NAM9 nuclear suppressor of mitochondrial ochre mutations in Saccharomyces cerevisiae codes for a protein homologous to S4 ribosomal proteins from chloroplasts, bacteria, and eucaryotes.

We report the genetic characterization, molecular cloning, and sequencing of a novel nuclear suppressor, the NAM9 gene from Saccharomyces cerevisiae, which acts on mutations of mitochondrial DNA. The strain NAM9-1 was isolated as a respiration-competent revertant of a mitochondrial mit mutant which carries the V25 ochre mutation in the oxi1 gene. Genetic characterization of the NAM9-1 mutation has shown that it is a nuclear dominant omnipotent suppressor alleviating several mutations in all four mitochondrial genes tested and has suggested its informational, and probably ribosomal, character. The NAM9 gene was cloned by transformation of the recipient oxi1-V25 mutant to respiration competence by using a gene bank from the NAM9-1 rho o strain. Orthogonal-field alternation gel electrophoresis analysis and genetic mapping localized the NAM9 gene on the right arm of chromosome XIV. Sequence analysis of the NAM9 gene showed that it encodes a basic protein of 485 amino acids with a presequence that could target the protein to the mitochondrial matrix. The N-terminal sequence of 200 amino acids of the deduced NAM9 product strongly resembles the S4 ribosomal proteins from chloroplasts and bacteria. Significant although less extensive similarity was found with ribosomal cytoplasmic proteins from lower eucaryotes, including S. cerevisiae. Chromosomal inactivation of the NAM9+ gene is not lethal to the cell but leads to respiration deficiency and loss of mitochondrial DNA integrity. We conclude that the NAM9 gene product is a mitochondrial ribosomal counterpart of S4 ribosomal proteins found in other systems and that the suppressor acts through decreasing the fidelity of translation.

Incipient mitochondrial evolution in yeasts. I. The physical map and gene order of Saccharomyces douglasii mitochondrial DNA discloses a translocation of a segment of 15,000 base-pairs and the presence of new introns in comparison with Saccharomyces cerevisiae.

We have determined the physical and genetic map of the 73,000 base-pair mitochondrial genome of a novel yeast species Saccharomyces douglasii. Most of the protein and RNA-coding genes known to be present in the mitochondrial DNA of Saccharomyces cerevisiae have been identified and located on the S. douglasii mitochondrial genome. The nuclear genomes of the two species are thought to have diverged some 50 to 80 million years ago and their nucleo-mitochondrial hybrids are viable but respiratorily deficient. The mitochondrial genome of S. douglasii displays many interesting features in comparison with that of S. cerevisiae. The three mosaic genes present in both genomes are quite different with regard to their structure. The S. douglasii COXI gene has two new introns and is missing the five introns of the S. cerevisiae gene. The S. douglasii cytochrome b gene has one new intron and lacks two introns of the S. cerevisiae gene. Finally, the L-rRNA gene of S. douglasii, like that of S. cerevisiae, has one intron of which the structure is different. Another salient feature of the S. douglasii mitochondrial genome reported here is that the gene order is different in comparison with S. cerevisiae mitochondrial DNA. In particular, a segment of approximately 15,000 base-pairs including the genes coding for COXIII and S-rRNA has been translocated to a position between the genes coding for varl and L-rRNA.

The role of GABA in morphine abstinence in rats.

GABA mimetics such as baclofen, aminooxyacetic acid (AOAA) and diazepam decreased naloxone-precipitated abstinence wet dog shakes in morphine dependent rats. The GABA antagonists bicuculline and picrotoxin were not effective in small doses. The inhibition of wet dog shakes by baclofen, AOAA and diazepam was not reversed by bicuculline. This suggests that baclofen, AOAA and diazepam may inhibit morphine wet dog shakes not by direct GABA receptor stimulation. Inhibition by baclofen of wet dog shakes was reversed by 5-hydroxytryptophan (5-HTP) suggesting that the inhibiting effects of GABA mimetics are mediated by serotoninergic neurons.

Noradrenergic and behavioural effects of naloxone injected in the locus coeruleus of morphine-dependent rats and their control by clonidine.

Naloxone HCl (10 micrograms/0.5 ml) was injected in the locus coeruleus (LC) of morphine-dependent rats and the behavioural manifestations of morphine withdrawal and the cortical levels of 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (MHPG-SO4) were measured 30 min later. Naloxone precipitated a withdrawal syndrome and raised cortical MHPG-SO4 in animals made dependent by ascending doses of morphine for 11 days. An injection of clonidine intraperitoneally (200 micrograms/kg) or in the LC (5 micrograms/0.5 microliter) blocked most aspects of the withdrawal syndrome except jumping and had no effect on the naloxone-induced rise in cortical MHPG-SO4. The findings confirm the hypothesis that the LC is one of the sites where naloxone and clonidine, respectively, precipitate and reduce the narcotic withdrawal syndrome but argue against a role of noradrenergic neurons originating in the LC and innervating the cortex in the ability of clonidine to suppress some aspects of withdrawal syndrome precipitated by naloxone in morphine-dependent animals.

The effect of different lesions of the median raphe on morphine analgesia.

The effect of different manipulations of the nucleus medianus raphe (MR) on morphine analgesia was investigated in rats using the tail-immersion test. Electrolytic lesions of this structure antagonized morphine analgesia, while injections of 5,7-dihydroxytryptamine (to destroy serotonergic neurons) or ibotenic acid (to destroy cell bodies) in the medianus raphe did not alter the effect of morphine. Injection of naloxone (0.5 and 0.1 micrograms) in the MR antagonized morphine analgesia. These results suggest the importance of this structure for morphine analgesia in this test, although the substrates within the nucleus that mediate this action are still unknown.

Different effects of zimelidine on the reinforcing properties of d-amphetamine and morphine on conditioned place preference in rats.

The possibility that serotoninergic mechanisms control the reinforcing properties of d-amphetamine and morphine was investigated in rats, using zimelidine, a potent and selective serotonin uptake blocker and the conditioned place preference test design. Zimelidine dihydrochloride 20 mg/kg did not cause place aversion and did not modify the place preference induced by 5 mg/kg morphine hydrochloride. Place preference induced by 5 mg/kg d-amphetamine sulphate was completely blocked by pretreatment with zimelidine. The results suggest that the reinforcing properties of d-amphetamine, but not of opioid agonists, may be reduced by agents which increase serotonin transmission in the brain.

The effect of intracerebroventricular 5,7-dihydroxytryptamine on morphine analgesia is time-dependent.

The analgesic effect of morphine in the tail immersion test was studied in rats three and ten days after intracerebroventricular 5,7-dihydroxytryptamine (5,7-DHT) given to selectively destroy serotonergic neurons. Morphine analgesia was reduced three but not ten days after the neurotoxin. Ten days after 5,7-DHT, the inhibiting effect of metergoline, a serotonin antagonist, on morphine analgesia was still present, suggesting that functional recovery of the serotonergic system may partly explain the different results.

Functional nuclear suppressor of mitochondrial oxi2 mutations in yeast.

A semidominant nuclear suppressor, called nam6, of oxi2-V276 mitochondrial mutation has been isolated and characterized. The nuclear character of nam6 was proved by its retention in rho degree strains, lack of mitotic segregation in diploids and meiotic 2:2 segregation in tetrads. The specificity of nam6 was tested on 315 mit- mutations of four mitochondrial genes (oxi1, oxi2, oxi3, and cob-box). It suppresses clearly only three mutations in the oxi2 gene, restoring partially or completely cytochrome aa3 formation. The results suggest a functional character of the suppression.

Mitochondrial and nuclear mitoribosomal suppressors that enable misreading of ochre codons in yeast mitochondria : I. Isolation, localization and allelism of suppressors.

A systematic search for suppressors of mutations which cause a deficiency in the splicing of mitochondrial RNA has been undertaken. These splicing mutations were localized in the mRNA-maturase coding sequence of the second intron of the cob-boxgene, i.e. in the box3locus. A total of 953 revertants (mostly spontaneous in origin) were isolated and their genetic nature (nuclear vs. mitochondrial) and phenotype characterized.Most revertants were mitochondrially determined and displayed a wild-type phenotype. A mitochondrial suppressor unlinked with the box3 (-)target mutation was uncovered among the revertants displaying a pseudo-wild phenotype: out of 26 revertants analyzed, derived from 7 different box3(-) mutants only one such suppressor mutation mim3-1 was found. It was localized by rho(-) deletion mapping in the region between the oxi2 and oxi3 gene, within (or in the vicinity) the gene specifying the 15S ribosomal RNA.Nuclear suppressors were isolated from seven different box3 (-)mutants. All were recessive and had a pseudo-wild phenotype. Three such suppressors nam3-1, nam3-2 and nam3-3 were investigated more extensively. Tetrad analysis has shown that they are alleles of the same nuclear locus NAM3 and mitotic analysis has shown that they do not segregate mitotically.

Mitochondrial and nuclear mitoribosomal suppressors that enable misreading of ochre codons in yeast mitochondria : II. Specificity and extent of suppressor action.

We describe studies on the action spectra of the mitochondrial suppressor mim3-1 and the three alleles of nuclear suppressor nam3. Their specificity of action was tested on 516 mit (-) mutations located in different mitochondrial genes. The degree of suppression was quantified by the extent of cytochrome oxidase and cytochrome b synthesis. We show that the four suppressors are allele-specific gene-nonspecific informational suppressors. They would act by changing the structure of the small mitoribosomal subunit which would decrease fidelity of translation enabling misreading of some but not all ochre codons. The implications of the results on the role of intron encoded maturases are discussed.

The role of central serotoninergic neurotransmission in the morphine abstinence syndrome in rats.

It was found that 200 mg/kg of DL-p-chlorophenylalanine (PCPA) and 2 mg/kg of methergoline drugs inhibiting central serotoninergic transmission-decreased naloxone precipitated abstinence wet dog shakes in morphine-dependent rats. The inhibitory effect of PCPA was reversed by 200 mg/kg of 5-hydroxy-L-tryptophan (5-HTP) but not by the same amount of L-tryptophan (TP). 5-HTP alone (200 mg/kg) increased wet dog shakes epissodes, whereas TP alone in the same dosage practically did not have any influence on the wet dog shakes in morphine-dependent rats. These findings suggest that brain serotonin may play some role in the expression of wet dog shakes in morphine-dependent rats.

The influence of pimozide, haloperidol, alpha-methyl-p-tyrosine and reserpine on the development of morphine dependence in rats.

It was found that pimozide, haloperidol and alpha-methyl-p-tyrosine (alpha-MT), drugs inhibiting central catecholaminergic (CA) transmission, increased the development of morphine dependence measured as wet dog shakes in rats. Reserpine inhibiting CA and serotoninergic (5-HT) transmission, decreased the development of morphine dependence in rats. The inhibitory effect of reserpine was reversed by 5-hydroxytryptophan (5-HTP), but not by L-dihydroxyphenylalanine (L-DOPA). These results suggest that wet dog shakes might develop as a result of regulatory adaptation in the 5-HT neurotransmitter system.

Mitochondrial mutagenesis in Saccharomyces cerevisiae : V. Frequencies of different mit (-) mutants and loss of their mit (+) alleles in rho (-) clones.

Four types of mit (-) mutations induced with manganese are found in the following relative proportions: oxi3 (-) > cob-box (-) > oxi2 (-) [Symbol: see text] oxi1 (-1). The frequences of loss of their respective mit (+) alleles in manganese-induced rho (-)] primary and secondary clones follow the same order. The possible interdependence between these two sets of data is discussed.

Participation of serotonin in development of morphine dependence in mice and rats.

The effect of compounds inhibiting serotonergic transmission (reserpine, cyproheptadine, LSD) and stimulating it (imipramine, fluoxetine, 5-hydroxytryptophan) on the development of morphine dependence in mice and rats was investigated. Reserpine inhibited the development of morphine dependence in mice and rats. A combined treatment with 5-hydroxytryptophan and reserpine reversed the inhibitory action of reserpine on development of morphine dependence in mice. Cyproheptadine did not affect and LSD potentiated the formation of morphine dependence in mice. Imipramine potentiated the development of dependence in mice and rats and fluoxetine in mice. The results suggest that a decrease in the function of serotonergic system inhibits the development of morphine dependence, while stimulation of this system potentiates the development of the dependence in mice and rats. It seems that the retention of activity of presynaptic serotonin nerve endings is of importance in the development of dependence.

Effect of regulatory mutations of sulphur metabolism on the levels of cysteine- and homocysteine-synthesizing enzymes in Neurospora crassa.

1. Regulation of four enzymes involved in cysteine and homocysteine synthesis, i.e. cysteine synthase (EC 4.2.99.8), homocysteine synthase (EC 4.1.99.10), cystathionine beta-synthase (EC 2.1.22) and gamma-cystathionase (EC 4.4.1.1) was studied in the wild type and sulphur regulatory mutants of Neurospora crassa. 2. Homocysteine synthase and cystathionine beta-synthase were found to be regulatory enzymes but only the former is under control of the cys-3 - scon system regulating several enzymes of sulphur metabolism, including gamma-cystathionase. 3. The results obtained with the mutants strongly suggest that homocysteine synthase plays a physiological role as an enzyme of the alternative pathway of methionine synthesis. Cysteine synthase activity was similar in all strains examined irrespective of growth conditions. 4. The sconc strain with derepressed enzymes of sulphur metabolism showed an increased pool of sulphur amino acids, except for methionine. Particularly characteristic for this pool is a high content of hypotaurine, a product of cysteine catabolism.

Recombinational analysis of OXI1 mutants and preliminary analysis of their translation products in S. cerevisiae.

Recombinational analysis of oxil mutants was performed using a and a mutant strains with the same mitochondrial and nuclear backgrounds, derived from strain 777-3A.In spite of minor inconsistencies the overall map of oxi1 mutations can be constructed on the basis of wild-type recombinant frequencies in the two-point oxi1 (-) (-) x oxi1 (-) crosses. The frequencies of wild-type recombinants varied in a wide range from 0.003% to 16%, reaching the maximal values expected for unlinked mitochondria) markers. No distinct clusters of mutants were observed.The analysis of translation products of oxil mutants showed that all but one of the oxil mutants studied are connected with the conspicuous changes of the polypeptide band corresponding to subunit 11 of cytochrome c oxidase in electrophoresis on polyacrylamide gels. The exceptional G565 mutant showed no conspicuous change in subunit II, but lacked subunit I of cytochrome c oxidase.Various oxi1 mutants seemed to carry premature chain termination mutations. Most of them show a correlation between the length of the putative fragment of subunit II synthesized and the position on the genetic map. The direction of translation is from the V2 to the V60 mutation. The V2 mutation is proximal to cap and V60 proximal to the par locus.