Dysregulated responses to emotions among abstinent heroin users: correlation with childhood neglect and addiction severity.

The aim of this paper was to investigate the subjective responses of abstinent heroin users to both neutral and negative stimuli and the related hypothalamus-pituitary-adrenal reactions to emotional experience in relationship to their perception of childhood adverse experiences. Thirty male abstinent heroin dependents were included in the study. Emotional responses and childhood neglect perception were measured utilizing the State-Trait Anxiety Inventory Y-1 and the Child Experience of Care and Abuse Questionnaire. Neutral and unpleasant pictures selected from the International Affective Picture System and the Self-Assessment Manikin procedure have been used to determine ratings of pleasure and arousal. These ratings were compared with normative values obtained from healthy volunteers used as control. Blood samples were collected before and after the experimental sessions to determine both adrenocorticotropic hormone and cortisol plasma levels. Basal anxiety scores, cortisol and adrenocorticotropic hormone levels were higher in abstinent heroin users than in controls. Tests showed that anxiety scores did not change in controls after the vision of neutral slides, whilst they did in abstinent heroin addicts, increasing significantly; and increased less significantly after the unpleasant task, in comparison to controls. Abstinent heroin users showed significantly higher levels of parent antipathy and childhood emotional neglect perception than controls for both the father and the mother. Plasma adrenocorticotropic hormone and cortisol levels did not significantly increase after unpleasant slide set viewing among addicted individuals, because of the significantly higher basal levels characterizing the addicted subjects in comparison with controls. Multiple regression correlation showed a significant relationship between childhood neglect perception, arousal reaction, impaired hypothalamus-pituitary-adrenal axis response and addiction severity. Early adverse experiences seem to affect the entire interaction between hyper-arousal, reduced hormonal response to stress and addiction severity. Our findings, although obtained in a small number of subjects, indicate a significant link between the perception of parental style/care/support during childhood and the ability to cope with stressful emotional stimuli in adulthood and addiction severity.

Supervised daily consumption, contingent take-home incentive and non-contingent take-home in methadone maintenance.

Methadone maintenance therapy (MMT) has been found effective in treating heroin addiction. Serious consideration should be given to the modality of methadone distribution, as it influences not only treatment outcome but the attitudes of policy makers and the community, too. On one hand, the choice of take-home methadone removes the need for daily attendance at a methadone clinic, which seems to improve patients' quality of life. On the other, this method, because of its lack of supervision and the absence of strict consumption monitoring, runs the risk of methadone misuse and diversion. In this study, we compared A) supervised daily consumption, B) contingent take-home incentives and C) non-contingent take-home in methadone maintenance in three groups of heroin-addicted patients attending three different MMT programmes. Retention rates at 12 months were significantly higher in contingent take-home patients (group B) than in those with supervised daily consumption (group A) and the non-contingent take-home (group C). Retention rates were higher in group A than in group C patients. Compared to patients in groups A and B, those in group C showed fewer negative urinalyses and higher rates of self-reported diversion and episodes of crime or violence. Results indicate a more positive outcomes following take-home methadone associated with behavioural incentives and other measures that aim to facilitate treatment compliance than those following daily supervised consumption. By contrast, non-contingent take-home methadone given to non-stabilized patients is associated with a high rate of diversion, along with more crime episodes and maladaptive behaviours.

Adverse childhood experiences (ACEs), genetic polymorphisms and neurochemical correlates in experimentation with psychotropic drugs among adolescents.

Epidemiological and clinical data show frequent associations between adverse childhood experiences (ACEs) and substance abuse susceptibility particularly in adolescents. A large body of evidences suggests that the possible dysregulation of neuroendocrine responses as well as neurotransmitters function induced by childhood traumatic experiences and emotional neglect could constitute one of the essential biological changes implementing substance abuse vulnerability. Moreover, genotype variables and its environment interactions have been associated with an increased risk for early onset substance abuse. In this paper we present several data that support the hypothesis of the involvement of hypothalamus-pituitary-adrenal (HPA) axis in mediating the combined effect of early adverse experiences and gene variants affecting neurotransmission. The presented data also confirm the relationship between basal plasma levels of cortisol and ACTH, on the one hand, and retrospective measures of neglect during childhood on the other hand: the higher the mother and father neglect (CECA-Q) scores are, the higher the plasma levels of the two HPA hormones are. Furthermore, such positive relationship has been proved to be particularly effective and important when associated with the "S" promoter polymorphism of the gene encoding the 5-HTT transporter, both in homozygote and heterozygote individuals.

Relevance of perceived childhood neglect, 5-HTT gene variants and hypothalamus-pituitary-adrenal axis dysregulation to substance abuse susceptibility.

The hypotheses of (1) gene x environment interaction in the susceptibility to experiment with drugs and (2) hypothalamus-pituitary-adrenal (HPA) axis involvement in mediating the effects of early adverse experiences and gene variants affecting serotonin function on substance abuse vulnerability were tested by investigating in 187 healthy adolescents the possible relevance of 5-HTT "S" polymorphism, childhood parental neglect reported retrospectively and HPA axis function to the susceptibility to experiment with illicit drugs. Higher frequency of the 5-HTT SS genotype seems to be associated with an increased susceptibility to use illegal psychotropic drugs among the adolescents. At the same time, reduced maternal care perception was found to represent a key intermediate factor of the association between SS polymorphism and drug use, suggesting that genetic factors and parental behavior concur to drug use susceptibility. Our results also confirm the relationship between basal plasma levels of cortisol and adrenocorticotropic hormone (ACTH) on the one hand, and retrospective measures of neglect during childhood: the higher the mother and father neglect CECA-Q scores, the higher the plasma levels of the two HPA hormones. Such positive relationship has been proved to be particularly effective and important when associated to the S-allele, both in homozygote and heterozygote individuals. However, when tested together with genotype and parental neglect, the effect of HPA hormones such as cortisol and ACTH was not found to improve significantly the explanatory power of the risk model.

Childhood neglect and parental care perception in cocaine addicts: relation with psychiatric symptoms and biological correlates.

UNLABELLED: Childhood neglect and poor child-parent relationships have been reported to increase substance use disorders susceptibility. Stressful environmental factors, including emotional neglect, could affect individual personality traits and mental health, possibly inducing stable changes in hypothalamic-pituitary-adrenal (HPA) axis and brain mono-amine function, in turn involved in addictive behavior vulnerability. Therefore, we decided to investigate homovanillic (HVA) and prolactin (PRL) plasma levels, as expression of possible changes in dopamine function, ACTH and cortisol plasma levels, as measures of HPA axis function, and concomitant psychiatric symptoms profile in abstinent cocaine addicts, in relationship to their childhood history of neglect and poor parental care perception. METHODS: Fifty abstinent cocaine dependent patients, and 44 normal controls, matched for age and sex, were submitted to a detailed psychiatric assessment (DSM IV criteria). All patients and controls completed the Symptoms Check List-90 (SCL-90) and the Buss Durkee Hostility Inventory (BDHI), to evaluate psychiatric symptoms frequency and aggressiveness levels. The Childhood Experience of Care and Abuse-Questionnaire (CECA-Q) and Parental Bonding Instrument (PBI) have been used to retrospectively investigate parent-child relationships. Blood samples were collected to determine HVA, PRL, ACTH and cortisol basal plasma levels. RESULTS: Cocaine addicted individuals in general showed significantly lower HVA, and higher PRL, ACTH and cortisol basal levels respect to controls. In particular, neuroendocrine changes characterized cocaine addicts with childhood history of neglect and low perception of parental care. Obsessive-compulsive, depression and aggressiveness symptoms have been found related to poor parenting, inversely associated to HVA levels and directly associated to PRL, ACTH and cortisol levels. CONCLUSIONS: These findings suggest the possibility that childhood experience of neglect and poor parent-child attachment may partially contribute to a complex neurobiological derangement including HPA axis and dopamine system dysfunctions, playing a crucial role in addictive and affective disorders susceptibility.

Homovanillic acid (HVA) plasma levels inversely correlate with attention deficit-hyperactivity and childhood neglect measures in addicted patients.

BACKGROUND: Attention deficit hyperactivity disorder (ADHD) seems to be a risk condition for substance use disorders, possibly in relationship to common neurobiological changes, underlying both addictive and externalising behaviour susceptibility. Although this vulnerability has been primarily attributed to gene variants, previous studies suggest that also adverse childhood experiences may influence neurotransmission, affecting in particular brain dopamine (DA) system and possibly concurring to the development of behavioural disorders. Therefore, we decided to investigate ADHD symptoms and plasma concentrations of the DA metabolite homovanillic acid (HVA) in abstinent addicted patients, in comparison with healthy control subjects, evaluating whether ADHD scores were related with HVA levels, as expression of DA turnover, and whether HVA values, in turn, were associated with childhood emotional neglect. METHODS: Eighty-two abstinent drug dependent patients, and 44 normal controls, matched for age and sex, completed the Wender Utah Rating Scale (WURS), measuring ADHD symptoms, and the Childhood Experience of Care and Abuse Questionnaire (CECA-Q). Blood samples were collected to determine HVA plasma levels. RESULTS: Addicted individuals showed significantly higher ADHD scores and lower HVA levels respect to control subjects. ADHD scores at WURS in addicted patients negatively correlated with plasma HVA values. In turn, plasma HVA levels were inversely associated with childhood neglect measures, reaching statistical significance with "mother-antipathy" and "mother neglect" scores. CONCLUSIONS: These findings suggest the possibility that childhood experience of neglect and poor mother-child attachment may have an effect on central dopamine function as an adult, in turn contributing to both ADHD and substance abuse neurobiological vulnerability.

Human kappa opioid receptor gene (OPRK1) polymorphism is associated with opiate addiction.

Variants of the opioid receptors are the obvious candidates underlying addiction. The kappa opioid receptor (KOR) system seems to play a role in stress responsivity, opiate withdrawal and responses to psycho-stimulants, inhibiting mesolimbic dopamine. KOR gene polymorphisms have been reported to contribute to predisposition to voluntary alcohol-drinking behavior in experimental animals. In humans, the 36G > T single nucleotide polymorphism (SNP) on KOR gene, that was recently identified, has been found associate with substance dependence, with inconclusive findings. In the present study, 106 heroin addicts (West European, Caucasians) and 70 healthy control subjects matched for race and gender, with no history of substance use disorder, have been genotyped. The frequency of KOR 36G > T SNP was significantly higher among heroin-dependent individuals compared with control subjects (Fisher's exact = 0.044; Pearson chi(2) = 4.2734, P = 0.039; likelihood ratio chi(2) tests = 4.6156, P = 0.032). Although KOR silent polymorphisms may apparently have no consequences on mRNA transcription, post-transcriptional mechanisms, such as mRNA stability, translation efficiency, and regulability may impair the function of kappa receptors system, with increased risk for substance use disorders. In specific, the neurobiological changes induced by mu-kappa opioid imbalance could underlie vulnerable personality traits and risk behavior.

Perceived parenting behavior in the childhood of cocaine users: relationship with genotype and personality traits.

Low parental care during childhood, a pattern characteristic of an "affectionless control" rearing style was frequently reported in the history of addicted individuals. Parents' childrearing regimes and children's genetic predispositions, with their own behavioral characteristics, have been seen to be closely interwoven, probably affecting children's development and addictive behavior susceptibility. In the present study, parents care perception, aggressive personality traits, and genotype (serotonin transporter promoter gene--5-HTTLPR) have been investigated in cocaine users and healthy control subjects. PBI scores (maternal and paternal care) were lower and BDHI scores (aggressiveness) higher in cocaine users in comparison with controls and significant differences in the perception of either paternal or maternal care were observed between cocaine users and non-users. The short-short (SS) genotype frequency was significantly higher among cocaine users compared with control subjects (P = 0.04). Logistic regression proves that persons bearing the SS genotype have a risk of becoming cocaine user almost three times higher than those having the LL genotype. Estimations of the effects of other factors potentially affecting the risk of being cocaine addicted clearly prove the significant impact of aggressiveness: the highest the score, the highest the risk of becoming cocaine user. Moreover, paternal and maternal care perception significantly improve the fit of the model (the log likelihood decreases passing from -105.9 to -89.8, LR test = 32.17, P-value = 0.0000). Each unit increase in the PBI score yields a significant 12% and 10% decrease of the risk of becoming cocaine user, respectively for paternal and maternal care. Interestingly, once controlled for the PBI score, the relative risk associated to the SS genotype drops strikingly and becomes no longer statistically significant. On the whole, our preliminary data suggest that the association between 5-HT transporter polymorphism and psycho-stimulant use may be mediated by mother-child relationship and parental attachment perception, both being environmental and genetic factors involved in the proneness to substance use disorders, particularly in aggressive-antisocial individuals.

Association of the serotonin transporter promoter polymorphism with smoking behavior among adolescents.

Serotonin transporter promoter polymorphism (5-HTTLPR) genotype was previously found associated with smoking behavior, difficulty in quitting smoking, and nicotine addiction; with non-replicated findings and contrasting results. Aim of the present study was to evaluate the possible association between 5-HTTLPR genotype and smoking behavior among adolescents, in relationship with psychological characteristics. Two hundred and ten Caucasian high school students (aged 14-19 years); 103 non-smokers, who have never smoked nicotine; and 107 tobacco smokers have been genotyped. Aggressiveness levels and temperamental traits were measured in both smokers and non-smokers, respectively, utilizing Buss-Durkee Hostility Inventory (BDHI) and Cloninger Three-Dimensional Personality Questionnaire (TPQ). Data about school performance have been also collected. The short-short (SS) genotype frequency was significantly higher among smokers compared with non-smokers (P = 0.023). The odds ratio for the SS genotype versus the long-long (LL) genotype frequency was 1.17 [95% CL (0.30-2.05)], when smokers were compared with non-smokers. The SS genotype frequency was significantly higher among heavy smokers with early onset, compared with moderate smokers with late onset (P = 0.042). BDHI irritability scores, NS scores at TPQ, and school failure frequency were significantly higher in smokers than in non-smokers. Multivariate model-fitting analysis evidenced a significantly greater relationship of genotype with irritability levels (BDHI scores) (0.34, P < 0.001) and temperament traits (NS scores) (0.36, P < 0.001), than with school performance (rate of school under-achievements) (0.18, P < 0.05) and nicotine smoking (number of cigarettes) (0.24, P < 0.01). Accordingly, factor-analysis showed that gene polymorphism contributes more directly to BDHI scores and NS scores (0.73; 0.71) than to smoking behavior and school under-achievement (0.54; 0.51). Our data suggest that a decreased expression of the gene encoding the 5-HTT transporter, due to "S" promoter polymorphism, may be associated with smoking behavior among adolescents and increased risk to develop nicotine dependence, possibly in relationship to personality traits, temperamental characteristics, and school under-achievements.

Serotonin transporter promoter polymorphism genotype is associated with temperament, personality traits and illegal drugs use among adolescents.

Serotonin transporter promoter polymorphism (5-HTTLPR) genotype was previously found associated with substance use disorders, particularly in the subjects with comorbid antisocial behavior, and with temperament and personality traits at risk for substance abuse. Aim of the present study was to evaluate the possible association between 5-HTTLPR genotype and the availability to experiment illegal drugs among adolescents, in relationship with psychological characteristics. 216 caucasian high school students (aged 14-19 ys), 125 abstinent subjects, who have never experimented psychotropic drugs, and 91 experimenters of illegal drugs have been genotyped. Aggressiveness levels and temperamental traits were measured in both abstinent subjects and experimenters utilizing respectively Buss-Durkee-Hostility-Inventory (BDHI) and Cloninger Three-dimensional Personality Questionnaire (TPQ). Data about school performance have been also collected. The short-short (SS) genotype frequency was significantly higher among experimenters compared with abstinent subjects (p = 0.001). The odds ratio for the SS genotype vs the long-long (LL) genotype frequency was 4.67, 95% Cl (1.97-11.04), when experimenters were compared with abstinent students. The SS genotype frequency was significantly higher among aggressive/novelty seeker (NS) experimenters with poor school achievements, compared with drugs experimenters without aggressiveness and school failure (p = 0.02). When evaluated on the entire sample, BDHI mean total scores, NS scores at TPQ and school failure frequency were significantly higher in SS individuals, in comparison with LL subjects. Our data suggest that a decreased expression of the gene encoding the 5-HTT transporter, due to "S" promoter polymorphism, may be associated with an increased availability to experiment illegal drugs among adolescents, particularly in the subjects with more consistent aggressiveness, NS temperament and learning disabilities.

Analysis of monoamine oxidase A (MAO-A) promoter polymorphism in male heroin-dependent subjects: behavioural and personality correlates.

The promoter of the monoamine oxidase A (MAO-A) gene was analysed to test whether length variation of the repeat polymorphism contributes to variation in individual vulnerability to aggressive-criminal behaviour, and liability to heroin dependence. The repeat number of the MAO-A polymorphism was assessed in 199 male subjects of Italian descent, a sample comprising 95 healthy subjects and 104 heroin-dependent subjects including 52 addicted individuals with violent behaviour and antisocial personality disorder. The frequency of the low-activity 3-repeat allele was significantly higher in violent offenders among heroin addicts, compared to addicted individuals without antisocial behaviour (34.6 vs. 15.4%; p<0.03) and controls (18.9%; p<0.05). No significant difference was evidenced in the frequencies of the MAO-A alleles between heroin-dependent subjects in general and control subjects. High activity 4-repeat allele frequency was significantly higher in addicted individuals without antisocial behavior compared to antisocial-aggressive heroin-dependent subjects (76.9 vs. 55.8%; p<0.02). Buss Durkee Hostility Inventory (BDHI) mean total scores were significantly higher in heroin addicts than in controls (p<0.001), and in antisocial-violent heroin addicts in comparison with addicted individuals without antisocial behaviour (p<0.005). Among heroin addicts BDHI irritability, suspiciousness and resentment subscales scores were found significantly higher in low activity 3-repeat allele subjects than in high activity alleles subjects (p<0.001; p<0.05; p<0.05, respectively). No association was found between MAO-A polymorphism and suicide history. Our findings suggest that the low-activity 3-repeat allele of the MAO-A promoter polymorphism confers increased susceptibility to antisocial-violent behavior and aggressiveness, rather than drug dependence per se, in heroin-dependent males.

Association between low-activity serotonin transporter genotype and heroin dependence: behavioral and personality correlates.

In previous studies, serotonin (5-HT) system disturbance was found involved in a variety of behavioral disorders, psychopathologies, and substance use disorders. A functional polymorphism in the promoter region of the human serotonin transporter gene (5-HTTLPR) was recently identified and the presence of the short (S) allele found to be associated with a lower level of expression of the gene, lower levels of 5-HT uptake, type 2 alcoholism, violence and suicidal behavior. In the present study, 101 heroin addicts (males, West European, Caucasians) and 101 healthy control subjects matched for race and gender, with no history of substance use disorder, have been genotyped. Aggressiveness levels were measured in both heroin addicts and controls utilizing Buss-Durkee-Hostility-Inventory (BDHI). Data about suicide attempt and violent criminal behavior in subject history have been collected. The short-short (SS) genotype frequency was significantly higher among heroin dependent individuals compared with control subjects (P = 0.025). The odds ratio for the SS genotype versus the long-long (LL) genotype frequency was 0.69, 95% Cl (0.49-0.97), when heroin addicts were compared with healthy controls. The SS genotype frequency was significantly higher among violent heroin dependent individuals compared with addicted individuals without aggressive behavior (P = 0.02). BDHI mean total scores and suspiciousness and negativism subscales scores were significantly higher in SS individuals, in comparison with LL subjects, among heroin addicts. No association was found between SS genotype and suicide history. Our data suggest that a decreased expression of the gene encoding the 5-HTT transporter, due to "S" promoter polymorphism, may be associated with an increased risk for substance use disorders, particularly in the subjects with more consistent aggressiveness and impulsiveness.

Three target genes for the transcriptional activator Cat8p of Kluyveromyces lactis: acetyl coenzyme A synthetase genes KlACS1 and KlACS2 and lactate permease gene KlJEN1.

The aerobic yeast Kluyveromyces lactis and the predominantly fermentative Saccharomyces cerevisiae share many of the genes encoding the enzymes of carbon and energy metabolism. The physiological features that distinguish the two yeasts appear to result essentially from different organization of regulatory circuits, in particular glucose repression and gluconeogenesis. We have isolated the KlCAT8 gene (a homologue of S. cerevisiae CAT8, encoding a DNA binding protein) as a multicopy suppressor of a fog1 mutation. The Fog1 protein is a homologue of the Snf1 complex components Gal83p, Sip1p, and Sip2p of S. cerevisiae. While CAT8 controls the key enzymes of gluconeogenesis in S. cerevisiae, KlCAT8 of K. lactis does not (I. Georis, J. J. Krijger, K. D. Breunig, and J. Vandenhaute, Mol. Gen. Genet. 264:193-203, 2000). We therefore examined possible targets of KlCat8p. We found that the acetyl coenzyme A synthetase genes, KlACS1 and KlACS2, were specifically regulated by KlCAT8, but very differently from the S. cerevisiae counterparts. KlACS1 was induced by acetate and lactate, while KlACS2 was induced by ethanol, both under the control of KlCAT8. Also, KlJEN1, encoding the lactate-inducible and glucose-repressible lactate permease, was found under a tight control of KlCAT8.

FOG1 and FOG2 genes, required for the transcriptional activation of glucose-repressible genes of Kluyveromyces lactis, are homologous to GAL83 and SNF1 of saccharomyces cerevisiae.

The fog1 and fog2 mutants of the yeast Kluyveromyces lactis were identified by inability to grow on a number of both fermentable and non-fermentable carbon sources. Genetic and physiological evidences suggest a role for FOG1 and FOG2 in the regulation of glucose-repressible gene expression in response to a glucose limitation. The regulatory effect appears to be at the transcriptional level, at least for beta-galactosidase. Both genes have been cloned by complementation and sequenced. FOG1 is a unique gene homologous to GAL83, SIP1 and SIP2, a family of regulatory genes affecting glucose repression of the GAL system in Saccharomyces cerevisiae. However, major differences exist between fog1 and gal83 mutants. FOG2 is structurally and functionally homologous to SNF1 of S. cerevisiae and shares with SNF1 a role also in sporulation.

IMP2, a gene involved in the expression of glucose-repressible genes in Saccharomyces cerevisiae.

Two mutants carrying different deletions of the IMP2 coding sequence of Saccharomyces cerevisiae, delta T1, which encodes a protein lacking the last 26 C-terminal amino acids, and delta T2, which completely lacks the coding region, were analysed for derepression of glucose-repressible maltose, galactose, raffinose and ethanol utilization pathways in response to glucose limitation. The role of the IMP2 gene product in the regulation of carbon catabolite repressible enzymes maltase, invertase, alcohol dehydrogenase, NAD-dependent glutamate dehydrogenase (NAD-GDH) and L-lactate:ferricytochrome-c oxidoreductase (L-LCR) was also analysed. The IMP2 gene product is required for the rapid glucose derepression of all above-mentioned carbon source utilization pathways and of all the enzymes except for L-LCR. NAD-GDH is regulated by IMP2 in the opposite way and, in fact, this enzyme was released at higher levels in both imp2 mutants than in the wild-type strain. Therefore, the product of IMP2 appears to be involved in positive and negative regulation. Both deletions result in growth and catalytic defects; in some cases partial modification of the gene product yielded more dramatic effects than its complete absence. Moreover, evidence is provided that the IMP2 gene product regulates galactose- and maltose-inducible genes at the transcriptional level and is a positive regulator of maltase, maltose permease and galactose permease gene expression.

Allelism of IMP1 and GAL2 genes of Saccharomyces cerevisiae.

Cloning and characterization of the previously described Saccharomyces cerevisiae IMP1 gene, which was assumed to be a nuclear determinant involved in the nucleomitochondrial control of the utilization of galactose, demonstrate allelism to the GAL2 gene. Galactose metabolism does not necessarily involve the induction of the specific transport system coded by GAL2/IMP1, because a null mutant takes up galactose and grows on it. Data on galactose uptake are presented, and the dependence on ATP for constitutive and inducible galactose transport is discussed. These results can account for the inability of imp1/gal2 mutants to grow on galactose in a respiration-deficient background. Under these conditions, uptake was affected at the functional level but not at the biosynthetic level.

IMP2, a nuclear gene controlling the mitochondrial dependence of galactose, maltose and raffinose utilization in Saccharomyces cerevisiae.

The IMP2 gene of Saccharomyces cerevisiae is involved in the nucleo-mitochondrial control of maltose, galactose and raffinose utilization as shown by the inability of imp2 mutants to grow on these carbon sources in respiratory-deficient conditions or in the presence of ethidium bromide and erythromycin. The negative phenotype cannot be scored in the presence of inhibitors of respiration and oxidative phosphorylation, indicating that the role of the mitochondria in the utilization of the above-mentioned carbon sources in imp2 mutants is not at the energetical level. Mutations in the IMP2 gene also confer many phenotypic alterations in respiratory-sufficient conditions, e.g. leaky phenotype on oxidizable carbon sources, sensitivity to heat shock and sporulation deficiency. The IMP2 gene has been cloned, sequenced and disrupted. The phenotype of null imp2 mutants is indistinguishable from that of the originally isolated mutant.

Catabolite repression by galactose in overexpressed GAL4 strains of Saccharomyces cerevisiae.

Catabolite repression by galactose was investigated in several strains of Saccharomyces cerevisiae grown on different carbon sources. Galactose repressed as much as glucose; raffinose was less effective. Full derepression was achieved with lactate. The functions tested were L-lactate ferricytochrome c oxidoreductase, NAD-glutamate dehydrogenase, and respiration. Galactose repression was observed only in the GAL4 but not in the gal4 strain. The presence of multiple copies of the GAL4 gene enhanced the repression by galactose. Different alleles of the GAL4 gene and the copy number did not affect glucose repression.

Isolation and characterization of carbon catabolite repression mutants in Saccharomyces cerevisiae.

Two carbon catabolite repression mutants of S. cerevisiae were isolated and characterized. In spite of the selection procedure (red colonies after tetrazolium overlay at high glucose concentration) the mutants exhibited a respiration which was as repressed as that of the parental strain or even more repressed. When grown at high glucose concentration the mutants display hyper-repression of cytochrome aa3 and of certain mitochondrial enzymes (L- and D-lactate dehydrogenases) but not of others (malate dehydrogenase, succinate dehydrogenase), indicating the existence of separate control sites for the different genes involved in the mitochondrial biogenesis. The data obtained pointed out that the same mutation affects both repression and derepression. In addition, the mutation(s) give rise to the complete derepression of the cytoplasmic enzyme NAD-glutamate dehydrogenase at 10% glucose whereas the enzyme is normally repressed at 3% glucose. The results of the genetic analysis indicate the mitochondrial nature of the mutation(s).

RAG1 and RAG2: nuclear genes involved in the dependence/independence on mitochondrial respiratory function for growth on sugars.

The analysis of five independent isolates of Kluyveromyces lactis shows that CBS 2359, CBS 683 and CBS 4574 could grow in the presence of mitochondrial inhibitors (antimycin A, oligomycin or erythromycin) and that CBS 2360 and CBS 141 were unable to grow in the presence of drugs. The resistant growth was observed only on glucose and not on other fermentable carbon sources (galactose, lactose). The phenotype 'growth on glucose in the presence of mitochondrial inhibitors' was called Rag+. This phenotype was found to be controlled by two unlinked nuclear genes: RAG1 and RAG2. Either of their recessive alleles, rag1 and rag2, led to the Rag- phenotype (i.e. the failure of growth on glucose in the presence of antimitochondrial drugs). Rag- strains represent the case in which fermentative growth becomes absolutely dependent on the functioning of the normal respiratory chain.