Mitochondrial Genetics. Xii. an Oligomycin-Resistant Mutant Localized at a New Mitochondrial Locus in SACCHAROMYCES CEREVISIAE

Three antibiotic-resistance mutations were isolated from strain FL496–2B: two are independent Mendelian genes, one conferring both oligomycin and venturicidin resistance (oli^R₄₉₆) and the other conferring cycloheximide resistance (cyh^R₄₉₆). The third is a mitochondrial mutation, O^R₉, and confers a low level of oligomycin resistance to cells (in vivo) but not to the extracted mitochondrial ATPase (in vitro). This mutation is located on the mitochondrial DNA at a new locus [OLI4] linked to [OLI2] and independent from [OLI1] and [OLI3] and from the other mitochondrial loci.

All three mutations (O ^R₉, oli^R₄₉₆, cyh^R₄₉₆ ) were found without any selection, in the same prototrophic haploid strain, which contained unknown resistances to antibiotics.

Some physiological, genetical and biochemical properties of the mitochondrial mutation are described.

     

Circadian Rhythms in Neurospora crassa: The Role of Mitochondria

Energy metabolism and mitochondria have been discussed with respect to their role in the circadian rhythm mechanism for some time. Numerous examples of inhibitors that affect the mitochondria of plants and animals and microorganisms are known, which cause large phase shifts in the rhythms of these organisms. Analogous studies on the role of mitochondria in the Neurospora circadian rhythm mechanism have also been reported and summarized. This communication differs from previous studies on other organisms in that it will focus on two lines of evidence derived from studies on Neurospora strains carrying mutations affecting the mitochondria, (a) Strains whose growth rate is resistant to oligomycin (oli^t) owing to an altered protein in the F₀ sector of the mitochondrial ATPase, showed no phase shifts when pulsed with oligomycin. Control strains (oli⁸) showed large phase shifts when pulsed with oligomycin. This indicates that the phase-shifting effect of oligomycin is due to the direct inhibition of the mitochondrial ATPase and not some side effect of this inhibitor, (b) In Neurospora, many different strains are known that carry mutations in the nuclear or mitochondrial genome that affect mitochondrially localized proteins. Some of these, such as oli', [MI-3], or cya-5, showed shorter (≥ 19-h) periods compared with the normal (21.5-h) period. Others showed little or no change in period. Those mutant strains exhibiting shorter periods also contained ≥60% more mitochondrial protein per gram total protein in extracts compared with the normal strains. Assays of the level of a mitochondrial-specific protein, acyl carrier protein, showed that the cellular content of this protein was approximately doubled. A parallel set of studies on the effects of antimycin or chloramphenicol on Neurospora demonstrated that these inhibitors also produced shorter periods as well as increased amounts of mitochondrial proteins. These two new lines of evidence may be interpreted to indicate that in Neurospora either some part of the oscillator is localized to the mitochondria and/or that mitochondria exert their effect on the clock mechanism through their effects on biosynthetic pathways or by their contribution in determining ion gradients.     

Mitochondrial resistance to miconazole in Saccharomyces cerevisiae

Summary One mutant of mitochondrial origin resistant to miconazole has been isolated and characterized in S. cerevisiae. The mutation is linked to the locus oli1, the structural gene for subunit 9 of ATPase on mitochondrial DNA. Miconazole inhibited the mitochondrial ATPase of the wild type while the enzyme of the resistant mutant was insensitive to this effect. Levels of ATP decreased to one-third of the control in the wild type in the presence of miconazole, while they were unaffected in the mutant.Abbreviations MNNG N-methyl-N-nitrosoguanidine - Mic^s/Mic^r phenotypic sensitivity/resistance to miconazole - M ₁ ^R mitochondrial locus conferring miconazole resistance - rho⁺/rho^- grand/cytoplasmic petite - rho^o cytoplasmic petite deleted of all mitochondrial DNA - w⁺ mitochondrial locus conferring polarity of recombination     

Effect of oligomycin on coupling in isolated mitochondria from oligomycin-resistant mutants of Saccharomyces cerevisiae carrying an oligomycin-resistant ATP phosphohydrolase.

Mutations at either of the two OLI 1 and OLI 2 loci on mitochondrial DNA of Saccharomyces cerevisiae confer high oligomycin resistance to cell growth, but only moderate oligomycin resistance to the ATPase in isolated mitochondria, the degree of resistance being characteristic of each mutant. For the most highly resistant mutant (locus OLI 1) the moderate resistance of the ATPase reaction is considerably magnified at the coupling level in isolated mitochondria. For the mutant at locus OLI 2 the coupling properties exhibit the same oligomycin sensitivity as for the wild type, contrasting with the oligomycin resistance of the ATPase and of cell growth. A conformational hypothesis is proposed to account for this finding.     

Genetic determination of ubiquinol-cytochromec reductase

Summary In order to find new genetic loci on the yeast mitochondrial DNA, especially mutations affecting the structure and function of ubiquinol-cytochrome c reductase, 45 independently arisen mutants resistant to mucidin have been isolated after MnCl₂ mutagenesis. The majority of the mutants exhibited increased sensitivity to chloramphenicol, diuron and antimycin A, respectively. it was shown by several criteria that all mutants resulted from mutations localized on the mitochondrial DNA.The allelism tests revealed that these mutations fall into three distinct loci muc1, muc2 and muc3. Mutations at a new locus muc3 were correlated with the changes in the binding or inhibitory sites on the inner mitochondrial membrane. Multifactorial crosses involving the mucidin resistance mutations and mitochondrial mutations conferring resistance to chloramphenicol, erythromycin, oligomycin and diuron revealed that the studied mutations at the loci muc1, muc2 and muc3 did not significantly influence the process of mitochondrial recombination and its control by the mitochondrial locus . The locus muc1 was found to be allelic to the locus diu2. The locus muc2 which was found to be allelic to cob1 locus appears to be linked to the locus oli1 but unlinked to the loci , cap1, ery1 and muc1. The new locus muc3 appears to be weakly linked to the locus diu1 but unlinked to the loci , cap1, ery1, oli1 and muc1.The results are consistent with the gene order oli1-muc2-muc3-diu1-muc1-oli2 and suggest the participation of at least three mucidin resistance loci and one diuron resistance locus in the biogenesis of the bc ₁ complex of the mitochondrial respiratory chain.     

Is there a chloride ATPase in the gills of the shore crab Carcinus maenas?

Summary In gills of the shore crab Carcinus maenas an ATPase activity was found which was stimulated by bicarbonate and inhibited by low concentration of oligomycin and thiocyanate. This ATPase was activated by small hydrated alkali cations, i.e., activation was absent in the presence of Li⁺, small in the presence of Na⁺, and highest in the presence of K⁺ (K _m=4 mM). Inhibitor studies using ouabain, NEM, and vanadate suggest that this ATPase is different from (Na⁺+K⁺)-ATPase, the H⁺-ATPase of organelles, or an E ₁ E ₂-type ATPase represented by the H⁺/K⁺-ATPase in gastric mucosa. Results obtained by differential and density gradient centrifugation indicate that this ATPase is located in crab gill mitochondria, a location ruling out its direct participation in transepithelial ion transport. Since the ATPase lacked specific Cl^--activation it is not considered to be a Cl^- pump but a mitochondrial F ₁ F ₀-ATPase. Specific activities of mitochondrial ATPase and (Na⁺+K⁺)-ATPase were of comparable magnitude. Both ATPases were greatly increased in gills of crabs acclimated to brackish water (salinity 10) compared to crabs maintained in sea water (30). These results imply that low salinity-induced modifications in branchial tissues include mechanisms for active ion uptake as well as the elements for provision of cellular energy.Abbreviations ATPase adenosine triphosphatase - HEPES N-(2-hydroxyethyl)-1-piperazine-N(2-ethanesulfonic acid) - LDH lactate dehydrogenase - NADH reduced nicotinamide adenine dinucleotide - NEM Niethylmaleimide - PEP phosphoenolpyruvate - PK pyruvate kinase - TRIS TRIS (hydroxymethyl)aminomethane - S salinity     

A proton-translocating adenosine triphosphatase is associated with the peroxisomal membrane of yeasts

The association of an ATPase with the yeast peroxisomal membrane was established by both biochemical and cytochemical procedures. Peroxisomes were purified from protoplast homogenates of the methanol-grown yeast Hansenula polymorpha by differential and sucrose gradient centrifugation. Biochemical analysis revealed that ATPase activity was associated with the peroxisomal peak fractions which were identified on the basis of alcohol oxidase and catalase activity. The properties of this ATPase closely resembled those of the mitochondrial ATPase of this yeast. The enzyme was Mg²⁺-dependent, had a pH optimum of approximately 8.5 and was sensitive to N,N-dicyclohexylcarbodiimide (DCCD), oligomycin and azide, but not to vanadate. A major difference was the apparent K _m for ATP which was 4–6 mM for the peroxisomal ATPase compared to 0.6–0.9 mM for the mitochondrial enzyme.Cytochemical experiments indicated that the peroxisomal ATPase was associated with the membranes surrounding these organelles. After incubations with CeCl₃ and ATP specific reaction products were localized on the peroxisomal membrane, both when unfixed isolated peroxisomes or formaldehyde-fixed protoplasts were used. This staining was strictly ATP-dependent; in controls performed i) in the absence of substrate, ii) in the presence of glycerol 2-phosphate instead of ATP, or iii) in the presence of DCCD, staining was invariably absent. Similar staining patterns were observed in subcellular fractions and protoplasts of Candida utilis and Trichosporon cutaneum X4, grown in the presence of ethanol/ethylamine or ethylamine, respectively.Abbreviations MES 2-(N-Morpholino)ethanesulfonic acid - DCCD N,N-dicyclohexylcarbodiimide     

Nucleo-cytoplasmic interaction between oligomycin-resistant mutations in Saccharomyces cerevisiae

1.A single-gene nuclear mutant of Saccharomyces cerevisiae, isolated as oligomycin-resistant, exhibits in vivo cross-resistance to venturicidin and collateral sensitivity to Synthalin. All three compounds are inhibitors of mitochondrial oxidative phosphorylation. Oligomycin resistance and Synthalin sensitivity are recessive, while venturicidin resistance is dominant. 2. Acytoplasmic mutant, also isolated as oligomycin-resistant, shows collateral sensitivity to both Synthalin and venturicidin. All three traits undergo mitotic segregation in diploids formed by crossing mutant and normal halpoids. 3. A novel nucleocytoplasmic interaction is observed in diploids formed by crossing haploid strains containing the nuclear and the cytoplasmic mutations, respectively. The dominant venturicidin resistance determined by the nuclear gene undergoes mitotic segregation, which results from a suppression of the nuclear phenotype by the cytoplasmic mutation. When a diploid mitotic segregant contains primarily mutant-type mitochondria, venturicidin resistance is completely suppressed. In haploids containing both the nuclear and cytoplasmic mutations, suppression is only partial. 4. Oxidative phosphorylation and ATPase in mitochondrial fractions isolated fromcytoplasmic mutant cells are less sensitive to inhibition by oligomycin than normal, but in vitro sensitivity to venturicidin is not significantly changed. In similar mitochondrial fractions isolated from normal and nuclear mutant cells, no significant differences in sensitivity to either inhibitor are detected. 5. The molecular basis for the nucleocytoplasmic suppression of venturicidin resistance may involve participation of mitochondrial membrane, plasma membrane or both. Either mitochondria can undergo changes in venturicidin sensitivity upon isolation, or the molecular entity which controls access of venturicidin to the mitochondria resides outside of the organelles. 6. Our data establish that aspects of the response in vivo of both venturicidin and Snythalin are controlled by the mitochondrial genome. 7. The nucleocytoplasmic interaction described here is the first example in which a specific restricted mitochondrial mutation modifies the phenotypic expression of a nuclear gene.     

Biogenesis of mitochondria 23. The biochemical and genetic characteristics of two different oligomycin resistant mutants ofSaccharomyces cerevisiae under the influence of cytoplasmic genetic modification

Two classes ofSaccharomyces cerevisiae mutants resistant to oligomycin, an inhibitor of mitochondrial membrane bound ATPase are described. Biochemical analysis shows thatin vitro the mitochondrial ATPase of both types of mutant are sensitive to oligomycin.In vivo sensitivity of the mutants to oligomycin can be demonstrated following anaerobic growth of the cells, which grossly alters the mitochondrial membrane and renders the ATPase of the mutants sensitive to oligomycin. It is concluded that the mutation to oligomycin resistance in both mutant types is phenotypically expressed as a change in the mitochondrial membrane. The intact mitochondrial membrane in the wild type cell is freely permeable to oligomycin, whereas the resistant mutant is impermeable to oligomycin; alteration of the mitochondrial membrane during isolation of the organelle or physiological modification of the membranes of the mitochondria by anaerobic growth renders the membranes permeable.These mitochondrial membrane mutants differ in their cross-reference patterns and their genetics. One is resistant to oligomycin only, and behaves like previously reported cytoplasmic mutants. The other shows cross-resistance to inhibitors of mitochondrial protein synthesis as well as to oligomycin; although the mutant appears to arise from a single step mutation its genetic properties are complex and show part-nuclear and part-cytoplasmic characteristics. The implications of the observations are discussed.     

Nuclear and mitochondrial revertants of a mitochondrial mutant with a defect in the ATP synthetase complex

Summary Yeast strain 990 carries a mutation mapping to the oli1 locus of the mitochondrial genome, the gene encoding ATPase subunit 9. DNA sequence analysis indicated a substitution of valine for alanine at residue 22 of the protein. The strain failed to grow on nonfermentable carbon sources such as glycerol at low temperature (20°C). At 28°C the strain grew on nonfermentable carbon sources and was resistant to the antibiotic oligomycin. ATPase activity in mitochondria isolated from 990 was reduced relative to the wild-type strain from which it was derived, but the residual activity was oligomycin resistant. Subunit 9 (the DCCD-binding proteolipid) from the mutant strain exhibited reduced mobility in SDS-polyacrylamide gels relative to the wild-type proteolipid. Ten revertant strains of 990 were analyzed. All restored the ability to grow on glycerol at 20°C. Mitotic segregation data showed that eight of the ten revertants were attributable to mitochondrial genetic events and two were caused by nuclear events since they appeared to be recessive nuclear suppressors. These nuclear mutations retained partial resistance to oligomycin and did not alter the electrophoretic behavior of subunit 9 or any other ATPase subunit. When mitochondrial DNA from each of the revertant strains was hybridized with an oligonucleotide probe covering the oli1 mutation, seven of the mitochondrial revertants were found to be true revertants and one a second mutation at the site of the original 990 mutation. The oli1 gene from this strain contained a substitution of glycine for valine at residue 22. The proteolipid isolated from this strain had increased electrophoretic mobility relative to the wild-type proteolipid.Abbreviations DCCD dicyclohexylcarbodiimide - SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - SMP submitochondrial particles - mit^- mitochondrial point mutant     

Protoplast fusion mediated transfer of oligomycin resistance from Nicotiana sylvestris to Solanum tuberosum by intergeneric cybridization

Summary We have successfully bridged the intergeneric barriers between Nicotiana and Solanum with respect to chondriome transfer. To enable this transfer we utilized the donor-recipient protoplast-fusion procedure. Consequently protoplasts of a Nicotiana sylvestris line with putativly oligomycin-resistant mitochondria (line Oli ^R 38) were used as irradiated chondriome donors and iodoacetate-treated protoplasts of Solanum tuberosum cv. Desiree served as recipients. The plated fusion products as well as their derived colonies and calli were exposed to gradually increasing levels of oligomycin. The resulting plantlets had potato morphology and were analyzed with respect to their mitochondrial DNA and chloroplast DNA. Fifteen out of 50 regenerated plants were verified as true cybrids. Detailed analyses of one cybrid revealed chondriome components from the oligomycin-resistant donor line, Oli ^R 38, but retention of the plastome of potato. This cybrid was oligomycin-resistant as revealed by root-culture analysis. It was thus verified that due to selection, chondriome components could be transferred from a N. sylvestris donor into a cybrid having all the phenotypic features controlled by the nucleus of the recipient fusion partner (S. tuberosum).     

ATPase-Associated Oligomycin Resistance in Acanthamoeba castellanii1

ABSTRACT. The multiplication rate of “wild-type” (WT) populations of Acanthamoeba castellanii was inhibited 50% by ～3 μg otigomycin/ml; Oli^R2 an oligomycin resistant cell line, required ～27 μg/ml for the same inhibition. ATPase solubilized from Oli^R2 mitochondrial fractions required 3–10-fold higher concentrations of oligomycin than did identical WT fractions to achieve 50% inhibition of activity. Resistance was correlated with altered mitochondrial ATPase sensitivity to oligomycin.     

Extrachromosomal oligomycin-resistant mutants of the petite-negative yeast Kluyveromyces lactis. Properties of mitochondrial ATPase and cross-resistance to inhibitors of phosphoryl transfer reactions

Summary The mitochondrial ATPase from oligomycin-resistant mutants which map on different regions of an extrachromosomal DNA (01 and 011 class mutants) showed an increased resistance to oligomycin and venturicidin when assayed in vitro as compared to the sensitive strains.The resistance to oligomycin of the isolated mitochondrial ATPase from 01 class mutants was higher than that of the 011 class mutants.Cross resistance of the oligomycin-resistant mutants to the antibiotics peliomycin and ossamycin, which also inhibit phosphoryl transfer reactions in mitochondria (Walter et al., 1967), was observed, 01 mutants being more resistant to ossamycin than 011 class mutants. At the concentrations of peliomycin studied, no difference in sensitivity among both groups of oligomycin-resistant mutants could be detected.Mitochondrial respiration and isolated mitochondrial ATPase activity are sensitive to venturicidin, suggesting that the previously observed (Brunner et al., 1977) in vivo venturicidin resistance of K. lactis is probably due to an impairment of the influx of the drug at the level of the plasma membrane.     

Fusion of mitochondria with protoplasts in Saccharomyces cerevisiae.

Summary Protoplasts prepared from a neutral petite haploid BO60AF-1 (a ade2 arg4 leu2 trp C ^O E ^O O ^{O O O}) were mixed with mitochondria isolated from an oligomycin resistant respiring haploid AN^ROR 12D (a his4 leu2 thr4 C ^S E ^S O _II ^{R + +}) and treated with 30% polythylene glycol and CaCl₂. When the treated protoplasts were spread and incubated on selective agar plates, oligomycin resistant respiration-sufficient colonies appeared with low frequency. All of these colonies carried the mitochondrial genotype of C ^S E ^S O _II ^{R + +} and showed the same mating type and nutritional requirements as did BO60AF-1, thus evidencing the mitochondrial transfer into protoplasts. Recombination and transmission of the mitochondrial drug resistance markers were studied in crosses involving the strains issued from mitochondria accepted protoplasts.     

Mutants in yeast affecting ethidium bromide induced rho- formation and their effects on transmission and recombination of mitochondrial genes.

Summary A series of mutants called ebi, less inducible by ethidium bromide than the parental strain for the ^+– mutation have been isolated after E.M.S. mutagenesis. Some of the ebi mutants also show an important accumulation of ^– cells, in the absence of ethidium bromide. Ebi mutations are nuclearly inherited as shown by meiotic segregation. The effects of these mutants on the transmission and recombination of mitochondrial genes among the diploid progeny of crosses have been studied. Some of the ebi mutants show a non coordinated transmission of the oli1 mitochondrial marker with respect to other mitochondrial markers unexpected for homosexual crosses. This bias which is independent from will be discussed in relation to the segregation and recombination. No significant decrease of the frequency of recombinants has been detected.Abbreviations E.B. Ethidium bromide - E.M.S. Ethyl méthane sulfonate - C^S/C^R Allelic forms of the rib 1 locus conferring chloramphenicol sensitivity/resistance - E^S/E^R Allelic forms of the rib 3 locus conferring erythromycine sensitivity/resistance - O^R/O^R Allelic forms of the oli 1 locus conferring oligomycin sensitivity/resistance - P^S/P^R Allelic forms of the par 1 locus conferring paromomycine sensitivity/resistance - D^S/D^R Allelic forms of the diu 1 or diu 2 loci conferring diuron sensitivity/resistance - C^S/C^R Allelic forms of the mitochondrial locus - ⁺ grande or respiratory competent cells - ^– petite or cytoplasmic respiratory deficient cells     

Genetics of the mammalian oxidative phosphorylation system: characterization of a new oligomycin-resistant Chinese hamster ovary cell line.

The properties of a new type of oligomycin-resistant Chinese hamster ovary (CHO) cell line (Olir 2.2) are described in this paper. Olir 2.2 cells were approximately 50,000-fold more resistant to oligomycin than were wild-type CHO cells when tested in glucose-containing medium, but only 10- to 100-fold more resistant when tested in galactose-containing medium. Olir 2.2 cells grew with a doubling time similar to that of wild-type cells both in the presence or absence of oligomycin. Oligomycin resistance in Olir 2.2 cells was stable in the absence of drug. In vitro assays indicated that there was approximately a 25-fold increase in the resistance of the mitochondrial ATPase to inhibition by oligomycin in Olir 2.2 cells, with little change in the total ATPase activity. The electron transport chain was shown to be functional in Olir 2.2 cells. Olir 2.2 cells were cross-resistant to other inhibitors of the mitochondrial ATPase (such as rutamycin, ossamycin, peliomycin, venturicidin, leucinostatin, and efrapeptin) and to other inhibitors of mitochondrial functions (such as chloramphenicol, rotenone, and antimycin). Oligomycin resistance was expressed codominantly in hybrids between Olir 2.2 cells and wild-type cells. Cross-resistance to ossamycin, peliomycin, chloramphenicol, antimycin, venturicidin, leucinostatin, and efrapeptin was also expressed codominantly in hybrids. Fusions of enucleated Olir 2.2 cells with wild-type cells and characterization of the resulting cybrid clones indicated that resistance to oligomycin and ossamycin results from a mutation in both a nuclear gene and a cytoplasmic gene. Cross-resistance to efrapeptin, leucinostatin, venturicidin, and antimycin results from a mutation in only a nuclear gene.     

Transformation of Aspergillus nidulans with a cloned, oligomycin-resistant ATP synthase subunit 9 gene

Summary An allele (oliC31) of the A. nidulans oliC gene has been cloned using homology with the equivalent gene from N. crassa. OliC31 codes for an oligomycin-resistant, triethyltin-hypersensitive form of subunit 9 of the mitochondrial ATP synthase complex. Direct selection for oligomycin-resistance was possible following transformation of A. nidulans with the oliC31 gene. The phenotypes of transformants cultured in the presence of oligomycin were indicative of the position of integration of the transforming plasmid within the genome. Subsequent recombination events involving the integrated oliC31 gene were also apparent from altered levels of resistance to oligomycin or triethyltin. This gene should prove useful as a marker for transformation of strains lacking auxotrophic lesions and in gene replacement or disruption experiments.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Modification and Inheritance of Pleiotropic Cross Resistance and Collateral Sensitivity in SACCHAROMYCES CEREVISIAE

A meiotic segregant (oli^PR1) was isolated with a phenotype of multiple cross resistance and collateral sensitivity. Strain oli^PR1 has increased sensitivity to ethidium bromide, dequalinium chloride, acriflavin, paromomycin and neomycin, and increased resistance to oligomycin, rutamycin, venturicidin, triethyltin bromide, antimycin, carbonylcynamide-m-chlorophenylhydrazone, tetra-N-butylammonium bromide, dibenzyldimethylammonium chloride, triphenylmethylphosphonium bromide, chloramphenicol, carbomycin, tetracycline, triton-X-165 and cycloheximide. Single gene inheritance of the cross resistance and collateral sensitivity was shown by 2:2 parental ditype segregation and reversion of the complete phenotype by a spontaneous revertant. The locus conferring the oli^PR1 phenotype was mapped 11.7 units from an unspecified centromere. Antibiotic resistance showed incomplete dominance, with the level of hybrid resistance dependent upon the inhibitor tested. Resistant diploids that produced four resistant ascospores were the result of mitotic recombination prior to meiosis. A partial revertant phenotype (sensitive to all inhibitors except oligomycin, antimycin and carbonylcyanide-m-chlorophenylhydrazone) was shown to be due to a single nuclear gene causing partial suppression of oli^PR1. Anaerobic pretreatment, 37° and 0.5 M KCl were observed to reduce the growth of oli^PR1 when challenged with seven diverse inhibitors (antimycin, carbonylcyanide-m-chlorophenylhydrazone,-chloramphenicol, cycloheximide, oligomycin, triethyltin bromide, and triphenylmethylphosphonium bromide). Resistance to cycloheximide was not altered by the [rho—] state. A revertant of oli^PR1 (sensitive to the above inhibitors but resistant to ethidium bromide, paromycin and neomycin) showed anaerobic and temperature sensitization to ethidium bromide, paromomycin and neomycin. Continuous monitoring of oxygen uptake by the revertant after anaerobic pretreatment revealed that anaerobiosis sensitized respiratory adaptation of the revertant to neomycin. It is proposed that oli^PR1 is a mutation resulting in the alteration of plasma membrane premeability to many diverse inhibitors.     

Genetic engineering of drought-resistant tobacco plants by introducing the trehalose phosphorylase (TP) gene from <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis>

This study generated transgenic tobacco plants expressing trehalose phosphorylase of Pleurotus sajor-caju (PsTP) constitutively under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Sixteen transgenic lines were selected by genomic Southern blot analysis for further study. Unlike yeast TPS1-transformed or Escherichia coli TPS1-transformed tobacco or potato, all of the PsTP transgenic tobacco lines showed normal growth phenotypes both in the culture tubes and soil mixture. The study measured the trehalose contents of PsTP-transformed tobacco plants as well as the wild type and empty vector-transformed control plants. Results showed that the PsTP transformant contained 6.3molg^–1 of plant tissues, while the wild type and the control plants had only minimal levels of trehalose. Because this study detected a significant amount of trehalose in PsTP transgenic tobacco plants, it decided to carry out a bioanalysis of the PsTP transgenic tobacco plants by drought treatment by not watering the plants for over 10days. A significant difference in drought resistance was observed from the second nonwatering day between the transgenic and the control tobacco plants. The transgenic tobacco plants had normal growth and did not wither, while the wild type and the only empty vector-transformed control plants withered severely. Among all the transgenic lines, line 10-4 showed the strongest resistance to drought stress. It did not wither even after 10days without watering. In addition, when the drought resistance of PsTP transgenic tobacco plants was tested using detached leaves, most transgenic plants, except one line, showed better capacity to retain water than the empty vector-transformed transgenic plant.