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M G Darlison

Publications and source records attributed to M G Darlison.

At least 73 records · Page 4Linked to original sources
PubMed1990

Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.

We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha-bungarotoxin (alpha-Bgt) and kappa-bungarotoxin (kappa-Bgt). Reversible block of the expressed insect AChR by mecamylamine, d-tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha-Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA.

Amino Acid Sequence↗
PubMed1990

Amino acid receptors from insect muscle: electrophysiological characterization in Xenopus oocytes following expression by injection of mRNA.

Poly(A)+ Messenger ribonucleic acid (mRNA) was extracted from leg muscles of the locust Schistocerca gregaria and injected into oocytes of Xenopus laevis. After 5-10 days incubation, receptors for L-glutamate, L-quisqualate, DL-ibotenate and gamma-aminobutyric acid (GABA) were expressed. Agonist-induced currents were dose-dependent, and, in the concentration range 1 microM to 1 mM, generally had peak values of 50 nA. The responses to all agonists, apart from GABA, exhibited desensitization which could not be reversed even by prolonged washing with Ringer. Application of 100 microM GABA to oocytes voltage clamped at -60 mV produced a smooth inward current with a reversal potential of -22 +/- 1 mV, which is consistent with the involvement of chloride ions. At 100 microM, picrotoxin reversibly abolished this current, while 100 microM bicuculline had no effect. L-Glutamate elicited a smooth current with a reversal potential of -52 +/- 3 mV. L-Quisqualate elicited an inward current at -60 mV with a reversal potential of -9 +/- 2 mV; this current occasionally had an oscillatory component. The response to ibotenate comprised a smooth inward current with a reversal potential of -21 +/- 3 mV which was probably mediated by chloride ions.

Amino Acids↗
PubMed1990

Distinct regional expression of nicotinic acetylcholine receptor genes in chick brain.

Four genes (alpha 2, alpha 3, alpha 4 and beta 2) have been reported as encoding subunits of the nicotinic acetylcholine receptor (nAChR) in chicken brain. The mRNAs transcribed from these genes have here been localised to particular regions using in situ hybridisation histochemistry. The beta 2 mRNA was clearly the most abundant transcript, being widely distributed throughout the chick brain. In the cerebellum, all four mRNA species were present, although they showed different cellular patterns of distribution. Only alpha 2 mRNA and beta 2 mRNA were found in significant amounts in the optic tectum. In the lateral spiriform nucleus, while alpha 2 mRNA, alpha 4 mRNA and beta 2 mRNA were all very abundant, the alpha 4 mRNA was localised to a subgroup of neurons containing alpha 2 mRNA and beta 2 mRNA. This represents the first evidence that individual cells may express two different nAChR alpha subunit genes in vivo. The distributions of the 4 mRNA species showed few common features. This suggests that other neuronal nAChR genes remain to be identified, and that these 4 genes are not generally expressed in the same cells to constitute a single macromolecular complex. The results therefore provide evidence for nAChR heterogeneity in the central nervous system.

Animals↗
PubMed1989

Development expression of the genes encoding the four subunits of the chicken muscle acetylcholine receptor.

Subunit-specific DNA probes for the chicken muscle acetylcholine receptor have been used in conjunction with Northern blots and dot-blots based upon the SP6/T7 RNA polymerase reaction to quantitate changes in the steady-state mRNA levels of all four subunits during development. In pectoral muscle, maximal subunit transcript levels are observed at day 12 in ovo for alpha, days 12-16 in ovo for beta, day 14 in ovo for gamma, and days 12-14 in ovo for delta. Interestingly, two delta-subunit transcripts which have somewhat different patterns of temporal expression are detected. At the peak of expression of each subunit mRNA the absolute levels of the alpha-, beta-, and gamma-subunit transcripts are very similar (49.2, 66.0, and 70.7 attomoles of transcript/micrograms of poly(A)+ RNA, respectively) whereas that for the summed delta-subunit transcripts is significantly lower (17.8 attomoles/micrograms of poly(A)+ RNA). Further, this lower level of delta-subunit mRNA is observed from day 10 to day 16 of embryonic development. We conclude that subunit transcript availability is important in the synthesis of acetylcholine receptor protein during development and that the level of the delta-subunit transcript can be rate limiting in the expression of this receptor.

Amino Acid Sequence↗
PubMed1989

Differential distribution of GABAA receptor mRNAs in bovine cerebellum--localization of alpha 2 mRNA in Bergmann glia layer.

Using in situ hybridization histochemistry, we have demonstrated that 3 alpha subunit mRNAs of the GABAA receptor are present in different cell populations of the bovine cerebellum. While the alpha 1 mRNA is the most abundant and is present in granule cells, Purkinje cells and stellate/basket cells, the alpha 2 mRNA appears to be confined to the Bergmann glial cell layer. The alpha 3 mRNA is only expressed in the Golgi cells. This differential distribution of GABAA receptor mRNA subtypes suggests a previously unrecognized complexity of GABAergic transmission in the cerebellum.

Animals↗
PubMed1989

The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.

The mdx mouse is an X-linked myopathic mutant, an animal model for human Duchenne muscular dystrophy. In both mouse and man the mutations lie within the dystrophin gene, but the phenotypic differences of the disease in the two species confer much interest on the molecular basis of the mdx mutation. The complementary DNA for mouse dystrophin has been cloned, and the sequence has been used in the polymerase chain reaction to amplify normal and mdx dystrophin transcripts in the area of the mdx mutation. Sequence analysis of the amplification products showed that the mdx mouse has a single base substitution within an exon, which causes premature termination of the polypeptide chain.

Amino Acid Sequence↗
PubMed1989

Localization of GABAA receptor alpha-subunit mRNAs in relation to receptor subtypes.

The distribution of 3 GABAA receptor alpha-subunit mRNAs in various regions of bovine brain has been investigated using in situ hybridization. Whereas the alpha 2- and alpha 3-transcripts are of low abundance in all regions except striatum, the alpha 1-transcript is considerably enriched in the inferior colliculus, olfactory bulb and substantia nigra, and appears to be correlated with benzodiazepine type I receptor localization.

Animals↗
PubMed1989

Chromosomal localization of GABAA receptor subunit genes: relationship to human genetic disease.

Hybridization of GABAA receptor probes to human chromosomes in situ and to DNA from sorted human chromosomes has localized the genes encoding a beta subunit and three isoforms of the alpha subunit. The alpha 2 and beta genes are both located on chromosome 4 in bands p12-p13 and may be adjacent. The alpha 1 gene is on chromosome 5 (bands q34-q35) and the alpha 3 gene is on the X chromosome. The alpha 3 locus was mapped also on the mouse X chromosome using genetic break-point analysis in an interspecies pedigree. The combined results locate the human alpha 3 gene within band Xq28, in a location that makes it a candidate gene for the X-linked form of manic depression.

Animals↗
PubMed1989

Physical linkage of a GABAA receptor subunit gene to the DXS374 locus in human Xq28.

We report the physical linkage of the gene encoding one of the subunits of the GABAA receptor (GABRA3) to the polymorphic locus DXS374 on the human X chromosome at Xq28. X-linked manic depression and other psychiatric disorders have been mapped to this region, and thus GABRA3 is a potential candidate gene for these disorders. DXS374--and therefore GABRA3--lies distal to the fragile X locus at a recombination fraction of approximately .15.

DNA Probes↗
PubMed1988

Structural and functional basis for GABAA receptor heterogeneity.

When gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in vertebrate brain, binds to its receptor it activates a chloride channel. Neurotransmitter action at the GABAA receptor is potentiated by both benzodiazepines and barbiturates which are therapeutically useful drugs (reviewed in ref. 1). There is strong evidence that this receptor is heterogeneous. We have previously isolated complementary DNAs encoding an alpha- and a beta-subunit and shown that both are needed for expression of a functional GABAA receptor. We have now isolated cDNAs encoding two additional GABAA receptor alpha-subunits, confirming the heterogeneous nature of the receptor/chloride channel complex and demonstrating a molecular basis for it. These alpha-subunits are differentially expressed within the CNS and produce, when expressed with the beta-subunit in Xenopus oocytes, receptor subtypes which can be distinguished by their apparent sensitivity to GABA. Highly homologous receptor subtypes which differ functionally seem to be a common feature of brain receptors.

Amino Acid Sequence↗
PubMed1988

Localization of the mdx mutation within the mouse dystrophin gene.

We have mapped human and mouse X chromosome-specific genomic and cDNA probes through an interspecies Mus musculus/spretus pedigree which contains the mdx mutation. The positions of these markers relative to one another and to the mdx mutation were delineated. Using probes corresponding to segments of the human Duchenne muscular dystrophy (DMD) gene transcript, the position of a cross-hybridizing mouse equivalent gene (mDMD) was located. In more than 200 animals mapped, three were identified which show recombination within this mDMD gene. Analysis of these three animals shows that the mDMD gene is oriented with its 5' end centromeric and its 3' end telomeric on the mouse X chromosome. Furthermore, their recombinational breakpoints are on either side of the mdx mutation, thus providing the first unequivocal demonstration that the mdx mutation is located within the mDMD gene and defining limits within that gene between which the mutation must lie. Within that segment the evidence indicates that there is no major deletion of an exon as detectable by Southern blot analysis in mdx animals. The mdx mouse becomes important as an animal model for the study of the expression of the DMD gene and its developmental consequences, for transgenic and other corrective manipulations.

Animals↗
PubMed1988

Distinct GABAA receptor alpha subunit mRNAs show differential patterns of expression in bovine brain.

Specific oligonucleotide probes have been used to visualize the regional and cellular distribution of the mRNAs encoding three structurally distinct GABAA receptor alpha subunits in bovine brain. In situ hybridization analysis showed that these transcripts differ in distribution and in relative abundance. In frontal cortex the alpha 1 and alpha 2 transcripts are most abundant in layers II-IV, whereas the alpha 3 mRNA is most abundant in layers V and VI. In the hippocampal complex, the alpha transcripts are differentially distributed in the entorhinal cortex and subiculum. The alpha 2 transcript is enriched in the dentate gyrus and CA4/CA3 regions of the hippocampus. In the cerebellum, essentially only the alpha 1 transcript is detectable in granule cells, Purkinje cells, and stellate/basket cells. These results suggest that the different alpha subunits represent components of distinct GABAA receptor subtypes.

Animals↗
PubMed1987

Differential expression of nicotinic acetylcholine receptor genes in innervated and denervated chicken muscle.

We have studied the mRNAs encoding all four subunits of the acetylcholine receptor in 13-day embryonic, innervated and denervated chicken pectoral muscle. In all three states the transcript sizes of the alpha, beta-, gamma- and delta-subunit mRNAs were approximately 3.2, 2.8, 1.8 and 1.9 kb respectively. Denervation was found to result in a large increase in the steady-state levels of each mRNA compared with those in innervated muscle. This increase was 8- to 9-fold for the beta and delta subunits and approximately 12-fold for the alpha subunit. The evidence obtained shows a coordinate regulation of the acetylcholine receptor genes in response to denervation. Interestingly, no gamma-subunit transcript was detected in innervated muscle, while significant levels were detected in embryonic and denervated tissue. This evidence suggests that there exists in a non-mammalian muscle acetylcholine receptor an additional subunit, analogous to the bovine epsilon subunit, and shows that the extra-junctional receptor of adult denervated muscle is produced by the same set of mRNAs which produce the major form of the receptor in embryonic muscle.

Animals↗