Biomedical subjects
Publications and source records attributed to J Mallet.
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Hybrid zones can yield estimates of natural selection and gene flow. The width of a cline in gene frequency is approximately proportional to gene flow (sigma) divided by the square root of per-locus selection (square root of s). Gene flow also causes gametic correlations (linkage disequilibria) between genes that differ across hybrid zones. Correlations are stronger when the hybrid zone is narrow, and rise to a maximum roughly equal to s. Thus cline width and gametic correlations combine to give estimates of gene flow and selection. These indirect measures of sigma and s are especially useful because they can be made from collections, and require no field experiments. The method was applied to hybrid zones between color pattern races in a pair of Peruvian Heliconius butterfly species. The species are Müllerian mimics of one another, and both show the same changes in warning color pattern across their respective hybrid zones. The expectations of cline width and gametic correlation were generated using simulations of clines stabilized by strong frequency-dependent selection. In the hybrid zone in Heliconius erato, clines at three major color pattern loci were between 8.5 and 10.2 km wide, and the pairwise gametic correlations peaked at R approximately 0.35. These measures suggest that s approximately 0.23 per locus, and that sigma approximately 2.6 km. In erato, the shapes of the clines agreed with that expected on the basis of dominance. Heliconius melpomene has a nearly coincident hybrid zone. In this species, cline widths at four major color pattern loci varied between 11.7 and 13.4 km. Pairwise gametic correlations peaked near R approximately 1.00 for tightly linked genes, and at R approximately 0.40 for unlinked genes, giving s approximately 0.25 per locus and sigma approximately 3.7 km. In melpomene, cline shapes did not perfectly fit theoretical shapes based on dominance; this deviation might be explained by long-distance migration and/or strong epistasis. Compared with erato, sample sizes in melpomene are lower and the genetics of its color patterns are less well understood. In spite of these problems, selection and gene flow are clearly of the same order of magnitude in the two species. The relatively high per locus selection coefficients agree with "major gene" theories for the evolution of Müllerian mimicry, but the genetic architecture of the color patterns does not. These results show that the genetics and evolution of mimicry are still only sketchily understood.
A cDNA clone complementary to the rat brain glutamic acid decarboxylase mRNA was isolated from a rat brain cDNA expression library using an antibody specific to the enzyme. The cDNA insert has been shown to direct the synthesis of an active protein in Escherichia coli. In this study, the nucleotide sequence of this clone, which includes the complete coding region, is presented. The predicted protein is 593 amino acids in length. The first 557 residues display a 95% identity when compared with the corresponding cat sequence. However, the deduced amino acid sequence of the carboxy-terminal end of the rat protein, downstream of residue 557, is totally different from the cat, whereas it agrees with a published partial peptidic sequence of the rat protein.
Rats were submitted to a series of 10 daily electroconvulsive shocks (ECS). A first group of animals was killed 1 day after the last seizure and a second group 30 days later. Tyrosine hydroxylase (TH) activity was measured using an in vitro assay in the nucleus caudatus, anterior cortex, amygdala, substantia nigra, ventral tegmental area, and locus ceruleus. The mRNA corresponding to this enzyme (TH-mRNA) was evaluated using a cDNA probe at the cellular level in the ventral tegmental area, substantia nigra, and locus ceruleus. Met-enkephalin (MET)-immunoreactivity and the mRNA coding for the preproenkephalin (PPE-mRNA) were assayed in striatum and the central nucleus of the amygdala. The day after the last ECS an increase of TH activity was observed in the ventral tegmental area, locus ceruleus, and substantia nigra in parallel with a similar increase in the amygdala and striatum; in the anterior cortex TH activity remained unchanged. TH-mRNA was increased in the locus ceruleus, evidencing the presence in this structure of a genomic activation. The amounts of MET and PPE-mRNA were unaffected in the striatum but increased in the amygdala. Thirty days after the last ECS we observed a decrease of TH activity in the amygdala and of TH-mRNA amount in the ventral tegmental area. In the locus ceruleus TH-mRNA remained higher in treated animals than in controls whereas TH activity returned to control levels. These results demonstrate that a series of ECS induces an initial increase of the activity of mesoamygdaloid catecholaminergic neurons followed by a sustained decrease through alterations of TH gene expression which could mediate the clinical effect of the treatment.
Tyrosine hydroxylase, aromatic L-amino-acid decarboxylase, and dopamine beta-hydroxylase activities were studied in the developing fetal rat brain. A delay of 2-3 days between the detection of the tyrosine hydroxylase and the aromatic L-amino-acid decarboxylase and dopamine beta-hydroxylase activities was observed. For this reason, the expression of tyrosine hydroxylase mRNA was studied. Tyrosine hydroxylase mRNA was visualized in the whole brain from 13 days of gestation, but the largest increase of the expression was observed in the hypothalamus. These results are discussed in terms of the relative gene expressions of the three enzymes involved in the biosynthesis of catecholamines and phenolamines in nervous tissues.
Increased synaptic stimulation of sympathetic neurons in vivo causes a delayed increase in the activity and the amount of tyrosine hydroxylase (TH). To determine whether these changes result from an increase in the messenger RNA for TH, the rat preganglionic cervical sympathetic trunk was electrically stimulated unilaterally for 90 min, and 48 h later RNA was extracted from stimulated and contralateral control superior cervical ganglia. Northern blots probed with a cDNA for TH demonstrated that nerve stimulation produced about a 2.5-fold increase in the amount of TH mRNA in the ganglion. These results indicate that synaptic stimulation leads to an increase in TH mRNA, either by increasing the rate of transcription of the TH gene or by increasing the stability of its mRNA.
The 5' end mapping of rat tryptophan hydroxylase (TPH) mRNA indicated a diversity in 5'-untranslated regions. Corresponding sequences were isolated by a variant of the Polymerase Chain Reaction, recently designated as 'anchor PCR', and a 'cRNA enrichment' procedure. The latter circumvents the limitations of 'anchor PCR', which failed to yield minor TPH sequences: this novel strategy allows purification of specific DNA fragments by elimination of the unspecific products, generated by the PCR, which prevent further amplification. Analysis of TPH sequences strongly suggests that TPH mRNAs are synthesized from at least two promoters, the proximal one exhibiting two 'CCAAT homologies'.
The existence of GABAergic neurons in the rat suprachiasmatic nucleus (SCN) was demonstrated by three specific markers; mRNA coding for glutamic acid decarboxylase (GAD) and visualized by in situ hybridization using a 35S-labelled cDNA probe, and GAD protein and GABA were identified by immunocytochemistry using specific antisera. In situ hybridization demonstrated well labelled GAD mRNA positive cells throughout SCN, and GABA and GAD immunoreactive cells showed similar distributions. These results indicate that GABA is a transmitter of a large portion of the SCN neuronal population.
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We have isolated cDNA clones believed to encompass the full-length coding sequences for a subunit of bovine monoamine oxidase type A (MAO-A). The clones code for an apoprotein of 527 amino acid residues corresponding to a molecular mass of 59,806 Da. The inferred protein sequences show an overall similarity of 68% with partial amino acid sequences of bovine type B MAO (about 41% of the total sequence), as well as a greater similarity (greater than 90%) with some regions including that for the published sequence of the flavin-binding region. Sequence comparisons indicate that these two forms of MAO are encoded by distinct genes. Comparison of this sequence with other flavoenzymes showed similarity with regions associated with non-covalent flavin-binding sites. Analysis of mRNAs coding for MAO enzymes showed a heterogeneity of transcripts consistent with several different forms of monoamine oxidase.
A cDNA clone encoding the complete sequence of an active rat choline acetyltransferase (ChoAcTase; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) has been isolated. Analysis of the deduced amino acid sequence reveals 85% and 31% identity with the porcine and Drosophila melanogaster enzymes, respectively. To further elucidate the molecular basis of neurotransmitter-related phenotypic plasticity, the expression of ChoAcTase mRNA was compared with that of tyrosine hydroxylase [TH; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], in neurons from superior cervical ganglia grown in the following conditions: 1) normal medium, 2) high K+ medium, and 3) normal medium supplemented with 50% muscle-conditioned medium (CM). TH mRNA was expressed in all three media; its level rose in high K+ and decreased strikingly in the presence of CM. ChoAcTase mRNA could be visualized in CM, but fell to undetectable levels in normal and high K+ media. These results suggest that translational or post-translational mechanisms do not play a major role for the modulation of neurotransmitter-associated phenotype.
In a previous study, we characterized two tryptophan hydroxylase mRNAs (TPH mRNAs) in the pineal gland. However, we failed to detect these species in the raphe by Northern blot experiments. Here, we report by S1 nuclease analysis and in situ hybridization that these two TPH mRNAs, as well as a third species, are expressed both in pineal gland and in raphe. In both tissues, the three mRNAs are transcribed predominantly from the same promoter. Strikingly, from the results of S1 maping analysis, it was observed that the total level of TPH mRNA per tissue is at least 150 times lower in the raphe than in the pineal gland. In contrast, TPH antigen as quantified by immunoblot experiments is about threefold more abundant per raphe than per pineal gland. TPH mRNA from one raphe and one pineal gland yield in vitro about the same amount of TPH antigen, suggesting that the discrepancy in the ratios of TPH mRNA and TPH antigen between the raphe and the pineal gland results, at least in part, from a difference in the translation efficiency of TPH mRNAs in the two structures.
The enzyme choline acetyltransferase catalyses the biosynthesis of the neurotransmitter acetylcholine and constitutes a specific marker of cholinergic system. To date, there is very limited information about the structure of the mammalian enzyme. More detailed understanding of this enzyme is particularly desirable because of the importance of the cholinergic system in neurotransmission, as well as the possible involvement of this system in certain neurological disorders. In this article, recent studies concerning the isolation of a cDNA encoding the complete sequence of the porcine enzyme are reported and the potential applications of this probe are discussed.
GFAP mRNA levels were quantified by Northern blot analysis using a human GFAP (glial fibrillary acidic protein) cDNA probe in association with immunocytochemistry. Ten days after a unilateral lesion of substantia nigra with 6-hydroxydopamine (6-OHDA), GFAP mRNA level is increased 1.4-fold in the ipsilateral striatum; thereafter it declined continuously to reach the control level 4 months later. This effect contrasted with the lower and more sustained increase of preproenkephalin (PPE) mRNA, a marker of neuronal target of nigrostriatal pathway. Following ibotenic acid-induced neuronal degeneration of the neostriatum in the rat, we observed a sharp elevation of the GFAP transcripts (4-fold) as soon as 2 days after the lesion both in the striatum and in the substantia nigra. Whereas in the striatum GFAP mRNA level already declined at 5 days postlesion, it remained stable in the substantia nigra. In comparison GFAP immunoreactivity was slightly delayed. No obvious modification was observed in the contralateral side to the lesion whatever the denervation condition studied. Implication of these results on the understanding and the therapeutic approach of glial scarring is discussed.
Little is known about the molecular events mediating neurotransmitter release, a crucial step in synaptic transmission. In this paper, the biosynthesis and release of L-beta-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine were analyzed in three heterologous cell lines after retroviral-mediated gene transfer of tyrosine hydroxylase (EC 1.14.16.2), the rate-limiting enzyme in catecholamine synthesis. A recombinant retrovirus encoding human tyrosine hydroxylase type I as well as neomycin-resistance gene was used to infect a fibroblast (NIH 3T3), a neuroblastoma (NS20 Y), and a neuroendocrine (AtT-20) cell line. After selection in the presence of neomycin and in tyrosine-free medium, high levels of exogenous tyrosine hydroxylase activity were detected in extracts of the three cell lines. High-performance liquid chromatography of cell extracts and culture supernatants confirmed that the three cell lines hydroxylated tyrosine to form L-DOPA and released this metabolite into the culture medium. Interestingly, the neuroendocrine cell line AtT-20 synthesized not only L-DOPA but also dopamine. Evoked secretion studies established that AtT-20 cells released the transmitter upon depolarization in a regulated, calcium-dependent way. We discuss the implication of this approach for the analyses of neurotransmitter release as well as in the context of degenerative disorders such as Parkinson disease.
Frequency-dependent selection against rare forms can maintain clines. For weak selection, s, in simple linear models of frequency-dependence, single locus clines are stabilized with a maximum slope of between square root of s/square root of 8 sigma and square root of s/square root of 12 delta, where sigma is the dispersal distance. These clines are similar to those maintained by heterozygote disadvantage. Using computer simulations, the weak-selection analytical results are extended to higher selection pressures with up to three unlinked genes. Graphs are used to display the effect of selection, migration, dominance, and number of loci on cline widths, speeds of cline movements, two-way gametic correlations ("linkage disequilibria"), and heterozygote deficits. The effects of changing the order of reproduction, migration, and selection, are also briefly explored. Epistasis can also maintain tension zones. We show that epistatic selection is similar in its effects to frequency-dependent selection, except that the disequilibria produced in the zone will be higher for a given level of selection. If selection consists of a mixture of frequency-dependence and epistasis, as is likely in nature, the error made in estimating selection is usually less than twofold. From the graphs, selection and migration can be estimated using knowledge of the dominance and number of genes, of gene frequencies and of gametic correlations from a hybrid zone.
It has been suggested that a form of inherited dystonia responsive to levodopa might be due to an abnormality of tyrosine hydroxylase gene. This hypothesis has been tested using a cDNA tyrosine hydroxylase gene probe in three families with this disorder. No evidence for genetic linkage between the disease and tyrosine hydroxylase loci was found; it is possible that the disorder results from a post-transcriptional defect confined to the brain.