Phyllobacterium catacumbae sp. nov., a member of the order 'Rhizobiales' isolated from Roman catacombs.

Two strains were isolated from tuff, a volcanic rock that forms the walls of the Roman Catacombs of Saint Callixtus in Rome, Italy. A polyphasic approach using nutritional and physiological tests, reactions to antibiotics, fatty acid profiles, DNA base ratios, DNA-DNA reassociation and 16S rRNA gene sequence comparisons showed that the two isolates belong to a novel species within the genus Phyllobacterium. The species Phyllobacterium catacumbae sp. nov. is proposed. The type strain is CSC19(T) (=CECT 5680(T)=LMG 22520(T)).

Thermomonas haemolytica gen. nov., sp. nov., a gamma-proteobacterium from kaolin slurry.

Four aerobic, gram-negative bacterial strains isolated from kaolin slurry used in the production of paper were subjected to a polyphasic analysis and characterization to determine their taxonomic position. Analysis of the 16S rDNA sequences of the four strains revealed that they represent a new lineage within the gamma-Proteobacteria, related to the genera Xanthomonas, Pseudoxanthomonas, Stenotrophomonas, Luteimonas, Xylella and Rhodanobacter. Analysis of the quinone system, the polyamines, the fatty acids and the polar lipids revealed a combination of characteristics that is unique and not described for the phylogenetic relatives. The four strains contain a ubiquinone Q-8, spermidine as the major polyamine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine as the predominant polar lipids, and a fatty acid profile with predominantly iso-branched fatty acids. The G+C content of the genomic DNA was determined to be within the narrow range 67.1-68.7 mol%. Determination of DNA relatedness, as well as riboprint band patterns and amplified fragment length polymorphism profiles, clearly demonstrated that the four strains are members of a single species. Antibiotic-susceptibility patterns were identical for the four strains. Although showing a high degree of similarites in physiological and biochemical patterns, each of the four strains could be distinguished from the others on the basis of a few biochemical characteristics. On the basis of the estimates of phylogenetic relationships derived from the 16S rDNA sequence analyses, the observed chemotaxonomic characteristics and other phenotypic traits, a new genus, Thermomonas gen. nov., and species, Thermomonas haemolytica sp. nov., are proposed for the strains A50-7-3T (= DSM 13605T = LMG 19653T), B 50-7-1 (= DSM 13598 = LMG 19655), D50-7-1 (= DSM 13610 = LMG 19656) and B50-8-1 (= DSM 13599 = LMG 19654), with strain A50-7-3T as the type strain.

The sequence of a 54.7 kb fragment of yeast chromosome XV reveals the presence of two tRNAs and 24 new open reading frames.

A 54,719 bp fragment from the right arm of Saccharomyces cerevisiae chromosome XV has been sequenced from the inserts of two cosmids (pEOA213 and pEOA217). The computer analysis of this sequence has revealed the presence of eight known genes (CKA2, CYC1, ALG8, TCM1, TMP1, UFE1, RTS2 and ASE1) and four open reading frames (ORFs) with strong homologies with known yeast genes (MLP1, SIS2 and HBS1 and the allantoin permease). The characteristics of the other ORFs and of the corresponding proteins do not allow postulation of a precise function. Several have features reminiscent of cytoskeleton or motor elements (keratin-like, myosin-like) and several others have characteristics of proteins which interact with DNA (extremely basic, b-Zip structure and/or acidic domains). Two tRNAs (tRNA(Lys) and tRNA(Pro)) have also been identified on this fragment. Many of these ORFs present similarities with ORFs located on chromosome XI, indicating some information reshuffling between the two chromosomal fragments.

Cloning of the ASN1 and ASN2 genes encoding asparagine synthetases in Saccharomyces cerevisiae: differential regulation by the CCAAT-box-binding factor.

Two new yeast genes, named ASN1 and ASN2, were isolated by complementation of the growth defect of an asparagine auxotrophic mutant. Genetical analysis indicates that these two genes are allelic to the asnA and asnB loci described previously. Simultaneous disruption of both genes leads to a total asparagine auxotrophy, while disruption of asn1 or asn2 alone has no effect on growth under tested conditions. Nucleotide sequences of ASN1 and ASN2 revealed striking similarities with genes encoding asparagine synthetase (AS) from other organisms. Regulation of ASN1 and ASN2 expression was studied using lacZ fusions and both genes were found to be several times less expressed in the absence of the transcription activator Gcn4p. The HAP complex, another transcription factor that binds to CCAAT-box sequences, was shown to specifically affect ASN1 expression. Hap2p and Hap3p subunits of the HAP complex are required for optimal expression of ASN1, while the Hap4p regulatory subunit, which is required for regulation by the carbon source, plays a minor role in this process. Consistent with the weak effect of Hap4p, the carbon source does not significantly affect expression of ASN1. Our results show that the role of the HAP complex is not limited to activation of genes required for respiratory metabolism.

A genetic screen to isolate genes regulated by the yeast CCAAT-box binding protein Hap2p.

We have developed a screening method to isolate yeast genes regulated by a specific transcription activator. The screen is based on the use of expression libraries in which the lacZ reporter gene is placed under control of yeast regulatory elements. Two partially representative libraries, constructed by different methods, were used to isolate genes regulated by the yeast CCAAT-box binding protein Hap2p. Among 26 fusions shown to be regulated by Hap2p only CYT1 was known to be regulated by this activator. Sequence analysis revealed that most of the remaining regulated fusions are in new yeast genes, while some are in previously characterized yeast genes (PTP1, RPM2, SDH1). Optimal expression of these three genes also requires Hap3p and Hap4p and is regulated by carbon source. Hap2p was known to regulate expression of genes involved in Krebs cycle, electron transport and heme biosynthesis. Our results suggest that Hap2p could play a more general role by regulating other mitochondrial processes such as protein import and phosphate transport (PTP1) or maturation of mitochondrial tRNAs (RPM2). Among the remaining regulated fusions, two of them correspond to open reading frames (ORFs) on chromosomes III and XI whose nucleotide sequences have been entirely determined. The use of this approach to functionally analyse ORFs of unknown function is discussed.

Structure and regulation of SDH3, the yeast gene encoding the cytochrome b560 subunit of respiratory complex II.

Using an expression library, we have isolated yeast genes activated in the presence of the yeast CCAAT box-binding protein HAP2. One of these genes, SDH3, encodes the cytochrome b560 subunit of respiratory complex II. The SDH3 protein contains three potential transmembrane domains and is more than 30% identical to bovine cytochrome b560 and to a mitochondrially encoded protein from Marchantia polymorpha. Disruption of SDH3 shows that this gene is required for growth on non-fermentable carbon sources. Expression of SDH1, SDH3, and SDH4 is activated in the presence of the HAP2 transcriptional activator.

The DNA sequence analysis of the HAP4-LAP4 region on chromosome XI of Saccharomyces cerevisiae suggests the presence of a second aspartate aminotransferase gene in yeast.

The nucleotide sequence of a 19,000 base pair region from the left arm of chromosome XI of Saccharomyces cerevisiae has been determined and analysed. It covers the HAP4-GFA1-LAP4 loci already described. As expected HAP4, GFA1 and LAP4 genes have been found and six new open reading frames (ORFs) with a coding capacity of more than 100 amino acid residues have been identified. One of them (YKL461) shows a high degree of identity with an aspartate aminotransferase gene. This raises the question of a second aspartate aminotransferase gene in yeast. A second ORF (YKL462) shows features compatible with a membranous localization. The other ORFs do not show a similarity with any known gene. A member of the highly repetitive 'CAT' DNA sequence is present.

DNA sequence analysis of a 17 kb fragment of yeast chromosome XI physically localizes the MRB1 gene and reveals eight new open reading frames, including a homologue of the KIN1/KIN2 and SNF1 protein kinases.

We report in this paper the sequence of a part of chromosome XI of Saccharomyces cerevisiae. This 17 kbp nucleotide sequence represents the right half of cosmid pUKG151 and contains nine open reading frames, YKL453, 450, 449, 448, 445, 443, 442, 441 and the 5' part of YKL440. YKL440 was previously identified as the MBR1 gene and plays a role in mitochondrial biogenesis. YKL443 is a homologue of the yeast serine-rich protein (SRP1), while YKL453 presents strong homologies with the KIN1/KIN2/SNF1 kinase family. It must be pointed out that the size of this gene is well above average for yeast.

Identification of nuclear genes which participate to mitochondrial translation in Saccharomyces cerevisiae.

The mitochondrial protein synthesis presents specific features and uses specific components different from their cytoplasmic counterparts. Since most genes which code for these components are localized in the chromosomes and only a small number are encoded by the mitochondrial DNA, it is important to identify and characterize the nuclear genes involved in this process. In order to do this, we have used a genetic screening which implies the selection and study of nuclear suppressors of mitochondrial mutations (or the reverse situation) which affect the mitochondrial protein synthesis. Three mutations have been used for this purpose. Two of them (ts 1398, cs 909) impair the mitochondrial ribosome; they were used to characterize new interacting components as well as two genes, MBR1 and MBR2, which control the assembly or the regulation of other genes involved in mitochondrial protein synthesis. The third mutation (ts 932), blocks the 3'-end maturation of the mitochondrial aspartyl tRNA. A nuclear suppressor has been obtained which presents all the characteristics of a mutation in the gene encoding the enzyme responsible for this process.

Tyrosine hydroxylase and dopamine beta-hydroxylase inductions evoked by reserpine in the superior cervical ganglion of developing eu- and hypothyroid rats.

This study was designed to determine the effect of neonatally-produced hypothyroidism on reserpine-elicited tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (D beta H) induction in the superior cervical ganglion (SCG) in rats. Some rats were rendered hypothyroid from birth by daily treatment with propylthiouracil (PTU). Some hypothyroid rats received replacement therapy with triiodothyronine (T3). Some rats received PTU for 20 days, beginning at 90 days of age. Some rats were not treated and served as controls. TH and D beta H activities were assayed at 30, 50 and 110 days of age. Basal TH activity in the SCG for rats made hypothyroid as neonates was significantly lower than for controls at all ages tested; basal D beta H activity for these rats was lower than for controls at 30 and 50 days of age, but by 110 days was not different from that for controls. Basal TH activity for rats made hypothyroid as adults was intermediate between that for controls and rats made hypothyroid from infancy. Injecting control rats with reserpine produces a robust TH induction in the SCG at each age tested, and a strong D beta H induction at 50 and 110 days of age. Reserpine-evoked TH and D beta H inductions in rats made hypothyroid as adults were not different from those seen in controls. In contrast, rats made hypothyroid from infancy showed virtually no evidence of a reserpine-provoked TH or D beta H induction at any age tested. TH and D beta H inductions for hypothyroid rats given T3 replacement were completely normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal tyrosine hydroxylase activation in the developing rat: influence of the thyroid status.

Adrenal tyrosine hydroxylase activation was elicited in developing control, hypo- and hyperthyroid rats by insulin-hypoglycaemia. Rats were deeply anaesthetized with chloroform at a low concentration, since intrinsic tyrosine hydroxylase activation was very low with this technique, as compared to Ketamine injection or chloroform at a high concentration. The study of time-course of tyrosine hydroxylase activation showed that the maximum value was observed 2 h after insulin administration. In control animals, tyrosine hydroxylase activation increased between 4 and 20 days, and then decreased. Hypothyroidism is associated with a decreased tyrosine hydroxylase activation between 4 and 50 days, as compared to controls and hyperthyroidism with an increased activation between 6 and 30 days. While tyrosine hydroxylase from saline-treated rats exhibits two different forms (with two apparent Km values for the cofactor), enzyme from insulin-treated animals was present in a single form with a Km corresponding to the low Km value of the saline-injected rats. At 6 and 14 days, hypothyroidism increases tyrosine hydroxylase Km values as compared to euthyroid animals.

Influence of neonatal hypothyroidism on adrenal tyrosine hydroxylase activation in the young rat.

1. Adrenal TH activation was elicited in young rats (aged 4, 6 and 14 days) by insulin hypoglycaemia. In the control rats, TH activation varied between 125 and 147% above basal values. 2. Neonatal hypothyroidism induced by PTU treatment impaired TH activation. Compensatory treatment with T3 to the PTU-treated young rats led to a return to control activation.

Tyrosine content, influx and accumulation rate, and catecholamine biosynthesis measured in vivo, in the central nervous system and in peripheral organs of the young rat. Influence of neonatal hypo- and hyperthyroidism.

The influence of neonatal hypo- and hyperthyroidism on different aspects of tyrosine metabolism in the hypothalamus, striatum, brainstem, adrenal glands, heart and brown adipose tissue (BAT) were studied in 14-day old rats. The synthesis rate of catecholamines (CA) was also determined in vivo after the injection of labelled tyrosine. Hypothyroidism increases tyrosinaemia and endogenous tyrosine concentration in the hypothalamus and BAT. Hyperthyroidism decreases tyrosinaemia and endogenous tyrosine levels in the striatum, adrenals and heart. The accumulation rate of tyrosine determined 30 min after an intravenous injection of the labelled amino acid has been determined in the organs, together with the influx of the amino acid, determined within 20s. Hypothyroidism increases tyrosine accumulation rate in all the organs studied, and tyrosine clearance is decreased in the striatum and brainstem; together with an increased tyrosinaemia, this leads to a normal influx. The influx of tyrosine is increased in the hypothalamus. Hyperthyroidism decreases tyrosine accumulation rate in all the organs except the adrenals. These results indicate that the thyroid status of the young rat can influence tyrosine uptake mechanisms, without modifying an organ's tyrosine content. The fact that hypothyroidism increases tyrosine influx in the hypothalamus without modifying it in the brainstem and striatum reflects an heterogeneous reactivity to the lack of thyroid hormones in different brain structures. Neonatal hypothyroidism decreases the CA synthesis rate in the striatum, the heart and the interscapular brown adipose tissue, while synthesis was enhanced in the brainstem and the adrenals. It is likely that these variations in CA synthesis are due to thyroid hormone modulation of tyrosine hydroxylase activity, the enzyme which catalyses the rate limiting step in CA biosynthesis.

Effects of insulin on brain serotonin in the young rat: influence of thyroid status.

Thyroid status has been shown to modify the adrenal catecholaminergic response to insulin. The influence of thyroid status on the brain serotonergic response to insulin is the subject of the present report. Newborn rats were divided into three groups: euthyroid, hypothyroid (propylthiouracil given to the suckling mother), and hypothyroid-treated with triiodothyronine (T3) as replacement therapy. At 14 days of age, the animals in each group received either insulin (10 IU/kg SC) or saline. Levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were determined in several brain regions. Hypothyroidism induced increases in hypothalamic 5-HT and 5-HIAA and in cortical 5-HIAA levels. The elevations in 5-HIAA levels were reversed by T3. Insulin treatment-induced increases in 5-HIAA levels in all brain regions of both the hypothyroid and the T3-replaced rats. Thyroid status thus influences the serotonergic response to insulin in the young rat, but contrary to what occurred in adrenals for catecholamines, hypothyroidism enhances the central serotonergic response to insulin.

Adrenal dopamine content of the developing rat. Influence of the thyroid status and insulin-hypoglycaemia.

Neonatal hypothyroidism accelerates the postnatal evolution of the adrenal dopamine (DA) content and concentration in the young rat. In 14-day-old control animals, insulin-hypoglycaemia leads to a transient increase in adrenal dopamine content up to 159% above the value in saline-injected animals. This increase is already at its maximum 1 h after insulin administration and lasts 7 h. In the hypothyroid animals, the DA increase evoked by hypoglycaemia is impaired in magnitude (74%) and duration (4 h).

Effect of progesterone on the testosterone and estradiol levels in the hypothalamus of neonatal rats.

Pregnant female rats were injected with progesterone or oil from 18.5 up to 21.5 days of gestation. Rat pups were delivered by caesarean section, and pups delivered from progesterone-treated mothers were injected with 100 micrograms of progesterone and pups from oil-treated mothers were injected with oil. Two hours after delivery, pups were killed and hypothalamic testosterone and estradiol were determined by RIA. Control males had substantially higher concentrations of testosterone and estradiol in hypothalamus than control females. Treatment with progesterone did not affect the accumulation of testosterone in the hypothalamic tissue of neonatal males, but completely prevented the formation of estradiol in hypothalamic tissue. This result suggests that progesterone can inhibit hypothalamic aromatase activity in the neonate and may explain why progesterone can protect against some of the neural defeminizing effects of neonatally administered androgens.

Adrenal dopamine beta-hydroxylase induction in the young rat: influence of thyroid hormones.

The adrenals of 14-day-old control and hypothyroid rats have been stimulated by insulin-induced hypoglycaemia, and the subsequent induction in dopamine beta-hydroxylase (DBH) has been studied. In control rats, DBH induction was maximum 48 h after insulin administration. Hypothyroidism completely suppressed DBH induction.

Adrenal medullary responses to insulin-induced hypoglycaemia in the young rat. Influence of thyroid hormones.

The adrenal medulla of normal, hypothyroid and hyperthyroid young rats was stimulated by insulin-induced hypoglycaemia. In normal rats, insulin-induced adrenal epinephrine secretion increases during the first 10 days of post-natal life. Hypothyroidism retards the development of adrenal response; hyperthyroidism facilitates the development of this response. At 14 days, when insulin-induced adrenal epinephrine depletion is the same for all groups, the recovery of adrenal catecholamines stores after depletion is linear and takes less than 48 h. Recovery rate is slightly slower for hyperthyroid rats than for either hypothyroid or control rats at 14 days. Following epinephrine depletion, adrenal tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) activities are increased for a few days in the control rats, corresponding to a transsynaptic induction. Hypothyroidism impairs TH induction and completely suppresses DBH induction; hyperthyroidism impairs TH induction, but has no effect on DBH induction. These data show that the various processes related to CA synthesis, in the adrenal medulla of the developing rat, are controlled in different ways by the thyroid hormones.

Adrenal phenylethanolamine-N-methyltransferase activity in the young rat: influence of thyroid hormones and insulin-induced hypoglycemia.

The postnatal evolution of adrenal phenylethanolamine-N-methyltransferase (PNMT) activity is slightly accelerated by hypothyroidism and slowed by hyperthyroidism. In 14-day-old rats a single stimulation of the adrenals by insulin-induced hypoglycemia does not lead to any change in enzymatic activity. If the stimulation is repeated for 4 days, it leads to a net increase in adrenal PNMT activity in the control rats, while no increase occurs in hypo- or hyperthyroid animals.

[Influence of neonatal hypothyroidism on the response of the adrenal medulla of young rats to a physiological stimulation]. / Influence de l'hypothyroïdie néo-natale sur la réponse des médullo-surrénales du jeune rat à une stimulation physiologique.

The adrenal medulla of control and hypothyroid young rats was stimulated through insulin hypoglycaemia. In the control animals, the intensity of the depletion of the adrenal epinephrine, which occurs in response to hypoglycaemia, increases up to 10 days of age. Neonatal hypothyroidism results in a retarded evolution. Following epinephrine depletion, the activities of adrenal tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) are transitorily increased in the 14 day-old controls; these increases are the consequence of transsynaptic inductions. Hypothyroidism decreases TH induction and completely suppresses that of DBH.