Download Chloroplast genes for components of the ATP synthase complex

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-