PCR isolation and cloning of novel splice variant mRNAs from known drug target genes

Pei, Jin; Fu, Glenn K.; Wilson, Amy; Yang, Junming; Chien, David; Hawkins, Phillip R; Au-Young, Janice; Stuve, Laura L.

doi:10.1016/j.ygeno.2003.09.023

Cited by 16 publications

(12 citation statements)

References 15 publications

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“…Alternative splicing has been demonstrated for most of the GABA A receptor subunit isoforms indicating complexity in gene expression (Jin et al, 2004;Tian et al, 2005). From Figure 1 it can be seen that there is considerable conservation and linkage disequilibrium extending across long stretches of sequence in the chromosome 4 and 5 clusters but less so in the chromosome 15 cluster.…”

Section: Association Between Gaba a Receptor Genes And Alcoholism In mentioning

confidence: 98%

“…However, no functional polymorphism has as yet been found There are numerous alternative splicing isoforms, most of which differ in their 3' end after exon 3 (Figure 2) and these may be implicated in function. Within the brain there are four major isoforms with alternative 5' (exon 1A or IB) and 3' exons (exon 9A or 10) as well as minor isoforms lacking exon 4 or exon 8 (Tian et al, 2005;Jin et al, 2004). The functional significance of these isoforms is at present unknown.…”

Section: Gaba a Receptor Chromosome 4 Gene Clustermentioning

confidence: 99%

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The role of GABAA receptors in the development of alcoholism

Enoch

2008

Pharmacology Biochemistry and Behavior

197

181

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Alcoholism is a common, heritable, chronic relapsing disorder. GABA A receptors undergo allosteric modulation by ethanol, anesthetics, benzodiazepines and neurosteroids and have been implicated in the acute as well as the chronic effects of ethanol including tolerance, dependence and withdrawal. Medications targeting GABA A receptors ameliorate the symptoms of acute withdrawal. Ethanol induces plasticity in GABA A receptors: tolerance is associated with generally decreased GABA A receptor activation and differentially altered subunit expression. The dopamine (DA) mesolimbic reward pathway originating in the ventral tegmental area (VTA), and interacting stress circuitry play an important role in the development of addiction. VTA GABAergic interneurons are the primary inhibitory regulators of DA neurons and a subset of VTA GABA A receptors may be implicated in the switch from heavy drinking to dependence. GABA A receptors modulate anxiety and response to stress; important elements of sustained drinking and relapse. The GABA A receptor subunit genes clustered on chromosome 4 are highly expressed in the reward pathway. Several recent studies have provided strong evidence that one of these genes, GABRA2, is implicated in alcoholism in humans. The influence of the interaction between ethanol and GABA A receptors in the reward pathway on the development of alcoholism together with genetic and epigenetic vulnerabilities will be explored in this review.

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Section: Association Between Gaba a Receptor Genes And Alcoholism In mentioning

confidence: 98%

Section: Gaba a Receptor Chromosome 4 Gene Clustermentioning

confidence: 99%

The role of GABAA receptors in the development of alcoholism

Enoch

2008

Pharmacology Biochemistry and Behavior

197

181

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“…However, we also wanted to assess the value of the method in identifying new AS events within medically important genes. We performed a manual search for new events within a set of 162 known drug target genes (Jin et al 2004). We used as reference the same two databases mentioned above, namely, the Ensembl transcript database (build 34) and the H-Invitational database.…”

Section: Automatic Detection Of New As Eventsmentioning

confidence: 99%

ASEtrap: A biological method for speeding up the exploration of spliceomes

Thill¹

2006

Genome Res.

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Alternative splicing (AS) of pre-messenger RNA is a major mechanism for generating protein diversity from a limited number of genes in higher eukaryotes, and it constitutes a central mode of genetic regulation. Thus, efficient methods are needed to systematically identify new AS events at a genomic scale across different tissues, stages of development, and physiological or pathological conditions in order to better understand gene expression. To fulfill this goal, we have designed the ASEtrap, which is a cloning procedure for producing AS libraries that is based on a single-stranded trap consisting of an ssDNA-binding protein. In this paper, we have applied our approach to the construction of an AS library and a Control library from human placenta. By analyzing 9226 and 9999 sequences of the AS and Control libraries, respectively, we show that internal AS events (events that can be identified by the sole resources provided by either the AS or the Control library) and the discovery rate of new AS events measured at early stages of sequencing were nine to 10 times higher in the former than in the latter. Moreover, by performing a search for new AS events within a group of 162 known drug target genes, we identified six new events in six genes, and we observed that they all were discovered exclusively through the AS library. Thus, it appears that ASEtrap has the potential to greatly facilitate the determination of the total complement of splice variants expressed in human, as well as other organisms.

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“…These two residues flank a novel alternative exon (16,17). When the residues are mutated, oligomerization of ␥2 with ␣2 and ␤1 is prevented, and cell surface access of ␥2 is abolished.…”

Section: ␥-Aminobutyric Acid Type a (Gaba A )mentioning

confidence: 99%

Serine 171, a Conserved Residue in the γ-Aminobutyric Acid Type A (GABAA) Receptor γ2 Subunit, Mediates Subunit Interaction and Cell Surface Localization

Pei

Walther

Zhang

et al. 2004

Journal of Biological Chemistry

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Serine 171 in the GABA A receptor ␥2 subunit is highly conserved in the ligand-gated ion channel superfamily. In this paper, we report that mutating serine 171 within ␥2 to glycine or cysteine prevents the interaction of ␥2 with ␣2 and ␤1 when these subunits are co-expressed in human embryo kidney 293 cells, resulting in intracellular retention of ␥2. Structure analysis based on a three-dimensional homology model of ␥2 (Ernst, M., Brauchart, D., Boresch, S., and Sieghart, W. (2003) Neuroscience 119, 933-943) reveals that serine 171 may play a critical role in the formation and stabilization of an exposed turn structure that is part of the subunit interaction site. Mutation of serine 171 in the ␥2 subunit could therefore result in alteration of the structure of the subunit interaction site, preventing correct subunit assembly. ␥-Aminobutyric acid type A (GABA A )1 receptors are members of the ligand-gated ion channel superfamily, which also includes nicotinic acetylcholine (ACh), glycine, glutamate, and 5-hydroxytryptamine type 3 (5HT3) receptors. Members of this superfamily share significant sequence similarity, implying a common evolutionary origin (2).GABA A receptors are the major inhibitory receptors in the brain and are the targets of clinically important drugs such as benzodiazepines, barbiturates, neurosteroids, and anesthetics (3). Several distinct classes of GABA A receptor subunits have been identified (3-8). Heterologous expression of recombinant GABA A receptor subunits in cultured cells demonstrates that the combination of ␣ and ␤ subunits is sufficient for creating the GABA binding sites to elicit GABA-gated Cl Ϫ currents (9). Benzodiazepines bind to a distinct site on the GABA A receptor and positively modulate GABA-gated C Ϫ current (10). Formation of the benzodiazepine binding site requires the presence of a third component, the ␥ subunit (9, 10). Typical native GABA A receptors have been proposed to be pentameric transmembrane proteins formed by two ␣, two ␤, and one ␥ subunits (9). Assembly of the hetero-pentamers occurs in the endoplasmic reticulum and requires the interaction between subunit-specific contact sites. Access to the cell surface is restricted to the correctly assembled GABA A receptors (11-15).We identified previously two highly conserved residues, serine 171 and tyrosine 172, in the ␥2 subunit. These two residues flank a novel alternative exon (16,17). When the residues are mutated, oligomerization of ␥2 with ␣2 and ␤1 is prevented, and cell surface access of ␥2 is abolished. In the present study, using single amino acid substitutions, we further demonstrate that serine 171 within ␥2 plays a critical role in mediating the subunit interaction and cell surface expression of ␥2, whereas tyrosine 172, as well as other residues surrounding Ser-171/ Tyr-172, exhibit little or no effect on the function of the ␥2 subunit. Mapping of the ␥2 protein sequence onto the threedimensional homology model of the extracellular component of the GABA A receptor generated from the ACh-binding prote...

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PCR isolation and cloning of novel splice variant mRNAs from known drug target genes

Cited by 16 publications

References 15 publications

The role of GABAA receptors in the development of alcoholism

The role of GABAA receptors in the development of alcoholism

ASEtrap: A biological method for speeding up the exploration of spliceomes

Serine 171, a Conserved Residue in the γ-Aminobutyric Acid Type A (GABAA) Receptor γ2 Subunit, Mediates Subunit Interaction and Cell Surface Localization

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