Eur. J. Biochem. Y3, 581 -586 (1979)
Poly(adeny1ic acid)-Containing RNA of EugZena gracilis during Chloroplast Development 2. Transcriptional Origin of the Different RNA Gerard VERDIER Departcmcnt de Biologie Genkrale et Appliquee (Laboratoire Associe au Centre National de la Recherche Scientifique), Universirt. Lyon I, Villeurbanne (Received February 3/0ctober 30, 1978)
The transcriptional origin of the polyadenylated RNA has been studied in dark-grown cultures of Euglena gracilis and during light-induced chloroplast development. Complementary DNA (cDNA) to poly(A)-containing RNA from different preparations (isolated chloroplasts and total cell polyribosomes from the wild-type Z strain or from the aplastidic mutant W3BUL) were synthesized and used for hybridization experiments. a) In Euglena cells illuminated for 0, 1 or 24 h, 7- 10 % of the total cell poly(A)-containing RNA are transcribed from chloroplast DNA. However, when RNA is extracted from isolated chloroplasts, this proportion of poly(A)-containing RNA is about two times higher (20%); in this case, the poly(A)-containing RNA could belong to a specific class and be transcribed from 18-20% of the double-strand chloroplast DNA. b) In the dark-grown cultures or in the greening cultures, the major part of the total cell poly(A)-containing RNA is transcribed from the non-repetitive sequences of nuclear DNA. The isolated chloroplast preparations contain also a high proportion of poly(A)-containing RNA from nuclear origin. c) About 10% of the total cell poly(A)-containing RNA of the wild-type strain, absent in the poly(A)-containing RNA of the aplastidic mutant, appear hybridizable to the nuclear genome of this mutant. Thus the transcription of some of the RNA encoded by nuclear DNA could be controlled by the presence of the chloroplast. Moreover, the fact that isolated chloroplast preparations appear to be enriched in such poly(A)-containing RNA from nuclear origin, suggests that these RNA are located at the level of the chloroplast. Three genomes are present in the Euglena gracilis. The nuclear DNA represents most of the total cell DNA and is composed of repetitive and unique sequences [l]. The chloroplast DNA corresponds to about 6 2 of the total cell DNA with a major component as a circular duplex molecule of molecular weight 92 x lo6 [ 2 ] . Finally the mitochondria1 DNA has a kinetic complexity 2-4 times lower than that of the chloroplast DNA [3,4]. The chloroplast morphogenesis stimulated by the light is developped concurrently with protein synthesis occurring both in the cytoplasm and in the chloroplast [5 - 71. The genomic information is transcribed as messenger RNA (mRNA), some of which contains polyadenylic acid poly(A) sequences [8 - 101 and corresponds to different frequency classes; changes
in the polyadenylated RNA distribution have been studied during the light-induced chloroplast development (preceding paper). In this report we attempt to measure the fractions of the poly(A)-containing RNA populations transcribed either by the nuclear DNA or by the chloroplast DNA. For this analysis, DNA (cDNA) complementary to different poly(A)-containing RNA preparations was synthesized and used in hybridization experiments with the nuclear DNA or with the chloroplast DNA. In order to determine the fraction of the chloroplast genome represented as RNA transcripts, the chloroplast DNA was hybridized to a large excess of poly(A)-containing RNA. Finally we have analyzed the meaning of poly(A)-containing RNA from nuclear origin present in the isolated chloroplast preparations.
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MATERIALS AND METHODS Strains and Cultures Euglenia gracilis wild-type Z strain was grown in darkness on a phosphate/carbon-limited medium [ll], then transferred to a 0.03 M KCl solution at the beginning of the stationary phase ; illumination was begun three days later, as described previously (preceding paper). The aplastidic mutant W3BUL was grown in the light on the same medium and the cultures were used in a late logarithmic phase. Preparation and Fractionation of Polysomal R N A and Chloroplast R N A ; Transcription of Polyadenylated R N A
Polysomal RNA were extracted by the phenol/ chloroform technique from total cellular polyribosomes as described previously [lo]. Chloroplasts of 24-h-illuminated cultures were purified by differential centrifugation, which was preferred to flotation procedure as discussed previously (preceding paper) ; these chloroplast preparations were used to extract total chloroplast RNA by the phenol/chloroform method. Poly(A)-containing RNA from either total polysomal RNA or chloroplast RNA were isolated by chromatography on oligo(dT)-cellulose as described previously (preceding paper). DNA complementary to poly(A)-containing RNA was synthesized using RNA-dependent DNA polymerase prepared from avian myeloblastosis virus (supplied by Dr R. Williamson, St Mary's Hospital, London) as described in the preceding paper. Preparations of Chloroplast and Nuclear DNA, and Radioiodination of Chloroplast Preparations
Chloroplast DNA was extracted from isolated chloroplasts by the phenol/chloroform method [ 101 and separated on an alkaline CsCl gradient into light and heavy components. In hybridization experiments the light component (e = 1.686 g/cm3) was used, since it contains sequences complementary to the majority of the chloroplast DNA transcripts [12], the heavy component (e = 1.701 g/cm3) corresponding mainly to sequences complementary to chloroplast ribosomal RNA [12,13]. The light component of DNA was sonicated to an average size of 500-600 bases and was treated with 0.3 M NaOH in 37 "C for 16 h to hydrolyze any contaminating RNA. After neutralization and precipitation with ethanol, the DNA was desalted by passage through Sephadex G-50. The nuclear DNA was prepared from the aplastidic W3BUL mutant by the same procedure. The light component of chloroplast DNA was labelled in vitro with ['Z51]iodine according to the Orosz and Wetmur procedure [14]. The 1251-labelled
Euglena Polyadenylated RNA Transcription
chloroplast DNA had a specific activity of 4 x lo6 counts min-l pg-'. After labelling, the single-strand I-labelled DNA had an apparent average molecular weight of 100000. Samples and Abbreviations Used for Annealing Experiments
Poly(A)-containing RNA isolated from total polysomal RNA of wild-type Z strain in the dark [ZoTpoly(A)-containing RNA] or after illumination for 1 h [ZIT-poly(A)-containing RNA] and 24 h [Z24Tpoly(A)-containing RNA] was used to synthesize corresponding complementary DNA : ZoT-cDNA, ZIT-CDNA, Z24T-cDNA. Poly(A)-containing RNA obtained from total RNA of chloroplasts isolated after 24 h of illumination [Z~ch-poly(A)-containingRNA] was used to synthesize complementary DNA (ZZ4Ch-cDNA). W3-poly(A)-containing RNA corresponded to poly(A)-containing RNA from total polysomal preparations of W3BUL mutant. W3-cDNA was the corresponding complementary DNA. Unlabelled nuclear DNA was obtained from the W3BUL mutant (W3-DNA), and Chl-DNA corresponded to unlabelled light component of chloroplast DNA. 'z51-labelledChl-DNA: light component of chloroplast DNA labelled by ['251]iodine. Hybridization Kinetics
Three types of annealing techniques have been used: hybridization of cDNA with an excess of unlabelled poly(A)-containing RNA; hybridization of cDNA with an excess of unlabelled DNA (Chl-DNA or W3-DNA) ;hybridization of '251-labelledChl-DNA with an excess of unlabelled poly(A)-containing RNA. Samples of cDNA (0.2 ng) or 'z51-labelled ChlDNA (0.5 ng) were mixed with appropriate amounts of unlabelled poly(A)-containing RNA (from 50 ng to 8 pg) or DNA (200 ng of Chl-DNA or 1-8 pg of W3-DNA), lyophilized and dissolved into 2 1.11 of 0.24 M sodium phosphate buffer (pH 6 4 , 1 mM EDTA. Each sample was sealed in a silicon-coated glass capillary. The cDNA/poly(A)-containing RNA samples were incubated at 68 "C. However, before incubation, capillaries with cDNA/DNA or lZ5Ilabelled Chl-DNA/poly(A)-containing RNA mixtures were boiled for 5 min and immediately transferred to a 68 "C bath. At the end of each incubation period required to reach the desired value of rot [initial RNA concentration (mol nucleotides/l) x time (s)] or of cot [initial DNA concentration (mol nucleotides/l) x time (s)l, the amount of cDNA or 1251-labelled Chl-DNA in hybrids was assayed using S1 nuclease as described previously (preceding paper).
G. Verdier
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RESULTS Annealing o j cDNA with Poly(A)-Containing RNA
To determine the degree of sequence homology between the RNA of the wild-type Z strain and the RNA of the aplastidic mutant W3BUL, we examined the hybridization of the cDNA of one preparation with the poly(A)-containing RNA of the heterologous preparation. For this investigation, Z24T-cDNA corresponding to the most complex poly(A)-containing RNA population that we have described previously (preceding paper) was used, whereas the Z24Ch-cDNA was tested to detect the sequences from nuclear origin in the poly(A)-containing RNA extracted from isolated chloroplast. The results are shown in Fig. 1 ; to facilitate comparison we have included in the figure the corresponding homologous reactions. The reaction of Z24T-cDNA to an excess of W3poly(A)-containing RNA reached a plateau of 49 compared with 79 % hybridization observed in the homologous Z24T-cDNA/Z24T-poly(A)-containing RNA annealing. Therefore, we conclude that 49/79 or 62% of the hybridizable Z24T-cDNA is complementary to W3-poly(A)-containing RNA. These common sequences seem to belong to the second and
x,
60
._ 50 40
' 3 0
1
20 10
third classes of the homologous Z ~ ~ T - C D N A / Z U T poly(A)-containing RNA hybridization : no hybridization corresponded to classes 1 and 4. The annealing of Z24Ch-cDNA with Ws-poly(A)containing RNA demonstrated that 50 % of the cDNA was hybridized for the highest rot values; no hybridization was observed in the range of the vot values of the first and fourth classes of the homologous Z~4Ch-cDNA/Z~~Ch-poly(A)-containing RNA reaction. Annealing of the Z ~ ~ T - C D Nwith A the WJ-DNA
To estimate, after 24 h of illumination, the proportions of poly(A)-containing RNA which was transcribed from the nuclear genome, the DNA from the aplastidic mutant W3BUL was used as nuclear DNA (Fig. 2). Total cell DNA, which corresponds for the major part to the nuclear DNA, is composed 18 :G by highly repetitive sequences, 40% by middle repetitive sequences and 36 % by non-repetitive sequences : this last fraction was reassociated in the range of cot values from 100-10000 mol 1-' s with cotliz = 2000mol 1-' s [l]. In order to determine which frequency classes of DNA sequences were represented in the cDNA population, we have hybridized the cDNA to a vast excess of DNA [15]. Our results show that at a cot value of 7600 mol I-' s at least 72% of the Z24T-cDNA was annealed with the W3-DNA (Fig. 2). About two log units of cot (from 100- 10000 moll-' s) were covered by the kinetic reaction : this result indicates that only one class of the WYDNA, corresponding to non-repetitive DNA sequences (cotl12 of the reaction = 2300 mol I s), was represented in the Z24T-cDNA.
n
Annealing of cDNA with Chl-DNA
2
1
4
3
90
00
The kinetic complexity of the Euglena chloroplast DNA is 92 x lo6 [2] or approximately 130 x lo3 base
-
70 -
60 -
*O 70
50-
10-2
10'
100
10'
102
103
4.103
r, t (rnol I-'s)
Fig. 1. Heterologous hybridizations of an excess of W3-poly(A)containing RNA with ( A ) ZZ~T-CDNA ( x - - x j~ or ( ~ B ) ZzaChcDNA ( x ~x ). See Materials and Methods, and abbreviation Homologous reaction of ZZ~T-CDNA and paragraph. (0-0) Z24T-poly(A)-containing RNA (A) or Zz4Ch-cDNA and ZzKhpoly(A)-containing RNA (B) (see preceding paper). The results are expressed as semi-logarithmic plots of the percentage of hybridized cDNA against rot values (moll-' s). 1 , 2, 3, 4: levels of the diffirent transitions of homologous hybridizations
i2"
1
"11
L
20 lo
0 1
10'
. .
* I
102
103
104
c a t (rnol I-' s )
Fig.2. Hybridization kinetics of Z Z ~ T - c D N Awith an excess of W3DNA (see Materials and Methodsj. The results are expressed as semi-logarithmic plots of the percentage of hybridized cDNA against cot values (moll-' s )
584
Euglena Polyadenylated RNA Transcription
-
.1" n N
ar ._ n
'= D
10
L a
8
5 -
4
z
0
6
Y
s
I
0 1 10-2
,
!
100
10'
I
lo-'
c o t (mot
2
0 lo-'
100
10'
I02
r o t (rnol K ' S )
IK'S)
Fig. 3. Hyhridization kinetics of'ZoT-cDNA ( x . . . . x ), Z , T-cDNA IS---.), Z Z ~ T - C D N (0A ---O) or Zz4Ch-cDNA ( b e ) with an excess of Chl-DNA (see Materials and Methods). Results are expressed as described in Fig. 2
Fig. 4. Hybridization kinetic of' '251-lahelled Cld-DNA with Z24Chpofy(A)-containing R N A . The resuits, expressed as semi-logarithmic plots of the percentage of hybridized 'ZsI-labelled Chl-DNA against rot values, were corrected for D N A . DNA renaturation in the absence of RNA
pairs. Recent studies have shown that the 16-S and 23-S rRNA genes are each present on a 5.6 x lo3base-pair repeated DNA segment [16,17]; this rRNA region is present 3 times as a tandem repeat on the genome [16,17]. These results are consistent with previous reports [13,18]. This rRNA region corresponds to only 17000 base pairs or 13 % of the chloroplast DNA while the rest of chloroplast DNA must correspond to unique sequences as shown by physical mapping [16]. More than 80% of the chloroplast DNA renature at cot values under 10 [12,19,20]. The ZoT-cDNA, ZIT-cDNA, Z24T-cDNA and Z24Ch-cDNA were hybridized to an excess of ChlDNA (e = 1.686 g/cm3, see Materials and Methods) (Fig. 3). From 7- 10 % of the various cDNA species synthesized from total cell poly(A)-containing RNA were annealed with the Chl-DNA ; however, about 20% of the Z24Ch-DNA was found to hybridize to Chl-DNA. Under the same conditions the proportions of the W3-cDNA which hybridize to the Chl-DNA corresponded to the background values of the reactions (1.5-2.5%). These results show that specific RNA sequences were transcribed from the chloroplast DNA and that these sequences correspond, in all the poly(A)-containing RNA isolated from different stages of chloroplast development, to a low proportions of these RNA populations.
containing-RNA . DNA and DNA . DNA duplexes. The fraction of poly(A)-containing RNA . DNA hybrids may be estimated by the difference between the duplex DNA appearing in the presence of an RNA excess and that attributable to DNA . DNA renaturation in the absence of poly(A)-containing RNA. The extent of DNA . DNA reassociation in these reactions was calculated by renaturing '251-labelled Chl-DNA in the absence of RNA at concentrations and time periods comparable to those used for the poly(A)containing RNA/DNA hybridization : the fraction of '251-labelled Chl-DNA renatured as a function of cot represented a maximum of 8.2%. In the poly(A)containing RNA/DNA hybridization, after correction for DNA . DNA renaturation, approximately 9 - 10 % of single-strand chloroplast DNA was saturated by Z24Ch-poly(A)-containing RNA. If we assume that only one strand of the DNA molecule is transcribed into RNA, 18-20% of the genomic information is present as polyadenylated RNA transcripts at this stage of the chloroplast development.
Hybridization ~ f ~ ~ ~ I - L a b eChl-DNA lled with Z24Ch-Poly( A )-Containing R N A In order to measure the amount of the chloroplast genome which is expressed as RNA transcripts, '251-labelled Chl-DNA was hybridized to an excess of polyadenylated RNA from isolated chloroplast preparation (Fig. 4). The fraction of '251-labelled Chl-DNA after S1 assay represents both poly(A)-
DISCUSSION Expression of the Chloroplast Genome of Euglena gracilis
We have found that the poly(A)-containing RNA extracted from chloroplast preparations were transcribed from 18-20% of the chloroplast genome, representing thus a minimal estimate of the chloroplast DNA transcription program. However, when chloroplasts were isolated from different stages of the plastid morphogenesis, one chloroplast RNA preparation might contain RNA sequences absent from another chloroplast preparation. Moreover, the messenger RNA of the large subunit of ribulose-l,5-bisphosphate
G. Verdier
carboxylase does not contain poly(A) tracts [21- 231 and we have also demonstrated that light induces synthesis of polysomal RNA, probably of messenger function, and devoid of poly(A) sequences [lo]. Our data can be compared with earlier reports. According to Chelm and Hallick [20], 32% of the chloroplast genome is complementary to nucleotide sequences in total cell RNA of Euglena gracilis illuminated for 24 h ; subtracting from this value the fractions of chloroplast DNA complementary to rRNA @ - l o%) [12] and to tRNA (0.7%) [24,25], it appears that the transcription for messenger RNA might represent 21 -23 % of the chloroplast genome. This value would be in good agreement with our findings. However, according to Rawson and Boerma [12, 191, the extent of the chloroplast genome transcription corresponds to 43 % for total cell RNA free of rRNA and tRNA. This value is higher than our results and those of Chelm and Hallick [20]. The difference between these two types of results could be explained by different culture conditions or by the interference with the polyadenylated RNA population, in Rawson and Boerma’s experiments, [12,19] of a significant fraction of mRNA devoid of poly(A) sequences. If such is the case, we must assume that at least a part of poly(A)-containing RNA could represent different RNA species absent in non-poly(A)-containing RNA. We have suggested previously a similar possibility [lo]. It is clear that a conclusive evaluation will only be obtained by the study of the RNA devoid of poly(A) sequences. The Transcriptional Origin the Poly(A)-Containing R N A Extracted from Isolated Chloroplasts of
The proportions of RNA sequences transcribed from chloroplast DNA were two times higher in the Z24-Ch-cDNA than in the other cDNA synthesized from total cell poly(A)-containing RNA isolated at different stages of chloroplast development (Fig. 3). However, these RNA sequences from chloroplast genome origin represented only 20% of the ZuChcDNA. Therefore, about 80 % of the polyadenylated RNA population extracted from isolated chloroplasts is probably transcribed from the nuclear genome. This result is not surprising, since the complexity of the Z24Ch-poly(A)-containing RNA had been found higher than the kinetic complexity of the chloroplast DNA (preceding paper). Moreover, about 50 % of the Z24Ch-poly(A)-containing RNA sequences were also present in the poly(A)-containing RNA population of the W3BUL mutant; the other half of the Zz4Chpoly(A)-containing RNA corresponded to abundant sequences, which were absent from WJ-poly(A)containing RNA population. Thus, it appears that
585
30 ”/, of the Z24Ch-poly(A)-containing RNA would correspond to abundant sequences, transcribed from nuclear genome but absent from W3-poly(A)-containing RNA sequences. Before separation of poly(A)-containing RNA, our preparations of chloroplast RNA were found to be contaminated by at most 10% cytoplasmic ribosomal RNA (preceding paper). To explain the presence of 80 % of poly(A)-containing RNA sequences transcribed from the nucleus in the poly(A)-containing RNA from chloroplast preparations, we may suppose that the contamination of these preparations by cytoplasmic poly(A)-containing RNA was higher than the contamination at rRNA level. If such is the case, the Z2&h-poly(A)-containing RNA population would be enriched in a fraction of abundant sequences, originating from the cytoplasmic RNA contaminants and representing up to 30 % of the total Z24Ch-poly(A)containing RNA. Alternatively another possibility could be that a high proportion of RNA from nuclear origin is bound to chloroplast envelopes in vivo; such results have been described in Nicotiana [26], in which the ribosomes bound to the outside of chloroplast membrane are thought to be responsible for the synthesis of a particular kind of chloroplast protein [27]. A third alternative is a possible migration of nuclearencoded RNA within the chloroplast structures; a similar situation has been described previously in Chlamydomonas reinhardi for a peptide of the chloroplast membrane which would be synthesized into the chloroplast from nuclear transcripts [28]. Recently, mRNA involved in spinach chloroplast synthesis in vitro has been shown to be coded partly by chloroplast DNA and partly by nuclear DNA (Guignery and Duranton, personal communication). The Transcriptional Origin ofthe Total Cell Poly(A)-Containing R N A during Greening Transcription from the Chloroplast Genome. A fraction of the poly(A)-containing RNA population was transcribed from chloroplast genome in the darkgrown cultures as well as in the cultures illuminated for 1 or 24 h. Although the cDNA obtained from these poly(A)-containing RNA preparations hybridized to Chl-DNA in almost equivalent proportions (7 - 10 %), these values do not prove that the corresponding sequences are identical. On the other hand, our results do not allow us to determine accurately whether the cDNA hybridized to Chl-DNA belongs to the same frequency classes or to different frequency classes. Moreover, Rawson and Boerma [19] have reported that at later stages of chloroplast development (25 - 50 h in the light) total cell RNA contains transcripts from 5 % of chloroplast DNA not previously transcribed.
586
Transcriptionfrom the Nucleur Genome. While the chloroplast morphogenesis is going on after 24 h of illumination [29], the major part of the total cell poly(A)-containing RNA appears to be from nuclear origin. At least, 72% of the Z24T-cDNA was hybridized to unique sequences of the W3-DNA. Moreover, 62 % of the ZZ4T-cDNAcorresponded to overlapping sequences with the W3-poly(A)-containing RNA. Therefore, a minimum of 10 % of poly(A)-containing RNA would represent specific sequences in the wildtype strain after illumination. Although these specific sequences belong to the nuclear DNA of the W3BUL mutant, they do not seem transcribed in this strain. Similarly, we have described previously that 11 % of the ZZ4T-poly(A)-containing RNA corresponded to a special class of abundant RNA sequences, absent from the dark-grown cultures (preceding paper). A possible conclusion could be that the presence of the chloroplast in the cell induces the activity of a specific fraction of the nuclear genome; this fraction would be made of RNA transcripts corresponding to, at least, 10% of the total cell poly(A)-containing RNA and 30 % of the poly(A)-containing RNA extracted from isolated chloroplasts. Therefore, we might conclude that the isolated chloroplast preparations are enriched in these specific transcripts. This would be a strong .argument to prove that these transcripts are at the level of the chloroplast and that they do not represent only a contamination of the chloroplast preparations by the RNA from cytoplasmic location. I am grateful to Professor V. Nigon for his helpful criticism. I thank Drs Heizmann, Crouse and Stutz for their generous gifts of E. gracilis chloroplast DNA. I thank also Mrs Schwob for her technical assistance and Dr Godet for critically reading the manuscript. I wish to thank also Mrs B. Szafranek for excellent secretarial work.
REFERENCES 1. Rawson, J. R. Y. (1975) Biochim. Biophys. Acta, 402, 171 - 178. 2. Manning, J. E. d Richards, 0. C. (1972) Biochemistry, 11, 2036 - 2043.
G . Verdier : Euglena Polyadenylated RNA Transcription 3. Edelman, M., Epstein, H. T. Sr Schiff, J. A. (1966) J . Mot. Biol. 17, 463 - 469. 4. Crouse, E. J., Vandrey, J. Sr Stutz, E. (3974) FEBS Lett. 42, 262 - 266. 5. Smillie, R. M., Evans, W. R. Sr Lyman, H. (1963) Brookhaven Symp. Biol. 16, 89-108. 6. Schiff, J. A. (1974) Proc. 3rd Int. Congr. Photosyntlzesis (Avron, M., ed.) pp. 1691 - 1717, Elsevier, Amsterdam. 7. Schwartzbach, S . D., Schiff, J. A. d Klein, S. (1976) Planfa, 131, 1-9. 8. Verdier, G., Trabuchet, G., Heizmann, P. Sr Nigon, V. (1973) Biochim. Biophys. Acra, 312, 528 - 539. 9. Sagher, D., Edelman, M. Sr Jakob, K. M. (1974) Biochim. Biophys. Acta, 349, 32-38. 10. Verdier, G . (1975) Biochim. Biophys. Acta, 407,91-98. 11. Freyssinet, G., Heizmann, P., Verdier, G., Trabuchet, G. Sr Nigon, V. (1 972) Physiol. Yeg. 10, 421 -442. 12. Rawson, J. R. Y. Sr Boerma, C. L. (1977) Biochem. Biophys. Res. Commun. 74, 932-918. 13. Kopecka, H., Crouse, E. J. Sr Stutz, E. (1977) Ew. J. Biochem. 72,525 -535. 14. Orosz, J. M. B Wetmur, J. B. (1974) Biochemistry, 13, 54675473. 15. Hastie, N. D. Sr Bishop, J. 0. (1976) Cell, 9, 761 -774. 16. Gray, P. W. d Hallick, R. B. (1978) Biochemistry, 17, 284289. 17. Jenni, B. Jt Stutz, E. (1978) Eur. J . Biochem. 88, 127-134. 18. Stutz, E. & Vandrey, J. P. (1971) FEBS Lett. 17,277-280. 19. Rawson, J. R. Y. d Boerma, C. L. (1976) Biochemistry, 15, 588 - 592. 20. Chelm, B. K. Sr Hallick, R. B. (1976) Biochemistry, 15, 593599. 21. Wheeler, A. M. Sr Hartley, M. R. (1975) Nature (Lond.) 257, 66 - 67. 22. Sagher, D., Grosfeld, H. Sr Edelman, M. (1976) Proc. Nut2 Acad. Sci. U.S.A. 73, 722 - 726. 23. Howell, S. H., Heizmann, P., Gelvin, S. B Walker, L. L. (1977) Plant Physiol. 59, 464-470. 24. Schwartzbach, S. D., Hecker, L. L. d Barnett, W. E. (1976) Proc. Nail Acad. Sci. U.S.A. 73. 1984-1988. 25. Gruol, D. J. B Haselkorn, R. (1976) Biochim. Biophys. Acta, 447,82 - 95. 26. Lerbs, S. B Wollgiehn, R. (1975) Biochem. Physiol. Pjlanzen. ( B P P ) 168, 167-174. 27. Apel, K . Jt Schweiger, H. G. (1973) Eur. J. Biochem. 38, 373383. 28. Kretzer, F., Ohad, I. Sr Bennoun, P. (1976) in Genetics and Biogenesis of Chloroplasts and Mitochondria (Bucher, Th. et al., eds) pp. 25 - 32, Elsevier North Holland Biomedical Press, Amsterdam. 29. Salvador, G., Lefort-Tran, M., Nigon, V. Sr Jourdan, F. (1971) Exp. Cell Res. 64, 457-462.
G. Verdier, Departement de Biologie GenCrale et Appliquee, Universite Claude-Bernard, 43 Boulevard du 11-Novembre 1918, F-69621 Villeurbanne, France
Poly(adeny1ic acid)-Containing RNA of EugZena gracilis during Chloroplast Development 2. Transcriptional Origin of the Different RNA Gerard VERDIER Departcmcnt de Biologie Genkrale et Appliquee (Laboratoire Associe au Centre National de la Recherche Scientifique), Universirt. Lyon I, Villeurbanne (Received February 3/0ctober 30, 1978)
The transcriptional origin of the polyadenylated RNA has been studied in dark-grown cultures of Euglena gracilis and during light-induced chloroplast development. Complementary DNA (cDNA) to poly(A)-containing RNA from different preparations (isolated chloroplasts and total cell polyribosomes from the wild-type Z strain or from the aplastidic mutant W3BUL) were synthesized and used for hybridization experiments. a) In Euglena cells illuminated for 0, 1 or 24 h, 7- 10 % of the total cell poly(A)-containing RNA are transcribed from chloroplast DNA. However, when RNA is extracted from isolated chloroplasts, this proportion of poly(A)-containing RNA is about two times higher (20%); in this case, the poly(A)-containing RNA could belong to a specific class and be transcribed from 18-20% of the double-strand chloroplast DNA. b) In the dark-grown cultures or in the greening cultures, the major part of the total cell poly(A)-containing RNA is transcribed from the non-repetitive sequences of nuclear DNA. The isolated chloroplast preparations contain also a high proportion of poly(A)-containing RNA from nuclear origin. c) About 10% of the total cell poly(A)-containing RNA of the wild-type strain, absent in the poly(A)-containing RNA of the aplastidic mutant, appear hybridizable to the nuclear genome of this mutant. Thus the transcription of some of the RNA encoded by nuclear DNA could be controlled by the presence of the chloroplast. Moreover, the fact that isolated chloroplast preparations appear to be enriched in such poly(A)-containing RNA from nuclear origin, suggests that these RNA are located at the level of the chloroplast. Three genomes are present in the Euglena gracilis. The nuclear DNA represents most of the total cell DNA and is composed of repetitive and unique sequences [l]. The chloroplast DNA corresponds to about 6 2 of the total cell DNA with a major component as a circular duplex molecule of molecular weight 92 x lo6 [ 2 ] . Finally the mitochondria1 DNA has a kinetic complexity 2-4 times lower than that of the chloroplast DNA [3,4]. The chloroplast morphogenesis stimulated by the light is developped concurrently with protein synthesis occurring both in the cytoplasm and in the chloroplast [5 - 71. The genomic information is transcribed as messenger RNA (mRNA), some of which contains polyadenylic acid poly(A) sequences [8 - 101 and corresponds to different frequency classes; changes
in the polyadenylated RNA distribution have been studied during the light-induced chloroplast development (preceding paper). In this report we attempt to measure the fractions of the poly(A)-containing RNA populations transcribed either by the nuclear DNA or by the chloroplast DNA. For this analysis, DNA (cDNA) complementary to different poly(A)-containing RNA preparations was synthesized and used in hybridization experiments with the nuclear DNA or with the chloroplast DNA. In order to determine the fraction of the chloroplast genome represented as RNA transcripts, the chloroplast DNA was hybridized to a large excess of poly(A)-containing RNA. Finally we have analyzed the meaning of poly(A)-containing RNA from nuclear origin present in the isolated chloroplast preparations.
582
MATERIALS AND METHODS Strains and Cultures Euglenia gracilis wild-type Z strain was grown in darkness on a phosphate/carbon-limited medium [ll], then transferred to a 0.03 M KCl solution at the beginning of the stationary phase ; illumination was begun three days later, as described previously (preceding paper). The aplastidic mutant W3BUL was grown in the light on the same medium and the cultures were used in a late logarithmic phase. Preparation and Fractionation of Polysomal R N A and Chloroplast R N A ; Transcription of Polyadenylated R N A
Polysomal RNA were extracted by the phenol/ chloroform technique from total cellular polyribosomes as described previously [lo]. Chloroplasts of 24-h-illuminated cultures were purified by differential centrifugation, which was preferred to flotation procedure as discussed previously (preceding paper) ; these chloroplast preparations were used to extract total chloroplast RNA by the phenol/chloroform method. Poly(A)-containing RNA from either total polysomal RNA or chloroplast RNA were isolated by chromatography on oligo(dT)-cellulose as described previously (preceding paper). DNA complementary to poly(A)-containing RNA was synthesized using RNA-dependent DNA polymerase prepared from avian myeloblastosis virus (supplied by Dr R. Williamson, St Mary's Hospital, London) as described in the preceding paper. Preparations of Chloroplast and Nuclear DNA, and Radioiodination of Chloroplast Preparations
Chloroplast DNA was extracted from isolated chloroplasts by the phenol/chloroform method [ 101 and separated on an alkaline CsCl gradient into light and heavy components. In hybridization experiments the light component (e = 1.686 g/cm3) was used, since it contains sequences complementary to the majority of the chloroplast DNA transcripts [12], the heavy component (e = 1.701 g/cm3) corresponding mainly to sequences complementary to chloroplast ribosomal RNA [12,13]. The light component of DNA was sonicated to an average size of 500-600 bases and was treated with 0.3 M NaOH in 37 "C for 16 h to hydrolyze any contaminating RNA. After neutralization and precipitation with ethanol, the DNA was desalted by passage through Sephadex G-50. The nuclear DNA was prepared from the aplastidic W3BUL mutant by the same procedure. The light component of chloroplast DNA was labelled in vitro with ['Z51]iodine according to the Orosz and Wetmur procedure [14]. The 1251-labelled
Euglena Polyadenylated RNA Transcription
chloroplast DNA had a specific activity of 4 x lo6 counts min-l pg-'. After labelling, the single-strand I-labelled DNA had an apparent average molecular weight of 100000. Samples and Abbreviations Used for Annealing Experiments
Poly(A)-containing RNA isolated from total polysomal RNA of wild-type Z strain in the dark [ZoTpoly(A)-containing RNA] or after illumination for 1 h [ZIT-poly(A)-containing RNA] and 24 h [Z24Tpoly(A)-containing RNA] was used to synthesize corresponding complementary DNA : ZoT-cDNA, ZIT-CDNA, Z24T-cDNA. Poly(A)-containing RNA obtained from total RNA of chloroplasts isolated after 24 h of illumination [Z~ch-poly(A)-containingRNA] was used to synthesize complementary DNA (ZZ4Ch-cDNA). W3-poly(A)-containing RNA corresponded to poly(A)-containing RNA from total polysomal preparations of W3BUL mutant. W3-cDNA was the corresponding complementary DNA. Unlabelled nuclear DNA was obtained from the W3BUL mutant (W3-DNA), and Chl-DNA corresponded to unlabelled light component of chloroplast DNA. 'z51-labelledChl-DNA: light component of chloroplast DNA labelled by ['251]iodine. Hybridization Kinetics
Three types of annealing techniques have been used: hybridization of cDNA with an excess of unlabelled poly(A)-containing RNA; hybridization of cDNA with an excess of unlabelled DNA (Chl-DNA or W3-DNA) ;hybridization of '251-labelledChl-DNA with an excess of unlabelled poly(A)-containing RNA. Samples of cDNA (0.2 ng) or 'z51-labelled ChlDNA (0.5 ng) were mixed with appropriate amounts of unlabelled poly(A)-containing RNA (from 50 ng to 8 pg) or DNA (200 ng of Chl-DNA or 1-8 pg of W3-DNA), lyophilized and dissolved into 2 1.11 of 0.24 M sodium phosphate buffer (pH 6 4 , 1 mM EDTA. Each sample was sealed in a silicon-coated glass capillary. The cDNA/poly(A)-containing RNA samples were incubated at 68 "C. However, before incubation, capillaries with cDNA/DNA or lZ5Ilabelled Chl-DNA/poly(A)-containing RNA mixtures were boiled for 5 min and immediately transferred to a 68 "C bath. At the end of each incubation period required to reach the desired value of rot [initial RNA concentration (mol nucleotides/l) x time (s)] or of cot [initial DNA concentration (mol nucleotides/l) x time (s)l, the amount of cDNA or 1251-labelled Chl-DNA in hybrids was assayed using S1 nuclease as described previously (preceding paper).
G. Verdier
583
RESULTS Annealing o j cDNA with Poly(A)-Containing RNA
To determine the degree of sequence homology between the RNA of the wild-type Z strain and the RNA of the aplastidic mutant W3BUL, we examined the hybridization of the cDNA of one preparation with the poly(A)-containing RNA of the heterologous preparation. For this investigation, Z24T-cDNA corresponding to the most complex poly(A)-containing RNA population that we have described previously (preceding paper) was used, whereas the Z24Ch-cDNA was tested to detect the sequences from nuclear origin in the poly(A)-containing RNA extracted from isolated chloroplast. The results are shown in Fig. 1 ; to facilitate comparison we have included in the figure the corresponding homologous reactions. The reaction of Z24T-cDNA to an excess of W3poly(A)-containing RNA reached a plateau of 49 compared with 79 % hybridization observed in the homologous Z24T-cDNA/Z24T-poly(A)-containing RNA annealing. Therefore, we conclude that 49/79 or 62% of the hybridizable Z24T-cDNA is complementary to W3-poly(A)-containing RNA. These common sequences seem to belong to the second and
x,
60
._ 50 40
' 3 0
1
20 10
third classes of the homologous Z ~ ~ T - C D N A / Z U T poly(A)-containing RNA hybridization : no hybridization corresponded to classes 1 and 4. The annealing of Z24Ch-cDNA with Ws-poly(A)containing RNA demonstrated that 50 % of the cDNA was hybridized for the highest rot values; no hybridization was observed in the range of the vot values of the first and fourth classes of the homologous Z~4Ch-cDNA/Z~~Ch-poly(A)-containing RNA reaction. Annealing of the Z ~ ~ T - C D Nwith A the WJ-DNA
To estimate, after 24 h of illumination, the proportions of poly(A)-containing RNA which was transcribed from the nuclear genome, the DNA from the aplastidic mutant W3BUL was used as nuclear DNA (Fig. 2). Total cell DNA, which corresponds for the major part to the nuclear DNA, is composed 18 :G by highly repetitive sequences, 40% by middle repetitive sequences and 36 % by non-repetitive sequences : this last fraction was reassociated in the range of cot values from 100-10000 mol 1-' s with cotliz = 2000mol 1-' s [l]. In order to determine which frequency classes of DNA sequences were represented in the cDNA population, we have hybridized the cDNA to a vast excess of DNA [15]. Our results show that at a cot value of 7600 mol I-' s at least 72% of the Z24T-cDNA was annealed with the W3-DNA (Fig. 2). About two log units of cot (from 100- 10000 moll-' s) were covered by the kinetic reaction : this result indicates that only one class of the WYDNA, corresponding to non-repetitive DNA sequences (cotl12 of the reaction = 2300 mol I s), was represented in the Z24T-cDNA.
n
Annealing of cDNA with Chl-DNA
2
1
4
3
90
00
The kinetic complexity of the Euglena chloroplast DNA is 92 x lo6 [2] or approximately 130 x lo3 base
-
70 -
60 -
*O 70
50-
10-2
10'
100
10'
102
103
4.103
r, t (rnol I-'s)
Fig. 1. Heterologous hybridizations of an excess of W3-poly(A)containing RNA with ( A ) ZZ~T-CDNA ( x - - x j~ or ( ~ B ) ZzaChcDNA ( x ~x ). See Materials and Methods, and abbreviation Homologous reaction of ZZ~T-CDNA and paragraph. (0-0) Z24T-poly(A)-containing RNA (A) or Zz4Ch-cDNA and ZzKhpoly(A)-containing RNA (B) (see preceding paper). The results are expressed as semi-logarithmic plots of the percentage of hybridized cDNA against rot values (moll-' s). 1 , 2, 3, 4: levels of the diffirent transitions of homologous hybridizations
i2"
1
"11
L
20 lo
0 1
10'
. .
* I
102
103
104
c a t (rnol I-' s )
Fig.2. Hybridization kinetics of Z Z ~ T - c D N Awith an excess of W3DNA (see Materials and Methodsj. The results are expressed as semi-logarithmic plots of the percentage of hybridized cDNA against cot values (moll-' s )
584
Euglena Polyadenylated RNA Transcription
-
.1" n N
ar ._ n
'= D
10
L a
8
5 -
4
z
0
6
Y
s
I
0 1 10-2
,
!
100
10'
I
lo-'
c o t (mot
2
0 lo-'
100
10'
I02
r o t (rnol K ' S )
IK'S)
Fig. 3. Hyhridization kinetics of'ZoT-cDNA ( x . . . . x ), Z , T-cDNA IS---.), Z Z ~ T - C D N (0A ---O) or Zz4Ch-cDNA ( b e ) with an excess of Chl-DNA (see Materials and Methods). Results are expressed as described in Fig. 2
Fig. 4. Hybridization kinetic of' '251-lahelled Cld-DNA with Z24Chpofy(A)-containing R N A . The resuits, expressed as semi-logarithmic plots of the percentage of hybridized 'ZsI-labelled Chl-DNA against rot values, were corrected for D N A . DNA renaturation in the absence of RNA
pairs. Recent studies have shown that the 16-S and 23-S rRNA genes are each present on a 5.6 x lo3base-pair repeated DNA segment [16,17]; this rRNA region is present 3 times as a tandem repeat on the genome [16,17]. These results are consistent with previous reports [13,18]. This rRNA region corresponds to only 17000 base pairs or 13 % of the chloroplast DNA while the rest of chloroplast DNA must correspond to unique sequences as shown by physical mapping [16]. More than 80% of the chloroplast DNA renature at cot values under 10 [12,19,20]. The ZoT-cDNA, ZIT-cDNA, Z24T-cDNA and Z24Ch-cDNA were hybridized to an excess of ChlDNA (e = 1.686 g/cm3, see Materials and Methods) (Fig. 3). From 7- 10 % of the various cDNA species synthesized from total cell poly(A)-containing RNA were annealed with the Chl-DNA ; however, about 20% of the Z24Ch-DNA was found to hybridize to Chl-DNA. Under the same conditions the proportions of the W3-cDNA which hybridize to the Chl-DNA corresponded to the background values of the reactions (1.5-2.5%). These results show that specific RNA sequences were transcribed from the chloroplast DNA and that these sequences correspond, in all the poly(A)-containing RNA isolated from different stages of chloroplast development, to a low proportions of these RNA populations.
containing-RNA . DNA and DNA . DNA duplexes. The fraction of poly(A)-containing RNA . DNA hybrids may be estimated by the difference between the duplex DNA appearing in the presence of an RNA excess and that attributable to DNA . DNA renaturation in the absence of poly(A)-containing RNA. The extent of DNA . DNA reassociation in these reactions was calculated by renaturing '251-labelled Chl-DNA in the absence of RNA at concentrations and time periods comparable to those used for the poly(A)containing RNA/DNA hybridization : the fraction of '251-labelled Chl-DNA renatured as a function of cot represented a maximum of 8.2%. In the poly(A)containing RNA/DNA hybridization, after correction for DNA . DNA renaturation, approximately 9 - 10 % of single-strand chloroplast DNA was saturated by Z24Ch-poly(A)-containing RNA. If we assume that only one strand of the DNA molecule is transcribed into RNA, 18-20% of the genomic information is present as polyadenylated RNA transcripts at this stage of the chloroplast development.
Hybridization ~ f ~ ~ ~ I - L a b eChl-DNA lled with Z24Ch-Poly( A )-Containing R N A In order to measure the amount of the chloroplast genome which is expressed as RNA transcripts, '251-labelled Chl-DNA was hybridized to an excess of polyadenylated RNA from isolated chloroplast preparation (Fig. 4). The fraction of '251-labelled Chl-DNA after S1 assay represents both poly(A)-
DISCUSSION Expression of the Chloroplast Genome of Euglena gracilis
We have found that the poly(A)-containing RNA extracted from chloroplast preparations were transcribed from 18-20% of the chloroplast genome, representing thus a minimal estimate of the chloroplast DNA transcription program. However, when chloroplasts were isolated from different stages of the plastid morphogenesis, one chloroplast RNA preparation might contain RNA sequences absent from another chloroplast preparation. Moreover, the messenger RNA of the large subunit of ribulose-l,5-bisphosphate
G. Verdier
carboxylase does not contain poly(A) tracts [21- 231 and we have also demonstrated that light induces synthesis of polysomal RNA, probably of messenger function, and devoid of poly(A) sequences [lo]. Our data can be compared with earlier reports. According to Chelm and Hallick [20], 32% of the chloroplast genome is complementary to nucleotide sequences in total cell RNA of Euglena gracilis illuminated for 24 h ; subtracting from this value the fractions of chloroplast DNA complementary to rRNA @ - l o%) [12] and to tRNA (0.7%) [24,25], it appears that the transcription for messenger RNA might represent 21 -23 % of the chloroplast genome. This value would be in good agreement with our findings. However, according to Rawson and Boerma [12, 191, the extent of the chloroplast genome transcription corresponds to 43 % for total cell RNA free of rRNA and tRNA. This value is higher than our results and those of Chelm and Hallick [20]. The difference between these two types of results could be explained by different culture conditions or by the interference with the polyadenylated RNA population, in Rawson and Boerma’s experiments, [12,19] of a significant fraction of mRNA devoid of poly(A) sequences. If such is the case, we must assume that at least a part of poly(A)-containing RNA could represent different RNA species absent in non-poly(A)-containing RNA. We have suggested previously a similar possibility [lo]. It is clear that a conclusive evaluation will only be obtained by the study of the RNA devoid of poly(A) sequences. The Transcriptional Origin the Poly(A)-Containing R N A Extracted from Isolated Chloroplasts of
The proportions of RNA sequences transcribed from chloroplast DNA were two times higher in the Z24-Ch-cDNA than in the other cDNA synthesized from total cell poly(A)-containing RNA isolated at different stages of chloroplast development (Fig. 3). However, these RNA sequences from chloroplast genome origin represented only 20% of the ZuChcDNA. Therefore, about 80 % of the polyadenylated RNA population extracted from isolated chloroplasts is probably transcribed from the nuclear genome. This result is not surprising, since the complexity of the Z24Ch-poly(A)-containing RNA had been found higher than the kinetic complexity of the chloroplast DNA (preceding paper). Moreover, about 50 % of the Z24Ch-poly(A)-containing RNA sequences were also present in the poly(A)-containing RNA population of the W3BUL mutant; the other half of the Zz4Chpoly(A)-containing RNA corresponded to abundant sequences, which were absent from WJ-poly(A)containing RNA population. Thus, it appears that
585
30 ”/, of the Z24Ch-poly(A)-containing RNA would correspond to abundant sequences, transcribed from nuclear genome but absent from W3-poly(A)-containing RNA sequences. Before separation of poly(A)-containing RNA, our preparations of chloroplast RNA were found to be contaminated by at most 10% cytoplasmic ribosomal RNA (preceding paper). To explain the presence of 80 % of poly(A)-containing RNA sequences transcribed from the nucleus in the poly(A)-containing RNA from chloroplast preparations, we may suppose that the contamination of these preparations by cytoplasmic poly(A)-containing RNA was higher than the contamination at rRNA level. If such is the case, the Z2&h-poly(A)-containing RNA population would be enriched in a fraction of abundant sequences, originating from the cytoplasmic RNA contaminants and representing up to 30 % of the total Z24Ch-poly(A)containing RNA. Alternatively another possibility could be that a high proportion of RNA from nuclear origin is bound to chloroplast envelopes in vivo; such results have been described in Nicotiana [26], in which the ribosomes bound to the outside of chloroplast membrane are thought to be responsible for the synthesis of a particular kind of chloroplast protein [27]. A third alternative is a possible migration of nuclearencoded RNA within the chloroplast structures; a similar situation has been described previously in Chlamydomonas reinhardi for a peptide of the chloroplast membrane which would be synthesized into the chloroplast from nuclear transcripts [28]. Recently, mRNA involved in spinach chloroplast synthesis in vitro has been shown to be coded partly by chloroplast DNA and partly by nuclear DNA (Guignery and Duranton, personal communication). The Transcriptional Origin ofthe Total Cell Poly(A)-Containing R N A during Greening Transcription from the Chloroplast Genome. A fraction of the poly(A)-containing RNA population was transcribed from chloroplast genome in the darkgrown cultures as well as in the cultures illuminated for 1 or 24 h. Although the cDNA obtained from these poly(A)-containing RNA preparations hybridized to Chl-DNA in almost equivalent proportions (7 - 10 %), these values do not prove that the corresponding sequences are identical. On the other hand, our results do not allow us to determine accurately whether the cDNA hybridized to Chl-DNA belongs to the same frequency classes or to different frequency classes. Moreover, Rawson and Boerma [19] have reported that at later stages of chloroplast development (25 - 50 h in the light) total cell RNA contains transcripts from 5 % of chloroplast DNA not previously transcribed.
586
Transcriptionfrom the Nucleur Genome. While the chloroplast morphogenesis is going on after 24 h of illumination [29], the major part of the total cell poly(A)-containing RNA appears to be from nuclear origin. At least, 72% of the Z24T-cDNA was hybridized to unique sequences of the W3-DNA. Moreover, 62 % of the ZZ4T-cDNAcorresponded to overlapping sequences with the W3-poly(A)-containing RNA. Therefore, a minimum of 10 % of poly(A)-containing RNA would represent specific sequences in the wildtype strain after illumination. Although these specific sequences belong to the nuclear DNA of the W3BUL mutant, they do not seem transcribed in this strain. Similarly, we have described previously that 11 % of the ZZ4T-poly(A)-containing RNA corresponded to a special class of abundant RNA sequences, absent from the dark-grown cultures (preceding paper). A possible conclusion could be that the presence of the chloroplast in the cell induces the activity of a specific fraction of the nuclear genome; this fraction would be made of RNA transcripts corresponding to, at least, 10% of the total cell poly(A)-containing RNA and 30 % of the poly(A)-containing RNA extracted from isolated chloroplasts. Therefore, we might conclude that the isolated chloroplast preparations are enriched in these specific transcripts. This would be a strong .argument to prove that these transcripts are at the level of the chloroplast and that they do not represent only a contamination of the chloroplast preparations by the RNA from cytoplasmic location. I am grateful to Professor V. Nigon for his helpful criticism. I thank Drs Heizmann, Crouse and Stutz for their generous gifts of E. gracilis chloroplast DNA. I thank also Mrs Schwob for her technical assistance and Dr Godet for critically reading the manuscript. I wish to thank also Mrs B. Szafranek for excellent secretarial work.
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G . Verdier : Euglena Polyadenylated RNA Transcription 3. Edelman, M., Epstein, H. T. Sr Schiff, J. A. (1966) J . Mot. Biol. 17, 463 - 469. 4. Crouse, E. J., Vandrey, J. Sr Stutz, E. (3974) FEBS Lett. 42, 262 - 266. 5. Smillie, R. M., Evans, W. R. Sr Lyman, H. (1963) Brookhaven Symp. Biol. 16, 89-108. 6. Schiff, J. A. (1974) Proc. 3rd Int. Congr. Photosyntlzesis (Avron, M., ed.) pp. 1691 - 1717, Elsevier, Amsterdam. 7. Schwartzbach, S . D., Schiff, J. A. d Klein, S. (1976) Planfa, 131, 1-9. 8. Verdier, G., Trabuchet, G., Heizmann, P. Sr Nigon, V. (1973) Biochim. Biophys. Acra, 312, 528 - 539. 9. Sagher, D., Edelman, M. Sr Jakob, K. M. (1974) Biochim. Biophys. Acta, 349, 32-38. 10. Verdier, G . (1975) Biochim. Biophys. Acta, 407,91-98. 11. Freyssinet, G., Heizmann, P., Verdier, G., Trabuchet, G. Sr Nigon, V. (1 972) Physiol. Yeg. 10, 421 -442. 12. Rawson, J. R. Y. Sr Boerma, C. L. (1977) Biochem. Biophys. Res. Commun. 74, 932-918. 13. Kopecka, H., Crouse, E. J. Sr Stutz, E. (1977) Ew. J. Biochem. 72,525 -535. 14. Orosz, J. M. B Wetmur, J. B. (1974) Biochemistry, 13, 54675473. 15. Hastie, N. D. Sr Bishop, J. 0. (1976) Cell, 9, 761 -774. 16. Gray, P. W. d Hallick, R. B. (1978) Biochemistry, 17, 284289. 17. Jenni, B. Jt Stutz, E. (1978) Eur. J . Biochem. 88, 127-134. 18. Stutz, E. & Vandrey, J. P. (1971) FEBS Lett. 17,277-280. 19. Rawson, J. R. Y. d Boerma, C. L. (1976) Biochemistry, 15, 588 - 592. 20. Chelm, B. K. Sr Hallick, R. B. (1976) Biochemistry, 15, 593599. 21. Wheeler, A. M. Sr Hartley, M. R. (1975) Nature (Lond.) 257, 66 - 67. 22. Sagher, D., Grosfeld, H. Sr Edelman, M. (1976) Proc. Nut2 Acad. Sci. U.S.A. 73, 722 - 726. 23. Howell, S. H., Heizmann, P., Gelvin, S. B Walker, L. L. (1977) Plant Physiol. 59, 464-470. 24. Schwartzbach, S. D., Hecker, L. L. d Barnett, W. E. (1976) Proc. Nail Acad. Sci. U.S.A. 73. 1984-1988. 25. Gruol, D. J. B Haselkorn, R. (1976) Biochim. Biophys. Acta, 447,82 - 95. 26. Lerbs, S. B Wollgiehn, R. (1975) Biochem. Physiol. Pjlanzen. ( B P P ) 168, 167-174. 27. Apel, K . Jt Schweiger, H. G. (1973) Eur. J. Biochem. 38, 373383. 28. Kretzer, F., Ohad, I. Sr Bennoun, P. (1976) in Genetics and Biogenesis of Chloroplasts and Mitochondria (Bucher, Th. et al., eds) pp. 25 - 32, Elsevier North Holland Biomedical Press, Amsterdam. 29. Salvador, G., Lefort-Tran, M., Nigon, V. Sr Jourdan, F. (1971) Exp. Cell Res. 64, 457-462.
G. Verdier, Departement de Biologie GenCrale et Appliquee, Universite Claude-Bernard, 43 Boulevard du 11-Novembre 1918, F-69621 Villeurbanne, France
