Search PubMed⌕ Search

Biomedical subjects

M G Darlison

Publications and source records attributed to M G Darlison.

At least 37 records · Page 2Linked to original sources
PubMed1996

Conservation of gamma-aminobutyric acid type A receptor alpha 6 subunit gene expression in cerebellar granule cells.

The gamma-aminobutyric acid type A receptor cDNAs encoding the alpha 6 subunit homologues from chicken and goldfish have been cloned and sequenced. These proteins exhibit 83 and 75% identity, respectively, to the rat alpha 6 polypeptide. In situ hybridization has demonstrated that, as in mammals, the avian and teleost fish alpha 6 subunit genes are predominantly expressed in cerebellar granule cells. Correspondingly, flunitrazepam-non-displaceable binding of [3H]Ro 15-4513 (a benzodiazepine partial inverse agonist), which is a major characteristic of gamma-aminobutyric acid type-A receptors that contain the alpha 6 polypeptide, is also mainly found for cerebellar granule cells of fish and chick. The conservation of this expression pattern suggests that gamma-aminobutyric acid type A receptors possessing the alpha 6 subunit are of fundamental importance for cerebellar function and that the corresponding gene regulatory elements, e.g., granule cell-specific enhancers, have also been conserved.

Amino Acid Sequence↗
PubMed1995

Localization of the rho 1- and rho 2-subunit messenger RNAs in chick retina by in situ hybridization predicts the existence of gamma-aminobutyric acid type C receptor subtypes.

We have amplified partial complementary DNAs for the chicken gamma-aminobutyric acid type C (GABAC) receptor rho 1 and rho 2 subunits using the polymerase chain reaction. These nucleotide sequences have been utilized to design specific oligonucleotide probes for the in situ hybridization localization of the corresponding messenger RNAs (mRNAs) in the 1-day-old chick retina. Although both transcripts are found almost exclusively in the inner nuclear layer, their distributions differ markedly. From the locations of the hybridization signals, we deduce that the rho 1-subunit mRNA is present mainly in bipolar cells and that the rho 2-subunit mRNA is present in both amacrine and horizontal cells. These results suggest that the rho 1 and rho 2 subunits frequently occur in different receptor complexes and, therefore, that subtypes of the GABAC receptor exist.

Amino Acid Sequence↗
PubMed1995

Isolation and sequence analysis of the chicken GABAA receptor alpha 1-subunit gene promoter.

A genomic clone containing the 5'-flanking sequence of the chicken GABAA receptor alpha 1-subunit-encoding gene (GabR alpha 1) was isolated and characterized. An intron was found to interrupt the 5'-untranslated region. The transcription start point (tsp) was determined by primer extension, RNase protection and the amplification of chick brain first-strand cDNA. DNA sequence analysis revealed a number of putative transcriptional regulatory motifs, including a TATA box 30 nucleotides upstream from the tsp, and that this region is a CpG island. While there is conservation between the chicken and human GabR alpha 1 sequences, the chicken GabR alpha 1 promoter has a different structure to those reported for the GABAA receptor beta 3- and delta-subunit-encoding genes.

Animals↗
PubMed1995

Channel gating in the absence of agonist by a homo-oligomeric molluscan GABA receptor expressed in Xenopus oocytes from a cloned cDNA.

We have previously described the isolation of a complementary DNA (cDNA) from the freshwater mollusc Lymnaea stagnalis encoding a polypeptide that exhibits approximately 50% identity to the beta-subunits of vertebrate gamma-aminobutyric acid (GABA) type A (GABAA) receptor. When expressed in Xenopus laevis oocytes from in vitro-transcribed RNA, the snail subunit forms functional homo-oligomeric receptors possessing chloride-selective ion channels. In recordings from voltage-clamped oocytes held at -60 mV, GABA induced an inward current, whereas application of the chloride-channel blocker picrotoxin (in the absence of agonist) elicited an apparent outward current. Single channel recordings obtained from cell-attached patches have revealed a single population of approximately 20 pS channels, with an open probability greater than 90% (at a pipette potential of -100 mV) in the absence of GABA. The relationship between single channel current and pipette potential was linear over the studied range (-100 mV to +60 mV), but the open probability was less for hyperpolarizations than for depolarizations. The spontaneous channel openings were blocked by micromolar concentrations of picrotoxin. Functional hetero-oligomeric receptors were formed when the molluscan subunit was co-expressed in oocytes with the bovine GABAA receptor alpha 1-subunit, but the channels gated by these receptors did not open spontaneously.

Animals↗
PubMed1995

Schizophrenia and GABAA receptor subunit genes.

Alterations in gamma-aminobutyric acid (GABA) neurotransmission have been indirectly implicated in the pathogenesis of schizophrenia. Using nine multiplex pedigrees, we tested for linkage between schizophrenia and simple sequence repeat polymorphisms for the GABAA receptor alpha 1, alpha 2, alpha 4, alpha 5, alpha 6, beta 1 and beta 3 subunit genes. Evidence of linkage was not found when assuming either autosomal dominant or autosomal recessive inheritance. The non-parametric sib pair test also did not reveal significant evidence of deviation from expected segregation ratios.

Genes, Dominant↗
PubMed1994

Further evidence for clustering of human GABAA receptor subunit genes: localization of the alpha 6-subunit gene (GABRA6) to distal chromosome 5q by linkage analysis.

GABAA receptors are hetero-oligomeric ion-channel complexes that are composed of combinations of alpha, beta, gamma, and delta subunits and play a major role in inhibitory neuro-transmission in the mammalian brain. We report here a microsatellite polymorphism within the human alpha 6-subunit gene (GABRA6). Mapping of this marker in a human-hamster hybrid cell-line panel and typing of the repeat in the Centre d'Etude du Polymorphisme Human (CEPH) reference families enabled the localization of this gene to chromosome 5q and established its linkage to the GABAA receptor alpha 1-subunit gene (GA-BRA1) with a maximum lod score (Zmax) of 39.87 at a theta of 0.069 (males) and 0.100 (females). These results reveal the clustering of GABRA6, GABRA1, and the GABAA receptor gamma 2-subunit gene (GABRG2) on distal chromosome 5q.

Animals↗
PubMed1994

Developmental up-regulation and agonist-dependent down-regulation of GABAA receptor subunit mRNAs in chick cortical neurons.

We have used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze the expression of GABAA receptor subunit genes in cultured neurons from the chick embryo cerebral cortex. During maturation of the neurons between day 2 and day 8 in culture, levels of the alpha 1 subunit transcript (per ng total RNA) increased 3.8 +/- 0.3 fold, while those for the beta 2S and beta 4S subunits increased 2.4 +/- 0.4 and 1.8 +/- 0.2 fold, respectively. The accumulation of the beta 4 S subunit mRNA was more rapid than those encoding either the alpha 1 or beta 2S polypeptides. After 4 days in culture the beta 4S subunit transcript level reached 105 +/- 7.7% of that found after 8 days, while the corresponding amounts for the alpha 1 and beta 2S subunit mRNAs were 50 +/- 7.1% and 44 +/- 10.7%, respectively. On the other hand, no significant differences were observed in the level of either the gamma 1 or the gamma 2S subunit mRNA during development in vitro. Likewise, the ratios of the large/small splice variants (beta 2 = 0.16 +/- 0.02; beta 4 = 0.57 +/- 0.02; gamma 2 = 0.30 +/- 0.06) did not show detectable changes during this period. To study the down-regulation of the mRNAs, a single dose of 100 microM GABA was added to the culture medium. After 7 days of exposure to GABA, the levels of transcripts for the alpha 1, beta 2, beta 4, gamma 1, and gamma 2 subunits and their splice variants (where present) were all reduced by 47-65% compared to untreated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1994

Alternative splicing of a 51-nucleotide exon that encodes a putative protein kinase C phosphorylation site generates two forms of the chicken gamma-aminobutyric acidA receptor beta 2 subunit.

Complementary DNAs that encode two forms of the chicken gamma-aminobutyric acid type A (GABAA) receptor beta 2 subunit have been isolated. These polypeptides differ by the presence of (beta 2L) or absence (beta 2S) of 17 amino acids, which contain a possible target for phosphorylation by protein kinase C, in the large intracellular loop between the third and fourth membrane-spanning domains. The extra sequence in the chicken beta 2L subunit is not found in previously published GABAA receptor beta 2-subunit sequences. Analysis of genomic DNA has revealed that the two beta 2-subunit mRNAs arise by alternative splicing of a novel 51-nucleotide exon. Although the two beta 2-subunit transcripts appear to be present in 1-day-old chick brain at similar steady-state levels, we have been unable to detect an mRNA for the long form of the beta 2 subunit in either the bovine or the rat. Because the various GABAA receptor genes are thought to have arisen by duplication of a common ancestor, our data, taken together with that on the gamma 2 subunit, which occurs in two forms that arise by alternative splicing of a 24-nucleotide exons, suggest that the coding region of the primordial gene or one of its very early descendants contained 10 exons, not nine as previously thought.

Alternative Splicing↗
PubMed1994

Sequence of a Drosophila ligand-gated ion-channel polypeptide with an unusual amino-terminal extracellular domain.

We report the isolation of a full-length clone from a Drosophila melanogaster head cDNA library that encodes a 614-residue polypeptide that exhibits all of the features of a ligand-gated chloride-channel/receptor subunit. This polypeptide, which has been named GRD (denoting that the polypeptide is a GABAA and glycine receptor-like subunit of Drosophila), displays between 33 and 44% identity to vertebrate GABAA and glycine receptor subunits and 32-37% identity to the GABAA receptor-like polypeptides from Drosophila and Lymnaea. It is interesting that the large amino-terminal, presumed extracellular domain of the GRD protein contains an insertion, between the dicysteine loop and the first putative membrane-spanning domain, of 75 amino acids that is not found in any other ligand-gated chloride-channel subunit. Analysis of cDNA and genomic DNA reveals that these residues are encoded by an extension of an exon that is equivalent to exon 6 of vertebrate GABAA and glycine receptor genes. The gene (named Grd) that encodes the Drosophila polypeptide has been mapped, by in situ hybridization, to position 75A on the left arm of chromosome 3.

Amino Acid Sequence↗
PubMed1994

Analysis of GABAA receptor subunit genes in multiplex pedigrees with manic depression.

The gamma-aminobutyric acid (GABA) neurotransmitter system has been implicated in the pathogenesis of manic depression. Tests of this hypothesis can now be carried out due to the recent characterization of simple sequence repeat polymorphisms for the GABAA receptor alpha 1, alpha 2, alpha 4, alpha 5, alpha 6, beta 1, beta 3 and gamma 2 subunit genes. Using both parametric and non-parametric methods, we tested for linkage between manic depression and these polymorphisms in six multi-generational pedigrees. No evidence of linkage was found.

Adolescent↗
PubMed1993

Molecular cloning reveals the existence of a fourth gamma subunit of the vertebrate brain GABAA receptor.

We have isolated a cDNA, from the chicken, that encodes a fourth type of gamma subunit of the vertebrate brain GABAA receptor. The mature polypeptide (which we name gamma 4) displays 67%, 69% and 70% identity, respectively, to the rat gamma 1, gamma 2 and gamma 3 subunits. In the developing chicken brain, the gamma 4-subunit mRNA is first detected at embryonic day 13; the transcript level then increases progressively during embryogenesis. In situ hybridization reveals that the gamma 4-subunit mRNA is abundant in several brain regions, including the ectostriatum, nucleus rotundus and hyperstriatum ventrale, which are involved in visual processing and learning.

Amino Acid Sequence↗
PubMed1993

A novel invertebrate GABAA receptor-like polypeptide. Sequence and pattern of gene expression.

A full-length complementary DNA has been isolated, from the fresh-water mollusc Lymnaea stagnalis, that encodes a polypeptide (which we have named zeta) that exhibits between 30% and 40% identity to vertebrate GABAA and glycine receptor subunit sequences. The locations of seven introns have been determined in the corresponding gene and six of these occur at similar relative positions as those found in vertebrate GABAA receptor genes. RNase protection studies have revealed that the transcript for this Lymnaean polypeptide is present at highest levels in the adult nervous system but that it can also be detected in peripheral tissues.

Amino Acid Sequence↗
PubMed1993

Direct sequencing of lambda DNA from crude lysates using an improved linear amplification technique.

We describe an improved method for directly sequencing lambda (lambda) DNA that has been isolated from either crude cleared lysates or plate lysates. This protocol does not require that the DNA be obtained from bacteriophage particles that have been purified by caesium chloride centrifugation. Nanogram quantities of lambda DNA are unidirectionally amplified using a radioactively-labelled oligonucleotide primer, and Thermus aquaticus (Taq) DNA polymerase, in the presence of T4 gene 32 protein (gp 32). The amplification/sequencing reactions are then incubated with terminal deoxynucleotidyl transferase (TdT) and all four deoxynucleotide triphosphates to elongate any prematurely-arrested products. This procedure, which is a modification of a previously-published method, results in a significant improvement in the quality and amount of DNA sequence information that can be obtained from lambda templates. Although it was developed to sequence DNA directly from lambda EMBL3 recombinants, it can also be used with cosmid DNA, M13 and plasmid DNA, and polymerase chain reaction (PCR) amplification products, yielding excellent ladders in each case. In addition, our method resolves the nucleotide sequences of double-stranded plasmid templates that are difficult to determine by conventional dideoxynucleotide sequencing protocols because of 'stalling', in which bands appear at the same position in all four lanes.

Bacteriophage lambda↗
PubMed1993

Channels formed by M2 peptides of a putative glutamate receptor subunit of locust.

A cDNA encoding part of a polypeptide (Loc1) that exhibits similarity to the corresponding portion of the rat GluR1 subunit has been identified by screening an amplified locust cDNA library. This polypeptide is deduced to be missing about 200 amino acids of the amino-terminus and about 100 amino acids of the carboxy-terminus. cDNAs encoding two other glutamate receptor-like polypeptides (Loc2 and Loc3), which both exhibit good sequence homology with Loc1, have also been identified. So far, there is no evidence for 'flip' and 'flop' variants of Loc1, 2 and 3. A 27-mer peptide including the M1 sequence of Loc1 and a 25-mer peptide including the M2 sequence of this putative glutamate receptor subunit have been synthesised and incorporated into artificial bilayers. Channel openings, of minimum conductance 20 pS, were seen more frequently with the M2 peptide. These studies are designed to lead to the isolation of full-length cDNAs for Loc1, 2 and 3 and to the electrophysiological characterisation of their ion transport properties.

Amino Acid Sequence↗
PubMed1993

Molluscan ligand-gated ion-channel receptors.

In this chapter we introduce the reader to the structures of the different types of ligand-gated ion-channel receptor, and the numerous receptor subtypes that have recently been revealed to exist, in both invertebrate and vertebrate species, by the application of molecular biological methods. We then review some of the data in support of the existence, in molluscs, of such receptor/channel complexes for gamma-aminobutyric acid, glutamate and acetylcholine. Finally, recent results from our laboratory on the cloning and expression of complementary DNAs, from the pond-snail Lymnaea stagnalis, that encode GABA(A) and glutamate receptor subunits will be described.

Amino Acid Sequence↗
PubMed1993

Sequence and novel distribution of the chicken homologue of the mammalian gamma-aminobutyric acidA receptor gamma 1 subunit.

cDNAs have been cloned that encode the chicken (Gallus domesticus) gamma-aminobutyric acidA receptor gamma 1 subunit, the mature sequence of which shares 90, 79, and 69% identity with those of the rat gamma 1, gamma 2, and gamma 3 subunits, respectively. In situ hybridization reveals that there are pronounced differences in the regional and cellular localizations of the corresponding gamma-aminobutyric acidA receptor gamma-subunit mRNA compared with that of the gamma 2-subunit mRNA in 1-day-old chick brain. The absence of the gamma 1-subunit transcript in certain chick brain nuclei of visual and auditory pathways, in which gamma 2-subunit mRNA is present, points to differences in the functional roles of receptors containing one or other of these polypeptides. Certain cells in other brain regions appear to contain both gamma 1- and gamma 2-subunit mRNAs, suggesting that they either have two gamma-aminobutyric acidA receptor subtypes or possess receptors incorporating two different gamma subunits. We have also found contrasts in the distribution patterns, in homologous brain regions, of the chicken gamma 1-subunit mRNA and the rat gamma 1-subunit mRNA. These data may reflect different functional roles of the chicken and rat gamma 1 subunits.

Amino Acid Sequence↗
PubMed1992

Unusual effects of benzodiazepines and cyclodiene insecticides on an expressed invertebrate GABAA receptor.

We have previously reported [(1991) EMBO J. 10, 3239-3245] the sequence of an invertebrate gamma-aminobutyric acid (GABA) type A (GABAA) receptor polypeptide which forms homo-oligomeric GABA-gated, bicuculline-sensitive, chloride-ion channels upon heterologous expression. We now demonstrate that the benzodiazepines Ro5-4864 (4'-chlorodiazepam) and diazepam, that are active at mammalian peripheral benzodiazepine sites, and not those benzodiazepines specific for central sites, directly active the homo-oligomeric receptor and evoke larger maximal responses than those elicited by GABA. In addition, members of the cyclodiene class of insecticides block the channel of the receptor in a manner indistinguishable from that of picrotoxin.

Animals↗