PCR-BASED HOMOLOGY PROBING REVEALS A FAMILY OF GABA RECEPTOR-LIKE GENES IN DROSOPHILA-MELANOGASTER
PCR-BASED HOMOLOGY PROBING REVEALS A FAMILY OF GABA RECEPTOR-LIKE GENES IN DROSOPHILA-MELANOGASTER
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DOI：
10.1016/0965-1748(94)90029-9
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发表时间：
1994-04-01
影响因子：
3.8
通讯作者：
SODERLUND, DM
中科院分区：
文献类型：
--
作者：
HENDERSON, JE;KNIPPLE, DC;SODERLUND, DM
采用基于聚合酶链反应（PCR）的同源性探针技术，从黑腹果蝇基因组DNA中筛选编码脊椎动物GABA受体和甘氨酸受体亚基基因保守氨基酸特征基序的序列。该方法产生了三个离散的扩增序列元件（命名为LCCH 1、LCCH 2和LCCH 3），其含有开放阅读框，并且与脊椎动物配体门控氯离子通道基因的相应区域具有>40%的氨基酸序列同一性。分离对应于每个元件的基因组DNA克隆并测序，并分析对应于脊椎动物基因的第二（M2）和第三（M3）跨膜结构域的预测氨基酸序列与已知序列的同一性或相似性。LCCH 1与已知的D.而LCCH 2和LCCH 3为新发现的D.与配体门控氯离子通道基因家族的其他成员表现出结构相似性的黑腹动物序列。LCCH 2在M2和M3中与该家族的所有其他已知成员同样不同（46-49%氨基酸同一性），因此可能代表该家族中的新亚基或受体类别。LCCH 2通过原位杂交定位于3号染色体左臂上的细胞遗传学区域75 A。LCCH 3与哺乳动物（79%氨基酸同源性）和蜗牛（96%氨基酸同源性）GABA受体β亚基密切相关，因此可能是D.这个亚单位纲的黑腹动物。LCCH 3通过原位杂交定位于X染色体上的细胞遗传学区域13 F。相应的LCCH 1，LCCH 2和LCCH 3的基因组DNA克隆的序列分析表明，每个转录单位缺乏至少一个内含子-外显子剪接点被发现是哺乳动物和蜗牛GABA受体亚基基因之间的保守。这些结果为D.在结构上与脊椎动物的配体门控氯离子通道基因家族相关。
A polymerase chain reaction (PCR)-based homology probing strategy was employed to screen Drosophila melanogaster genomic DNA for sequences encoding a conserved amino acid 'signature motif known to be present in vertebrate GABA receptor and glycine receptor subunit genes. This approach yielded three discrete amplified sequence elements (designated LCCH1, LCCH2, and LCCH3) that contained open reading frames and >40% amino acid sequence identity to the corresponding regions of vertebrate ligand-gated chloride channel genes. Genomic DNA clones corresponding to each element were isolated and sequenced, and predicted amino acid sequences corresponding to the second (M2) and third (M3) transmembrane domains of vertebrate genes were analyzed for identity or similarity to known sequences. LCCH1 was identical to the Rdl gene, a known GABA receptor subunit gene from D. melanogaster, whereas LCCH2 and LCCH3 were novel D. melanogaster sequences that exhibited structural similarity to other members of the ligand-gated chloride channel gene family. LCCH2 was equally divergent in M2 and M3 (46-49% amino acid identity) from all other known members of this family and may therefore represent a new subunit or receptor class within this family. LCCH2 was localized by in situ hybridization to cytogenetic region 75A on the left arm of chromosome 3. LCCH3 was closely related to mammalian (79% amino acid identity) and snail (96% amino acid identity) GABA receptor beta subunits and may therefore be the homologue in D. melanogaster of this subunit class. LCCH3 was localized by in situ hybridization to cytogenetic region 13F on the X chromosome. Sequence analysis of genomic DNA clones corresponding to LCCH1, LCCH2, and LCCH3 showed that each transcription unit lacked at least one of the intron-exon splice junctions found to be conserved between mammalian and snail GABA receptor subunit genes. These results provide evidence for the existence of a diverse family of genes in D. melanogaster that are structurally related to the ligand-gated chloride channel gene family of vertebrates.