Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe

In previous papers we have reported the characterisation of mitochondrial mutator mutants of Schizosaccharomyces pombe. In contrast to nuclear mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome. Since an efficient biolistic transformation system for fission yeast mitochondria is not yet available, we relocated the mitochondrial urf a gene to the nucleus. As host strain for the ectopic expression, we used the nonsense mutant ana ^r -6, which carries a premature stop codon in the urf a gene. The phenotype of this mutant is characterised by continuous segregation of progeny giving rise to fully respiration competent colonies, colonies that show moderate growth on glycerol and a fraction of colonies that are unable to grow on glycerol. The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic expression of the urf a gene. Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types of expression cassettes containing either the mitochondrial version of the urf a gene (mt-urf a) or a standard genetic code version (nc-urf a; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae. We show that the expression of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana ^r -6, whereas the expression of the nc-urf a gene completely restores the wild-type (non-mutator) phenotype. The significant similarity of the urf a gene to the mitochondrial var1 gene of S. cerevisiae and homologous genes in other yeasts suggests that the urf a gene product might be a ribosomal protein with a dual function in protein synthesis and maintenance of mitochondrial DNA integrity. Received: 13 May 1997 / Accepted: 14 January 1998     

Map-based cloning of a novel rice cytochrome P450 gene CYP81A6 that confers resistance to two different classes of herbicides

Development of hybrid rice has greatly contributed to increased yields during the past three decades. Two bentazon-lethal mutants 8077S and Norin8m are being utilized in developing new hybrid rice systems. When the male sterile lines are developed in such a mutant background, the problem of F1 seed contamination by self-seeds from the sterile lines can be solved by spraying bentazon at seedling stage. We first determined the sensitivity of the mutant plants to bentazon. Both mutants showed symptoms to bentazon starting from 100 mg/l, which was about 60-fold, lower than the sensitivity threshold of their wild-type controls. In addition, both mutants were sensitive to sulfonylurea-type herbicides. The locus for the mutant phenotype is bel for 8077S and bsl for Norin8m. Tests showed that the two loci are allelic to each other. The two genes were cloned by map-based cloning. Interestingly, both mutant alleles had a single-base deletion, which was confirmed by PCR-RFLP. The two loci are renamed bel ^a (for bel) and bel ^b (for bsl). The wild-type Bel gene encodes a novel cytochrome P450 monooxgenase, named CYP81A6. Analysis of the mutant protein sequence also revealed the reason for bel ^a being slightly tolerant than bel ^b . Introduction of the wild-type Bel gene rescued the bentazon- and sulfonylurea-sensitive phenotype of bel ^a mutant. On the other hand, expression of antisense Bel in W6154S induced a mutant phenotype. Based on these results we conclude that the novel cytochrome P450 monooxygenase CYP81A6 encoded by Bel confers resistance to two different classes of herbicides. Gang Pan and Xianyin Zhang contributed equally to this work.     

Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe

In previous papers we have reported the characterisation of mitochondrial mutator mutants of Schizosaccharomyces pombe. In contrast to nuclear mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome. Since an efficient biolistic transformation system for fission yeast mitochondria is not yet available, we relocated the mitochondrial urf a gene to the nucleus. As host strain for the ectopic expression, we used the nonsense mutant ana ^r -6, which carries a premature stop codon in the urf a gene. The phenotype of this mutant is characterised by continuous segregation of progeny giving rise to fully respiration competent colonies, colonies that show moderate growth on glycerol and a fraction of colonies that are unable to grow on glycerol. The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic expression of the urf a gene. Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types of expression cassettes containing either the mitochondrial version of the urf a gene (mt-urf a) or a standard genetic code version (nc-urf a; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae. We show that the expression of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana ^r -6, whereas the expression of the nc-urf a gene completely restores the wild-type (non-mutator) phenotype. The significant similarity of the urf a gene to the mitochondrial var1 gene of S. cerevisiae and homologous genes in other yeasts suggests that the urf a gene product might be a ribosomal protein with a dual function in protein synthesis and maintenance of mitochondrial DNA integrity.     

A dominant interfering mutation in RAS1 of Saccharomyces cerevisiae

A mutant allele of RAS1 that dominantly interferes with the wild-type Ras function in the yeast Saccharomyces cerevisiae was discovered during screening of mutants that suppress an ira2 disruption mutation. A single amino acid substitution, serine for glycine at position 22, was found to cause the mutant phenotype. The inhibitory effect of the RAS1 ^Ser22 gene could be overcome either by overexpression of CDC25 or by the ira2 disruption mutation. These results suggest that the RAS1^Ser22 gene product interferes with the normal interaction of Ras with Cdc25 by forming a dead-end complex between Ras1^Ser22 and Cdc25 proteins.     

Cloning of colicin E1 tolerant tolC (mtcB) gene of Escherichia coli K12 and identification of its gene product

Summary The normal 24-h period of the circadian rhythms of locomotor activity and eclosion of Drosophila melanogaster is altered by changes in per gene dosage. Females with only one dose of per ⁺ or per ^s (the 19-h short-period mutant allele) or per ¹ (the 29-h long-period mutant allele) have periods which are about 1–2 h longer than the corresponding females with 2 doses. Females with 3 doses of per ⁺ and males with 2 doses of per ⁺ or per ^s have periods which are 1/2 to 1 h shorter than the corresponding individuals without the extra dose. Males with three per ⁺ doses have periods which are about 1.5 h shorter than wild-type males; additional per ⁺ doses do not shorten period further. The observation that decreased per dosage lengthens period while increased dosage shortens period suggests that the long- and short-period mutations alter period by respectively decreasing and increasing per gene or gene product activity. The per ⁺ dosage results and the complementation behavior of per ^s indicate that the hypermorphic phenotype of per ^s results from increased activity of the per ^s gene product rather than an overproduction of per ⁺ product. This is the first report of such a mutant action in Drosophila.     

<Emphasis Type="Italic">rpoS</Emphasis> Gene Expression in Carbon-Starved Cultures of the Polyhydroxyalkanoate-Accumulating Species <Emphasis Type="Italic">Pseudomonas oleovorans</Emphasis>

The expression of the rpoS gene during PHA depolymerization was monitored in Pseudomonas oleovorans GPo1 and its mutant defective in PHA degradation by analyzing the tolerance to oxidative and thermal stresses and the RpoS intracellular content. An increase in the tolerance to H₂O₂ and heat shock was observed coincidentally with PHA degradation. Western blotting experiments performed in carbon-starved cultures showed that the RpoS levels were higher in the wild type than in the mutant strain. Complementation of the phaZ mutation restores the wild-type RpoS levels. These results suggest a probable association between PHA depolymerization and the stress tolerance phenotype controlled by RpoS.     

In vivo cloning ad characterization of mutations of the regulatory locus ompR of Escherichia coli K12

Summary The product of the ompR gene of E. coli K12 is a positive regulatory protein, which is needed for the expression of the major outer membrane proteins OmpC and OmpF in E. coli K12. A simple in vivo technique was used to transfer three ompR mutations (ompR101, ompR472, ompR4) onto a multicopy plasmid carrying the wild-type ompR gene. The resulting clones were transformed into wild type and corresponding mutant back-grounds to analyze their effects on ompC and ompF expression. All of the cloned ompR mutant alleles exhibited a dominant OmpC^- phenotype in an ompR ⁺background. In addition negative complementation of ompF expression was observed between chromosomal ompR4 and multicopy ompR101 alleles. The results suggest an interaction between different OmpR molecules, and thereby support the idea that OmpR can exist as a multimeric protein.     

Injection of wild-type cytoplasm and poly(A)+ RNA provokes phenotype rescue in spätzle mutant Drosophila embryos

Summary spätzle (spz), a maternal effect gene of Drosophila, is involved in the establishment of the dorso-ventral axis during embryogenesis. Eggs from females lacking the spz gene product develop into completely dorsalized embryos, i.e. the ventral and lateral pattern elements fail to develop. Upon injection of either cytoplasm or poly(A)⁺ RNA from early wild-type embryos, spz embryos develop lateral pattern elements represented by Filzkörper and in the case of injected cytoplasm additional ventral pattern elements represented by ventral setae. Wild-type cytoplasm retains the rescuing activity longer than the poly(A)⁺ RNA fraction does, and cytoplasm is always more effective in provoking the rescue than poly(A)⁺ RNA. Mosaic females containing spz germ cells surrounded by spz ⁺ tissues were generated by pole cell transplantations; a mutant genotype in the germ cells is sufficient to produce all aspects of the spz mutant phenotype, suggesting that the maternal source of spz gene product is the germ line.     

Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes

Summary When a non-selected DNA sequence was added during the transformation of amdS320 deletion strains of Aspergillus nidulans with a vector containing the wild-type amdS gene the AmdS⁺ transformants were cotransformed at a high frequency. Cotransformation of an amdS320, trpC801 double mutant strain showed that both the molar ratio of the two vectors and the concentration of the cotransforming vector affected the cotransformation frequency. The maximum frequency obtained was defined by the gene chosen as selection marker for transformation. Cotransformation was used to induce a gene replacement in A. nidulans. An amdS320 strain was transformed to AmdS⁺ and cotransformed with a DNA fragment containing a fusion between a non-functional A. nidulans trpC gene and the Escherichia coli lacZ gene. Ten AmdS⁺, LacZ⁺ transformants with a Trp^– mutant phenotype were selected. All of these strains could be transformed with a functional copy of the A. nidulans trpC gene, but only two strains yielded TrpC⁺ transformants which, with a low frequency, had a LacZ^– phenotype. These latter transformants had also lost the AmdS⁺ phenotype. Southern blotting analysis of DNA from these transformants confirmed the inactivation of the wild-type trpC gene, but revealed that amdS vector sequences were also involved in the gene replacement events.     

Analysis of Some Barley Chlorophyll Mutants and Their Response to Temperature Stress

Six barley chlorophyll (Chl) mutants, viridis, flavoviridis, chlorina, xanhta, lutea, and albina, differed in the contents of Chl (a+b) and carotenoids (Cars). In accordance with their Chl-deficient phenotype, the Chl a and b and Car contents of mutants decreased from viridis to albina, only xantha had the same or even higher concentration of Cars as the wild type plant. The albina mutant completely lacked and xantha had a significantly reduced photosynthetic activity. We found quantitative differences in protein contents between wild type and mutant plants, with the lowest concentration per fresh mass in the albina mutant. Chl fluorescence analysis revealed that heat-treated barley leaves of both the wild type and Chl mutants had a lower photosystem 2 efficiency than the untreated ones. With ³⁵S-methionine labelling and SDS-PAGE we found that six to nine de novo synthetized proteins appeared after heat shock (2 h, 42 °C) in the wild type and Chl mutants. In albina the expression of heat shock proteins (HSPs) was reduced to 50 % of that in the wild type. Hence mainly albina mutants, with a completely destroyed proteosynthetic apparatus of the chloroplasts, are able to synthesize a small set of HSPs. The albina mutant is a very useful tool for the study of different gene expression of chloroplast and nuclear DNA.     

Disruption of <Emphasis Type="Italic">RCI2A</Emphasis> leads to over-accumulation of Na<Superscript>+</Superscript> and increased salt sensitivity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na⁺ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na⁺ ions in the cell and shows increased Na⁺ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na⁺ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na⁺ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na⁺ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO₃, Na₂SO₄, KCl, KNO₃, K₂SO₄ or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl₂, MgCl₂, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na⁺ and K⁺ ions in plants, and contributes to salt tolerance.     

A deletion spanning the promoter and first exon of the hair cycle-specific ASIP transcript isoform in black and tan rabbits

Black and tan animals have tan-coloured ventral body surfaces separated by sharp boundaries from black-coloured dorsal body surfaces. In the a^t mouse mutant, a retroviral 6 kb insertion located in the hair cycle-specific promoter of the murine Asip gene encoding agouti signalling protein causes the black and tan phenotype. In rabbits, three ASIP alleles are thought to exist, including an a^t allele causing a black and tan coat colour that closely resembles the mouse black and tan phenotype. The goal of our study was to identify the functional genetic variant causing the rabbit a^t allele. We performed a WGS-based comparative analysis of the ASIP gene in one black and tan and three wt agouti-coloured rabbits. The analysis identified 75 a^t-associated variants including an 11 kb deletion. The deletion is located in the region of the hair cycle-specific ASIP promoter and thus in a region homologous to the site of the retroviral insertion causing the a^t allele in mice. We observed perfect association of the genotypes at this deletion with the coat colour phenotype in 49 rabbits. The comparative analysis and the previous knowledge about the regulation of ASIP expression suggest that the 11 kb deletion is the most likely causative variant for the black and tan phenotype in rabbits.     

Phenotypic suppression of the Drosophila mitochondrial disease-like mutant tko by duplication of the mutant gene in its natural chromosomal context

A mutation in the Drosophila gene technical knockout (tko^25t), encoding mitoribosomal protein S12, phenocopies human mitochondrial disease. We isolated three spontaneous X-dominant suppressors of tko^25t (designated Weeble), exhibiting almost wild-type phenotype and containing overlapping segmental duplications including the mutant allele, plus a second mitoribosomal protein gene, mRpL14. Ectopic, expressed copies of tko^25t and mRpL14 conferred no phenotypic suppression. When placed over a null allele of tko, Weeble retained the mutant phenotype, even in the presence of additional transgenic copies of tko^25t. Increased mutant gene dosage can thus compensate the mutant phenotype, but only when located in its normal chromosomal context.     

The role of the functional sites of the merlin tumor suppressor in <Emphasis Type="Italic">Drosophila Spermatogenesis</Emphasis>

The Merlin gene of Drosophila is homologous to the human Neurofibromatosis 2 (NF2) gene, an important regulator of proliferation and endocytosis of cell receptors. It was earlier shown that the Thr⁵⁵⁹ residue of the Drosophila Merlin protein was homologous to Ser⁵¹⁸ of the human protein (which was already known to undergo phosphorylation); hence, it was assumed that Thr⁵⁵⁹ of Drosophila also was a substrate of phosphorylation. The mutant Merlin proteins MerT^559D (an analog of the phosphorylated form) and MerT^559A (a nonphosphorylated form) were constructed and tested, under the conditions of ectopic expression, for the ability to correct the spermatogenesis defects induced by the Mer4 mutation. The mutant form MerT^559D was demonstrated to restore the abnormal nebenkern phenotype induced by this mutation, whereas the MerT^559A substituted form did not restore this phenotype. Ectopic expression o the wild-type Merlin protein, MerT^559A mutant form, and mycMer^345–635 truncated protein in a normal genotype resulted in the abnormal nebenkern phenotype, whereas this phenotype was not observed in the case of ectopic expression of the Mer^T559D analog of the phosphorylated form. Ectopic expression of the mycMer³, mycMer^ΔBB, and mycMer^1–379 truncate variants led to disturbance of meiotic cytokinesis.     

AtCPK23 functions in <Emphasis Type="Italic">Arabidopsis</Emphasis> responses to drought and salt stresses

Calcium-dependent protein kinases (CDPKs) are unique serine/threonine kinases in plants and there are 34 CDPKs in Arabidopsis genome alone. Although several CDPKs have been demonstrated to be critical calcium signaling mediators for plant responses to various environmental stresses, the biological functions of most CDPKs in stress signaling remain unclear. In this study, we provide the evidences to demonstrate that AtCPK23 plays important role in Arabidopsis responses to drought and salt stresses. The cpk23 mutant, a T-DNA insertion mutant for AtCPK23 gene, showed greatly enhanced tolerance to drought and salt stresses, while the AtCPK23 overexpression lines became more sensitive to drought and salt stresses and the complementary line of the cpk23 mutant displayed similar phenotype as wild-type plants. The results of stomatal aperture measurement showed that the disruption of AtCPK23 expression reduced stomatal apertures, while overexpression of AtCPK23 increased stomatal apertures. The alteration of stomatal apertures by changes in AtCPK23 expression may account, at least in partial, for the modified Arabidopsis response to drought stress. In consistent with the enhanced salt-tolerance by disruption of AtCPK23 expression, K⁺ content in the cpk23 mutant was not reduced under high NaCl stress compared with wild-type plants, which indicates that the AtCPK23 may also regulate plant K⁺-uptake. The possible mechanisms by which AtCPK23 mediates drought and salt stresses signaling are discussed.     

A novel fission yeast gene, kms1 +, is required for the formation of meiotic prophase-specific nuclear architecture

In the meiotic prophase nucleus of the fission yeast Schizosaccharomyces pombe, chromosomes are arranged in an oriented manner: telomeres cluster in close proximity to the spindle pole body (SPB), while centromeres form another cluster at some distance from the SPB. We have isolated a mutant, kms1, in which the structure of the meiotic prophase nucleus appears to be distorted. Using specific probes to localize the SPB and telomeres, multiple signals were observed in the mutant nuclei, in contrast to the case in wild-type. Genetic analysis showed that in the mutant, meiotic recombination frequency was reduced to about one-quarter of the wild-type level and meiotic segregation was impaired. This phenotype strongly suggests that the telomere-led rearrangement of chromosomal distribution that normally occurs in the fission yeast meiotic nucleus is an important prerequisite for the efficient pairing of homologous chromosomes. The kms1 mutant was also impaired in karyogamy, suggesting that the kms1 ⁺ gene is involved in SPB function. However, the kms1 ⁺ gene is dispensable for mitotic growth. The predicted amino acid sequence of the gene product shows no significant similarity to known proteins. Received: 5 September 1996 / Accepted: 21 November 1996     

Anti-H-2 hybridoma antibodies as immunoselective agents

Using immunoselection with an H-2K^k-specific monoclonal antibody following mutagenesis on an (H-2 ^k/H-2^d) F₁ cell line we have obtained variants that do not react with the selecting monoclonal antibody but continue to react with other monoclonal antibodies directed against the same gene product. The mutants fall into two classes based on their serological profile. This phenotype is suggestive of a structural mutation in the selected gene. If the genetic change involved is a point mutation (as opposed to a deletion), one should be able to obtain revertants. Using the fluorescence-activated cell sorter, we have been able to obtain from one of the monoclonal-antibody-nonseactive mutants cells that do bind the selecting antibody. In order to prove that the presumptive revertant is not a contaminant wild-type cell that inadvertantly got mixed into the resistant mutant, we first introduced an outside marker, resistance to the purine analogue 2-amino-6-mercaptopurine (6-thioguanine), into the monoclonal-antibody-resistant mutant. The revertants obtained using the cell sorter continue to express the nonselective phenotype of resistance to 6-thioguanine, showing that they are not wild-type cells. In addition, their serological characteristics are different from those of either the wild-type cells or the hybrid oma-resistant mutants from which they were derived. Based on the serological analyses, it would seem that we have isolated at least three variant forms of the H-2K^k-gene product.