Stable Diploids Research Articles

Selecting Schizosaccharomyces pombe Diploids.

Here we describe procedures for the selection of diploid Schizosaccharomyces pombeade6-M210/ade6-M216 heteroallelic complementation is widely used to select for Ade+ diploids. Such diploids will readily sporulate when starved of nitrogen. For some investigations, stable diploids are preferable (e.g., for genetic complementation tests), and in these cases mating an h- strain with an h90 mat2-Pi-102 strain can be used to prevent sporulation. When ade6-M210/ade6-M216 mutations impact on, or show synthetic interactions with, the gene of interest, two different auxotrophic markers can be used to select complementation.

Flow Cytometry-based Purification of <em>S. cerevisiae</em> Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) (1). S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions (2). Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function. Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells (3-8). Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated. Reports of purification of yeast zygotes describe protocols based on their sedimentation properties (9); however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Reversibility of replicative senescence in Saccharomyces cerevisiae: Effect of homologous recombination and cell cycle checkpoints

Primary human somatic cells grown in culture divide a finite number of times, exhibiting progressive changes in metabolism and morphology before cessation of cycling. This telomere-initiated cellular senescence occurs because cells have halted production of telomerase, a DNA polymerase required for stabilization of chromosome ends. Telomerase-deficient Saccharomyces cerevisiae cells undergo a similar process, with most cells arresting growth after approximately 60 generations. In the current study we demonstrate that senescence is largely reversible. Reactivation of telomerase (EST2) expression in the growth-arrested cells led to resumption of cycling and reversal of senescent cell characteristics. Rescue was also observed after mating of senescent haploid cells with telomerase-proficient cells to form stable diploids. Although senescence was reversible in DNA damage checkpoint response mutants (mec3 and/or rad24 cells), survival of recombination-defective rad52 mutants remained low after telomerase reactivation. Telomere lengths in rescued est2 cells were initially half those of wildtype cells, but could be restored to normal by propagation for ∼70 generations in the presence of telomerase. These results place limitations on possible models for senescence and indicate that most cells, despite gross morphological changes and short, resected telomeres, do not experience lethal DNA damage and become irreversibly committed to death.

Isogenic auxotrophic mutant strains in the Aspergillus fumigatus genome reference strain AF293.

Aspergillus fumigatus is a ubiquitous fungus that is a frequent opportunistic pathogen in immunosuppressed patients. Because of its role as a pathogen, it is of considerable experimental interest. A set of auxotrophic isogenic strains in the A. fumigatus genome reference strain AF293 has been developed. Using molecular genetic methods, arginine and lysine auxotrophs were made by deletion of argB and lysB, respectively. Transformation of these auxotrophic strains with plasmids carrying argB or lysB, respectively, results in efficient integration at these loci. Finally, these strains are able to form stable diploids, which should further facilitate analysis of gene functions in this fungus. Furthermore, the development of this isogenic set of auxotrophic strains in the AF293 background will enable investigators to study this important opportunistic human pathogen with greater facility.

A New Method for Production of Combinatorial Libraries by Mating of Saccharomyces cerevisiae Cells.

There are two kinds of mating types in yeast cells, a-type and α-type, and they can make a diploid, which carries both the genes originated from a-type and α-type. We propose a new method to obtain a larger size of combinatorial libraries by mating these yeast cells. By transforming libraries of genes of heavy and light chains to a- and α-types respectively, for example, it should be possible to obtain a larger size of libraries of antibodies by mating them. To confirm our method, the expression vectors containing heavy chain gene (μ) and light chain genes (κ and λ) of human antibodies were transformed into a-type and α-type strains of S. cerevisiae, respectively, and these transformants were mated by mixing the cultured cells of both types. The effects of media, cell concentration and the ratio of a-type cells to α-type cells on the mating efficiency were studied. The direct contact between a- and α-type cells was essential to initiate mating of cells, and the probability of contact among cells determined the mating efficiency, when cells are well nourished. Stable diploids were formed by the combination of plasmids of the 2 μm types, as well as the combination between the 2 μm type and the ARS1 type.

The 78,000 M(r) intermediate chain of Chlamydomonas outer arm dynein isa WD-repeat protein required for arm assembly.

We have isolated and sequenced a full-length cDNA clone encoding the 78,000 Mr intermediate chain (IC78) of the Chlamydomonas outer arm dynein. This protein previously was shown to be located at the base of the solubilized dynein particle and to interact with alpha tubulin in situ, suggesting that it may be involved in binding the outer arm to the doublet microtubule. The sequence predicts a polypeptide of 683 amino acids having a mass of 76.5 kD. Sequence comparison indicates that IC78 is homologous to the 69,000 M(r) intermediate chain (IC69) of Chlamydomonas outer arm dynein and to the 74,000 M(r) intermediate chain (IC74) of cytoplasmic dynein. The similarity between the chains is greatest in their COOH-terminal halves; the NH(2)-terminal halves are highly divergent. The COOH-terminal half of IC78 contains six short imperfect repeats, termed WD repeats, that are thought to be involved in protein-protein interactions. Although not previously reported, these repeated elements also are present in IC69 and IC74. Using the IC78 cDNA as a probe, we screened a group of slow-swimming insertional mutants and identified one which has a large insertion in the IC78 gene and seven in which the IC78 gene is completely deleted. Electron microscopy of three of these IC78 mutants revealed that each is missing the outer arm, indicating that IC78 is essential for arm assembly or attachment to the outer doublet. Restriction fragment length polymorphism mapping places the IC78 gene on the left arm of chromosome XII/XIII, at or near the mutation oda9, which also causes loss of the outer arm. Mutants with defects in the IC78 gene do not complement the oda9 mutation in stable diploids, strongly suggesting that ODA9 is the structural gene for IC78.

The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation.

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

The CRY1 Gene in Chlamydomonas reinhardtii: Structure and Use as a Dominant Selectable Marker for Nuclear Transformation

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

Restriction fragment length polymorphisms (RFLPs) and the relationships of some host‐adapted isolates of Verticillium dahliae

The relationships of two host‐adapted pathotypes of Verticillium dahliae have been examined at the molecular level using restriction fragment length polymorphisms. Isolates obtained from and adapted to Mentha×piperita (peppermint), which were presumed to be haploid, formed a distinct subspecific group (referred to as M) related to the previously described non‐host‐adapted subspecific group A of V. dahliae. The limited molecular variation found among the four group M isolates was not related to geographic origin. Isolates from several cruciferous hosts (and one from Beta vulgaris (sugar beet)), which are thought to be natural, stable diploids, formed another distinct group (referred to as D) that was markedly different from all previously described subspecific groupings in both V. dahliae and V. alboatrum. This group of isolates might better be regarded as a separate species. Again, only limited variation was found within the D group. Polymorphisms revealed by two probes distinguished two isolates derived from Brassica rapa (Chinese cabbage) from the six other isolates (four from Brassica napus (oilseed rape) and one each from Raphanus raphanistrum (wild radish) and Beta vulgaris).

Relationship between DNA Content and Spore Volume in Sixteen Isolates of Verticillium lecanii and Two New Diploids of V. dahliae (= V. dahliae var. longisporum Stark)

Summary: Sixteen wild-type isolates of Verticillium lecanii from different insects and non-insect hosts, soils and locations exhibited a wide range in spore volumes, from 2.18 μm3 to 18.25 μm3, as recorded by Coulter counter analysis. A cold hydrolysis technique for Feulgen DNA microdensitometry indicated that all the isolates were haploid. Two wild-type strains of V. dahliae, from rape and sugarbeet from Sweden, were shown to be stable diploids by the criteria of conidial volume and relative DNA content. After treatment with p-fluorophenylalanine, both V. dahliae strains produced haploid segregants with half the conidial volume and relative DNA content of the original parents. Both diploid strains are regarded as new records for V. dahliae var. longisporum (Stark).

Somatic cell fusion of Trichoderma reesei resulting in new genetic combinations

A selection method has been developed for the isolation of recombinant strains of Trichoderma reesei QM 9414. The method is based on somatic hybridization via anastomosis or protoplast fusion, and on the difference in growth rate of the resulting heterokaryons and synkaryons. The more intensive growth of the synkaryons as due to allelic complementation of adenine-requiring auxotrophic strains mutated in the adenylosuccinate synthetase gene. The synkaryons appeared is energetically growing spots in the heterokaryotic background. Stable diploids could not be isolated, which points to the transient nature of the diploid state in this species.

Azacytidine-induced tumorigenesis of CHEF/18 cells: correlated DNA methylation and chromosome changes.

5-Azacytidine (azaC), a drug that induces decreased methylation of DNA in mammalian cells, was shown previously to induce differentiation of mesenchymal cell types in CHEF/18 cells (Chinese hamster embryo fibroblasts). This paper describes the effectiveness of azaC in inducing tumorigenicity in CHEF/18 cells, previously shown to be nontumorigenic stable diploids. A short exposure of growing cells to 3 microM azaC induced tumor-forming ability in CHEF/18 stem cells. Pre-adipocyte clones and subclones derived from CHEF/18 by prior treatment with azaC were also found to be tumorigenic. Pre-adipocytes previously induced by insulin in the absence of azaC were mostly nontumorigenic, but one clone produced tumors and gave rise to both tumorigenic and nontumorigenic subclones. Karyotype analysis of 41 clones and subclones from azaC-induced and insulin-induced pre-adipocytes revealed a complete correlation between tumor-forming ability and the presence of trisomy for chromosome 3q. In addition, the tumorigenic and tumor-derived lines were demethylated at specific C-C-G-G sites in the preproinsulin, Ha-ras, and Ki-ras genes as revealed by blot hybridization to Msp I- and Hpa II-digested DNAs, whereas the nontumorigenic lines resembled the CHEF/18 controls. This three-way correlation between tumorigenicity, trisomy for 3q, and specific demethylation suggests that decreased DNA methylation may be involved both in differentiation and in tumorigenicity, and that azaC may induce chromosomal aberrations as well as altering DNA methylation.

Karyoduction in Saccharomyces cerevisiae

We introduced the nuclei isolated from the respiration-sufficient killer strain of the yeast Saccharomyces cerevisiae into the yeast protoplasts prepared from the respiration-deficient non-killer strain with the aid of polyethylene glycol. The resulting karyoductants were respiration-deficient non-killers. Nuclear staining with ethidium bromide or DAPI and tetrad analysis of the karyoductants presented evidence that the nuclei introduced into protoplasts were fused to the resident nuclei, leading to stable diploids. This technique termed karyoduction will be useful in the study of nucleo—cytoplasmic relationship in yeast and other organisms.

Uniflagellar mutants of chlamydomonas: Evidence for the role of basal bodies in transmission of positional information

A series of uniflagellar mutants isolated following mutagenesis of Chlamydomonas reinhardtii (strain 137c) with ICR-191 show a remarkable positional phenotype. The flagellum that fails to develop is cis to the eyespot in more than 95% of the cells examined. Both the positional and the uniflagellar phenotypes are transmitted stably through mitotic and meiotic divisions, and in backcrosses the meiotic segregation is two mutant to two wild-type progeny. Four of the mutants, uni1, uni2, uni3 and uni4, have been studied extensively. They appear to be alleles of a single gene locus or to be closely linked (≤0.06 map units). The characteristic expression of the uniflagellar defect in cells under different growth conditions or in stable diploids indicates that the mutations alter the rate of development of the flagellum in the cis-eyespot flagellum. Electron microscopic studies suggest that the developmental defect resides in the basal body. Extensive recombination analysis to 33 nuclear markers representing the 16 linkage groups failed to establish linkage. The uni mutants, however, showed linkage to four unmapped mutant loci. Mutations for each of these loci also affect flagellar assembly.

Zygote formation and recombination between like mating types in the yeast Saccharomycopsis lipolytica by protoplast fusion

Protoplasts from auxotrophic strains of the alkane yeast, Saccharomycopsis (Candida) lipolytica, will hybridize despite identity in mating type. Fusion products following regeneration and selection form stable prototrophic diploids, and recombinant progeny can be obtained either through the parasexual or the sexual cycle. These results confirm that mating type alleles of this yeast control only the initial steps in the mating sequence, cell recognition and agglutination, but not karyogamy and meiosis.

Genetic analysis of products of protoplast fusion in Saccharomyces cerevisiae.

Protoplasts of Saccharomyces cerevisiae were prepared from two different haploid strains both of mating type a, which carried different nuclear (ade1, ura1, his4, leu2 and thr4) and mitochondrial (ρ, ω, CR, ER and OR) markers, and were fused with the aid of polyethylene glycol. Cells of fused products (prototrophs) displayed phenotype of mating type a and were crossed to mating type α/α diploids having auxotrophic markers, e.g. trp1. On sporulation of the resulting hybrid clones, as a rule, there were three tetrad types for mating types, i.e. (I) 4non-maters, (II) 2a:2α and (III) a:α: 2non-maters. The relative frequencies of these three tetrad types were close to the ones theoretically predicted from a/a/α/α tetraploids, suggesting that the fusion products were a/a diploids. Auxotrophic markers involved in these crosses, which were located on four different chromosomes, were also segregated to yield the tetrad distributions expected from the parentages. Consequently, it was concluded that the protoplast fusion proceeded to karyogamy to produce stable diploids. A study of mitochondrial recombination demonstrated that the fusion products accepted the mitochondrial genome (the polar gene ω as well as the drug resistance genes) from one parent of ρ+, but not from another of neutral petite.

Linkage analysis of developmental mutations in aggregation-deficient mutants of Dictyostelium discoideum.

Simple parasexual genetic techniques have been employed to extend the linkage analysis initiated in an earlier study (Coukell, 1975) of developmental mutations (agg mutations) in 40 independently isolated aggregation-deficient mutants of Dictyostelium discoideum. Using these techniques, agg mutations in 28 of the 40 mutants have been assigned to 4 linkage groups: 16 in group II, 1 in group III, 10 in group IV, and 1 in group VI. None of the agg mutations analyzed appear to map in linkage group I. In addition, a new temperature-sensitive growth locus, designated tsgJ, was mapped in group III. It was also found that diploid strains of D. discoideum are readily induced to undergo haploidization when grown on 0.1% p-fluorophenylalanine (PFP) at 25.5 degrees C. Growth of diploid strains on PFP had no effect on the type of segregant classes obtained (i.e. PFP does not induce mitotic crossing-over), the subsequent growth and/or development of the segregants, or the ability of the segregants to reform stable diploids.

A cytoplasmic gene for partial suppression of a nuclear pleiotropic respiratory deficient mutant in the petite negative yeast Schizosaccharomyces pombe.

The nuclear pleiotropic respiratory-deficient mutant pet1 (previously M126) exhibits cytochromes aa3 and b deficiencies accompanied by loss of the oligomycin-sensitivity of the mitochondrial ATPase. The mutant pet1, unable to grow on glycerol, growth on glucose. The latter phenotypic trait symbolized by ANAS-D, exhibits a high frequency (2 to 4 X 10(5)) Of spontaneous suppression into Antimycin A-resistant strains. Mutagenesis with MnCl2 increases by a factor of 10(2) the frequency of ANAR-D derivatives. This suppression is partial since none of the suppressed strains is able to grow on glycerol even when respiratory functions and cytochromes activities are restored as in the pet1 [SUP2] strain. In the latter strain it is concluded that the extralocus suppressor gene [SUP2] is responsible for the ANAR-D trait. Tetrad analysis in a cross homozygous for pet1 demonstrates a non-Mendelian segregation pattern for the SUP2 suppressor gene. In stable diploids, homozygous for pet1, the [SUP2] suppressor exhibits a mitotic segregation pattern. Furthermore the transmission of the [SUP2] gene is decreased by ethidium bromide treatment. Therefore, the [SUP2] suppressor gene responsible for partial suppression of the nuclear pleiotropic phenotype in mutant pet1 is of cytoplasmic heredity.

Extrachromosomal inheritance in Schizosaccharomyces pombe

In crosses of [ANTr8] with auxotrophic strains, resistance to antimycin segregates almost 50:50 in random spore analysis with a slight preponderance for the sensitivity allele. Tetrad analysis, however, shows all possible types of tetrads (2:2; 3:1; 1:3; 4:0; 0:4 resistant versus sensitive) with an excess of 2:2 segregations and sectoring of colonies on antimycin medium indicating an extrachromosomal mode of inheritance. The overall ratio of resistant versus sensitive spores is the same as compared with random spore data. Using a mutant blocked in meiosis (mei 1) mitotic segregation of stable diploids is achieved, leading to a ratio of 20% resistant to 80% sensitive clones. Possible reasons for the bias in transmission of the resistance determinant is discussed.

Studies on the size of the diploid region in Bacillus subtilis merozygotes from strains carrying the trpE26 mutation.

Simultaneous selection of transformants for trpE26 and a second unliked marker of B. subtilis in many cases yields double heterogenotic clones. Several chromosome areas analyzed in this way were found to be involved in the diploid condition. Diploids for areas on the left hand side of trpE26 on the map (and as near as the aroB locus) are in general unstable while stable merodiploids can be obtained for areas on the right hand side of this marker (as far as the ilvA locus). Merozygotes for regions other than the "aromatic segment" are also formed by transformation of already diploid (stable and unstable) clones. Stable diploids give rise to new heterogenotes only for markers on the right hand side of trpE26. Through reversion of untransformed markers in unstable and stable diploids it was found that these clones are homodiploid for loci situated at a long distance from (or between) the areas which were involved in the transformation. This indicates that the diploid state covers a continuous segment of the chromosome, the length of which can be determined. The segregation pattern of unstable multiple merodiploids suggests that exchange of genetic material must take place between the two homologous regions. The data presented are in agreement with the hypothesis that the merodiploids possess a very long duplication on their chromosome. In the case of the stable clones this duplication is shorter.