Struve I, Rausch T, Bernasconi P, Taiz L
Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz 95064.
J Biol Chem. 1990 May 15;265(14):7927-32.
We investigated the 5'-upstream region of the gene encoding the catalytic subunit of the V-type H(+)-ATPase in Daucus carota. A genomic sublibrary was screened with a cDNA probe, and a 4-kilobase genomic clone was obtained covering the first two exons and about 3 kilobases of the 5'-upstream sequence. The intron/exon boundaries matched established consensus sequences. Within 240 base pairs (bp) upstream of the initiation codon three putative TATA boxes were found. Ribonuclease protection and primer extension analysis indicated that the three TATA boxes corresponded to two major and one minor transcription start sites. The flanking sequences of the two more proximal TATA boxes were nearly identical. Additional sequence motifs with putative regulatory function are two CCAAT boxes, an Sp1-binding consensus sequence, and long (TATA)n stretches within 800 bp of the 5'-upstream sequence. Transcriptional fusions to the beta-glucuronidase reporter gene were made for two different promoter constructs, and the resulting plasmids were mobilized into Agrobacterium tumefaciens. The analysis of beta-glucuronidase activities in the transformed carrot calli showed that 240 bp of the upstream sequence, including all three TATA boxes, led to low but detectable beta-glucuronidase expression; however, the larger construct, which included the putative Sp1-binding sequence and the (TATA)n stretches, led to an approximately 6-fold higher beta-glucuronidase expression. Histochemical analysis of beta-glucuronidase activity in the transformed calli showed no preferential expression in any specific cell type, in keeping with the presumed "housekeeping" character of the V-type H(+)-ATPase catalytic subunit gene.
我们研究了胡萝卜中V型H(+)-ATP酶催化亚基编码基因的5'-上游区域。用cDNA探针筛选基因组亚文库，获得了一个4千碱基的基因组克隆，它覆盖了前两个外显子和大约3千碱基的5'-上游序列。内含子/外显子边界与已确立的共有序列相匹配。在起始密码子上游240个碱基对（bp）内发现了三个推定的TATA框。核糖核酸酶保护和引物延伸分析表明，这三个TATA框对应于两个主要转录起始位点和一个次要转录起始位点。两个更靠近近端的TATA框的侧翼序列几乎相同。具有推定调控功能的其他序列基序是两个CCAAT框、一个Sp1结合共有序列以及5'-上游序列800 bp内的长（TATA）n序列段。针对两种不同的启动子构建体构建了与β-葡萄糖醛酸酶报告基因的转录融合体，并将所得质粒导入根癌农杆菌。对转化的胡萝卜愈伤组织中β-葡萄糖醛酸酶活性的分析表明，包括所有三个TATA框的240 bp上游序列导致低水平但可检测到的β-葡萄糖醛酸酶表达；然而，包含推定的Sp1结合序列和（TATA）n序列段的更大构建体导致β-葡萄糖醛酸酶表达提高了约6倍。对转化愈伤组织中β-葡萄糖醛酸酶活性的组织化学分析表明，在任何特定细胞类型中均无优先表达，这与V型H(+)-ATP酶催化亚基基因假定的“管家”特性相符。
