LéJohn H B, Cameron L E, Yang B, Rennie S L
Department of Microbiology, University of Manitoba, Winnipeg, Canada.
J Biol Chem. 1994 Feb 11;269(6):4523-31.
The gene for an NAD-specific glutamate dehydrogenase (NAD-GDH) that is allosterically activated by NADP+ (non-substrate) was cloned, and its physical structure and nucleotide sequence was determined. The gene consists of 9 introns and 10 exons; the 10th and largest exon, which is 1863 nucleotides long, is at the 3'-end of the gene. The shortest exon of 33 base pairs is the first and is located at the 5'-end of the gene. The large exon is in perfect register along the complementary strand with a heat shock 70 (HSP)-like protein gene. The NAD-GDH gene is inducible with L-glutamine, just as the HSP 70-like protein gene (LéJohn, H.B., Cameron, L.E., Yang, B., MacBeath, G., Barker, D.S., and Williams, S.A. (1994) J. Biol. Chem. 269, 4513-4522). The phenomenon of anti-parallel coupling of two genes is named antisense gene pair. By Northern and Western blotting techniques, we obtained indirect evidence that the gene is expressed in vivo. The gene encodes a protein of M(r) 118,740 which consists of 1063 amino acid residues. The 5' and 3' borders of the gene display typical but unproven promoter motifs of CCAAT, TATAAT, and AAATAAAA polyadenylation signal bounded by a pyrimidine-rich transcription termination-type format. Restriction endonuclease site mapping of all the genomic clones isolated that carry most or all of the gene, and of the genome itself, gave hybridization patterns that are consistent with the interpretation that the organism, Achlya klebsiana, has only one form of the gene. 3'-End-labeling of a 5.2-kb XbaI DNA fragment (carrying the antisense gene pair) that was then asymmetrically cleaved to produce two single 3'-end-labeled pieces that were used as probes on L-glutamine-induced cell poly(A)+ RNA, showed that the end-labeled DNA equivalent to the HSP 70-like protein mRNA hybridized to a 3.4-kb transcript and the end-labeled DNA equivalent to the NAD-GDH mRNA hybridized to a 2.4-kb transcript.
克隆了一种由NADP⁺（非底物）变构激活的NAD特异性谷氨酸脱氢酶（NAD-GDH）基因，并确定了其物理结构和核苷酸序列。该基因由9个内含子和10个外显子组成；第10个也是最大的外显子，长度为1863个核苷酸，位于基因的3'端。最短的外显子为33个碱基对，是第一个外显子，位于基因的5'端。这个大的外显子与一个热休克70（HSP）样蛋白基因的互补链完全对齐。NAD-GDH基因可被L-谷氨酰胺诱导，就像HSP 70样蛋白基因一样（LéJohn，H.B.，Cameron，L.E.，Yang，B.，MacBeath，G.，Barker，D.S.，和Williams，S.A.（1994）J. Biol. Chem. 269，4513 - 4522）。两个基因反平行偶联的现象被称为反义基因对。通过Northern和Western印迹技术，我们获得了该基因在体内表达的间接证据。该基因编码一种分子量为118,740的蛋白质，由1063个氨基酸残基组成。该基因的5'和3'边界显示出典型但未经证实的启动子基序，即CCAAT、TATAAT和AAATAAAA多聚腺苷酸化信号，其边界为富含嘧啶的转录终止型结构。对所有分离出的携带该基因大部分或全部的基因组克隆以及基因组本身进行限制性内切酶位点图谱分析，得到的杂交模式与以下解释一致：该生物体，即克氏壶菌，只有一种形式的该基因。对一个5.2 kb的XbaI DNA片段（携带反义基因对）进行3'端标记，然后将其不对称切割以产生两个单链3'端标记的片段，将其用作L-谷氨酰胺诱导的细胞多聚（A）⁺ RNA的探针，结果表明，与HSP 70样蛋白mRNA等效的末端标记DNA与一个3.4 kb的转录本杂交，与NAD-GDH mRNA等效的末端标记DNA与一个2.4 kb的转录本杂交。
