Translational regulation of gene expression in chloroplasts and mitochondria.
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Chloroplasts↗
Biomedical subjects
N W Gillham
Publications and source records attributed to N W Gillham.
Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation.
The respiratory deficient dum-1 mutant of Chlamydomonas reinhardtii fails to grow in the dark because of a terminal 1.5 kb deletion in the linear 15.8 kb mitochondrial genome, which affects the apocytochrome b (CYB) gene. In contrast to the wild type where only mitochondrial genomes of monomer length are observed, the dum-1 genomes are present as a mixture of monomer and dimer length molecules. The mutant dimers appear to result from head-to-head fusions of two deleted molecules. Furthermore, mitochondrial genomes of dum-1 were also found to be unstable, with the extent of the deletion varying among single cell clones from the original mutant population. The dum-1 mutant also segregates, at a frequency of ca. 4% per generation, lethal minute colonies in which the original deletion now extends at least into the adjacent gene encoding subunit four of NAD dehydrogenase (ND4). We have used the dum-1 mutant as a recipient to demonstrate stable mitochondrial transformation in C. reinhardtii employing the biolistic method. After 4 to 8 weeks dark incubation, a total of 22 respiratory competent colonies were isolated from plates of dum-1 cells bombarded with C. reinhardtii mitochondrial DNA (frequency 7.3 x 10(-7)) and a single colony was isolated from plates bombarded with C. smithii mitochondrial DNA (frequency 0.8 x 10(-7)). No colonies were seen on control plates (frequency < 0.96 x 10(-9)). All transformants grew normally in the dark on acetate media; 22 transformants were homoplasmic for the wild-type mitochondrial genome typical of the C. reinhardtii donor. The single transformant obtained from the C. smithii donor had a recombinant mitochondrial genome containing the donor CYB gene and the diagnostic HpaI and XbaI restriction sites in the gene encoding subunit I of cytochrome oxidase (COI) from the C. reinhardtii recipient. The characteristic deletion fragments of the dum-1 recipient were not detected in any of the transformants.
Animals↗
A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11.
We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C. reinhardtii. Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase. Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time. L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit. Thiostrepton-resistant mutants of Bacillus megaterium and B. subtilis lack L11, show reduced ribosome activity, and have slow growth rates. Similarities between the thiostrepton-resistant mutants of bacteria and C. reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E. coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.
Animals↗
Chloroplast transformation in Chlamydomonas.
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Animals↗
The carboxyl-terminal extension of the D1 protein of photosystem II is not required for optimal photosynthetic performance under CO2- and light-saturated growth conditions.
Synthesis of the photosystem II D1 protein as a precursor with a carboxyl-terminal extension occurs in almost all eukaryotic photosynthetic organisms examined so far, as well as in cyanobacteria. Processing of the D1 precursor has been recently postulated to play a regulatory role in the light-dependent migration of photosystem II units from the unstacked to the stacked thylakoids (Bowyer, J. M., Packer, J. C. L., McCormack, B. A., Whitelegge, J. P., Robinson, C., and Taylor, M. A. (1992) J. Biol. Chem. 267, 5424-5433). To test this hypothesis, site-directed mutagenesis and chloroplast transformation have been used to create a "preprocessed" mutant Chlamydomonas strain which synthesizes mature D1 protein directly. We have found that this strain is indistinguishable from wild type in terms of photosynthetic performance and cell doubling time under CO2- and light-saturated photoautotrophic growth conditions.
Animals↗
Nonrandom distribution of chloroplast recombination events in Chlamydomonas reinhardtii: evidence for a hotspot and an adjacent cold region.
Intermolecular recombination of Chlamydomonas chloroplast genes has been analyzed in sexual crosses and following biolistic transformation. The pattern and position of specific exchange events within 15 kb of the 22-kb inverted repeat have been mapped with respect to known restriction fragment length polymorphism markers that distinguish the chloroplast genomes of the interfertile species Chlamydomonas reinhardtii and Chlamydomonas smithii. Recombinant progeny were selected from two- and three-factor crosses involving point mutations conferring herbicide (dr) and antibiotic resistance (er and spr) in the psbA, 23S and 16S ribosomal RNA genes, respectively. Exchange events were not randomly distributed over the 15-kb region, but were found to occur preferentially in a 0.7-kb sequence spanning the 3' end of the psbA gene and were much less common in an adjacent region of ca. 2.0 kb. These findings are corroborated by data showing that the dr mutation is unlinked genetically (3% recombination/kb) to the er and spr rRNA mutations, which are themselves linked and show ca. 1% recombination/kb. This discrepancy is significant since the dr-er and er-spr intervals are about the same length (ca. 7 kb). During chloroplast transformation, the 0.7-kb recombination hotspot also functions as a preferential site for exchange events leading to the integration of donor psbA gene sequences. The 0.7-kb hotspot region contains four classes of 18-37-bp direct repeats also found in other intergenic regions, but no open reading frame. Using deletion constructs in a chloroplast transformation assay, the hotspot was localized to a 500-bp region that lacks most of these repeats, which suggests that the repeats themselves are not responsible for the increased recombination frequency. Within this region, a 400-bp sequence is highly conserved between the chloroplast genomes of C. reinhardtii and C. smithii and includes several structural motifs characteristic of recombination hotspots in other systems.
Animals↗
Targeted disruption of chloroplast genes in Chlamydomonas reinhardtii.
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 non-photosynthetic 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.
Animals↗
A nuclear mutant of Chlamydomonas that exhibits increased sensitivity to UV irradiation, reduced recombination of nuclear genes, and altered transmission of chloroplast genes.
Meiotic progeny of Chlamydomonas reinhardtii normally receive chloroplast genomes only from the mt+ parent. However, exceptional zygotes, which transmit the chloroplast genomes of both parents or, more rarely, only those of the mt- parent, arise at a low frequency. Mutations at the mt(+)-linked mat-3 locus were found previously to elevate the transmission of chloroplast genomes from the mt- parent, resulting in a much higher than normal frequency of exceptional zygotes. In this paper we demonstrate that an ultraviolet-sensitive nuclear mutation mapping at the uvsE1 locus, which is unlinked to mating type, also promotes chloroplast genome transmission from the mt- parent. This mutant, which was previously shown to reduce recombination of nuclear genes in meiosis, acts synergistically with the mat-3-3 mutation to produce an extremely high frequency of exceptional zygotes. Through the use of restriction fragment length polymorphisms existing in the chloroplast genomes of C. reinhardtii and the interfertile strain C. smithii, we show that chloroplast DNA fragments from the mt- parent normally begin to disappear shortly after zygote formation. However, this process appears to be blocked totally in the absence of wild-type uvsE1 and mat-3 gene products. Our findings are consistent with the hypothesis that both gene products contribute to the mechanism responsible for uniparental inheritance of the chloroplast genome from the mt+ parent.
Alleles↗
Cotranscription of the wild-type chloroplast atpE gene encoding the CF1/CF0 epsilon subunit with the 3' half of the rps7 gene in Chlamydomonas reinhardtii and characterization of frameshift mutations in atpE.
We have characterized two independently isolated point mutants in Chlamydomonas reinhardtii, ac-u-a-1-15 and FUD 17, mapping to the chloroplast ac-u-a locus which corresponds to the atpE gene. Both mutants have a single A:T base pair deletion in a sequence of 6 A:T base pairs at nucleotide positions 102 to 107. This causes a frameshift, altering the coding sequence for the next 8 amino acids and creating a termination codon at amino acid position 44, 98 amino acids from the C-terminus of the protein. Assembly of the ATP synthase is impaired in the mutants; less than 5% of the wild-type level of alpha and beta subunits and no gamma or epsilon subunits are associated with thylakoid membranes of the mutants. The genes encoding the beta and epsilon subunits of the chloroplast ATP synthase from C. reinhardtii are not cotranscribed, in contrast to all other photosynthetic organisms examined to date. Four transcripts, of approximately 1.7, 2.9, 3.3 and 7.0 x 10(3) nucleotides (nt), are found for the atpE gene. S1 nuclease mapping of the 1.7 x 10(3) nt transcript shows that the atpE gene message is preceded by a leader of about 1250 nt. DNA sequence analysis of this region revealed a 159 bp open reading frame corresponding to the 3' half of the rps7 gene, encoding the S7 protein of the small subunit of the chloroplast ribosome. Only the 5' portion of this gene is located in the opposite unique sequence region of the C. reinhardtii chloroplast genome where the rps7 gene was previously mapped by heterologous hybridization.
Amino Acid Sequence↗
Transformation of chloroplast ribosomal RNA genes in Chlamydomonas: molecular and genetic characterization of integration events.
Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.
Chlamydomonas↗
The chloroplast ribosome comes of age. The Translational Apparatus of Photosynthetic Organelles: a NATO Advanced Research Workshop, Grenoble, France, July 3-6, 1990.
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Animals↗
Chloroplast ribosomal protein gene rps12 of Chlamydomonas reinhardtii. Wild-type sequence, mutation to streptomycin resistance and dependence, and function in Escherichia coli.
The chloroplast rps12 gene encoding ribosomal protein S12 from wild-type as well as from streptomycin-resistant and -dependent mutants of Chlamydomonas reinhardtii was cloned and sequenced. At least six mRNA transcripts ranging in size from 800 to 4800 nucleotides were found to contain the rps12 coding sequence. As in Escherichia coli and Euglena, the C. reinhardtii rps12 gene is continuous, in contrast to its trans-spliced structure in higher plants. However, rps12 of C. reinhardtii is no longer immediately adjacent to the rps7 gene encoding ribosomal protein S7 as it is in other organisms. The deduced amino acid sequence of C. reinhardtii S12 protein shows strong homologies (48-79% identity) to S12 protein sequences of other organisms, but it has extra amino acid residues at its C terminus. Single base pair changes at different sites result in streptomycin-resistant or -dependent mutants with amino acid changes identical to comparable mutations in the E. coli S12 protein. The chloroplast rps12 gene is also expressed under the control of its own promoter in E. coli cells, and the C. reinhardtii S12 protein produced assembles into the E. coli ribosomes where it appears to function efficiently.
Amino Acid Sequence↗
Molecular characterization of two point mutants in the chloroplast atpB gene of the green alga Chlamydomonas reinhardtii defective in assembly of the ATP synthase complex.
Two point mutants of Chlamydomonas reinhardtii, previously found by recombination and complementation analysis to map in the chloroplast atpB gene encoding the beta subunit of the CF1/CF0 ATP synthase, are here shown to be missense alterations near the 5' end of that gene. One mutant (ac-u-c-2-9) has a change at amino acid position 47 of the beta subunit from leucine (CTA) to arginine (CGA). In the second mutant (ac-u-c-2-29), the codon AAA (lysine) is changed to AAC (asparagine) at position 154. Spontaneous revertants of each mutant were isolated that restore the original wild type base pair. Northern analysis of total RNA and in vivo pulse labeling followed by immunoprecipitation reveals that both mutant atpB genes are transcribed and translated normally. However, immunoblots show that the amount of beta subunit associated with mutant thylakoids is only approximately 3% of that seen in wild type and that the CF1 alpha and gamma subunits are missing entirely. The disruption of ATP synthase complex assembly in these mutants is much more severe than in Escherichia coli beta subunit gene point mutants, which retain significant amounts of alpha and beta subunits on their membranes (Noumi, T., Oka, N., Kanazawa, H., and Futai, M. (1986) J. Biol. Chem. 261, 7070-7075). These results support the hypothesis that there are differences in assembly of the ATP synthase between E. coli and chloroplasts. In particular they indicate that beta must be present for assembly of the alpha and gamma subunits of CF1 onto chloroplast membranes.
Base Sequence↗
Electrophoretic and immunological comparisons of chloroplast and prokaryotic ribosomal proteins reveal that certain families of large subunit proteins are evolutionarily conserved.
Antibodies to individual chloroplast ribosomal (r-)proteins of Chlamydomonas reinhardtii synthesized in either the chloroplast or the cytoplasm were used to examine the relatedness of Chlamydomonas r-proteins to r-proteins from the spinach (Spinacia oleracea) chloroplast, Escherichia coli, and the cyanobacterium Anabaena 7120. In addition, 35S-labeled chloroplast r-proteins from large and small subunits of C. reinhardtii were co-electrophoresed on 2-D gels with unlabeled r-proteins from similar subunits of spinach chloroplasts, E. coli, and Anabaena to compare their size and net charge. Comigrating protein pairs were not always immunologically related, whereas immunologically related r-protein pairs often did not comigrate but differed only slightly in charge and molecular weight. In contrast, when 35S-labeled chloroplast r-proteins from large and small subunits of a closely related species C. smithii were coelectrophoresed with unlabeled C. reinhardtii chloroplast r-proteins, only one pair of proteins from each subunit showed a net displacement in mobility. Analysis of immunoblots of one-dimensional SDS and two-dimensional urea/SDS gels of large and small subunit r-proteins from these species revealed more antigenic conservation among the four species of large subunit r-proteins than small subunit r-proteins. Anabaena r-proteins showed the greatest immunological similarity to C. reinhardtii chloroplast r-proteins. In general, antisera made against chloroplast-synthesized r-proteins in C. reinhardtii showed much higher levels of cross-reactivity with r-proteins from Anabaena, spinach, and E. coli than did antisera to cytoplasmically synthesized r-proteins. All spinach r-proteins that cross-reacted with antisera to chloroplast-synthesized r-proteins of C. reinhardtii are known to be made in the chloroplast (Dorne et al. 1984b). Four E. coli r-proteins encoded by the S10 operon (L2, S3, L16, and L23) were found to be conserved immunologically among the four species. Two of the large subunit r-proteins, L2 and L16, are essential for peptidyltransferase activity. The third (L23) and two other E. coli large subunit r-proteins (L5 and L27) that have immunological equivalents among the four species are functionally related to but not essential for peptidyltransferase activity.
Blotting, Western↗
Antibiotic resistance mutations in the chloroplast 16S and 23S rRNA genes of Chlamydomonas reinhardtii: correlation of genetic and physical maps of the chloroplast genome.
Mutants resistant to streptomycin, spectinomycin, neamine/kanamycin and erythromycin define eight genetic loci in a linear linkage group corresponding to about 21 kb of the circular chloroplast genome of Chlamydomonas reinhardtii. With one exception, all of these mutants represent single base-pair changes in conserved regions of the genes encoding the 16S and 23S chloroplast ribosomal RNAs. Streptomycin resistance can result from changes at the bases equivalent to Escherichia coli 13, 523, and 912-915 in the 16S gene, or from mutations in the rps12 gene encoding chloroplast ribosomal protein S12. In the 912-915 region of the 16S gene, three mutations were identified that resulted in different levels of streptomycin resistance in vitro. Although the three regions of the 16S rRNA mutable to streptomycin resistance are widely separated in the primary sequence, studies by other laboratories of RNA secondary structure and protein cross-linking suggest that all three regions are involved in a common ribosomal neighborhood that interacts with ribosomal proteins S4, S5 and S12. Three different changes within a conserved region of the 16S gene, equivalent to E. coli bases 1191-1193, confer varying levels of spectinomycin resistance, while resistance to neamine and kanamycin results from mutations in the 16S gene at bases equivalent to E. coli 1408 and 1409. Five mutations in two genetically distinct erythromycin resistance loci map in the 23S rDNA of C. reinhardtii, at positions equivalent to E. coli 2057-2058 and 2611, corresponding to the rib3 and rib2 loci of yeast mitochondria respectively. Although all five mutants are highly resistant to erythromycin, they differ in levels of cross-resistance to lincomycin and clindamycin. The order and spacing of all these mutations in the physical map are entirely consistent with our genetic map of the same loci and thereby validate the zygote clone method of analysis used to generate this map. These results are discussed in comparison with other published maps of chloroplast genes based on analysis by different methods using many of the same mutants.
Base Sequence↗
Relationship between Gene Dosage and Gene Expression in the Chloroplast of Chlamydomonas reinhardtii.
Expression of three chloroplast genes encoding proteins of different chloroplast complexes and the rRNA gene has been examined in cells having reduced numbers of chloroplast genomes as a result of growth in the presence of the thymidine analog 5-fluorodeoxyuridine. While accumulation of total mRNA for rpl2 (ribosomal protein L-1), rbcL (ribulose bisphosphate carboxylase large subunit) and atpA (alpha-subunit of ATP synthase) declined with gene copy number, the levels of translatable mRNA and rates of synthesis of these three proteins were largely unaffected. Accumulation of rRNA declined less precipitously than mRNA levels for the three proteins in response to the reduction in chloroplast genome number. Chlamydomonas appears to compensate for reductions in the number of chloroplast genomes at several different levels. Populations of cells with only one-fourth the wild-type amount of chloroplast DNA per cell on average have half the normal level of chloroplast ribosomes and nearly normal rates of CO(2) fixation and levels of specific chloroplast encoded proteins. These results suggest that normal cells accumulate a large excess of transcripts for chloroplast genes and that levels of expression of these genes are regulated by posttranscriptional mechanisms.
Journal Article↗
Chloroplast transformation in Chlamydomonas with high velocity microprojectiles.
Bombardment of three mutants of the chloroplast atpB gene of Chlamydomonas reinhardtii with high-velocity tungsten microprojectiles that were coated with cloned chloroplast DNA carrying the wild-type gene permanently restored the photosynthetic capacity of the algae. In most transformants of one of the mutants, a fragment with a 2.5-kilobase deletion was restored to normal size by a homologous replacement event; in about 25 percent of the transformants the restored restriction fragment was 50 to 100 base pairs smaller or larger than that of wild type. About one-fourth of the transformants of this mutant contained unintegrated donor plasmid when first examined. This plasmid persisted in four different transformants after 65 cell generations of continuous liquid culture but was lost from all transformants maintained on plates of selective medium. The restored wild-type atpB gene remains in all transformants as an integral part of the chloroplast genome and is expressed and inherited normally.
Chlamydomonas↗
Chloroplast ribosomal protein L-18 in Chlamydomonas reinhardtii is processed during ribosome assembly.
Chloroplast ribosomal protein L-18 is made in the cytoplasm as a precursor, imported into the chloroplast, and processed to the mature form in two steps. We report here that the intermediate produced following the first processing step associates specifically with a ribosomal complex migrating with the chloroplast ribosome large subunit peak in sucrose gradients, and is then processed into mature L-18. This processing event is slowed down in mutant cells deficient in synthesis of non-ribosomal proteins in the chloroplast. Thus the second processing step of L-18 occurs during ribosome assembly, depends on one or more nonribosomal proteins made in the chloroplast, and may be required for the maturation of the 50 S ribosome subunit. The mature L-18 protein shows extensive sequence homology at its amino-terminus to Escherichia coli ribosomal protein L27, which is located at the interface between 30 S and 50 S subunits and is involved in the formation of the peptidyl-tRNA binding site.
Amino Acid Sequence↗