Plastome mutants

Summary Since the first reports seventy-five years ago on the non-Mendelian inheritance of variegation in plants, chloroplast gene mutations have been useful for genetical and physiological investigations. The mutations have been shown to affect the chloroplast translational apparatus, photosystem I, photosystem II, the cytochrome f/b₆ complex, carbon fixation, or the ATP synthase. They arose spontaneously or were induced by mutagens or by the action of nuclear mutator genes. Alterations of chloroplast DNA include point mutations, deletions, duplications, and inversions. In 1909, Correns discovered uniparental transmission of chloroplasts when he observed the maternal inheritance of a chlorophyll deficiency inMirabilis jalapa. At the same time, Baur (1909) reported crosses ofPelargonium zonale in which the offspring inherited chloroplasts from both parents (biparental transmission) with variegated leaves resulting as the green and white plastids sorted out. since the experiments of Baur and Correns, many non-Mendelian mutants have been isolated in both higher plants and algae (for reviews see Hagemann, 1964; Kirk and Tilney-Bassett, 1978; Gillham, 1978). Some of these are mitochondrial traits, including cytoplasmic male sterility in maize and several other plants (Hanson and Conde, 1985; Pring and Lonsdale, 1985). Several other traits have been tentatively identified as mitochondrial since their inheritance pattern differs from that of both nuclear and chloroplast genes, including the deformed leaf (“falsifolia”) syndrome ofOenothera (Stubbe, 1970), non-chromosomal stripe of maize (Coe, 1983), and inChlamydomonas, photoautotropism (Wiseman et al., 1977) and a minute colony phenotype (Alexander et al., 1974). A far larger number of extranuclear mutations affect the plastome (plastid genome). Among the algae,Euglena gracilis (Russell and Lyman, 1982),Scenedesmus obliquus (Bishop, 1982) andChlorella (Galling, 1982) have yielded interesting mutants, but unlikeChlamydomonas, they are not known to undergo sexual reproduction, and thus the Mendelian or non-Mendelian nature of the mutations has not been determined. Most of the plastome mutations which have been characterized have been isolated in higher plant lines or fromChlamydomonas.     

Monokaryotic chloroplast mutation has no effect on non-Mendelian transmission of chloroplast and mitochondrial DNA in Chlamydomonas species

Summary. We studied whether the monokaryotic chloroplast (moc) mutation affects the transmission of chloroplast and mitochondrial DNA in Chlamydomonas species. We used a previously isolated moc mutant from our cell line G33, which had only one large chloroplast nucleus. To obtain zygotes we crossed the mutant cells with wild-type cells, and mutant cells with receptive mates (females [mt+] with males [mt–]). In these zygotes, we recorded preferential dissolution of mt– parental chloroplast nuclei and fusion of the two cell nuclei. Antibiotic-resistance markers of chloroplast DNA were maternally transmitted in all crosses. PCR analysis of the cytochrome b (cob) gene sequence showed that the mitochondrial DNA was paternally transmitted to offspring. These results suggest that the moc mutation did not affect the organelle DNA transmission.Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa, 903-0213, Japan.     

Mapping of chloroplast genes involved in chloroplast ribosome biogenesis in Chlamydomonas reinhardtii

Summary Chloroplast gene mutations which confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii have been tested for allelism and mapped by recombination analysis of progeny from biparental zygote clones. Thirty-one independently isolated streptomycin resistant mutants have chloroplast ribosomes which are resistant to this drug in an assay based on misreading of isoleucine in response to a poly U template, and comprise one nuclear and four chloroplast gene loci. Four mutants resistant to spectinomycin, and three mutants resistant to neamine and kanamycin, which have chloroplast ribosomes resistant to their respective antibiotics in poly U directed phenylalanine incorporation, appear to map in a single chloroplast gene locus. Representative alleles of this nr/spr locus, the four streptomycin resistance loci, and two chloroplast gene loci for erythromycin resistance, have been analyzed in a series of parallel crosses to establish the following map order for these seven genes in the chloroplast genome: er-u-la-er-u-37-nr-u-2-1/spr-u-1-H-4-sr-u-2-23-sr-u-2-60-sr-u-sm3-sr-u-sm2. These seven genes may constitute a ribosomal region within the chloroplast genome of Chlamydomonas comparable to the ribosomal gene clusters in bacteria.     

Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii.

Summary We have developed an efficient procedure for the disruption of Chlamydomonas chloroplast genes. Wild-type C. reinhardtii cells were bombarded with microprojectiles coated with a mixture of two plasmids, one encoding selectable, antibiotic-resistance mutations in the 16S ribosomal RNA gene and the other containing either the atpB or rbcL photosynthetic gene inactivated by an insertion of 0.48 kb of yeast DNA in the coding sequence. Antibiotic-resistant transformants were selected under conditions permissive for growth of nonphotosynthetic mutants. Approximately half of these transformants were initially heteroplasmic for copies of the disrupted atpB or rbcL genes integrated into the recipient chloroplast genome but still retained photosynthetic competence. A small fraction of the transformants (1.1% for atpB; 4.3% for rbcL) were nonphotosynthetic and homoplasmic for the disrupted gene at the time they were isolated. Single cell cloning of the initially heteroplasmic transformants also yielded nonphotosynthetic segregants that were homoplasmic for the disrupted gene. Polypeptide products of the disrupted atpB and rbcL genes could not be detected using immunoblotting techniques. We believe that any nonessential Chlamydomonas chloroplast gene, such as those involved in photosynthesis, should be amenable to gene disruption by cotransformation. The method should prove useful for the introduction of site-specific mutations into chloroplast genes and flanking regulatory sequences with a view to elucidating their function.     

Recombination of Chlamydomonas chloroplast DNA occurs more frequently in the large inverted repeat sequence than in the single-copy regions

Summary It is well documented that chloroplast DNA (cpDNA) recombination occurs at a relatively high frequency during sexual reproduction of unicellular green algae from the Chlamydomonas genus. Like the cpDNAs of most land plants, those of Chlamydomonas species are divided into two single-copy regions by a large inverted repeat sequence, part of which encodes the chloroplast rRNA genes. In the present study, we scored the inheritance of polymorphic loci spanning the entire chloroplast genome in hybrids recovered from reciprocal interspecific and F₁ crosses between Chlamydomonas eugametes and C. moewusii, and from these data, estimated the density of recombination junctions within each region of recombinant cpDNAs. Our results indicate that recombination junctions occur at highly variable frequencies across the three main domains of the chloroplast genome. The large inverted repeat sequence was found to exhibit at least a five-fold higher density of recombination junctions compared to one of the singlecopy regions, whereas junctions in the latter region were five-fold more abundant relative to those in the other single-copy region. This marked difference in the densities of recombination junctions implies that the extent of genetic linkage between two given chloroplast loci will depend not only on their physical distance, but also on their locations within the genome.     

Mutants of Chlamydomonas reinhardtii with physical alterations in their chloroplast DNA

We have isolated nonphotosynthetic (acetate-requiring) mutants with physical alterations in chloroplast DNA following growth of haploid cells in the chloroplast specific mutagen 5-fluorodeoxyuridine (FdUrd) or treatment of FdUrd-grown diploid cells with X rays. About one-third of the nonphotosynthetic mutations resulting from FdUrd treatment alone show simple deletions. All eight of the mutants examined so far which were obtained with FdUrd plus X rays have deletions that are accompanied by rearrangements, including inversions or duplications. All the alterations extend into one of the two inverted repeat regions of the chloroplast genome which contain the ribosomal RNA cistrons. However, Southern hybridization experiments reveal that the rRNA cistrons are not deleted but instead are contained in new fragments. The relocated rRNA cistrons appear to be functional, since the mutants have normal levels of chloroplast ribosomes. In most cases the deletions and rearrangements are symmetrical and affect both inverted repeats in a similar fashion. An exception is the mutant ac-u-c-2–43, which lacks one inverted repeat region almost completely, including an entire set of rRNA genes. Three additional mutants, which fail to recombine with ac-u-c-2–43 to give photosynthetically competent cells, have smaller deletions in the same region of the genome. These physical mapping studies have allowed us to place the ac-u-c locus itself in a region of unique sequence DNA in a fragment, Ba10, which also includes the right-hand end of one inverted repeat.     

Conservation and Structural Divergence of Organellar DNA and Gene Expression in Non-Photosynthetic Plastids During Ontogenetic Differentiation and Phylogenetic Adaptation

Plastids of non-photosynthetic cells or tissues, such as chromoplasts or leukoplasts, which develop during the course of ontogenetic differentiation contain DNA which is identical to chloroplast DNA with respect to size, organization and gene content. Also in ribosome-deficient bleached plastids, produced in leaves by experimental treatments or mutation, chloroplast DNA remains unaltered. The chloroplast DNA of various bleached mutant strains of Euglena has suffered major deletions or rearrangements, but is, however, never totally lost. Also leukoplasts of parasitic higher plants contain DNA. In the organellar DNA of several parasitic plants photosynthetic genes are conserved. In the heterotrophic flagellate Astasia and in the holoparasite Epifagus virginiana (Orobanchaceae) the size of the plastid DNA is greatly reduced by major deletions and most or all photosynthetic genes or genes related to the chloroplastic respiratory chain are lost. The residual plastid genomes have, however, retained genes for RNAs, tRNAs and ribosomal polypeptides and these are transcribed, although plastidic RNA-polymerase genes are lost in Epifagus. These findings demand the existence of a nuclear-encoded RNA-polymerase. The relevance of the conservation of plastid DNA and of plastidic gene expression in non-photosynthetic cells is discussed, remains, however, at present elusive. Open reading frames of unknown function might be of particular significance for non-photosynthetic plastids.     

The mechanism of maternal inheritance in Chlamydomonas: Biochemical and genetic studies

Summary The cytoplasmic linkage group of Chlamydomonas shows maternal inheritance, i.e. preferential transmission of cytogenes from the female (m t ⁺) parent and loss of the corresponding male (m t ^–) genome in sexual crosses. The mechanism of this process is postulated to be enzymatic modification of chloroplast DNA of the female to protect it from a restriction enzyme which degrades the chloroplast DNA of the male parent in the zygote soon after fusion. Genetic, biochemical and physical data bearing on this hypothesis are summarized and discussed.This paper is dedicated, with great admiration and affection, to Professor Marcus M. Rhoades, whose enthusiasm and curiosity contributed so much to my own scientific development, whose openness to biochemical and molecular interpretations of genetic data, played an exemplary role in focussing my own approach to research and whose fundamental studies of cytoplasmic inheritance in maize (Rhoades 1933, 1946) were directly responsible for my determination to tackle this difficult and perplexing area of research.     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^– mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.     

The mitochondrial genome of Chlamydomonas. II. Genetic analysis of non-mendelian obligate photautotrophic mutants

Summary Among a collection of obligate photoautotrophic (dark-dier,dk) mutants isolated inChlamydomonas reinhardtii, two have been found which are inherited in crosses to wild type in a non-Mendelian, biparental and apparently random fashion. F₁ progeny include not only cells which show thedk and wildtype parental phenotypes but also many which possess intermediate phenotypes between wild type anddk. When F₁ progeny withdk, intermediate or wild-type phenotype were backcrossed to wild type, thedk phenotype continued to be inherited in a biparental and random fashion. Upon selection, neither mutant formed stable clones producing onlydk progeny, suggesting that the two mutants segregatedk and wild-type progeny somatically and that the homozygousdk condition may be lethal. The biparental transmission of these two non-Mendeliandk mutations resembles the transmission of acriflavin-inducedminute mutations ofChlamydomonas and is distinct from the uniparentally inherited chloroplast mutations of this alga. Both thedk andminute mutations may alter mitochondrial DNA and thereby alter mitochondrial functions.     

Molecular and genetic analysis of the chloroplast ATPase of chlamydomonas

Summary We have carried out a molecular and genetic analysis of the chloroplast ATPase in Chlamydomonas reinhardtii. Recombination and complementation studies on 16 independently isolated chloroplast mutations affecting this complex demonstrated that they represent alleles in five distinct chloroplast genes. One of these five, the ac-u-c locus, has been positioned on the physical map of the chloroplast DNA by deletion mutations. The use of cloned spinach chloroplast ATPase genes in heterologous hybridizations to Chlamydomonas chloroplast DNA has allowed us to localize three or possibly four of the ATPase genes on the physical map. The beta and probably the epsilon subunit genes of Chlamydomonas CF₁ lie within the same region of chloroplast DNA as the ac-u-c locus, while the alpha and proteolipid subunit genes appear to map adjacent to one another approximately 20 kbp away. Unlike the arrangement in higher plants, these two pairs of genes are separated from each other by an inverted repeat.     

Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker

Reverse-genetic studies of chloroplast genes in the green alga Chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. We have constructed a series of vectors for the targeted insertion and expression of foreign genes in the Chlamydomonas chloroplast genome. Using these vectors we have developed a novel selectable marker based on the bacterial gene aphA-6, which encodes an aminoglycoside phosphotransferase. The aphA-6 marker allows direct selection for transformants on medium containing either kanamycin or amikacin. The marker can be used to inactivate or modify specific chloroplast genes, and can be used as a reporter of gene expression. The availability of this marker now makes possible the serial transformation of the chloroplast genome of Chlamydomonas. Received: 26 October 1999 / Accepted: 28 December 1999     

Hybrid plasmids containing an active thymidine kinase gene of Herpes simplex virus 1.

The gene for the thymidine kinase (TK) of Herpes simplex virus type 1 (HSV-1) is located in the KpnI m and BamHI p fragments of the genome (Wigler et al., Cell 11, 223-232 (1977)). These fragments have been inserted into the EcoRI and BamHI sites, respectively, of plasmid pBR322, and propagated in E.coli. The TK gene contained in the recombinant plasmids was shown to be biologically active when introduced into TK- mouse L cells. Detailed restriction site maps of the BamHI p fragment have been constructed and the approximate location of the TK gene has been determined. Mouse cells transformed with cloned HSV-1 tk+ DNA produced HSV-1-specific thymidine kinase; superinfection with HSV-1 tk- virus increased the level of TK activity tenfold, suggesting that the BamHI p sequences present in transformed cells respond to virus-encoded regulatory gene product(s).     

Metabolism of 5-fluorouracil in sensitive and resistant Novikoff hepatoma cells.

N1-S1/FdUrd Novikoff hepatoma cells, which lack thymidine kinase activity, are resistant to 5-fluorouracil (FUra) as well as 5-fluorodeoxyuridine (FdUrd), suggesting that the pathway, FUra leads to FdUrd leads to FdUMP, is utilized for the conversion of FUra to FdUMP. However, the inhibition of thymidylate synthetase activity, the presumed target of FdUMP, by 1 X 10(-4) M FUra in intact N1-S1 Novikoff hepatoma cells, which have significant levels of thymidine kinase activity, is completely eliminated by 5 X 10(-4) M hydroxyurea, which is a potent inhibitor of ribonucleotide reductase. These results imply that the formation of ribonucleotides and does not involve thymidine kinase. This apparent dichotomy can be explained by the fact that, in addition to the well known lack of thymidine kinase activity, [14C]FUra conversion to ribonucleotides is greatly depressed in the N1-S1/FdUrd cells. Hence, the formation of FdUMP from FUra in Novikoff hepatoma cells apparently proceeds primarily via the intermediate formation of ribonucleotides. The decreased conversion of FUra to ribonucleotides in N1-S1/FdUrd cells decreases not only the ability of the analog to inhibit DNA synthesis, but also its effect on RNA metabolism. FUra, at a concentration of 1 X 10(-5) M, inhibits rRNA maturation in N1-S1 cells, but not in N1-S1/FdUrd cells. Since N1-S1/FdUrd cells are completely resistant to 1 X 10(-5) M FUra, whereas N1-S1 cells are completely inhibited by 1 X 10(-5) M FUra, even in the presence of 1 X 10(-4) M thymidine, the effects of FUra on RNA metabolism appear to contribute significantly to the cytotoxicity of the analog at higher drug concentrations.     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^? mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.     

Chloroplast DNA base substitutions: an experimental assessment

An experimental assessment was carried out to determine directly the frequency and types of spontaneous base substitutions that occur in chloroplast DNA. A target site within the chloroplast 16S rRNA gene of the green alga Chlamydomonas reinhardtii was chosen for the assay. Mutations at this site were known to confer spectinomycin resistance and simultaneously result in the loss of an AatII cleavage site. In the experiments reported here, base substitutions at any individual base occurred at a frequency in the range of 0.9–11 per 10⁹ viable cells plated. Four new mutations that confer resistance to spectinomycin were identified at the target site in the Chlamydomonas chloroplast 16S rRNA gene. When the relative rates of transition and transversion mutations were quantified, a bias toward transversions was observed. The prominence of A/T C/G transversions in the observed mutation spectrum suggests that oxidative damage may be the major cause of base substitution mutations within the chloroplast.     

Nuclear and chloroplast transformation inChlamydomonas reinhardtii: strategies for genetic manipulation and gene expression

Transformation of the nuclear, chloroplast, and mitochondrial genomes can now be accomplished inChlamydomonas reinhardtii. Many biosynthetic pathways are carried out in the chloroplast, and efforts to manipulate these pathways will require that gene products be directed to this compartment. Chloroplast proteins are encoded in either the chloroplast or nuclear genome. In the latter case they are synthesized in the cytoplasm and imported post-translationally into the chloroplast. Thus, strategies for expressing foreign genes or overexpressing endogenous genes whose products reside in the chloroplast could involve either genome. This paper reviews the present status of transformation methodology for the nuclear and chloroplast genomes inChlamydomonas. Considerations for expressing gene products in the chloroplast are discussed. Experimental evidence for homologous recombination during transformation of the nuclear genome is presented.