True reversion of a mutation in the chloroplast gene encoding the large subunit of ribulosebisphosphate carboxylase/oxygenase in <i>Chlamydomonas</i>

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True reversion of a mutation in the chloroplast gene encoding the large subunit of ribulosebisphosphate carboxylase/oxygenase in

Chlamydomonas

SPREITZER, Robert Joséph, RAHIRE, Michèle, ROCHAIX, Jean-David

Abstract

The ribulosebisphosphate carboxylase/oxygenase-defective Chlamydomonas mutant, 10-6C, was the first mutant to be physically defined in chloroplast DNA. In this report, a photosynthesis-competent revertant of the 10-6C mutant has been found to result from true reversion within the chloroplast large-subunit gene. This result supports the original assignment of the 10-6C mutation within the large-subunit gene.

SPREITZER, Robert Joséph, RAHIRE, Michèle, ROCHAIX, Jean-David. True reversion of a mutation in the chloroplast gene encoding the large subunit of ribulosebisphosphate

carboxylase/oxygenase in Chlamydomonas . Current Genetics , 1985, vol. 9, no. 3, p.

229-231

DOI : 10.1007/BF00420316

Available at:

http://archive-ouverte.unige.ch/unige:150769

Disclaimer: layout of this document may differ from the published version.

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Current Genetics (1985) 9:229-231 Current Genetics

© Springer-Verlag 1985

True reversion of a mutation in the chloroplast gene encoding the large subunit of ribulosebisphosphate carboxylase/oxygenase in Chlamydomonas

Robert J. Spreitzer*, Michble Rahire, and Jean-David Rochaix

Department of Molecular Biology, University of Geneva, 1211 Geneva 4, Switzerland

Abstract.

The ribulosebisphosphate carboxylase/oxygen- ase-defective

mutant, 10-6C, was the first mutant to be physically defined in chloroplast DNA.

In this report, a photosynthesis-competent revertant of the 10-6C mutant has been found to result from true reversion within the chloroplast large-subunit gene. This result supports the original assignment of the 10-6C mutation within the large-subunit gene.

Key words:

Chloroplast gene reversion - Chloroplast genetics - Ribulose-1,5-bis- phosphate carb oxylase/oxygenase

Introduction

Ribulose-l,5-bisphosphate (RuBP) carboxylase/oxygen- ase limits the rate of photosynthesis, and consists of a nuclear-encoded small subunit and chloroplast-encoded large subunit, each present in eight copies in the holo- enzyme (reviewed by Miziorko and Lorimer 1983).

Genetic manipulation of this enzyme may be useful for increasing the photosynthetic rate. Multiple mutations affecting RuBP carboxylase/oxygenase may provide new information on nuclear/chloroplast gene interaction and chloroplast genetics in general.

Spreitzer and Mets (1980) recovered the uniparental- ly-inherited 10-6C mutant

* Present address:

Department of Agricultural Biochemistry, University of Nebraska, Lincoln, Nebraska 68583-0718, USA

R. J. Spreitzer

that had a lower isoelectric point for the RuBP carboxyl- ase/oxygenase large subunit. The enzyme had greatly reduced activities, but did not differ from normal enzyme in the ability to bind a substrate analog (Spreitzer et al.

1982). Dron et al. (1983) cloned and sequenced the large-subunit gene from 10-6C and wild type, and showed that the 10-6C gene differed by a single G-C to A-T transi- tion. This change would introduce a negatively-charged aspartic acid in place of a neutral glycine near one of the active sites (Hartman et al. 1978) and account for the increased negative charge on the large-subunit protein.

Mets and Geist (1983) demonstrated linkage between the 10-6C mutant phenotype and other markers on the

uniparental linkage group, thereby defining the first point of correlation between the genetic (re- viewed by Gillham 1978) and physical (reviewed by Rochaix 1981) maps of the chloroplast genome.

Photosynthesis-competent revertants of 10-6C were selected and analyzed for altered RuBP carboxylase/oxy- genase catalytic properties (Spreitzer et al. 1982). While no revertant enzyme was found to be different from wild-type, the demonstration of the ability to genetically manipulate RuBP carboxylase/oxygenase in

encourages further attempts along these lines. However, the physical basis of reversion was not known.

In the present investigation, we have determined the molecular basis of the reversion of 10-6C by studying one of the revertants. This is important for two reasons.

First, we must physically define the genetic variation

that we are attempting to exploit for the modification of

RuBP carboxylase/oxygenase. Secondly, the 10-6C mu-

tant provided the first point of correlation between the

genetic and physical maps of the chloroplast genome

(Dron et al. 1983; Mets and Geist 1983). The molecular

basis of reversion would substantiate the assignment of

the original mutation within the large-subunit gene.

230 R.J. Spreitzer et al. : True reversion of a chloroplast mutation extracted (Rochaix 1980). EcoRI-digested R4-7 and lambda 1149 DNA was ligated and packaged (Becket and Gold 1975).

Recombinant-phage plaques appeared on a lawn of Y1073. The plaques were screened (Benton and Davis 1977) using a nick- translated (Maniatis et al. 1975) internal R15.4 fragment of the large-subunit gene as a probe (Dron et al. 1982). DNA was iso- lated from positive plaques (Maniatis et al. 1982), and the large- subunit R15 fragment (Dron et al. 1982) was subcloned into pBR328 (Soberon et al. 1980). Recombinant plasmids were used to transform C600 (Mandel and Higa 1970), and colonies were screened for an ampicillin-resistant, chloramphenicol-sensitive phenotype. Plasmid DNA was isolated (Katz et al. 1973), and standard procedures were followed for R15 sub-fragment recov- ery, kinase-labeling and DNA sequencing (Maxam and Gilbert 1980). The procedure of Levis et al. (1980) was followed for 3' end labeling.

Results and discussion

Fig. 1. Sequence of the R4-7 revertant large-subunit gene. The substitution of GC in place of AT in the original mutant is in- dicated*. The area sequenced is indicated below a map of the large-subunit gene in which translated and untranslated (white) coding regions are shown (Dron et al. 1982). The HindIII frag- ment from position 1577 to position 2468 (Dron et al. 1982) was labeled at the 5' (Maxam and Gilbert 1980) or 3' ends (Levis et al. 1980). The fragment was digested with HphI and the largest labeled fragment was sequenced 3' to 5' ('-0 or 5' to 3' (t-0, as indicated in the figure. The sequence of the non-coding strand in the region of the original mutation is compared between wild type (2137 rot+), mutant 10-6C rat + and revertant R4-7 rat +

Materials and methods

Strains and culture conditions. The C. reinhardff R4-7 mt+ rever- tant grows normally in the absence of an exogenous carbon source in the light. It was selected following 5-fluorodeoxyuridine treatment and ethyl methanesulfonate mutagenesis of the light- sensitive, acetate-requiring 10-6C mt + mutant (Spreitzer et al.

1982). All photosynthesis-competent revertants of 10-6C mt+

are routinely maintained on 10 mM acetate medium (Spreitzer and Mets 1981) in the dark.

E. coli strains Y1073 hfl 150 (Young and Davis 1983) and C600 were used. Strains that contained recombinant plasmids were screened on 50 #g/ml chloramphenicol and maintained on 100 t~g/ml ampicillin. Bacteriophage lambda 1149, containing a single EcoRI site in the cI gene, was obtained from N. Murray and propagated by standard methods (Maniatis et al. 1982).

Gene cloning and sequencing. R4-7 mt+ cells were grown in 500 ml of acetate medium, broken in a Yeda press and DNA was

Dron et al. (1982, 1983) sequenced the C. reinhardii large-subunit gene from wild-type and the 10-6C RuBP carboxylase/oxygenase-defective mutant. Spreitzer et al.

(1982) recovered photosynthesis-competent revertants of 10-6C that were indistinguishable from wild type, both biochemically and genetically.

In the present investigation, we sequenced part of the large-subunit gene from the R4-7 revertant (Fig. 1).

Beginning at a HindlII site at nucleotide 1577 in the gene sequence (Dron et al. 1982), we sequenced the coding strand in a 3' to 5' direction for 230 nucleotides, and the non-coding strand in a 5' to 3' direction for 229 nucleotides. No difference was found between the R4-7 sequence and the wild-type large-subunit sequence pub- lished by Dron et al. (1982). Particular attention was given to position 1705 at which a single base change from GC to AT was reported as the basis for the 10-6C mutant phenotype (Dron et al. 1983). Since the R4-7 revertant was derived from the 10-6C mutant (Spreitzer et al. 1982), our results show that true reversion of AT to GC has now occurred to restore the wild-type pheno- type of R4-7. Thus, as shown in Fig. 1, glycine was replaced by aspartic acid in the 10-6C mutant, and then aspartic acid was replaced by glycine in the R4-7 rever- tant. While any of nine single-base changes could occur in the codon for aspartic acid to substitute eight different amino acids, only the true reversion reported here would substitute a glycine. This is not an unexpected result considering that the original mutation occurs in a highly conserved region of the large-subunit gene (Dron et al.

1982) that may represent a component of the active site of the holoenzyme (Hartman et al. 1978). Furthermore, the RuBP carboxylase/oxygenase enzyme present in every revertant of 10-6C is biochemically identical to the wild-type enzyme (Spreitzer et al. 1982).

The 10-6C mutant has greatly reduced RuBP carbo- xylase and oxygenase activities (Spreitzer et al. 1982),

R. J. Spreitzer et al.: True reversion of a chloroplast mutation 231

and has physical alterations in the large-subunit protein (Spreitzer and Mets 1980) and large-subunit gene (Dron et al. 1983). Results reported here and elsewhere (Spreitzer et al. 1982) show that true reversion of 10-6C restores all of these properties to the characteristics of wild type.

Our results are consistent with the sequence analysis of Dron et al. (1983), which defined the physical basis of the 10-6C mutant, and clearly demonstrate that genetic manipulation of the large-subunit gene can be achieved within the C. reinhardii chloroplast genome.

Acknowledgements. We thank M. Goldschmidt-Clermont for lambda packaging extract and Y. Epprecht for preparing the fig- ure. This work was supported by grant 3.258.0.82 from the Swiss National Science Foundation.

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Communicated by L. Mets Received June 28 / October 11, 1984