In both cases, the H+-PPase mRNA content was the highest in the elongating region (segment nos. Bleomycin sulfate pumps are essential for quick expansion of vacuoles. Previously we reported that, even in the young cells of the mung bean hypocotyl, the relative H+-PPase level on the basis of membrane protein content was the same as that of the mature cell (Maeshima, 1990). In this study we focused our attention on the developmental expression of the H+-PPase gene during expansion of vacuoles in elongating hypocotyls of mung bean. RNA-blot analysis revealed the down-regulation of H+-PPase gene expression in hypocotyl development, supporting the previous observations. Here we discuss the primary structure of H+-PPase and the physiological meanings of the regulation of H+-PPase level in growing plant cells. MATERIALS AND METHODS Seeds of mung bean (cv Wilczek) were soaked in 1 mm CaSO4 and then germinated at 26C in the dark. Hypocotyls from 3- and 5-d-old seedlings were cut into 1- and 1.5-cm segments, respectively. The segments were numbered from the hypocotyl apex, as shown in Figure ?Figure2A 2A (3-d-old hypocotyl, segment nos. 31C36; 5-d-old hypocotyl, segment nos. 51C59). To obtain leaves, mung beans were grown in garden soil for 3 weeks under long-day conditions (16/8 h, 28/20C, light/dark regime). The first, second, and third leaf pairs were harvested from the 3-week-old plants. Open in a separate window Figure 2 Amounts of RNA and DNA in segments of etiolated mung bean hypocotyls. A, Mung bean seedlings germinated for 3 and 5 d. Three- and 5-d-old hypocotyls were cut into 1- and 1.5-cm segments, respectively. B and C, Amounts of total RNA and total DNA, calculated on the basis of fresh weight (fw) of segment. D, Relative content of RNA on the basis of DNA content. cDNA Construction and Screening Total RNA was isolated from the hook portion of 3-d-old hypocotyls for construction of a cDNA library. The hypocotyl segments were CSH1 immediately frozen in liquid nitrogen, and then RNA was extracted by the phenol/SDS extraction method. RNA and DNA were precipitated by cold ethanol and resuspended in TE buffer (10 mm Tris-HCl, pH 8.0, and 1 mm EDTA). Then RNA was precipitated by 4 m LiCl (total RNA fraction). Poly(A+) RNA was purified with oligo(dT)-latex. A cDNA library was synthesized from 5 g of the poly(A+) RNA, ligated into phage vector (Uni-ZAP XR, Stratagene), and then packaged with a packaging extract (Gigapack II Gold, Stratagene). Recombinant phages from the library were blotted onto nylon membranes and then screened with a 32P-labeled DNA probe according to standard hybridization protocol (Sambrook et al., 1989). Positive clones were excised in vivo into the pBluescript SK(?) plasmid (Stratagene). DNA Preparation DNA was isolated from hypocotyl segments by the same method as RNA extraction. After RNA was removed by LiCl precipitation, the remaining supernatant was used as the DNA fraction. Samples and standard DNA were diluted to 0.5 mL with TE buffer and 10 ng/mL 4,6-diamidino-2-phenylindole dihydrochloride. Fluorescence of the DNA solution was measured with a fluorescence spectrophotometer set at 348 nm for excitation and 450 nm for emission, and then DNA content was calculated. Preparation of DNA Probes After treatment of mRNA with DNase, cDNAs were synthesized from the mRNAs with reverse transcriptase using oligo(dT)-16 primer. cDNA templates were amplified by PCR using DNA polymerase and gene-specific primers (forward, 5-ACTGGTTATGGTCTTGGTGGGT-3; reverse, 5-GGCAACATCTTGCACAGGGCTGT-3). The primers correspond to the consensus nucleotide sequences of H+-PPase cDNA of Bleomycin sulfate barley (Tanaka et al., 1993) and (Sarafian et al., 1992). The amplification protocol was 5 min at 95C (once), 1 min at 95C, 1 min at 55C, 2 min at 72C (40 cycles), and 7 min at 72C (once). Amplified DNA Bleomycin sulfate fragments (630 bp) were purified and ligated into the MV1184. The insert DNA was confirmed to be a cDNA fragment of H+-PPase by DNA sequencing. A DNA probe for subunit A of V-ATPase was prepared by PCR.