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Biomedical subjects

M G Darlison

Publications and source records attributed to M G Darlison.

At least 55 records · Page 3Linked to original sources
PubMed1992

Effects of subunit types of the recombinant GABAA receptor on the response to a neurosteroid.

When vertebrate brain poly(A)+ RNA is expressed in Xenopus oocytes the response of the GABA receptors formed is found to be inhibited allosterically by a neurosteroid, pregnenolone sulphate (PS). This negative modulation was reproduced after expressing RNAs encoding bovine GABAA receptor subunits in the combinations alpha i + beta 1, or alpha i + beta 1 + gamma 2 (where i = 1, 2 or 3). The characteristics of this inhibition vary significantly with the type of the alpha subunit (alpha 1, alpha 2, or alpha 3) used. When the bovine gamma 2L alternate form of the gamma 2 subunit was replaced by the human gamma 2S subunit, the behaviour was unchanged: the human gamma 2S subunit used is a newly-cloned form, which encodes a polypeptide with two amino acid differences from the human gamma 2 subunit previously described. The results of co-application of PS and 3 alpha-hydroxy-5 alpha-pregnan-ol-20-one, a neurosteroid which is a positive modulator of the GABAA receptor, indicate that these act at different sites on the receptor. PS also increases the desensitisation of the receptor by GABA. This effect, also, is alpha-subunit-type dependent and occurs by an acceleration of the fast phase of desensitisation.

Animals↗
PubMed1992

Invertebrate GABA and glutamate receptors: molecular biology reveals predictable structures but some unusual pharmacologies.

Determination of the sequences of invertebrate gamma-aminobutyric acid (GABA)-gated and glutamate-gated receptor/ion channels, through the application of recombinant DNA methods, is not just an academic exercise to effect evolutionary comparisons with the sequences of the corresponding vertebrate receptors. The isolation of DNA clones would provide the tools to investigate the exact locations and functional properties of these neurotransmitter receptors within simple nervous systems. In addition, since GABA receptors, at least, have been suggested to be the targets of certain pesticides, the availability of invertebrate receptor cDNAs might provide the agrochemical industry with the basis for 'high-throughput' screening methods for novel pesticidal compounds. Recently, the isolation of molluscan and Drosophila GABA receptor and glutamate receptor cDNAs, and the pharmacological properties of a GABA receptor expressed from one of these clones, have been reported. These studies should stimulate further research into the electrophysiology and pharmacology of native invertebrate ion channel proteins.

Animals↗
PubMed1992

Fine mapping of the human biglycan (BGN) gene within the Xq28 region employing a hybrid cell panel.

Human biglycan is a small proteoglycan that is expressed at high levels in the growing skeleton and in human skin at the cell surface of differentiating keratinocytes. The human gene for biglycan (BGN) has previously been mapped by in situ hybridization to the Xq27-q28 region. Employing somatic hybrid cell lines with human X chromosome breakpoints within this region, we performed a fine mapping of the gene within Xq28. Our results indicate that the biglycan gene is proximal to the red/green cone pigment genes, G6PD, and coagulation factor VIII and is distal to DXS304, DXS305, and GABRA3. The biglycan gene precisely maps to a region of the X chromosome, where, by comparative gene mapping, one would expect to find the gene for X-linked dominant chondrodysplasia punctata/ichthyosis/short stature (Happle) syndrome. Hence, BGN is a candidate gene for the Happle syndrome.

Animals↗
PubMed1992

Confirmation of the localization of the human GABAA receptor alpha 1-subunit gene (GABRA1) to distal 5q by linkage analysis.

The GABAA receptor is the major inhibitory neurotransmitter receptor in the mammalian brain. To date, 14 genes that encode subunits of this receptor have been identified; these appear to be scattered throughout the human genome and are under investigation as candidate loci for a number of neurological and psychiatric disorders. We report here a highly polymorphic (dC-dA)n repeat within the human alpha 1-subunit gene (GABRA1). Typing of this marker in the Centre d'Etude du Polymorphisme Humain (CEPH) panel of families confirms the previous assignment of the GABRA1 locus to the distal portion of chromosome 5q by demonstrating linkage to the markers CRI-L45 (D5S61) (Zmax = 11.00, theta max = 0.15), CRI-V1022 (D5S54) (Zmax = 7.25, theta max = 0.20), and CRI-P148 (D5S72) (Zmax = 5.71, theta max = 0.24).

Base Sequence↗
PubMed1992

Assembly of functional GABAA receptors in insect cells using baculovirus expression vectors.

We have constructed recombinant baculoviruses containing cDNAs encoding either the alpha 1- or the beta 1-subunit of the bovine GABAA receptor. In Spodoptera frugiperda (IPLB-Sf-21) cells infected with recombinant virus expressing either the alpha 1- or beta 1-subunit, or in cells co-infected with both viruses, functional GABAA receptors were detected by whole-cell electrophysiological recordings. The threshold for the responses mediated by the homo-oligomeric channels (alpha- or beta-) was 2-3 x 10(-6) M GABA, and for the co-infected cells was 8 x 10(-8) M GABA, suggesting that hetero-oligomeric channels formed in these cells. All GABA-induced currents were found to be inhibited by bicuculline and picrotoxin, potentiated by pentobarbital but were insensitive to benzodiazepines.

Animals↗
PubMed1991

Distribution of the GABAA receptor alpha 1- and gamma 2-subunit mRNAs in chick brain.

We have used sequence-specific oligonucleotide probes and in situ hybridisation histochemistry to examine the distribution of the GABAA receptor alpha 1- and gamma 2-subunit mRNAs in serial sections of 1-day-old chick brain. Both transcripts are present together, at high levels, in many brain regions. Differences are found, however, in the relative amounts of these mRNAs in two isthmic nuclei of the optic lobe, the deep cerebellar nuclei, and the dorsal thalamus. We therefore conclude that while the alpha 1 and gamma 2 subunits predominantly occur together in the same receptor complex, they may also be found separately in other GABAA receptor subtypes.

Animals↗
PubMed1991

Cloning of a cDNA that encodes an invertebrate glutamate receptor subunit.

A full-length cDNA which encodes a putative glutamate receptor polypeptide was isolated from the pond snail Lymnaea stagnalis, using a short stretch of exonic sequence and two variants of the polymerase chain reaction. In this first comparison of invertebrate and vertebrate glutamate receptor sequences, the mature molluscan polypeptide, which comprises 898 amino acids and has a predicted Mr of 100,913, displays between 37% and 46% amino-acid identity to the rat ionotropic glutamate receptor subunits, GluR1 to GluR6.

Amino Acid Sequence↗
PubMed1991

Sequence of a functional invertebrate GABAA receptor subunit which can form a chimeric receptor with a vertebrate alpha subunit.

The sequence of an invertebrate GABAA receptor subunit is described. This was deduced from a cDNA which was isolated from the mollusc Lymnaea stagnalis and which corresponds to a transcript of extremely low abundance. The cDNA was isolated using short exonic sequences from part of the corresponding gene in combination with a variant of the polymerase chain reaction (PCR) known as RACE (rapid amplification of cDNA ends). The mature polypeptide has a predicted molecular weight of 54,569 Daltons and exhibits approximately 50% identity to vertebrate GABAA receptor beta subunits. The six intron-exon boundaries determined to date in the molluscan gene occur at the same relative positions as those found in vertebrate GABAA receptor genes. Functional expression, in Xenopus oocytes, of the molluscan cDNA alone results in the formation of GABA-activated chloride ion channels that have a finite open probability even in the absence of agonist. These GABA-evoked currents can be reversibly blocked by the vertebrate GABAA receptor antagonist bicuculline. Surprisingly, the molluscan beta subunit is capable of replacing vertebrate beta subunits in co-expression experiments with the bovine GABAA receptor alpha 1 subunit. These findings suggest that invertebrate GABAA receptors exist in vivo as hetero-oligomeric complexes.

Amino Acid Sequence↗
PubMed1991

The chicken GABAA receptor alpha 1 subunit: cDNA sequence and localization of the corresponding mRNA.

We report the sequence of a complementary DNA (cDNA) that encodes the chicken GABAA receptor alpha 1 subunit, which is extremely homologous to mammalian alpha 1 subunits. The distribution of alpha 1 subunit transcripts is shown to correlate mainly, but not completely, with the previously-reported pattern of benzodiazepine type I (BZI) binding sites in the avian brain. These results suggest that the alpha 1 subunit may not necessarily be restricted to receptors having BZI pharmacology.

Amino Acid Sequence↗
PubMed1991

Genetic mapping of the mouse X chromosome in the region homologous to human Xq27-Xq28.

The four loci Gabra3, DXPas8, CamL1, and Bpa, located near the murine X-linked visual pigment gene (Rsvp), have been ordered using 248 backcross progeny from an interspecific mating of (B6CBA-Aw-J/A-Bpa) and Mus spretus. One hundred twenty backcross progeny have been analyzed at seven anchor loci spanning the X chromosome and form a regional mapping panel. An additional 128 progeny have been screened for recombination events between Cf-9 and Dmd. Eighteen recombinants between these loci have been detected in the 248 animals; all of the recombinants were screened at the other anchor loci to identify any double crossovers. Pedigree analysis using these recombinants strongly favors a gene order of (Cf-9)-Gabra3-(DXPas8, Bpa)-CamL1-(Rsvp, P3, Cf-8)-Dmd for the loci studied. Synteny with human Xq27-Xq28 is retained, although the relative order of some loci may differ between the two species.

Animals↗
PubMed1991

Conserved organization of gamma-aminobutyric acidA receptor genes: cloning and analysis of the chicken beta 4-subunit gene.

A series of genomic clones containing DNA that encodes the chicken gamma-aminobutyric acidA (GABAA) receptor beta 4 subunit have been isolated. These have been restriction mapped and partially sequenced to determine the structural organization and the size of the beta 4-subunit gene. This gene, which comprises nine exons, spans more than 65 kb. The organization of the chicken GABAA receptor beta 4-subunit gene has been compared to that of the murine GABAA receptor delta-subunit gene and to those of the genes that encode other members of the ligand-gated ion-channel superfamily, namely muscle and neuronal nicotinic acetylcholine receptors (AChRs). Although the positions of the intron/exon boundaries of GABAA receptor subunit genes are seen to be highly conserved, there are significant differences between the genes that encode GABAA receptor and AChR subunits. These results are discussed in relation to the proposal that this superfamily of ligand-gated ion-channel receptor genes arose by duplication of an ancestral receptor gene.

Amino Acid Sequence↗
PubMed1991

gamma-Aminobutyric acidA receptor heterogeneity is increased by alternative splicing of a novel beta-subunit gene transcript.

DNA sequences encoding two variants of a novel gamma-aminobutyric acidA (GABAA) receptor beta subunit were isolated from an embryonic chicken whole-brain cDNA library and a chicken genomic library. The coding regions of these variants only differ from each other by the absence or presence of 12 bp in the region that encodes the presumed intracellular loop between transmembrane domains M3 and M4; the encoded subunits have been named beta 4 and beta 4', respectively. The predicted mature polypeptides are 72-77% identical to the previously characterized mammalian and chicken beta 1, beta 2, and beta 3 subunits. Analysis of the beta 4-subunit gene reveals that the different transcripts encoding the two variants arise by the use of one of two 5'-donor splice sites that are separated by 12 bp. This is the first demonstration of alternative splicing of a GABAA receptor subunit gene transcript and represents a further mechanism for the generation of GABAA receptor heterogeneity.

Animals↗