L487. these experiments, CDP synthesized by CPS activity was utilized 13 occasions more efficiently for sp. L487, and genes from several species provided a way to examine the regulation of gene was cloned from pumpkin (gene. Previously, studies of GA-deficient mutants resulted in the cloning of genes from Arabidopsis (with a large deletion in the locus encoding CPS is still able to produce low amounts of GAs (Zeevaart and Talon, 1992). In a similar fashion, a maize deletion mutant with a knock-out for the gene encoding a putative CPS was shown to accumulate genes may be expressed in a herb species. In the present paper, for the first time to our knowledge, two genes from your same herb species, genes and the gene were compared during seedling development and in adult plants. Our data show that this genes are purely regulated in a different organ-specific and developmental manner. MATERIALS AND METHODS Plant Material Seeds of pumpkin (L. cv Riesenmelone gelb vernetzt) were obtained from van Waveren Pflanzenzucht (Rosdorf, Germany) courtesy of Professor Jan Graebe. Pumpkin seedlings were cultivated under continuous light (220 mol m?2 s?1) at 25C Ro 48-8071 fumarate on moist vermiculite. Adult (1-month-old) plants were grown under the same conditions. Immature seeds were harvested from field-grown plants, as explained by Yamaguchi et al. (1996). PCR Amplification of Pumpkin cDNA Fragments Degenerate primers were designed from your sequences SAYDTAWV (1F forward primer), FNGGVPN (3R reverse primer), and KHFERNG (5R reverse primer) (Ait-Ali et al., 1997). Double-stranded cDNA was synthesized from poly(A+) RNA isolated from cotyledons of immature pumpkin seeds, as explained by Yamaguchi et al. (1996). PCR was carried out as explained by Ait-Ali et al. (1997). Two PCR fragments of 0.89 kb (1F and 5R primers) and 0.64 kb (1F and 3R primers) were obtained and subcloned into the pCRII vector, as described by the manufacturer (TA Cloning, Invitrogen, San Diego, CA). We named the genes corresponding to the 0.89- and 0.64-kb PCR fragments and cDNAs Both 0.89- and 0.64-kb PCR fragments were used as the probes to screen a cDNA library prepared from immature pumpkin seeds (Yamaguchi et al., 1996). The probes were Ro 48-8071 fumarate labeled using the ECL Direct Nucleic Acid Labeling and Detection System (Amersham, Japan). Plaque lifts on nylon membranes (Hybond-ECL, Amersham) were hybridized in the buffer provided by the manufacturer at 42C. Washing was repeated three times at 42C with a buffer made up of 4 m urea, 0.5 SSC, and 0.4% (w/v) SDS, and then three times at room heat with 2 SSC buffer. All positive clones that were selected by library testing carried the sequence. Only one of the clones experienced the full-length ORF; however, it also contained a putative unspliced intron. To obtain a cDNA for functional expression in ORF is usually underlined), and reverse, 5-ATATTAGCGGCCGCTTGACAATACAACATGGCTG-3 (the strain JM109. To determine which part of the ORF is usually dispensable for the CPS activity, we subcloned several 5- and 3-truncated cDNAs in the EPSTI1 pGEX-4T-3 (Pharmacia) for functional expression in ORF and utilized for PCR reactions with the cDNA as the template. Cloning of cDNAs To isolate the full-length cDNA, cDNAs made up of the full-length ORF was carried out from male blossom bud cDNA by PCR with end-specific primers: forward, 5-ATATATGAATTCCATGTCCTCCTCCTCCTCTCTCT-3 (the ORF is usually underlined), and reverse, 5-ATAATTCTCGAGACAACATGGGTGTGTGGGTAGCTA-3 (the cDNAs were cloned into the pGEX-4T-3 for functional assay. For cloning, three forward primers made up of an cDNA as the template. DNA Sequencing DNA sequencing Ro 48-8071 fumarate was done with double-stranded DNA using a dye primer cycle sequencing kit (Applied Biosystems) and an ABI373A DNA Sequencer (Applied Biosystems). For sequencing of the cultures incubated for 22 h at 20C. Bacterial extracts in a CPS buffer (50 mm potassium phosphate, pH 8.0, 10% [w/v] glycerol, 2 mm DTT, and 5 mm MgCl2) were cleared by centrifugation at 12,000for 20 min at 4C. Rapid assays of CPS and KS activities were carried out as explained by Ait-Ali et al. (1997). Reactions were performed for 30 min at 30C in 200 L of the CPS buffer that contained either 1 kBq (75 GBq mmol?1) of [3H]GGDP (Amersham) or 1.5 kBq (74 GBq mmol?1) of [3H]CDP (a gift from Dr. T. Saito, Institute of Physical and Chemical Research, Saitama, Japan)..