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270 , - A - BIOCHEMICAL SOCIETY TRANSACTIONS Table 1. Effect of modulators on prostaglandin Eproductwn by WI-38fibroblast cells Monolayer cultures were incubated with lop1 of the modulating agent in 1ml of culture medium containing 0.1% bovine serum albumin for 60min at 37°C. Control samples were incubated with lop1 of vehicle. The medium was assayed by radioimmunoassay for prostaglandin E. Values are means ~ s . E . M .for quadruplicate incubations. Abbreviations : pOH-HgBzO, p-hydroxymercuribenzoate; A'-THC, A'-tetrahydrocannabinol. Addition , Prostaglandin E Stimulation 01M) (nslml) (%I 1.7 kO.1 Control pOH-HgBzO 25 2.9 f0.3 70.1 pOH-HgBzO 50 9.9k0.2 489.0 pOH-HgBzO 100 21 .O* 0.9 1160.0 26.6 1.4 1490.0 pOH-HgBzO 150 8 22.8 k0.7 1260.0 A'-THC medium. After a 60min exposure of 100jm-inhibitor with the fibroblasts, the liberated products were shown to be increased by 23% (P<0.0005) with a concomitant decrease in 14C-labelledphospholipids in the cells. Arachidonic acid, the major product, was enhanced by 54% and [14C]prostaglandin E2 increased by 113% over cells treated with vehicle alone. Furthermore, [ 14C]thromboxane A2 was increased 11%by the inhibitor. Inclusion of the inhibitor was found to potentiate the stimulatory response by 8 @+tetrahydrocannabinol on the release of 14C products from labelled fibroblasts by 71% over cells treated with cannabinoid alone. Therefore the enhanced amounts of arachidonate metabolites, namely prostaglandin E2 and thromboxane A2, obtained in these experiments could be attributed to the elevated amounts of arachidonate resulting from the blocking, by inhibitor, of the re-uptake of fatty acid via an acylating pathway. viability was monitored throughout these studies by DNA measurements (Burstein et al., 1982), and over 90% of the cells remained viable after exposure to the drugs. By using conditions described previously for the labelling of WI-38 fibroblasts and the release reaction (Burstein et al., 1982), the effects of p-hydroxymercuribenzoate were examined on the release of labelled products to the culture Burstein, S. & Hunter, S. A. (1981) J . Clin. Pharmacol. 21, 240s2488 Burstein, S., Hunter, S. A., Sedor, C. & Shulman, S . (1982) Biochem. Pharmacol. 31, 2361-2365 DeHaas, G. H., Postema, N. M., Nieuwenhuizen, W. & van Deenen, L. L. M. (1968) Biochim. Biophys. Acra 189, 103-118 Feinmark, S. J. & Bailey, J. M. (1982) J . Biol. Chem. 257, 2816282 1 Irvine, R. F. (1982) Biochem. J . 204, 3-16 Kroner, E. E., Peskar, B. A., Fischer, H. & Ferber, E. (1981) J. Biol. Chem. 256, 3690-3697 Wilson, D. B., Prescott, S. M. & Majerus, P. W. (1982) J . Biol. Chem. 257, 3510-3515 Chloroplast genes for components of the ATP synthase complex ALISON K. HUTTLY,* CHRISTOPHER J. HOWE,' CATHERINE M. BOWMAN,? TRISTAN A. DYER? and JOHN C. GRAY* *Botany School, University of Cambridge, Downing St., Cambridge CB2 3EA. U .K., and ?Plant Breeding Institute, Trumpington, Cambridge CB2 2LQ, U.K. The ATP synthase complex of higher-plant chloroplasts is composed of a number of different subunits, which are arranged in an extrinsic component, CF,, and an intrinsic one, CF,. There is evidence that subunits a, fl and E of CF, and subunit I11 of CF, are synthesized inside the chloroplast (Mendiola-Morgenthaler et al., 1976; Nelson et al., 1980; Doherty & Gray, 1980). Genes for these chloroplast-synthesized subunits have been located in pea (Pisum sativum cv. Feltham First) and wheat (Triticum aestivum cv. Mardler) chloroplast DNA, whose chloroplast genome arrangement differs. The wheat chloroplast genome is typical of most higher plants in having two copies of the 16s and 23s rRNA genes arranged in an inverted repeat (Bowman et al., 1981), whereas the chloroplast genome of pea has a single copy of the rRNA genes. In addition, hybridization data indicate extensive chromosomal rearrangements between the two types (Palmer & Thompson, 1981). To map the chloroplast genes, a cell-free coupled transcription-translation system derived from Escherichia coli (Bottomley & Whitfeld, 1979) was programmed with cloned chloroplast DNA restriction fragments, and L-[~%]methionine-labelled products were identified by immunoprecipitation with specific antibodies. The identity of immunoprecipitated polypeptides was confirmed by com- * Abbreviation: kbp, thousand base-pairs. petition of the immunoprecipitation by added unlabelled authentic material. By this means, the positions of genes for subunits a, p, E and I11 have been located in wheat (Fig. la), and a,fi and I11 in pea (Fig. 1b). In addition, the positions of genes for subunit a in wheat, and a, p, E and I11 in pea have been demonstrated by heterologous hybridization of cloned fragments of genes from other species to Southern blots of restriction-enzyme digests of wheat and pea chloroplast DNA. In both species, the genes for these subunits are arranged in two clusters, with the gene for p close to that for E and the gene for a close to that for subunit 111, but in wheat the clusters are 20kbp* apart, whereas in pea they are 50 kbp apart. The genes for fl and E subunits are close to, and transcribed divergently from, the gene for the large subunit of ribulose bisphosphate carboxylase (Koller et al., 1982; Oishi & Tewari, 1983). The genes for a and subunit I11 are 2 kbp apart, and are transcribed in the same direction as one another, on the opposite strand to that for p and E. The nucleotide sequence of the gene for subunit I11 has been determined in wheat and pea by inserting appropriate DNA fragments into bacteriophage M13 and using the dideoxynucleotide-chain-terminationmethod of Sanger et al. (1980). The nucleotide sequences of the pea and wheat genes show 90% homology; of 25 nucleotide differences in the coding region, 22 are in the third position of the codon, one is in the second and two are in the first. Only one of these changes results in an amino acid substitution: residue 47 is glycine in wheat and aspartate in pea. This change occurs in a hydrophilic region in the centre of a largely hydrophobic molecule. Both genes have a potential ribosome-binding site shortly before the ATG initiation codon, but there are no flanking sequences showing clear homology with prokaryotic promoter or terminator signals. It is possible that the gene is transcribed as part of a much larger 1984 27 1 604th MEETING, CAMBRIDGE Fig. 1, Restriction maps of wheat (a) and pea (b) chloroplast genomes The maps show restriction sites of PstI (P), SalGI (S) and BamHI (B) in wheat (Bowman et al., 1981) and of PstI (P) and SulGI (S) in pea (Palmer & Thompson, 1981). The positions and orientations of genes for the subunits of ATP synthase are indicated. Positions of the wheat genes have been published elsewhere (Howe et al., 1982a,b, 1983). Also indicated are the positions of the genes for the 16s and 23s rRNA (16S,23S: Bowmanetal., 1981; Palmer&Thompson, 1981), forthelargesubunit of ribulose bisphosphate carboxylase (LS) (Bowman et al., 1981; Oishi & Tewari, 1983), for the 32kDa photogene in pea (32 k; J. Palmer, unpublished work) and for cytochrome (cyt)fin pea (Willey et al., 1983). Arrows indicate the direction of transcription of the genes. transcript. In E. coli the ATP synthase genes form a single operon (Gibson et al., 1978; Downie et al., 1979) and the clustering of the chloroplast genes may reflect a prokaryotic origin. This work was supported by grants from the Science and Engineering Research Council and the Agricultural Research Council. Howe, C. J., Auffret, A. D., Doherty, A., Bowman, C. M., Dyer, T. A. & Gray, J. C. (19826) Proc. Natl. Acad. Sci. U S A . 79, 69036907 Howe, C. J., Bowman, C. M., Dyer, T. A. &Gray, J. C. (1983) Mol. Gen. Genet. 190, 51-55 Koller, B., Delius, H. & Dyer, T. A. (1982) Eur. J. Bwchem. 122, 17-23 Mendiola-Morgenthaler, L. R., Morgenthaler, J.-J. & Price; C. A. (1976) FEBS Lett. 62, 9 6 1 0 0 Nelson, N., Nelson, H. & Schatz, G. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1361-1364 Oishi, K. K. & Tewari, K. K. (1983) Mol. Cell Bwl. 3, 587- Bottomley, W. & Whitfeld, P. R. (1979) Eur. J. Biochem. 93, 31-39 Bowman. C. M., Koller, B., Delius, H. & Dyer, T. A. (1981) Mol. Gen. Genet. 183, 93-101 595 Doherty, A. & Gray, J. C. (1980) Eur. J . Biochem. lOS, 131-136 Downie, J. A., Senior, A. E.. Gibson, F. & Cox, G. 9. (1979) J . Palmer, J. D. & Thompson, W. F. (1981) Proc. Natl. Acud. Sci. U.S.A. 78, 5533-5537 Bacteriol. 137, 7 1 1-7 18 Gibson, F., Downie, 3. A., Cox, G. 9 . & Radik, J. (1978) J. Sanger, F., Coulson, A. R., Barrett, B. G., Smith, A. J. H. & Roe, B. A. (1980) J. Mol. Biol. 143, 161-178 Bacteriol. 134, 728-736 Howe, C. J., Bowman, C. M., Dyer, T. A. & Gray, J. C. (1982~) Willey, D. L., Huttly, A. K., Phillips, A. L. & Gray, J. C. (1983) Mol. Gen. Genet. 189, 85-89 Mol. Gen. Genet. 186, 525-530 Chloroplast genes for components of the cytochrome b-- complex from pea DAVID L. WILLEY, ANDREW L. PHILLIPS and JOHN C. GRAY Botany School, University of Cambridge, Downing Street. Cambridge CB2 3EA. U.K. Three components (cytochrome J cytochrome b563 and an M,-1 5 200 polypeptide) of the cytochrome kfcomplex have been shown to be synthesized on pea (Pisum sativum L.) chloroplast ribosomes (Doherty & Gray, 1979; Phillips & Gray, 1984). By analogy with other polypeptides synAbbreviation : bp, base-pair VOl. 12 thesized on chloroplast ribosomes, the genes for these components are most likely to be located in chloroplast DNA. The gene for cytochrome f has been located in chloroplast DNA by coupled transcription-translation (Willey et al., 1983) and a similar approach has been used here to locate the gene for the M,-15200 polypeptide. Both genes have been localized on small restriction fragments and their nucleotide sequences determined. Cloned restriction fragments of pea chloroplast DNA (Palmer & Thompson, 1981a) have been used in a cell-free coupled transcription-translation system from Escherichia coli (Bottomley & Whitfeld, 1979) and ~-[~~S]methionine-