Abstract
DNA has been isolated from barley chloroplasts and analyzed with restriction endonucleases PstI, SalI, PvuII and HindIII. Fragments obtained by digestion with PstI and HindIII have been inserted in the bacterial transformation vectors pBR325 and pBR322 and amplified in Escherichia coli. The inserts of the recombinant plasmids have been mapped with the same restriction enzymes as the intact chloroplast DNA yielding a physical map for the barley chloroplast genome. It is a circular molecule about 133,000 basepairs in size, which is equivalent to the chloroplast genomes of other gramineae such as wheat and maize. Like these it has an inverted repeat, of about 20,900 basepairs, containing the genes for the ribosomal RNAs. There are many similarities of the barley, wheat and maize genomes with respect to recognition sites for the enzymes PstI, SalI and PvuII. Heterologous hybridization with a probe containing parts of the genes for the large subunit of ribulose bisphosphate carboxylase and for the dicistronic mRNA for two ATPsynthetase CF; subunits, reveal that the position and organization of these genes in the barley chloroplast DNA are the same as found for maize and wheat.
Selected chloroplast DNA fragments isolated from the recombinant plasmids and covering about 80% of the genome have been used for hybridization to RNA purified from plastids of dark grown and 8 hours illuminated seedlings. The RNA was electrophoretically separated in denaturing agarose gels and subsequently Northern blotted onto DBM-paper. The DNA fragments were nick translated and hybridized to the filterbound RNA. A total of 70 transcripts ranging in size from 350 nucleotides to more than 6,000 were identified. Of these 11 were synthesized only after illumination of the seedlings. The transcripts encoding the large subunit of ribulose bisphosphate carboxylase and the ATP synthetase CF1 subunits β and ε have been tentatively identified. About 30 transcripts are large than 3,000 nucleotides, indicating either the existence of a large number of polycistronic mRNAs or various forms of precursor RNAs.
Article PDF
Similar content being viewed by others
Abbreviations
- bp:
-
basepairs
- DBM:
-
diazobenzyloxymethyl-
- DCCD:
-
dicyclohexylcarbodiimide
- DCMU:
-
dichlorophenyl-dimethyl-urea
- kb:
-
kilobases
- kbp:
-
kilobasepairs
- LS:
-
large subunit
- NBM:
-
nitrobenzyloxymethyl-
- NBPC:
-
nitrobenzyloxy-methylpyridinium chloride
- pHvC:
-
recombinant plasmids carrying Hordeum vulgare chloroplast DNA fragments
- RuBPCase:
-
ribulose bisphosphate carboxylase
- Genes: rbcL:
-
the gene for RuBPCase LS
- atpA:
-
the gene for ATP synthetase CF1 subunit ε
- atpH:
-
the gene for ATP synthetase CF0 subunit III
- psbA:
-
gene for a 32,000 dalton photosystem II protein
- rrs, rrl and rrf:
-
16S, 23S and 5S rRNA genes
- trn:
-
tRNA genes
- pet:
-
genes for photosynthetic electron transport proteins, cytochrome f
References
Alwine, J.C., D.J. Kemp, B.A. Parker, J. Reiser, J. Renart, G.R. Stark &G.M. Wahl: Detection of specific RNAs or specific fragments of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper. In: Methods in Enzymology 68, R. Wu, ed., pp 220–242 (1979) Academic Press New York and London.
Bedbrook, J.R. &L. Bogorad: Endonuclease recognition sites mapped on Zea mays chloroplast DNA. Proc. Natl. Acad. Sci. USA 73, 4309–4313 (1976)
Bedbrook, J., G. Link, D.M. Coen, L. Bogorad &A. Rich: Maize plastid gene expressed during photoregulated development. Proc. Natl. Acad. Sci. USA 75, 3060–3064 (1978)
Bibb, M.J., R.A. Van Etten, C.T. Wright, M.A. Walberg &D.J. Clayton: Sequence and gene organization of mouse mitochondrial DNA. Cell 26, 167–180 (1982)
Birnboim, H.C. &J. Doly: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Research 7, 1513–1520 (1979)
Bohnert, H.J., E.J. Crouse &J.M. Schmitt: Organization and expression of plastid genomes. In: Encycl. Plant Physiol. vol 14B, pp. 475–530, B. Parthier and D. Boulter eds., (1982) Springer Verlag, Berlin, Heidelberg
Bolivar, F.: Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique EcoRI sites for selection of EcoRI generated recombinant DNA molecules. Gene 4, 121–136 (1978)
Bottomley, W. &P.R. Whitfeld: Cell-free transcription and translation of total spinach chloroplast DNA. Eur. J. Biochem. 93, 31–39 (1979)
Bowman, C.M., B. Koller, H. Delius &T.A. Dyer: A physical map of wheat chloroplast DNA showing the location of the structural genes for the ribosomal RNAs and the large subunit of ribulose 1,5-bisphosphate carboxylase. Mol. gen. Genet. 183, 93–102 (1981)
Broach, J.R., J.F. Atkins, C. McGill &L. Chow: Identification and mapping of the transcriptional products of the yeast plasmid, 2 μ circle. Cell 16, 827–839 (1979)
Chu, N.M. &K.K. Tewari: Arrangement of the ribosomal RNA genes in chloroplast DNA of Leguminosae. Mol. gen. Genet. 186, 23–32 (1982)
Chu, N.M., K.K. Oishi &K.K. Tewari: Physical mapping of the pea chloroplast DNA and localization of the ribosomal RNA genes. Plasmid 6, 279–292 (1981)
Cohen, S.N., A.C.Y. Chang &L. Hsu: Non-chromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA 69, 2110–2114 (1972)
Driesel, A.J., J. Speirs &H.J. Bohnert: Spinach chloroplast mRNA for a 32,000 dalton polypeptide. Size and localization on the physical map of the chloroplast DNA. Biochim. Biophys. Acta. 610, 297–310 (1980)
Driesel, A.J., E.J. Crouse, K. Gordon, H.J. Bohneri, R.G. Herrmann, A. Steinmetz, M. Mubumbila, M. Keller, G. Burkard &J.H. Weil: Fractionation and identification of spinach chloroplast transfer RNAs and mapping of their genes on the restriction map of chloroplast DNA. Gene 6, 285–306 (1979)
Denhardt, D.T.: A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641–646 (1966)
Gordon, K.H.J., E.J. Crouse, H.J. Bohnert &R.G. Herrmann: Restriction endonuclease cleavage site map of chloroplast DNA from Oenothera parviflora (Euoenothera Plastome IV). Theor. Appl. Genet. 59, 281–296 (1981)
Gruissem, W., D.M. Prescott, B.M. Greenberg &R.B. Hallick: Transcription of E. coli and Euglena chloroplast tRNA gene clusters and processing of polycistronic transcripts in a Hela cell-free system. Cell 30, 81–92 (1982)
Guidelines for research involing DNA molecules Federal Register U.S. 47 (77) 17180–17198 (1982)
Guilley, H., R.K. Dudley, G. Jonard, E. Balazs &K.E. Richards: Transcription of cauliflower mosaic virus DNA: Detection of promotor sequences and characterization of transcripts. Cell 30: 763–773 (1982)
Howe, C.J., A.D. Auffret, A. Doherty, C.M. Bowman, T.A. Dyer &J.C. Gray: Location and nucleotide sequence of the gene for proton translocating subunit of wheat chloroplast ATP synthase. Proc. Natl. Acad. Sci. USA 79, 6903–6908 (1982)
Howe, C.J., C.M., Bowman, T.A. Dyer &J.C. Gray: Localization of wheat chloroplast genes for the beta and epsilon subunits of ATP synthase. Mol. gen. Genet. 186, 525–530 (1982)
Kannangara, C.G., S.P. Gough, B. Hansen, J.N. Rasmussen &D.J. Simpson: A homogenizer with replaceable razor blades for bulk isolation of active barley plastids. Carlsberg Res. Commun. 42, 431–440 (1977)
Koller, B., H. Delius &T.A. Dyer: The organization of the chloroplast DNA in wheat and maize in the region containing the LS gene. Eur. J. Biochem. 122, 17–23 (1982)
Kolodner, R. &K.K. Tewari: The molecular size and conformation of the chloroplast DNA from higher plants. Biochim. Biophys. Acta 402, 372–390 (1975)
Kolodner, R. &K.K. Tewari: Inverted repeats in chloroplast DNA from higher plants. Proc. Natl. Acad. Sci. USA 76, 41–45 (1978)
Krebbers, E.T., I.M. Larrinua, L. McIntosh &L. Bogorad: The maize chloroplast genes for the β and ε subunits of the photosynthetic coupling factor CF1 are fused. Nucl. Acids Res. 10, 4085–5002 (1982)
Lehrach, H., D. Diamond, J.M. Wozney &H. Boedtker: RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16, 4743–4752 (1977)
Link, G.: Phytochrome control of plastid mRNA in mustard (Sinapis alba L.). Planta 154, 81–86 (1982)
Malnoe, P., J.-D. Rochaix, N.H. Chua &P.-F. Spahr: Characterization of the gene and messenger RNA of the large subunit of ribulose 1,5-bisphosphate carboxylase in Chlamydomonas reinhardii. J. Mol. Biol. 133, 417–434 (1979)
Maniatis, T., E.F. Fritsch & J. Sambrook: Electrophoresis of RNA through gels containing formaldehyde. In: Molecular cloning. A laboratory manual. pp 202–203, Cold Spring Harbor Laboratory 1982.
McIntosh, L., C. Poulsen &L. Bogorad: Chloroplast gene sequence for the large subunit of ribulose bisophosphate carboxylase of maize. Nature 288, 556–560 (1980)
Oishi, K., T. Sumnicht &K.K. Tewari: Messenger ribonucleic acid transcript of pea chloroplast deoxyribonucleic acid. Biochemistry 20, 5710–5717 (1981)
Palmer, J.D. &W.F. Thompson: Clone banks of the mung bean, pea and spinach chloroplast genomes. Gene 15, 21–26 (1981)
Palmer, J.D. &W.F. Thompson: Chloroplast DNA rearrangements are more frequent when a large inverted repeat sequence is lost. Cell 29, 537–550 (1982)
Poulsen, C.: The in vivo transcriptional activity of the barley chloroplast genome. In: Structure and Function of Plant Genomes. O. Ciferri and L. Dure III, eds., Pleum Press, New York (1982), in press.
Reisfeld, A., A.K. Mattoo &M. Edelman: Processing of a chloroplast-translated membrane protein in vivo. Analysis of the rapidly synthesized 32000-dalton shield protein and its precursor in Spirodela oligorrhiza. Eur. J. Biochem. 124, 125–129 (1982)
Rigby, P.W.J., M. Dieckmann, C. Rhodes &P. Berg: Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 133, 237–251 (1977)
Schwarz, E., G. Scherer, G. Hobom &H. Kossel: Nucleotide sequence of cro, CII and part of the O gene in phage γ DNA. Nature 272, 410–414 (1978)
Schwarz, Z., S.O. Jolly, A. Steinmetz &L. Bogorad: Overlapping divergent genes in the maize chloroplast chromosome and in vitro transcription of the gene for tRNAHis. Proc. Natl. Acad. Sci. USA 78, 3423–3427 (1981)
Shinozaki, K. &M. Sugiura: Sequence of the intercistronic region between the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and the coupling factor β subunit gene. Nucl. Acids Res. 10, 4923–4934 (1982)
Shinozaki, K. &M. Sugiura: The nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose bisphosphate carboxylase-oxygenase. Gene 20, 91–102 (1982)
Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517 (1975)
Steinback, K.E., L. McIntosh, L. Bogorad &C. Arntzen: Identification of the triazine receptor protein as a chloroplast gene product. Proc. Natl. Acad. Sci. USA 78, 7463–7467 (1981)
Steinmetz, A., E.J. Gubbins &L. Bogorad: The anticodon of the maize chloroplast gene for tRNALeu is split by a large intron. Nucl. Acids Res. 10, 3027–3037 (1982)
Stiegler, G.L., H.M. Matthews, S.E. Bingham &R.B. Hallick: The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in Euglena gracilis chloroplast DNA: location, cloning and evidence for an intervening sequence. Nucl. Acids Res. 10, 3427–3444 (1982)
Takaiwa, F. &M. Sugiura: Nucleotide sequence of the 16S–23S spacer region in an rRNA gene cluster from tobacco chloroplast DNA. Nucl. Acids Res. 10, 2665–2676 (1982)
Takaiwa, F. &M. Sugiura: The complete nucleotide sequence of a 23-S rRNA gene from tobacco chloroplasts. Eur. J. Biochem. 124, 13–19 (1982)
Wahl, G.M., M. Stern &G.R. Stark: Efficient transfer of large DNA fragments to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc. Natl. Acad. Sci. USA 76, 3683–3687 (1979)
Westhoff, P., N. Nelson, H. Bunemann &R.G. Herrmann: Localization of genes for coupling factor subunits on the spinach plastid chromosome. Current Genetics 4, 109–120 (1981)
Wienand, U., Z. Schwarz &G. Feix: Electrophoretic elution of nucleic acids from gels adapted for subsequent biological tests. FEBS Letters 98, 319–323 (1979)
Zech, M., M.R. Hartley &H.J. Bohnert: Binding sites of E. coli DNA-dependent RNA polymerase on spinach chloroplast DNA. Current Genetics 4, 37–46 (1981)
Zurawski, G., W. Bottomley &P.R. Whitfeld: Structures of the genes for the β and ε subunits of spinach chloroplast ATPase indicate a dicistronic mRNA and an overlapping translation stop/start signal. Proc. Natl. Acad. Sci. USA 79, 6260–6264 (1982)
Zurawski, G., B. Perrot, W. Bottomley &P.R. Whitfeld: The structure of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase from spinach chloroplast DNA. Nucl. Acids Res. 9, 3251–3270 (1981)
Zurawski, G., H.J. Bohnert, P.R. Whitfeld &W. Bottomley: Nucleotide sequence of the gene for the 32,000-MR thylakoid membrane protein from Spinacia oleracea and Nicotiana debneyi predicts a totally conserved primary translation product of MR 38,800. Proc. Natl. Acad. Sci. USA 79, 7699–7703 (1982)
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Poulsen, C. The barley chloroplast genome: Physical structure and transcriptional activity in vivo. Carlsberg Res. Commun. 48, 57–80 (1983). https://doi.org/10.1007/BF02906170
Issue Date:
DOI: https://doi.org/10.1007/BF02906170