Murtagh J J, Eddy R, Shows T B, Moss J, Vaughan M
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Mol Cell Biol. 1991 Feb;11(2):1146-55. doi: 10.1128/mcb.11.2.1146-1155.1991.
Go alpha, (gene symbol GNA01), a member of the signal-transducing guanine nucleotide-binding (G) protein family, has been implicated in ion channel regulation. Some tissues contain multiple Go alpha mRNAs of different sizes that differ in the 3' untranslated regions (UTRs). Using sequence-specific 48-base oligonucleotides, two complementary to the different 3' UTRs and one complementary to the coding region, we investigated the origin of the multiple Go alpha transcripts, the organization of the Go alpha gene, the interspecies conservation of 3' UTRs, and the chromosomal localization of Go alpha. Oligonucleotides labeled to high specific activity by using terminal deoxynucleotidyltransferase each hybridized with a single band of restriction enzyme-digested mouse and human DNAs. In three of four digests of human DNA, the two probes specific for the different 3' UTRs hybridized with the same restriction fragment. Thus, these nucleotide sequences are in close proximity in the human genome. The order of the UTRs in the bovine, human, and mouse genomes was confirmed directly by polymerase chain reaction (PCR) amplification and sequencing. Hybridization of bovine oligonucleotide sequence with mouse and human genomic DNA indicated a high degree of interspecies sequence conservation: conservation was confirmed by PCR amplification and sequencing. Bands detected by both UTR probes, as well as the predominant bands detected by a bovine Go alpha cDNA, segregated with human chromosome 16 on Southern blot analysis of human-mouse somatic cell hybrids. We conclude that Go alpha mRNAs with different 3' UTRs arise by alternative splicing of transcripts from a single gene. The UTRs, which exhibit a high degree of interspecies conservation, may play a role in regulation of Go alpha expression during differentiation or in specific tissues. The use of oligonucleotide probes of the type described here represents a new strategy, potentially widely applicable for mapping and elucidating structural features of genes.
Goα（基因符号GNA01）是信号转导鸟嘌呤核苷酸结合（G）蛋白家族的成员，与离子通道调节有关。一些组织含有多种大小不同的Goα mRNA，它们在3'非翻译区（UTR）存在差异。我们使用序列特异性的48个碱基的寡核苷酸，其中两个与不同的3'UTR互补，一个与编码区互补，研究了多种Goα转录本的起源、Goα基因的组织、3'UTR的种间保守性以及Goα的染色体定位。通过末端脱氧核苷酸转移酶标记至高比活性的寡核苷酸，分别与经限制性内切酶消化的小鼠和人DNA的单一条带杂交。在人DNA的四次消化中的三次中，针对不同3'UTR的两个探针与相同的限制性片段杂交。因此，这些核苷酸序列在人类基因组中紧密相邻。通过聚合酶链反应（PCR）扩增和测序直接证实了牛、人和小鼠基因组中UTR的顺序。牛寡核苷酸序列与小鼠和人基因组DNA的杂交表明种间序列具有高度保守性：通过PCR扩增和测序证实了这种保守性。在人 - 小鼠体细胞杂种的Southern印迹分析中，两个UTR探针检测到的条带以及牛Goα cDNA检测到的主要条带与人类16号染色体分离。我们得出结论，具有不同3'UTR的Goα mRNA是由单个基因转录本的可变剪接产生的。UTR表现出高度的种间保守性，可能在分化过程中或特定组织中对Goα表达的调节中发挥作用。使用本文所述类型的寡核苷酸探针代表了一种新策略，可能广泛适用于基因的定位和阐明基因的结构特征。
