Abstract
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.
Highlights
We investigated the 5’-upstream region of the gene encoding the catalytic subunit of the V-type H+-ATP
Ribonuclease protection and primer extension analysis indicated that the three the putative Splbinding sequence and the (TATA) boxes corresponded to two major and one minor transcription start sites
Isolation and Sequence of the 5’-Region of the Carrot V-type H”-ATPase Catalytic Subunit Gene-Southern blot analysis of carrot genomic DNA suggested the presence of a single gene when probed with a 300-bp 5’-fragment of the cDNA (Fig. 1); only digestion with PstI yielded more than one band, probably due to an internal restriction site
Summary
IQ1 and analyzed by Southern blot with a 300-bp] cDNA fragment from the -5’ end of the sequence [17] as probe (prepared by random priming [18]) according to standard protocols [19]. For analysis of gene expression, the two fragments, 240 and 800, were released by HindIII-XbaI digest and cloned into the promoterless plasmid pB1 101 (Clontech), containing the B-glucuronidase reporter gene, to generate pB1 101-240 and pBI 101-800. Transformed cells were selected by kanamycin resistance; since the inserts were short compared with the vector and did not show up clearly on agarose gels, we confirmed the presence of both plasmids by Southern blot analysis with the 240.hp fragment as probe (obtained by random priming according to Ref. 18). The callus transformation procedure made it possible to assay for /j-glucuronidase expression in a number of different transformants for each promoter construct; in this way gene dosage and position effects are. at least to some extent, averaged out
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
