphae immersed in the agar swell considerably, filling completely with dense contents which stain deeply with iodine-potassium iodide. When fruiting begins, vacuoles appear in the swollen cells, and enlarge until the latter are devoid of dense contents. The swollen cells are thus organs of storage in preparation for fruiting. Observations suggest that the exhaustion of their contents is brought about by protoplasmic streaming motivated by vacuolar pressure. Similar storage cells develop in abundance in haploid colonies.