Gene controlled selection of mitochondria in Paramecium.

Summary The slow growing mutant cl₁ of Paramecium, previously described (Sainsard, Claisse and Balmefrezol, 1974) differs from wild-type by a single recessive nuclear mutation and by a particular mitochondrial phenotype (M^cl) that gene cl ₁ distinguishes from the wild-type mitochondrial phenotype (M⁺). A further analysis of these nucleo-mitochondrial interactions was carried out by confronting the genes cl ₁ and cl ₁ ⁺ with mixed populations of M⁺ and M^cl mitochondria obtained after cytoplasmic exchange at conjugation. The following results were obtained: 1. M⁺ and M^cl mitochondria introduced respectively into mutant and wild-type cells do not multiply easily; 2. when a mixed population (M⁺+M^cl) is established, both mitochondrial types are maintained during the growth of the F₁ heterozygous cl ₁/cl ₁ ⁺ clones; 3. when the nuclear segregation occurs in F₂, the formation of homozygotes cl ₁/cl ₁ or cl ₁ ⁺ /cl ₁ ⁺ is soon followed by the segregation of the two mitochondrial types, M^cl or M⁺, reconstituting the two parental nucleo-mitochondrial associations.This paper is dedicated to Professor T.M. Sonneborn on the occasion of his 70th birthday     

Genetic interactions in the control of mitochondrial functions in Paramecium

Summary The genetic and physiological properties of two nuclear mutants of Parameccium tetraurelia affecting mitochondrial properties, and first screened as resistant to tetrazolium (TTC) are described. The mutant TTC _64-1 ^R is strongly deficient in cytochrome c and the mutant TTC _66p ^R is partially deficient in cytochrome aa₃; both mutants display cyanide insensitive respiration in exponential growth phase. In the double mutant TTC _64-1 ^R -TTC _66p ^R /TTC _64-1 ^R -TTC _66p ^R the deficiency in cytochrome aa₃ due to the TTC _64-1 ^R mutation is suppressed. The mutation TTC _64-1 ^R does not suppress cytochrome aa₃ deficiencies due to mitochondrial mutations, but does interact with another nuclear mutation, cl ₁, (compatible only with mitochondria deficient in cytochrome oxidase) in such a way that the double mutant TTC _64-1 ^R -cl ₁/TTC _64-1 ^R -cl ₁ displays a normal amount of cytochrome aa₃. The possible mechanisms and physiological significance of these suppressive effects are discussed.Abbreviations TTC^R/TTC^S resistant/sensitive to tetrazolium - KCN^R/KCN^S cyanide insensitive/sensitive respiration - aa ₃ ^- /aa ₃ ⁺ deficient/normal amount of cytochrome aa₃ - c^-/c⁺ deficient/normal amount of cytochrome c     

Altered Kinetic Properties of Tyrosine-183 to Cysteine Mutation in Glutamine Synthetase of <Emphasis Type="Italic">Anabaena variabilis</Emphasis> Strain SA1 Is Responsible for Excretion of Ammonium Ion Produced by Nitrogenase

A L-methionine-D,L-sulfoximine-resistant mutant of the cyanobacterium Anabaena variabilis, strain SA1, excreted the ammonium ion generated from N₂ reduction. In order to determine the biochemical basis for the NH₄ ⁺-excretion phenotype, glutamine synthetase (GS) was purified from both the parent strain SA0 and from the mutant. GS from strain SA0 (SA0-GS) had a pH optimum of 7.5, while the pH optimum for GS from strain SA1 (SA1-GS) was 6.8. SA1-GS required Mn⁺² for optimum activity, while SA0-GS was Mg⁺² dependent. SA0-GS had the following apparent K _m values at pH 7.5: glutamate, 1.7 mM; NH₄ ⁺, 0.015 mM; ATP, 0.13 mM. The apparent K _m for substrates was significantly higher for SA1-GS at its optimum pH (glutamate, 9.2 mM; NH₄ ⁺, 12.4 mM; ATP, 0.17 mM). The amino acids alanine, aspartate, cystine, glycine, and serine inhibited SA1-GS less severely than the SA0-GS. The nucleotide sequences of glnA (encoding glutamine synthetase) from strains SA0 and SA1 were identical except for a single nucleotide substitution that resulted in a Y183C mutation in SA1-GS. The kinetic properties of SA1-GS isolated from E. coli or Klebsiella oxytoca glnA mutants carrying the A. variabilis SA1 glnA gene were also similar to SA1-GS isolated from A. variabilis strain SA1. These results show that the NH₄ ⁺-excretion phenotype of A. variabilis strain SA1 is a direct consequence of structural changes in SA1-GS induced by the Y183C mutation, which elevated the K _m values for NH₄ ⁺ and glutamate, and thus limited the assimilation of NH₄ ⁺ generated by N₂ reduction. These properties and the altered divalent cation-mediated stability of A. variabilis SA1-GS demonstrate the importance of Y183 for NH₄ ⁺ binding and metal ion coordination. Received: 3 July 2002 / Accepted: 29 July 2002     

A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA

We have isolated a Saccharomyces cerevisiae mutant that shows an increased tendency to form cytoplasmic petites (respiration-deficient ρ⁻ or ρ⁰ mutants) in response to treatment of cells growing on a solid medium with the DNA-damaging agent methyl methanesulfonate or ultraviolet light. The mutation in this strain, atm1-1, was found to cause a single amino acid substitution in ATM1, a nuclear gene that encodes the mitochondrial ATP-binding cassette (ABC) transporter. When the mutant cells were grown in liquid glucose medium, they accumulated free iron within the mitochondria and at the same time gave rise to spontaneous cytoplasmic petite mutants, as seen previously in cells carrying a mutation in a gene homologous to the human gene responsible for Friedreich's ataxia. Analysis of the effects of free iron and malonic acid (an inhibitor of oxidative respiration in mitochondria) on the incidence of petites among the mutant cells indicated that spontaneous induction of petites was a consequence of oxidative stress rather than a direct effect of either a defect in the ATM1 gene or the accumulation of free iron. We observed an increase in the incidence of strand breaks in the mitochondrial DNA of the atm1-1 mutant cells. Furthermore, we found that rates of induction of petites and accumulation of strand breaks in mitochondrial DNA were enhanced in the atm1-1 mutant by the introduction of another mutation, mhr1-1, which results in a deficiency in mitochondrial DNA repair. These observations indicate that spontaneous induction of petites in the atm1-1 mutant is a consequence of oxidative damage to mitochondrial DNA mediated by enhanced accumulation of mitochondrial iron. Received: 26 March 1999 / Accepted: 29 June 1999     

Induction of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> Pyruvate Decarboxylase Genes

A thalium chloride-resistant (TlCl^r) mutant strain and a sodium chloride-resistant (NaCl^r) mutant strain of the diazotrophic cyanobacterium Anabaena variabilis have been isolated by spontaneous and chemical mutagenesis by using TlCl, a potassium (K⁺) analog, and nitrosoguanidine (NTG), respectively. The TlCl^r mutant strain was found to be defective in K⁺ transport and showed resistance against 10 μM TlCl. However, it also showed sensitivity against NaCl (LD₅₀, 50 mM). In contrast, neither wild-type A. variabilis nor its NaCl^r mutant strain could survive in the presence of 10 μM TlCl and died even at 1 μM TlCl. The TlCl^r mutant strain exhibited almost negligible K⁺ uptake, indicating the lack of a K⁺ uptake system. High K⁺ uptake was, however, observed in the NaCl^r mutant strain, reflecting the presence of an active K⁺ uptake system in this strain. DCMU, an inhibitor of PS II, inhibited the K⁺ uptake in wild-type A. variabilis and its TlCl^r and NaCl^r mutant strains, suggesting that K⁺ uptake in these strains is an energy-dependent process and that energy is derived from photophosphorylation. This contention is further supported by the inhibition of K⁺ uptake under dark conditions. Furthermore, the inhibition of K⁺ uptake by KCN, DNP, and NaN₃ also suggests the involvement of oxidative phosphorylation in the regulation of an active K⁺ uptake system. The whole-cell protein profile of wild-type A. variabilis and its TlCl^r and NaCl^r mutant strains growing in the presence of 50 mM KCl was made in the presence and absence of NaCl. Lack of transporter proteins in TlCl^r mutant strain suggests that these proteins are essentially required for the active transport and accumulation of K⁺ and make this strain NaCl sensitive. In contrast, strong expression of the transporter proteins in NaCl^r mutant strain and its weak expression in wild-type A. variabilis is responsible for their resistance and sensitivity to NaCl, respectively. Therefore, it appears that the increased salt tolerance of the NaCl^r mutant strain was owing to increased K⁺ uptake and accumulation, whereas the salt sensitivity of the TlCl^r mutant strain was owing to the lack of K⁺ uptake and accumulation. Received: 7 March 2002 / Accepted: 8 April 2002     

Two Cell Division Cycle Mutants of SACCHAROMYCES CEREVISIAE Are Defective in Transmission of Mitochondria to Zygotes

Mutations in CDC genes of S. cerevisiae disrupt the cell cycle at specific stages. The experiments reported here demonstrate that two CDC genes, CDC5 and CDC27, are necessary for mitochondrial segregation as well as for nuclear division. The defect in the transmission of mitochondria was revealed by the examination of uninucleate and binucleate progeny of transient heterokaryons generated by using the kar1-1 mutation that disrupts nuclear fusion. One of the parents lacked mitochondrial DNA (ρ⁰) whereas the other parent had functional mitochondria (ρ⁺). When the parents of the heterokaryon were both wild-type (CDC), nearly all progeny received mitochondria at 21° and at 34°. Thirty-four of the 36 cdc mutations tested had no defect in transmission of mitochondria to zygotic progeny in crosses in which one parent was a cdc mutant and the other parent was not (CDC). However, the cdc5 and cdc27 mutations prevented the transmission of mitochondria to cdc progeny at 34° but not at 21°; CDC progeny received mitochondria at either temperature. This defect was observed in crosses of cdc5 or cdc27 by wild-type cells regardless of which parent donated mitochondria to the zygote. The defect in mitochondrial transmission cosegregated in meiotic tetrads with the defect in mitosis demonstrating that both are likely to be caused by the same temperature-sensitive mutation. These results indicate that the CDC5 and CDC27 gene products are essential in two motility-related processes: mitochondrial movement from the zygote to the progeny and in mitosis.—Furthermore, the results suggest that the function performed by the CDC5 and CDC27 gene products for mitochondrial transmission differ in some fundamental way from the function performed for mitosis. The function necessary for mitosis can be supplied to the cdc5 (or cdc27) nucleus by the CDC5 (or CDC27) nucleus in the same heterokaryon but the function necessary for mitochondrial transmission cannot. Perhaps the function needed for mitochondrial transmission must be performed in the cell cycle preceding the actual segregation of mitochondria whereas the function needed for nuclear segregation can be performed at the time that mitosis occurs.     

In vitro mitochondrial complementation in Neurospora crassa

Mitochondrial isoleucine-valine biosynthesis in strain 330a, an iv-1 mutant of Neurospora, is blocked at the dihydroxy acid dehydration step owing to a mutation in the nuclear structural gene for the specific enzyme dihydroxy acid dehydratase. Dehydratase purified from either the soluble or the mitochondrial fraction of wild-type Neurospora, and incubated in vitro with 330a mitochondria, restores valine synthesis from pyruvate-C ¹⁴ to wild type levels. Up to 29% of the restored synthesis could be attributed to the penetration of enzyme into the mitochondria. However, the bulk of the restored synthesis was found to be mediated via the secretion of dihydroxyvaline (DHV) by the mitochondria into the assay milieu, with subsequent enzymatic catalysis of this metabolite to ketovaline occurring outside the organelle. The ketovaline apparently diffuses back into mitochondria for final transamination to valine. This shunting of valine precursors in and out of mitochondria has been demonstrated to be the mechanism whereby two different populations of mitochondria isolated from mutants 330a and 305A (an iv-2 mutant lacking a functional reductoisomerase) can complement each other for the biosynthesis of valine, even when each population is enclosed in a separate dialysis bag. This observation provides the basis for a biochemical understanding of the growth complementation at the organismic level when these two iv-requiring mutants are cultured together in minimal medium.Work supported by grants GM 12323 and 2TO1 GM 00337 from the National Institutes of Health, USPHS, and a grant from the Robert A. Welch Foundation, Houston, Texas.     

Mutations by near-ultraviolet radiation in Escherichia coli strains lacking superoxide dismutase

Om wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic. However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O₂⁻), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant. When a sodA sodB double mutant contained a plasmid carrying katG⁺ HP-I catalase), mutation by NUV was reduced to wild-type (sodA⁺sodB⁺) levels. Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutations frequency by NUV was reduced to wild-type levels. This synergistic action of NUV and O₂⁻ suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants. Exposure to H₂O₂ induced a 2.8 fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG⁺ was introduced. These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH^. radicals, possibly by generating excess H₂O₂.     

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     

Sorption of Heavy Metal Cations by Corn Mitochondria and the Effects on Electron and Energy Transfer Reactions

The passive sorption of Pb⁺², Cd⁺², Zn⁺², Co⁺², Ni⁺², and Mn⁺² by isolated corn mitochondria was determined, and, except for Pb⁺², the maximum sorption for each cation was about 58 nmol per milligram of protein. Sorption of Pb⁺² was apparently ten times greater, but precipitation may have been the cause of this larger value. The effects of Pb⁺², Cd⁺², Zn⁺², Co⁺², and Ni⁺² on acceptorless rates of electron transport for three substrates were determined. Greater than 50% inhibitions of oxidation were observed for succinate after additions of >0.1 mM Cd⁺², Zn⁺², or Pb⁺²: for NADH after additions of >0.5 mM Cd⁺² or Zn⁺²; and for malate + pyruvate after additions of >0.1 mM Cd⁺². Some inhibition of the rate of substrate oxidation was observed for most cations at higher concentrations. Coupling, as measured by ADP/O ratios, was inhibited at lowest concentrations by Cd⁺² or Zn⁺² and at higher concentrations by Co⁺² or Ni⁺². Substantial swelling of mitochondria oxidizing succinate was observed following additions of O.1 mM Cd⁺² or Pb⁺², Correlations are drawn between the effects of Pb⁺², Cd⁺², Zn⁺², Co⁺², and Ni⁺² and their sorption to mitochondrial membranes.     

The Holliday junction resolvase SpCCE1 prevents mitochondrial DNA aggregation in Schizosaccharomyces pombe

SpCCE1 (YDC2) from Schizosaccharomyces pombe is a DNA structure-specific endonuclease that resolves Holliday junctions in vitro. To investigate the in vivo function of SpCCE1 we made an Spcce1::ura4 ⁺ insertion mutant strain. This strain is viable and, despite being devoid of the Holliday junction resolvase activity that is readily detected in fractionated extracts from wild-type cells, exhibits normal levels of UV sensitivity and spontaneous or UV-induced mitotic recombination. In accordance with the absence of a nuclear phenotype, we show by fluorescence microscopy that a SpCCE1-GFP fusion localises exclusively to the mitochondria of S. pombe. In Saccharomyces cerevisiae the homologue of SpCCE1, CCE1, is known to function in the mitochondria where its role appears to be to remove recombination junctions and thus facilitate mitochondrial DNA segregation. A similar function can probably be attributed to SpCCE1 in S. pombe, since the majority of mitochondrial DNA from the Spcce1::ura4 ⁺ strain is in an aggregated form apparently due to extensive interlinking of DNA molecules by recombination junctions. Surprisingly, this marked effect on the conformation of mitochondrial DNA results in little or no effect on proliferation or viability of the Spcce1::ura4 ⁺ strain. Possible explanations are discussed. Received: 28 October 1999 / Accepted: 28 March 2000     

A mutant of Paramecium with increased relative resting potassium permeability

Fast-2, a membrane mutant of Paramecium aurelia, is due to a single-gene mutation and has behavioral abnormalities. Intracellular recordings through changes of external solutions were made. The mutant membrane hyperpolarized when it encountered solutions with low K⁺ concentration. This hyperpolarization and other associated activities were best observed in Ca- or Na-solutions devoid of K⁺. Membrane potential was plotted against the concentration of K⁺ (0.5 to 16 mM) in solutions of fixed Na⁺ or Ca⁺⁺ concentration. The slopes of the curves for the mutant membrane were steeper than those for the wild type at the lower concentrations of K⁺. Inclusion of 2 mM tetraethylammonium chloride (TEA-Cl) counteracted the mutational effects. Spontaneous action potentials in Ba-solution and the electrically evoked action potentials in various solutions are normal in this mutant. We conclude that the resting permeability to K⁺ relative to the permeabilities to Na⁺ and Ca⁺⁺ has been increased by the mutation.     

Characterization of csi52, a Cs+ resistant mutant of Arabidopsis thaliana altered in K+ transport

Plant roots accumulate potassium from a wide range of soil concentrations, utilizing at least two distinct plasma membrane uptake systems with different affinities for the cation. Although details on the structure and function of these transporters are beginning to emerge many prominent questions remain concerning how these proteins function in planta. Such questions can be addressed through the use of well-defined transport mutants. Csi52, a caesium-insensitive mutant of Arabidopsis thaliana which is defective in potassium transport, is further characterized here using conventional electrophysiology, patch-clamp and radiometric approaches to identify the nature of the potassium transport lesion. Rb⁺ uptake experiments reveal a reduced uptake in csi52 in both the high- and low-affinity uptake range. Patch-clamp analysis indicates that the activity of the predominant inward rectifying channel observed in wild-type cells is extremely low in root protoplasts isolated from csi52, whereas outward rectifying channel activity is comparable between wild-type and mutant. Rb⁺ uptake studies show that in both wild-type and csi52 the high-affinity uptake pathway is considerably less sensitive to Cs⁺ than the low-affinity pathway with K_1/2 values for Cs⁺ of around 1.3 and 0.2 mM, respectively. Furthermore, K⁺ starvation leads to a larger relative increase in high-affinity K⁺ uptake in the mutant than the wild-type. The results demonstrate the Cs⁺ sensitivity of each individual uptake pathway is comparable in wild-type and csi52 but the high-affinity pathway is less Cs⁺ sensitive (in both wild-type and csi52). Therefore, the larger shift toward high-affinity uptake in the mutant compared with the wild-type under K⁺-starvation conditions will endow the mutant with a higher degree of overall Cs⁺ resistance. The data supply evidence for the hypothesis that the csi52 mutation lies within a gene that regulates the activity of several potassium transport systems and coordinates their relative contribution to overall root K⁺ uptake.     

A cold-sensitive cytoplasmic mutation of Saccharomyces cerevisiae affecting assembly of the mitochondrial 50S ribosomal subunit.

Summary A cytoplasmic mutant of Saccharomyces cerevisiae (E23-1) has been isolated that is resistant to erythromycin and cold sensitive for growth on nonfermentable carbon sources at 18°. Genetic analysis has shown that both of these properties probably result from a single mutation at the rib2 locus which maps close to or within the gene for the 21S rRNA of the mitochondrial 50S ribosomal subunit. Electrophoresis of total RNA extracted from purified mitochondria demonstrated that the 21S and 14S rRNA species from both mutant and wild-type cells were present in roughly equimolar quantities regardless of growth temperature. The mutant is therefore not defective in the synthesis of the 21S rRNA. Sucrose gradient analysis of the mitochondrial ribosomes in Mg²⁺-containing buffers revealed that approximate values for the ratio of 50S to 37S subunits were 1:1 for wild-type cells grown at either 18° or 32°, 0.5:1 for the mutant grown at 32° and 0.2:1 for the mutant grown at 18°. The subunit ratios were approximately 1:1 when Ca²⁺-containing buffers were used, however, In alls cases, 50S particles from the mutant grown at 18° lacked or contained markedly reduced amounts of two distinctive protein components that were present in the mutant at 32° and in the wild-type at both temperatures. In addition, no intact 21S RNA could be recovered from the mitochondrial ribosomes of the mutant grown at the restrictive temperature, even in the presence of Ca²⁺. These findings indicate that mitochondrial 50S ribosomal subunits produced by the mutant at 18° are structurally defective and raise the possibility that the defect results from an alteration in the gene for 21S rRNA.A preliminary report of this work was presented at the meeting on The Molecular Biology of Yeast, Cold Spring Harbor Laboratory, August 18–22, 1977     

Suppression of carboxy-terminal truncations of the yeast mitochondrial mRNA-specific translational activator PET122 by mutations in two new genes, MRP17 and PET127

Summary The PET122 protein is one of three Saccharomyces cerevisiae nuclear gene products required specifically to activate translation of the mitochondrially coded COX3 mRNA. We have previously observed that mutations which remove the carboxy-terminal region of PET122 block translation of the COX3 mRNA but can be suppressed by unlinked nuclear mutations in several genes, two of which have been shown to code for proteins of the small subunit of mitochondrial ribosomes. Here we describe and map two more new genes identified as allele-specific suppressors that compensate for carboxy-terminal truncation of PET122. One of these genes, MRP17, is essential for the expression of all mitochondrial genes and encodes a protein of M_r 17343. The MRP17 protein is a component of the small ribosomal subunit in mitochondria, as demonstrated by the fact that a missense mutation, mrp17-1, predicted to cause a charge change indeed alters the charge of a mitochondrial ribosomal protein of the expected size. In addition, mrp17-1, in combination with some mutations affecting another mitochondrial ribosomal protein, caused a synthetic defective phenotype. These findings are consistent with a model in which PET122 functionally interacts with the ribosomal small subunit. The second new suppressor gene described here, PET127, encodes a protein too large (M_r 95900) to be a ribosomal protein and appears to operate by a different mechanism. PET127 is not absolutely required for mitochondrial gene expression and allele-specific suppression of pet122 mutations results from the loss of PET127 function: a pet127 deletion exhibited the same recessive suppressor activity as the original suppressor mutation. These findings suggest the possibility that PET127 could be a novel component of the mitochondrial translation system with a role in promoting accuracy of translational initiation.     

Mechanism and role of furosemide-sensitive K+ transport in L cells: A genetic approach

Summary As part of a genetic study of the mechanisms for cation transport in cultured mammalian cells, two mouse fibroblastic cell lines have been compared with respect to unidirectional⁴²K⁺ influx. The cell lines areLM(TK ^–) andLTK-5, a mutant selected fromLM(TK ^–) by the ability to grow in medium containing 0.2mm K⁺. In both cell lines, the overall influx can be resolved into three components: (i) a ouabain- and vanadate-sensitive component ( ⁱ M_K ^f), presumably the Na/K pump, which is a saturable function of extracellular K⁺ with aK _1/2 of 1.3mm; (ii) a furosemide-sensitive component ( ⁱ M_k ^fx), also a saturable function of extracellular K⁺, with aK _1/2 of 6mm; and (iii) a diffusional component ( ⁱ M_k ^d); which is a linear function of extracellular K⁺.By several independent criteria, ⁱ M_k ^o and ⁱ M_k ^f appear to be distinct transport processes. First, as indicated above, they can be separated with the use of inhibitors. In addition, they can be separated genetically, since theLTK-5 mutant shows a threefold elevation in ⁱ M_k ^f with no change in ⁱ M_k ^o. And finally, extracellular Na⁺ has no effect on ⁱ M_k ^o, but stimulates ⁱ M_k ^f, a result consistent with the notion that ⁱ M_k ^f influx occurs by Na–K cotransport.Further experiments were directed towards understanding the nature of theLTK-5 mutation and the physiological role of ⁱ M_k ^f. LTK-5 differs from the parental cell line, not only in having an increased ⁱ M_k ^f, but also in having a large cell volume, a slow maximal growth rate, and an ability to grow at 0.2mm K⁺. The most straightforward interpretation — that the increased ⁱ M_k ^f is itself responsible—is unlikely since the addition of furosemide to the growth medium had no effect upon the growth rate or cell volume of the mutant at either normal or low extracellular K⁺ concentrations. It did, however, render the parent capable of growth at 0.2mm K⁺. Possible interpretations are discussed.     

Genetic and physiological evidence concerning the development of chemically sensitive voltage-dependent ionophores in L6 cells

The electrophysiological properties of a tissue culture muscle line, L₆, and a K⁺ resistant mutant (MK₁) derived from L₆ were determined to elucidate certain aspects of membrane differentiation and function. MK₁ was selected as a clone of myoblasts resistant to the toxic effects of 55 mM K⁺. The resting potentials of L₆ and MK₁ myoblasts and myotubes were K⁺ dependent and equal. The amplitudes of the action potentials were equal in normal medium, but 27.7 mM K⁺ interfered with or eliminated the ability of L₆ myotubes to produce action potentials. MK₁ myotubes produced nearly normal action potentials under these conditions. Thus, the K⁺ resistant myoblasts differentiate into myotubes which have an action potential generating mechanism much less sensitive to K⁺ than the normal mechanism. Also, both d-tubocurarine and α-bungarotoxin enhance the amplitude of the action potentials produced by L₆ myotubes in the presence of 27.7 mM K⁺; these compounds do not enhance the amplitude of the action potentials produced by MK₁ myotubes under the same conditions. It is proposed that as a consequence of differentiation a type of ionophore present in myoblasts becomes a voltage-dependent ionophore in myotubes. Furthermore, these voltage-dependent ionophores can be chemically sensitive.     

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     

Mitochondrial ribosomal RNA genes of yeast: Their mutations and a common nuclear suppressor

Summary Due to the absence of repetition of the rRNA genes in S. cerevisiae mitochondria, isolation of ribosomal mutants at the level of the rRNA genes is relatively easy in this system. We describe here a novel thermosensitive mutation, ts1297, localized by rho^- deletion mapping in (or very close to) the sequence corresponding to the small ribosomal RNA (15S) gene. Defective mutations of the small rRNA have not been reported so far.In the mutant, the amount of 15S rRNA and of the small ribosomal subunit, 37S, is reduced. The quantity of the large ribosomal RNA (21S), directly extracted from mitochondria, appears normal. However, the large ribosomal subunit, 50S, seems to be fragile and could be recovered only in the presence of Ca²⁺ in place of Mg²⁺. The 50S particles seem to be completely degraded under normal conditions of extraction with Mg²⁺.The thermosensitive phenotype of the ts1297 mutant is suppressed by a nuclear mutation SU₁₀₁. The SU₁₀₁ mutation had been originally isolated as a suppressor of another mitochondrial mutation, ts902, which is located within the 21S rRNA gene.These results suggest that the mitochondrial mutations ts1297 and ts902 are both involved in the interaction of the large and small ribosomal subunits.