The neutral rate of whole-genome duplication varies among yeast species and their hybrids.

Hybridization and polyploidization are powerful mechanisms of speciation. Hybrid speciation often coincides with whole-genome duplication (WGD) in eukaryotes. This suggests that WGD may allow hybrids to thrive by increasing fitness, restoring fertility and/or increasing access to adaptive mutations. Alternatively, it has been suggested that hybridization itself may trigger WGD. Testing these models requires quantifying the rate of WGD in hybrids without the confounding effect of natural selection. Here we show, by measuring the spontaneous rate of WGD of more than 1300 yeast crosses evolved under relaxed selection, that some genotypes or combinations of genotypes are more prone to WGD, including some hybrids between closely related species. We also find that higher WGD rate correlates with higher genomic instability and that WGD increases fertility and genetic variability. These results provide evidence that hybridization itself can promote WGD, which in turn facilitates the evolution of hybrids.

Mobility and transformation of CdSe/ZnS quantum dots in soil: Role of the capping ligands and ageing effect.

The increasing application of Quantum Dots (QDs) is cause of concern for the potential negative effects for the ecosystem, especially in soils that may act as a sink. In this study, soil leaching experiments were performed in quartz sand packed columns to investigate the behavior of core-shell CdSe/ZnS QDs coated with either small ligands (TGA-QDs) or more complex polymers (POAMA-QDs). Fluorescence emission was compared to mass spectrometric measurements to assess the nanoparticles (NPs) state in both the leachate (transported species) and porous media (deposited amounts). Although both QDs were strongly retained in the column, large differences were observed depending on their capping ligand stability. Specifically, for TGA-QDs elution was negligible and the retained fraction accumulated in the top-columns. Furthermore, 74% of the NPs were degraded and 38% of the Se was found in the leachate in non-NPs state. Conversely, POAMA-QDs were recovered to a larger extent (78.1%), and displayed a higher transport along the soil profile. Further experiments with altered NPs showed that homo-aggregation of the QDs prior injection determined a reduced mobility but no significant changes in their stability. Eventually, ageing of the NPs in the column (15 days) caused the disruption of up to 92% of the original QDs and the immobilization of NPs and metals. These results indicate that QDs will accumulate in top-soils, where transformations phenomena will determine the overall transport, persistency and degradation of these chemicals. Once accumulated, they may act as a source for potentially toxic Cd and Se metal species displaying enhanced mobility.

Tracing multi-isotopically labelled CdSe/ZnS quantum dots in biological media.

The strengths and limits of isotopically labelled Engineered Nanoparticles (spiked ENPs) spread in biological media have been assessed. Multi-spiked CdSe/ZnS quantum dots (QDs), measuring 7 nm and coated with thioglycolic acid (TGA), were synthesized and enriched in 68Zn, 77Se and 111Cd. These QDs were dispersed at very low concentrations (0.1 to 5000 ppt) in diverse biological matrices (synthetic saliva, synthetic urine, plasma and Dulbecco's phosphate buffered saline - DPBS growth medium) and the isotopic compositions were determined by HR-ICP-MS. The initial QDs concentrations were calculated to assess the limit of quantification (QD-LOQ) according to the matrix and the isotopically enriched element. The obtained results demonstrated the advantages of the isotopic labelling method in order to work at very low concentrations: the QD-LOQ values for the spiked Zn, Cd and Se originated from the QDs were 10, 0.3 and 6 ppt, respectively, which is below the conventional LOQ of the HR-ICP-MS used (30, 3 and 60 ppt for Zn, Cd and Se, respectively). Conversely, in complex matrices such as saliva, urine, plasma and DPBS growth medium, the QD-LOQ values increased significantly, with values ranging from 16 to 32 ppt for Cd, 446 to 10598 ppt for Zn and 1618 to 8317 ppt for Se. These QD-LOQs are dependent on factors as the elemental background concentration already present in the matrices, and the dilution factor. In this study, the QD-LOQs are expressed for the first time with respect to the background concentration in biological media (QD-RLOQ), which can be used to better assess and then predict the efficiency of the spiking method.

Isotopically Labeled Nanoparticles at Relevant Concentrations: How Low Can We Go? The Case of CdSe/ZnS QDs in Surface Waters.

Analytical barriers impose work at nanoparticles (NPs) concentrations orders of magnitude higher than the expected NPs concentrations in the environment. To overcome these limitations, the use of nontraditional stable isotope tracers incorporated in NPs (spiked-NPs) coupled with HR-ICP-MS has been proposed. The performance and efficiency of this analytical method was assessed in the case of quantum dots (QDs). Multi-isotopically labeled 111Cd77Se/68ZnS QDs were synthesized and their dissemination in natural aquatic matrices (river, estuarine and sea waters) was modeled at very low concentrations (from 0.1 to 5000 ppt). The QD limits of quantification (QD-LOQ) in each matrix were calculated according to the isotopic tracer. In ultrapure and simple medium (HNO3 2%), Zn, Cd, and Se originated from the QDs were quantifiable at concentrations of 10, 0.3, and 6 ppt, respectively, which are lower than the conventional HR-ICP-MS LOQs. In aquatic matrices, the QD-LOQs increase 10-, 130-, and 250-fold for Zn, Cd, and Se, respectively, but remain relevant of environmental concentrations (3.4 ppt ≤ QD-LOQs ≤ 2.5 ppb). These results validate the use of isotopically labeled ENPs at relevant concentrations in experimental studies related to either their fate, behavior, or toxicity in most aquatic matrices.

Neuropathology of NFHgp160 transgenic mice expressing HIV-1 env protein in neurons.

The physiopathology of HIV-1 dementia remains largely hypothetical. Although several sets of evidence point towards an indirect multicellular inflammatory pathway, gp120, one of the HIV-1 env products, was shown to be very cytotoxic for neurons in vitro. To explore a direct pathway in the physiopathology of dementia in AIDS, we developed transgenic mouse models carrying the HIV-1 env proteins gp 120 and gp 41 (gp 160) under the control of the human light neurofilament and murine heavy neurofilament promoters. To date, this is the first mouse model in which the HIV-1 env protein can be detected in neurons by immunohistochemistry. The expression is found in several brainstem and spinal cord gray structures and in the cerebellum in one of the mouse lines bearing the NFHgp160 transgene. The morphological findings at 3 months are subtle and are dominated by a watery, dendritic degeneration and a reactive gliosis. At 12 months, the evidence of neuronal degeneration and loss is present along with various degenerative phenomena involving synapses, dendrites and axons, including axonal swellings. Cytoskeletal abnormalities were found by immunohistochemistry. Chronic inflammation was also observed in the leptomeninges of the spinal cord and brainstem and in the cerebellar white matter. These models thus offer an exciting sequence of morphological findings initiated by the neuronal expression of the HIV-1 env proteins and offer a different tool to explore the neuronal dysfunction in AIDS.

Response of onion plants to arbuscular mycorrhizae : 2. Effects of nitrogen fertilization on biomass and bulb firmness.

The effects of N fertilization on growth and root colonization of preinoculated onion (Allium cepa L. cv. Improved Autumn Spice) were studied. Onion transplants, inoculated with either Glomus intraradices, G. versiforme or nothing at sowing, were grown under three levels of N in soils which had either been irradiated, irradiated and amended with nonmycorrhizal microflora, or not irradiated. Interactions between inoculation and soil treatment had a significant effect on dry biomass and final bulb diameter. Control plants cultivated in non-irradiated natural soil grew normally because of the presence of indigenous arbuscular mycorrhizae, but control plants in irradiated soils were stunted. There was no such difference among inoculated plants. In non-irradiated natural soil, bulbs of onions inoculated with G. intraradices or G. versiforme were significantly firmer than bulbs of control plants. Bulb firmness decreased as N fertilization level increased. In non-irradiated natural soil, tissue P concentration of onion plants preinoculated with either fungus was significantly higher than that of control plants. In all soil types, N, P, and Zn concentrations were higher in onion plants colonized by G. versiforme than in those colonized by G. intraradices. The opposite was true of Mn tissue concentration.

Agonist stimulation provokes dendritic and axonal dopamine D(1) receptor redistribution in primary cultures of striatal neurons.

To investigate the influence of neurotransmitter on G-protein-coupled receptor trafficking and compartimentalization in neurons, we have developed a model of primary neuronal cultures from fetal rat striatum on which we have studied the cellular and subcellular distribution and trafficking of the D(1) dopaminergic receptor. This receptor is known to be somatodendritic and axonal targeted in vivo, mostly to extrasynaptic locations. Immunohistochemical studies at the light and electron microscopic levels showed that, in cultures, the D(1) dopaminergic receptor is expressed in the absence of dopamine stimulation. The pattern of D(1) dopaminergic receptor immunostaining after stimulation by the D(1) dopaminergic receptor agonist SKF 82958 (1 microM) is dramatically modified with a decrease of the number of labeled D(1) dopaminergic receptor puncta (-40%) and an increase of their size in both dendrites (+120%) and axons (+240%). Seven hours after removal of the agonist, return to normal pattern was observed. The D(1) dopaminergic receptor antagonist SCH 23390 (2 microM) abolishes the effect of SKF 82958. Electron microscopy demonstrated, in dendrites, a translocation of the labeling from the plasma membrane to endosomes. Axonal D(1) dopaminergic receptor redistribution after acute stimulation indicates that the D(1) dopaminergic receptor is membrane targeted and responsive to stimulation. These results validate primary culture of striatal neurons to study subcellular localization and intraneuronal trafficking of G-protein-coupled receptors. This preparation will be useful to address various questions concerning the behavior and the trafficking of these receptors in neurons in relation to the neurotransmitter environment.

Developmental expression and localization of the prolactin receptor (PRL-R) gene in ewe mammary gland during pregnancy and lactation: estimation of the ratio of the two forms of PRL-R messenger ribonucleic acid.

In this study, we have analyzed the developmental expression of the prolactin receptor (PRL-R) gene in the ewe mammary gland during pregnancy and lactation. Using Northern and slot-blot analysis and in situ hybridization, we showed that the level of PRL-R mRNA in mammary epithelial cells increased during the second half of pregnancy, decreased at the end of pregnancy, and remained relatively stable during lactation with a level above that observed at the beginning of pregnancy. As shown by RNase protection assay, the ratio of the long to the short form of the PRL-R mRNA was always above 1. This ratio increased between Day 70 of pregnancy and term and decreased progressively during lactation. The high level of PRL-R mRNA before the induction of alphaS1-casein gene expression suggests that PRL may be involved in the growth and development of the mammary gland. More precisely, the increase of the ratio of the long to the short form of the PRL-R during lactogenesis suggests that the latter form may have a dominant negative action in the activation of milk protein gene transcription. Thus the long/short-form ratio of the PRL-R may play a key role in the shift between growth and differentiation of the mammary gland.

Ontogeny of the striatal neurons expressing the D2 dopamine receptor in humans: an in situ hybridization and receptor-binding study.

D2 dopamine receptor (D2R) gene expression was analyzed by in situ hybridization and D2R ligand autoradiography in the human striatum during ontogeny. D2R mRNA and ([3H]YM-09151-2)-binding sites were detected in the striatum from week 12 of fetal life. At this time, D2R mRNA and binding sites were predominant in the putamen and occurred in a pattern of clusters. D2R-binding sites displayed a similar pattern. The signal in the caudate nucleus was weak from weeks 12 to 16. From week 20 of fetal life, D2R mRNA and D2R-binding sites signals became intense in the ventral striatum. At birth, D2R mRNA became homogeneously distributed while D2R-binding sites kept an heterogeneously distribution. Comparative topological and temporal analysis of the D2R, enkephalin and D1 dopamine receptor (D1R) mRNAs showed a distinct developmental pattern for each mRNA. Before birth, the neurons expressing enkephalin and D1R mRNAs were preferentially distributed in the matrix and in the striosomes, respectively, while the neurons expressing D2R mRNA did not display a preferential localization. At birth, high levels of enkephalin mRNA were restricted to the matrix; D1R mRNA level was homogeneous throughout the striatum. D2R mRNA was heterogeneously distributed in the whole striatum with high signals located both in the striosomes and the matrix. These results demonstrate that functional D2R are expressed as early as week 12 in the striatum with a heterogeneous distribution. Our findings also demonstrate that, in contrast to what was expected from similar studies in rodents, D2R mRNA and enkephalin mRNA do not display identical, overlapping expression patterns in striatal neurons during human ontogeny.

An mRNA encoding a putative GABA-gated chloride channel is expressed in the human cardiac conduction system.

GABA-gated chloride channels are the main inhibitory neurotransmitter receptors in the CNS. Conserved domains among members of previously described GABAA receptor subunits were used to design degenerate sense and antisense oligonucleotides. A PCR product from this amplification was used to isolate a full-length cDNA. The predicted protein has many of the features shared by other members of the ligand-gated ion channel family. This channel subunit has significant amino acid identity (25-40%) with members of GABAA and GABAC receptor subunits and thus may represent a new subfamily of the GABA receptor channel. Although we cannot rule out that this clone encodes a receptor for an unidentified ligand, it was termed GABA chi. This gene is mainly expressed in placenta and in heart; however, placenta appears to express only an unspliced mRNA. In situ hybridization reveals that the GABA chi subunit mRNA is present in the electrical conduction system of the human heart. Our results suggest that novel GABA receptors expressed outside of the CNS may regulate cardiac function.

Ontogeny of the striatal neurons expressing the D1 dopamine receptor in humans.

We studied D1 dopamine receptor (D1R) gene expression in the human striatum during ontogeny by in situ hybridization, immunohistochemistry, and D1R ligand autoradiography. D1R mRNA, protein, and binding sites ([3H]SCH 23390) were detected in the striatum from week 12 of fetal life. At this time, D1R mRNA was predominant in the striosomal neurons; D1R immunoreactivity (D1R-IR) and D1R binding sites displayed a pattern similar to D1R mRNA. D1R-IR was essentially present in striosomal cell bodies and neuropil, whereas only a few cell bodies were detected in the matrix. From week 20 of fetal life, D1R gene expression developed in the matrix neurons as well, thus leading to an even D1R mRNA expression throughout striosomes and matrix compartments at birth. Comparative analysis of the expression of D1R and dynorphin mRNA show the same developmental patchy pattern up to week 26. Indeed, neurons expressing the D1R gene contain dynorphin mRNA; in contrast, they do not express the preproenkephalin A gene. At birth, the pattern of D1R mRNA expression level was sharply different from that of dynorphin (DYN) gene expression. High DYN mRNA expression was restricted to the striosomes, whereas high D1R mRNA expression was present in the whole striatum. These results demonstrate that, during human ontogeny, functional D1 receptors are expressed as early as week 12 in the striatum, developing initially in the striosomal neurons containing high dynorphin mRNA content. Toward the end of fetal life, there is a dissociation between D1R and DYN expression levels, suggesting that neuroanatomical or neurochemical modifications occur at this period, which may contribute to the regulation of the tone of the striatal D1R and DYN gene with topological specificity.

Multiple neuron-specific enhancers in the gene coding for the human neurofilament light chain.

To define DNA regions involved in the neuron-specific expression of the neurofilament light (NF-L) gene, we generated transgenic mice bearing different NF-L constructs. A 4.9-kilobase human NF-L fragment including -292 base pairs of 5'-flanking sequences contained sufficient elements for nervous system expression in transgenic mice. Deletion of introns 1 and 2 from this 4.9-kilobase DNA fragment resulted in reduced levels of transgene expression in the cortex, while deletion of intron 3 had little effect. Both introns 1 and 2 could act independently as enhancers to confer neuronal expression of the basal heat shock promoter (hsp68) fused to lacZ in transgenic mice. The hNF-L basal promoter (-292 base pairs) was found to contain elements for directing neuronal expression of either the lacZ reporter gene or an intronless hNF-L construct. Sequence comparison revealed that intron 1, intron 2, and the basal human NF-L promoter all contain an ETS-like motif, CAGGA, present in a variety of genes expressed in the nervous system.

Neuron specificity of the neurofilament light promoter in transgenic mice requires the presence of DNA unwinding elements.

Three reporter genes, the chloramphenicol acetyltransferase (CAT), the lacZ, and the intronless NF-L DNA, were used to test the activity of the proximal promoter region (-292 bp) of the human neurofilament light (hNF-L) gene in transgenic mice. Surprisingly, the hNF-L/CAT construct was highly sensitive to position effect, and its expression was found at low levels in several tissues of adult transgenic mice (Beaudet, L., Charron, G., Houle, D., Tretjakoff, I. Peterson, A., and Julien, J.-P. (1992) Gene (Amst.) 116, 205-214). In contrast, the hNF-L/lacZ or the hNF-L/intronless constructs were expressed exclusively in the nervous system during embryonic development and in adult animals. The DNA sequences analysis of the different reporter genes revealed the presence of matrix attachment regions (MARs) within the 3'-untranslated regions of all three transgenes. DNA unwinding elements were found within the MARs of lacZ and hNF-L gene constructs but not in the CAT gene construct. When this element was removed from the lacZ construct, expression of the hNF-L/lacZ transgene became susceptible to position effect and was no longer tissue-specific. These results indicate that DNA unwinding elements are essential for position effect independence conferred by MARs to the hNF-L basal promoter.

Ontogeny of the striatal neurons expressing neuropeptide genes in the human fetus and neonate.

The distribution patterns of neurons expressing mRNAs for four neuropeptides in the human striatum were studied during ontogeny by the use of in situ hybridization. The results of our study demonstrate that somatostatin, enkephalin, dynorphin, and substance P mRNAs are present in striatal neuronal populations from week 12 of fetal life. Each neuronal population undergoes a specific differentiation. Neurons containing somatostatin mRNA are scattered throughout the caudate-putamen up until birth. Neurons containing enkephalin, dynorphin, or substance P mRNAs evolve throughout fetal life in relation to caudate-putamen and patch-matrix compartmentalization. Neurons containing enkephalin mRNA (distinct from those containing substance P or dynorphin mRNAs) are present in the matrix from week 12 of fetal life. These neurons are preferentially distributed in the matrix and, at birth, display higher enkephalin mRNA content in the matrix than in the patches. Dynorphin mRNA is found in the caudate and putamen, preferentially in the patch neurons; nevertheless, a low level of dynorphin mRNA is also present in neurons of the caudate matrix. Substance P mRNA is initially restricted to caudate neurons. At birth, both substance P and dynorphin mRNAs are expressed at high levels in the patches. These results demonstrate that each neuropeptide gene is expressed during human fetal life in neurons with a specific topology and pace of development in relation to caudate-putamen and patch-matrix differentiation. These results also contribute evidence that neurochemical evolution of the striatal neuronal populations is not complete at birth in humans.

Down syndrome-critical region contains a gene homologous to Drosophila sim expressed during rat and human central nervous system development.

Many features of Down syndrome might result from the overdosage of only a few genes located in a critical region of chromosome 21. To search for these genes, cosmids mapping in this region were isolated and used for trapping exons. One of the trapped exons obtained has a sequence very similar to part of the Drosophila single-minded (sim) gene, a master regulator of the early development of the fly central nervous system midline. Mapping data indicated that this exonic sequence is only present in the Down syndrome-critical region in the human genome. Hybridization of this exonic sequence with human fetal kidney poly(A)+ RNA revealed two transcripts of 6 and 4.3 kb. In situ hybridization of a probe derived from this exon with human and rat fetuses showed that the corresponding gene is expressed during early fetal life in the central nervous system and in other tissues, including the facial, skull, palate, and vertebra primordia. The expression pattern of this gene suggests that it might be involved in the pathogenesis of some of the morphological features and brain anomalies observed in Down syndrome.

kappa-Opioid receptor in humans: cDNA and genomic cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system.

Using the mouse delta-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human mu- and kappa-opioid receptor genes. Their organization appears similar to that of the human delta receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The kappa gene was mapped at position q11-12 in human chromosome 8. A full-length cDNA encoding the human kappa-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical kappa 1 pharmacological profile and is negatively coupled to adenylate cyclase. The expression of kappa-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of kappa-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors.

Intragenic regulatory elements contribute to transcriptional control of the neurofilament light gene.

To date, no DNA regions involved in the neuron-specific expression of the neurofilament light gene (NF-L) have been defined using transfection assays in cultured cells. To identify those regulatory regions in the human NF-L gene, we generated transgenic mice with a construct containing the basal NF-L promoter (-292 to +15) fused to the cat gene and with three DNA fragments of 21.5, 7.6 and 4.9 kb each, including NF-L with different lengths of either 5'- or 3'-flanking sequences. We show that the proximal NF-L 5' region (0.3 kb) constitutes a weak promoter and that it lacks information to confer neural specificity. However, appropriate expression in the nervous system occurred when this minimal promoter was combined with either 7.3 or 4.6 kb of NF-L sequences downstream from the transcription start point. We conclude that the intragenic NF-L region contains cis-acting elements conferring cell-type-specific regulation on the basal activity of the NF-L promoter. Interestingly, AP-2 motifs were found within homologously placed introns of all three NF genes, as well as in the promoter regulatory regions of many neuronal genes. We propose that the acquisition of introns by an ancestral intronless IF gene may have contributed to the emergence of a lineage of IF genes expressed in the nervous system.

Origin of the two mRNA species for the human neurofilament light gene.

The human neurofilament light (hNF-L) gene yields two major species of mRNAs of 2.4 and 3.8 kilobases (kb) in size. To investigate the origin of these two mRNAs, we have analyzed in transgenic mice the expression of hNF-L DNA fragments including different lengths of 5'-flanking regions. The finding that the 3.8-kb mRNA species is produced by a hNF-L transgene that includes only the proximal promoter region (-0.3 kb) demonstrates that both the 2.4- and 3.8-kb mRNAs are derived from the same site of transcription initiation. Sequencing of the 3' untranslated region of the hNF-L gene revealed the presence of multiple AATAAA polyadenylation signals. We conclude from Northern blotting experiments using probes spanning various regions of the hNF-L gene that the 2.4- and 3.8-kb mRNAs originate from the selective use of polyadenylation signals located 1.4 kb apart.

Pineal rhythm of N-acetyltransferase activity and melatonin in the male badger, Meles meles L, under natural daylight: relationship with the photoperiod.

The rhythmicity of melatonin secretion and of pineal NAT activity was compared in male badger kept in natural daylight during two distinctly different photoperiods (January and June). The hormone and its enzyme follow the same pattern with a nighttime elevation and a low level during the day, demonstrating the presence of a nyctohemeral rhythm. The high correlation found between the NAT activity and the melatonin concentration suggests that NAT is the rate-limiting enzyme in melatonin synthesis in the badger. Peak amplitudes were similar under the two photoperiods. Melatonin secretion occurred in the first part of the night irrespective of the photoperiod. The rhythm of melatonin secretion is modified by the photoperiod. The duration of high nighttime levels varies; it is longer (8 h) when the night is long (16 h) in January, and shorter (6 h) when the night is short (8 h) in June. In the badger, differences in the duration of high level melatonin at night may reflect variations in day length and convey to the animal the photoperiodic information.

C21 steroids and transcortin-type protein during delayed implantation in the European badger Meles meles L.

A high-affinity corticosteroid-binding protein (CBG) roughly resembling a transcortin-type protein is present in badger plasma. Plasma CBG, corticosteroid and progesterone (P) concentrations were measured in relation to delayed implantation, true progestation and gestation. Two significant CBG increases were observed during pregnancy. The first, in the second half of embryonic diapause coincides with an increase in plasma corticosteroid concentration and the second, during true progestation and gestation, with an increase in P concentration. Relationship of CBG increases with pregnancy in badger are discussed.