Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria.

Interactions between primary producers and bacteria impact the physiology of both partners, alter the chemistry of their environment, and shape ecosystem diversity. In marine ecosystems, these interactions are difficult to study partly because the major photosynthetic organisms are microscopic, unicellular phytoplankton. Coastal phytoplankton communities are dominated by diatoms, which generate approximately 40% of marine primary production and form the base of many marine food webs. Diatoms co-occur with specific bacterial taxa, but the mechanisms of potential interactions are mostly unknown. Here we tease apart a bacterial consortium associated with a globally distributed diatom and find that a Sulfitobacter species promotes diatom cell division via secretion of the hormone indole-3-acetic acid, synthesized by the bacterium using both diatom-secreted and endogenous tryptophan. Indole-3-acetic acid and tryptophan serve as signalling molecules that are part of a complex exchange of nutrients, including diatom-excreted organosulfur molecules and bacterial-excreted ammonia. The potential prevalence of this mode of signalling in the oceans is corroborated by metabolite and metatranscriptome analyses that show widespread indole-3-acetic acid production by Sulfitobacter-related bacteria, particularly in coastal environments. Our study expands on the emerging recognition that marine microbial communities are part of tightly connected networks by providing evidence that these interactions are mediated through production and exchange of infochemicals.

Consequences of negative energy balance on follicular development and oocyte quality in primiparous sows†.

Metabolic demands of modern hybrid sows have increased over the years, which increases the chance that sows enter a substantial negative energy balance (NEB) during lactation. This NEB can influence the development of follicles and oocytes that will give rise to the next litter. To study effects of a lactational NEB on follicular development, we used 36 primiparous sows of which 18 were subjected to feed restriction (3.25 kg/day) and 18 were full-fed (6.5 kg/day) during the last 2 weeks of a 24.1 ± 0.3 day lactation. Feed restriction resulted in a 70% larger lactational body weight loss and 76% higher longissimus dorsi depth loss, but similar amounts of backfat loss compared to the full fed sows. These changes were accompanied by lower plasma insulin-like growth factor 1 (IGF1) and higher plasma creatinine levels in the restricted sows from the last week of lactation onward. Ovaries were collected 48 h after weaning. Restricted sows had a lower average size of the 15 largest follicles (-26%) and cumulus-oocyte complexes showed less expansion after 22 h in vitro maturation (-26%). Less zygotes of restricted sows reached the metaphase stage 24 h after in vitro fertilization and showed a higher incidence of polyspermy (+89%). This shows that feed restriction had severe consequences on oocyte developmental competence. Follicular fluid of restricted sows had lower IGF1 (-56%) and steroid levels (e.g., ß-estradiol, progestins, and androgens), which indicated that follicles of restricted sows were less competent to produce steroids and growth factors needed for oocytes to obtain full developmental competence.

Effect of pregnancy on endometrial expression of luteolytic pathway components in the mare.

Endometrial oxytocin receptors (OXTR) and prostaglandin-endoperoxide synthase 2 (PTGS2) are central components of the luteolytic pathway in cyclic mares, and their suppression is thought to be critical to luteal maintenance during early pregnancy. We examined the effect of pregnancy on endometrial expression of potential regulators of prostaglandin (PG) F2α secretion in mares. Expression of the nuclear progesterone receptor and oestrogen receptor ERα was high during oestrus, and depressed when progesterone was elevated; the opposite applied to the membrane progesterone receptor. PTGS2 was upregulated on Day 14 of dioestrus, but not pregnancy. Although OXTR mRNA expression was not elevated on Day 14 of dioestrus, protein abundance was; this increase in OXTR protein was absent on Day 14 of pregnancy. Intriguingly, gene and protein expression for PTGS2 and OXTR increased markedly between Days 14 and 21 of pregnancy suggesting that, although initial avoidance of luteolysis during pregnancy involves their suppression, this is a transient measure that delays rather than abolishes luteolytic pathway generation. The only oxytocin-PGF2α feedback loop component downregulated on both Days 14 and 21 of pregnancy was the PGF2α receptor we propose that downregulation of the PGF2α receptor uncouples the oxytocin-PGF2α feedback loop, thereby preventing generation of the large PGF2α pulses required for luteolysis.

Ketoconazole blocks progesterone production without affecting other parameters of cumulus-oocyte complex maturation.

Ketoconazole (KTZ) is widely used as a fungicide, but it is also known to target steroid hormone formation which may affect female reproductive health. Our study aims to investigate the effects of KTZ on in vitro matured bovine cumulus-oocyte complexes (COCs), as a model for female reproductive toxicity. Cumulus cells of in vitro maturing COCs produce progesterone and pregnenolone, but exposure to 10-6 M KTZ effectively blocked the synthesis of these hormones. Exposure to lower concentrations of KTZ (i.e. 10-7 M and 10-8 M) had no such effect on steroidogenesis compared to the 0.1 % v/v DMSO vehicle control. Classical parameters of in vitro COC maturation, such as oocyte nuclear maturation to the metaphase II stage and expansion of the cumulus investment, were not affected by any KTZ concentration tested. Apoptosis and necrosis levels were also not altered in cumulus cells or oocytes exposed to KTZ. Moreover, oocytes exposed to KTZ during maturation showed normal cleavage and early embryo development up to day 8 post fertilization; albeit a statistically significant decrease was observed in day 8 blastocysts produced from oocytes exposed to the lowest concentration of 10-8 M KTZ. When unexposed mature oocytes were fertilized, followed by embryo culture for 8 days under KTZ exposure, no adverse effects in embryo cleavage and blastocyst formation were observed. In conclusion, KTZ has no major impact on in vitro bovine oocyte maturation and blastocyst formation in our study, even at concentrations blocking steroidogenesis.

The origin of Indonesian cattle and conservation genetics of the Bali cattle breed.

Both Bos indicus (zebu) and Bos javanicus (banteng) contribute to the Indonesian indigenous livestock, which is supposedly of a mixed species origin, not by direct breeding but by secondary cross-breeding. Here, the analysis of mitochondrial, Y-chromosomal and microsatellite DNA showed banteng introgression of 10-16% in Indonesian zebu breeds with East-Javanese Madura and Galekan cattle having higher levels of autosomal banteng introgression (20-30%) and combine a zebu paternal lineage with a predominant (Madura) or even complete (Galekan) maternal banteng origin. Two Madura bulls carried taurine Y-chromosomal haplotypes, presumably of French Limousin origin. There was no evidence for zebu introgression in five populations of the Bali cattle, a domestic form of the banteng.

Bovine In Vitro Oocyte Maturation and Embryo Production Used as a Model for Testing Endocrine Disrupting Chemicals Eliciting Female Reproductive Toxicity With Diethylstilbestrol as a Showcase Compound.

Endocrine disrupting chemicals (EDCs) can interfere with normal hormonal action and regulation. Exposure of women to EDCs has been associated with adverse reproductive health outcomes. The assays currently used to identify EDCs that elicit female reproductive toxicity lack screening tests that address effects on the maturation of oocytes, a process that enables them to be fertilized and develop into embryos. Here, a screening method employing the bovine model of in vitro oocyte maturation and embryo production is described. Endpoints explored address important events in oocyte maturation and developmental competence acquisition. To test the method, the effects of the known human EDC diethylstilbestrol (DES; an estrogen receptor agonist) were evaluated in a range of concentrations (10-9 M, 10-7 M, 10-5 M). Bovine oocytes were exposed to DES during in vitro maturation (IVM) or embryos were exposed during in vitro embryo culture (IVC). The endpoints evaluated included nuclear maturation, mitochondrial redistribution, cumulus cell expansion, apoptosis, and steroidogenesis. DES-exposed oocytes were fertilized to record embryo cleavage and blastocyst rates to uncover effects on developmental competence. Similarly, the development of embryos exposed to DES during IVC was monitored to assess the impact on early embryo development. Exposure to 10-9 M or 10-7 M DES did not affect the endpoints addressing oocyte maturation or embryo development. However, there were considerable detrimental effects observed in oocytes exposed to 10-5 M DES. Specifically, compared to vehicle-treated oocytes, there was a statistically significant reduction in nuclear maturation (3% vs 84%), cumulus expansion (2.8-fold vs 3.6-fold) and blastocyst rate (3% vs 32%). Additionally, progesterone and pregnenolone concentrations measured in IVM culture media were increased. The screening method described here shows that bovine oocytes were sensitive to the action of this particular chemical (i.e., DES), albeit at high concentrations. In principle, this method provides a valuable tool to assess the oocyte maturation process and early embryo development that can be used for reproductive toxicity screening and possibly EDC identification. Further studies should include EDCs with different mechanisms of action and additional endpoints to further demonstrate the applicability of the bovine oocyte model for chemical risk assessment purposes and EDC identification.

Expression of progesterone and oestrogen receptors by early intrauterine equine conceptuses.

Progesterone and oestrogen play essential roles in the maintenance of pregnancy in eutherian mammals and are thought to exert their effects on the developing conceptus indirectly, via the endometrium. In some species, early embryos have themselves been shown to express steroid receptors, thereby suggesting that reproductive steroids may also influence embryonic development directly. The aim of this study was to determine whether early intrauterine equine conceptuses express either the classical intracellular progesterone (PR) and oestrogen receptors (ERalpha and ERbeta) or the more recently characterised membrane-bound progesterone receptors (PGRMC1 and mPR). Horse conceptuses recovered on days 7, 10 and 14 after ovulation (n=8 at each stage) were examined for steroid receptor mRNA expression using quantitative rtPCR. Where commercial antibodies were available (PR, ERbeta), receptor localisation was examined immunohistochemically in day 10, 12, 14, 15 and 16 conceptuses (n=2 at each stage). mRNA for PR, PGRMC1 and mPR was detected at all stages examined, but while PGRMC1 and mPR expression increased during the day 7-14 period, PR expression decreased. ERalpha mRNA was not detected at any stage examined, whereas ERbeta mRNA was detected in all day 14, some day 10 and no day 7 conceptuses. Immunoreactive ERbeta receptors were localised to the trophectoderm of day 14-16 conceptuses; PR were not detected immunohistochemically in conceptus tissue. In summary, this study demonstrates that equine conceptuses express mRNA and, in the case of ERbeta, protein for steroid hormone receptors during the period encompassing rapid conceptus growth, differentiation and maternal pregnancy recognition.

The interaction between TPH2 promoter haplotypes and clinical-demographic risk factors in suicide victims with major psychoses.

Tryptophan hydroxylase isoform 2 (TPH2) is a rate-limiting enzyme in the biosynthesis of serotonin (5-HT) and is predominantly localized in the brain. Previous studies have suggested that there is an association between serotonergic dysfunction in the brain and suicidality. This study was designed to examine whether the -473T > A and -8396G > C polymorphisms of the TPH2 gene may be associated with completed suicide in subjects with major psychoses from the Stanley Foundation Brain Bank sample. TPH2 genotypes were determined in 69 subjects with a diagnosis of schizophrenia or bipolar disorder, among which 22 died by suicide. Genomic DNA was amplified by polymerase chain reaction and typed by automated methods. Both markers were found to be in Hardy-Weinberg equilibrium and in strong linkage disequilibrium. No association with history of suicide was found for either polymorphism. Haplotype analysis with EHAP showed no association between completed suicide and haplotype distribution (chi2 = 1.877; 3 df; P = 0.598). Nor was there any association between suicide and these genetic markers even when clinical-demographic factors were considered as covariates in the haplotype analysis. These findings suggest that these 5' marker haplotypes in the TPH2 gene do not influence suicidal behaviour.

Expression of 3alpha- and 3beta-hydroxy steroid dehydrogenase mRNA in COCs and granulosa cells determines Zearalenone biotransformation.

Zearalenone (ZEA) is a mycoestrogen found in diverse food and feed materials, particularly in corn and small grains. Following ingestion, the parent zearalenone is converted predominantly into alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) by hepatic hydroxy steroid dehydrogenases (HSD). The present study demonstrated by standard RT-PCR the expression of 3alpha- and 3beta-HSD also in porcine cumulus oocyte complexes (COCs) and granulosa cells isolated form cumulus oocyte complexes. Analysis of the rate of bioconversion of zearalenone (ZEA) by the cultured granulose cells showed the extra-hepatic production of both hydroxy metabolites of ZEA with alpha-ZOL being the dominating metabolites as previously observed in incubations with liver microsomes. The endogenous steroids 5alpha-dihydrotestosterone (5alpha-DHT), and progesterone (PGTN), both known substrates for 3alpha-HSD inhibited the conversion of ZEA into alpha-ZOL. In the presence of pregnelonone (PGN), a major substrate for 3beta-HSD only a slight inhibitory effect on the apparent beta-ZOL formation could be observed. In conclusion, these data indicate that both 3alpha- and 3beta-HSDs are expressed in porcine COCs and GCs, whereas the biotransformation experiments confirm the involvement of these enzymes in the extra-hepatic biotransformation of ZEA.

Dopamine receptor pharmacology.

Dopamine receptors are the primary targets in the treatment of schizophrenia, Parkinson's disease, and Huntington's chorea, and are discussed in this review by Philip Seeman and Hubert Van Tol. Improved therapy may be obtained by drugs that selectively target a particular subtype of dopamine receptor. Most antipsychotic drugs block D2 receptors in direct correlation to clinical potency, except clozapine, which prefers D4 receptors. D1 and D2 receptors can enhance each other's actions, possibly through subunits of the G proteins. In schizophrenia, the D2 and D3 receptor density is elevated by 10%, while the D4 receptor density is elevated by 600%. Therefore, D4 receptors may be a target for future antipsychotic drugs. While antipsychotics originally helped to discover dopamine receptors, the five cloned dopamine receptors are now facilitating the discovery of selective antipsychotic and antiparkinson drugs.

Analysis of the D4 dopamine receptor gene variant in an Italian schizophrenia kindred.

BACKGROUND: Among the different dopamine receptors, the D4 dopamine receptor is of particular interest in schizophrenia because of its high affinity for the atypical neuroleptic clozapine. Recently, the gene for the D4 dopamine receptor has been cloned and a new and intriguing polymorphism has been described. Different versions of the receptor have varying affinity for clozapine, and thus variant froms of D4 with differing pharmacologic activity exist in the human population. Our hypothesis was that these variants play a role in susceptibility to psychotic illness. Thus, our objective was to test the D4 dopamine receptor genes for linkage to schizophrenia. METHODS: Our genetic linkage study was carried out in a large Italian kindred segregating schizophrenia. Diagnoses were made by using a structured clinical interview and a consensus diagnosis was established. For the computer analysis, 80 members of the family were constructed into a linked set of relatives with 15 of these individuals affected by schizophrenia. The functional variants of the D4 dopamine receptor gene were identified by a combination of Southern blot techniques and the polymerase chain reaction. The gene for tyrosine hydroxylase (TH) was also tested for linkage to schizophrenia in this family. Linkage analyses were done with both a single-locus and a two-locus model. RESULTS: Our results revealed significantly negative lod scores in the region of the D4 dopamine receptor gene and the TH gene. The application of different models of transmission for schizophrenia had an effect on the magnitude of the lod scores, but did not modify the direction of the results. CONCLUSIONS: Our results provide significant evidence for exclusion for linkage between schizophrenia and the dopamine D4 receptor gene and the TH gene under the models specified. Furthermore, we tabulated the distribution of D4 dopamine gene variants in the diseased vs healthy individuals in the family and the results showed that no specific form of the receptor gene is significantly associated with the presence of schizophrenia in the family. Our study does not exclude the possibility that regulatory elements of the D4 dopamine gene located elsewhere in the genome may be involved in the etiology of schizophrenia.

SH3 ligands in the dopamine D3 receptor.

It has recently been observed that G protein-coupled receptors (GPCRs) can interact with SH3 domains through polyproline motifs. These interactions appear to be involved in receptor internalization and MAPK signalling. Here we report that the third cytoplasmic loop of the dopamine D3 receptor can interact in vitro with the adaptor protein Grb2. While the amino- and carboxy-terminal SH3 domains of Grb2 separately did not interact with the D3 receptor loop, the interaction is at least partially maintained with a Grb2 mutant for the amino-terminal SH3 domain, but disrupted for a Grb2 mutant with a nonfunctional carboxy-terminal SH3 domain. The data indicate the need of structural integrity of the entire Grb2 protein for the interaction and dominant role of the carboxy-terminal SH3 domain in the interaction. Disruption of the PXXP motifs in the D3 receptor did not affect the interaction with Grb2. These results indicate that GPCRs may contain SH3 ligands that do not contain the postulated minimal consensus sequence PXXP.

Co-expression of human Kir3 subunits can yield channels with different functional properties.

To date, no comprehensive study has been done on all combinations of the human homologues of the Kir3.0 channel family, and the human homologue of Kir3.3 has not yet been identified. To obtain support for the contention that most of the functional data on non-human Kir3.0 channels can be extrapolated to human channels, we have cloned the human homologues of the Kir3.0 family, including the yet unidentified human Kir3.3, and the human Kir4.1. The expression pattern of these channels in various human brain areas and peripheral tissues, analysed by Northern blot analysis, allows for the existence of various homomeric and heteromeric forms of human Kir3.0 channels. Expression studies of all possible combinations in Xenopus oocytes indicated that in homomeric Kir3.2c and heteromeric Kir3.1/3.2c channels mediate, in our studies, inward currents with largest amplitude of any other Kir3.0 channel combinations, followed by heteromeric Kir3.1/3.4 and homomeric Kir4.1 channels. Channel combinations which include Kir3.3 are detrimental to the formation of functional channels. The co-expression experiments with different Kir channel subunits indicate the selective formation of certain channel combinations, suggesting that channel specificity is not solely dependent on spatial and temporal regulation of Kir subunit expression.

Expression of bone morphogenetic protein2 (BMP2), BMP4 and BMP receptors in the bovine ovary but absence of effects of BMP2 and BMP4 during IVM on bovine oocyte nuclear maturation and subsequent embryo development.

Bone morphogenetic proteins (BMPs) have been implicated in the regulation of ovarian follicular development and are promising candidates to apply in IVM and IVF protocols. We investigated the expression of BMP2, BMP4 and BMP receptors in bovine ovaries and the effects of BMP2 and BMP4 during oocyte maturation on bovine IVM. Reverse transcription polymerase chain reaction studies with antral follicles showed the expression of BMPR-IA, BMPR-IB, ActR-IA, ActR-IIB, BMPR-II and BMP4 mRNA in all follicular compartments, while BMP2 mRNA was generally restricted to theca and cumulus tissue. Immunohistochemistry demonstrated the presence of BMPR-II in oocytes and granulosa cells of preantral follicles but only in oocytes of antral follicles. The immunostaining of BMP2 and BMP4 was limited to theca interna and approximately 25% of oocytes of antral follicles. Exogenously added BMP2 or BMP4 to IVM medium did not affect oocyte nuclear maturation, cumulus cell expansion, nor blastocyst formation following IVF. It is concluded that a BMP-signaling system, consisting of BMP2, BMP4, type II and I receptors, is present in bovine antral follicles and that this system plays a role in development and functioning of these follicles rather than in final oocyte maturation and cumulus expansion.

Dopamine D4 receptor-mediated inhibition of cyclic adenosine 3',5'-monophosphate production does not affect prolactin regulation.

Under physiological conditions, PRL synthesis and secretion are predominantly under negative control by dopamine acting through dopamine D2 receptors present in the pituitary lactotroph cells. To investigate the role of D4 receptors in the regulation of PRL synthesis and secretion, we stably transfected the human D4 receptor complementary DNA into the somatomammotrophic cell line GH4C1. The pharmacological characteristics of D4 expressed in GH4C1 were in close agreement with previous D4 receptor studies in Chinese hamster ovary and COS-7 cells. In GH4C1 cells, activation of D4 receptor variants (D4.2, D4.4, and D4.7) resulted in a similar level of reduction in forskolin- and vasoactive intestinal peptide (VIP)-stimulated cAMP levels (33% and 50%, respectively). In addition, the forskolin-stimulated activity of cAMP response elements fused to the VIP promoter driving the lacZ reporter gene could be blocked by D4 activation. However, quinpirole treatment had a minimal effect on transiently expressed luciferase reporter gene driven by a proximal PRL promoter in one of the D4-expressing cell lines. In contrast, the dopamine D2short receptor expressing GH4ZR7 cells treated with quinpirole displayed a significant decrease (51.3 +/- 4.1%) in PRL promoter activity. VIP-stimulated PRL release was not affected by D4 receptor activation, whereas in GH4ZR7 cells, a significant decrease in VIP-stimulated PRL levels was observed. Neither PRL promoter activity nor PRL secretion levels were affected in control untransfected GH4C1 cells. From this study it appears that although the D4 receptor may be expressed in the anterior pituitary, it does not have a major effect on PRL promoter activity or PRL secretion in GH4C1 cells despite its ability to reduce cAMP production. This might explain why D4- over D2-preferring antipsychotics such as clozapine do not cause hyperprolactinemia.

Oxytocin gene expression in discrete hypothalamic magnocellular cell groups is stimulated by prolonged salt loading.

The effect of prolonged osmotic stimulation on the oxytocin (OT) mRNA levels of OT-producing neurons was investigated in separate hypothalamic nuclei of the rat. After drinking 2% NaCl for 2 weeks, a 2-fold increase in the OT mRNA content was found by Northern blot analysis of microdissected supraoptic nucleus (SON) and paraventricular nucleus (PVN). The same samples showed a similar change in vasopressin (VP) mRNA levels. This treatment resulted in a 70% depletion of both OT and VP from the neurointermediate lobe of the pituitary gland and a marked increase in OT and VP plasma levels. Analysis of the OT mRNA concentration by quantitative in situ hybridization showed that the SON, PVN, and anterior commissural nucleus responded similarly, with a 1.5-fold increase in OT mRNA after 7 days of osmotic stimulation. These results demonstrate that OT gene expression in the magnocellular neurons of the SON, PVN, and anterior commissural nucleus is sensitive to this osmotic stimulus, as is the VP gene of magnocellular neurons in the SON and PVN. The responsiveness of OT-producing neurons may point to a role for OT in the regulation of water and electrolyte balance.

Oxytocin and vasopressin gene expression in the hypothalamo-neurohypophyseal system of the rat during the estrous cycle, pregnancy, and lactation.

To study the state of hypothalamic expression of the oxytocin (OT) and vasopressin (VP) genes in different conditions of the female rat, OT and VP mRNA levels were determined during the estrous cycle, pregnancy, and lactation. OT and VP mRNA levels of the supraoptic nucleus were quantified by Northern blot and dot-blot analysis. OT and VP contents of the pituitary gland were determined by RIA. During the estrous cycle, OT mRNA levels of the supraoptic nucleus were significantly increased 1.5- to 2-fold at estrous relative to the other periods of the cycle. No significant cyclic variation was observed in VP mRNA levels. The OT content of the pituitary gland was significantly decreased at estrus and also the VP contents showed a cyclic variation with the lowest levels occurring at estrus and metestrus. At the last day of gestation a 3- and 2-fold increase of OT mRNA and VP mRNA, respectively, was measured, but no changes were observed at gestational day 12, 15, and 18. In rats lactating for 15 days, OT and VP mRNA levels were approximately 3-fold higher than during the estrous cycle. Concomitantly the OT and VP contents of the pituitary gland were decreased by approximately 50%. It is concluded that both OT and VP gene expression are stimulated just before birth and during lactation indicating that both genes are sensitive to common factors. A dissociation of the regulation of the OT and VP genes occurs in the estrous cycle.

Comparative pharmacological and functional analysis of the human dopamine D4.2 and D4.10 receptor variants.

The human dopamine D4 receptor is a D2-like receptor which is a target for most common neuroleptics. Previous investigations have shown that this receptor displays a large polymorphic variation in the third intracellular loop involving a variable number of direct imperfect tandem repeats (VNTR) of 16 amino acids. The shortest and longest repeat variants reported to date contain two and 10 repeat units (D4.2 and D4.10). No major pharmacological differences have been reported for the most common variants of this receptor (D4.2, D4.4 and D4.7), although the D4.7 was reported by us to display a slightly lower potency for dopamine in functional assays. Direct pharmacological and functional comparison of the longest and shortest variants in this study suggest no major discrepancies in pharmacological or functional profile between both receptors. Both receptors display, on average, a 15-fold and 90-fold lower potency for epinephrine and norepinephrine, respectively, compared with dopamine. We observed small increases in functional potency and affinity for dopamine and quinpirole at the D4.10 receptor variant compared with the D4.2 receptor. Our data indicate that there is no direct relationship between the length of the polymorphism and changes in pharmacology or functional activity. These findings are a suitable caution against the arbitrary pooling of D4 receptor VNTR genotypes in genetic studies, based on length.

The human tRNAVal gene family: organization, nucleotide sequences and homologous transcription of three single-copy genes.

At least 13 independent tRNAVal gene loci were detected in the human genome. Three of these genes were isolated and shown to occur only once in the haploid genome. No further functional tRNA genes are located on the isolated clones. Two tRNAVal genes encode the known major and minor tRNAVal isoacceptors, the third may be a pseudogene because a corresponding tRNAVal is not yet known. Comparison of extragenic sequences did not reveal significant homologies, indicating the separation of these genes early in vertebrate evolution. An Alu-type repeat was found in two of the clones within several hundred bp distance from the tDNA. All three genes are transcriptionally active in a HeLa nuclear extract. We show here for the first time that homologous in vitro transcription of mammalian tRNA genes strongly depends on extragenic control regions: interestingly, as a consequence of different flanking regions, the transcription efficiencies vary by an order of magnitude among the genes for the major and the minor tRNAVal and thus reflect the concentrations of these tRNAs in vivo.

Vasopressin gene expression is attenuated in the fetal Brattleboro rat.

Due to a genetic defect the homozygous Brattleboro rat is unable to synthesize vasopressin gene products but still transcribes a mutant vasopressin mRNA from the gene. To study the influence of vasopressin gene products on the development of vasopressin gene expression, vasopressin mRNA levels of the supraoptic and paraventricular nucleus were measured at fetal day 20, postnatal day 1, 15 and 30 in the Wistar rat and in the heterozygous and homozygous Brattleboro rat by Northern blot analysis and in situ hybridization. In the homozygous Brattleboro rat of fetal day 20 and postnatal day 1, no or minute amounts of vasopressin mRNA were detectable but vasopressin mRNA was readily detectable at postnatal day 15 and 30. The Wistar rat and heterozygous Brattleboro rat had abundant vasopressin mRNA at fetal day 20 with increasing amounts towards postnatal day 30. The results indicate that vasopressin gene expression in the development of the homozygous Brattleboro rat is attenuated, possibly due to the absence of vasopressin gene products.