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      Isolation, characterization, and localization of human genomic DNA encoding the β1 subunit of the GABAA receptor (GABRB1)

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      Genomics

      Elsevier BV

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          Abstract

          Genomic DNA that encodes the beta 1 subunit of the human gamma-aminobutyric acidA (GABAA) receptor was cloned and mapped. Exons and flanking introns (greater than 14 kb) were sequenced to determine the structural organization of the gene. The gene was localized on human chromosome 4, in bands p12-13. The beta 1 subunit is encoded by a relatively large gene (greater than 65 kb) on nine exons. In contrast to other conserved regions of the subunit polypeptide, the proposed channel-forming domain (M2) is derived from more than one exon. The organization of exons was compared with that of the genes that code for subunits of nicotinic acetylcholine receptors. There is no evidence for conservation of gene structure between these two members of the proposed gene superfamily. However, intron-exon junctions were found to be conserved precisely between subtypes of GABAA receptor subunits.

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          Most cited references 24

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          Organization and expression of eucaryotic split genes coding for proteins.

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            High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones.

            Cosmid clones containing human DNA inserts have been mapped on chromosome 11 by fluorescence in situ hybridization under conditions that suppress signal from repetitive DNA sequences. Thirteen known genes, one chromosome 11-specific DNA repeat, and 36 random clones were analyzed. High-resolution mapping was facilitated by using digital imaging microscopy and by analyzing extended (prometaphase) chromosomes. The map coordinates established by in situ hybridization showed a one to one correspondence with those determined by Southern (DNA) blot analysis of hybrid cell lines containing fragments of chromosome 11. Furthermore, by hybridizing three or more cosmids simultaneously, gene order on the chromosome could be established unequivocally. These results demonstrate the feasibility of rapidly producing high-resolution maps of human chromosomes by in situ hybridization.
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              Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries.

              A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzyme-labeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laser-scanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.
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                Author and article information

                Journal
                Genomics
                Genomics
                Elsevier BV
                08887543
                August 1991
                August 1991
                : 10
                : 4
                : 985-995
                Article
                10.1016/0888-7543(91)90189-L
                1655634
                © 1991

                https://www.elsevier.com/tdm/userlicense/1.0/

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