Chloroplast Genes In Chlamydomonas Research Articles

The sequence and structure of the 3'-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability.

A general characteristic of the 3'-untranslated regions (3' UTRs) of plastid mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops are RNA 3'-end processing signals and determinants of mRNA stability, not transcription terminators. Incubation of synthetic RNAs corresponding to the 3' UTRs of Chlamydomonas chloroplast genes atpB and petD with a chloroplast protein extract resulted in the accumulation of stable processing products. Synthetic RNAs of the petA 3' UTR and the antisense strand of atpB 3' UTR were degraded in the extract. To examine 3' UTR function in vivo, the atpB 3' UTR was replaced with the 3' UTR sequences of the Chlamydomonas chloroplast genes petD, petD plus trnR plus trnR, rbcL, petA and E. coli thrA by biolistic transformation of Chlamydomonas chloroplasts. Each 3' UTR was inserted in both the sense and antisense orientations. The accumulation of both total atpB mRNA and ATPase beta-subunit protein in all transformants was increased compared to a strain in which the atpB 3' UTR had been deleted. However, the level of discrete atpB transcripts in transformants containing the antisense 3' UTR sequences was reduced to approximately one-half that of transformants containing the 3' UTRs in the sense orientation. These results imply that both the nucleotide sequences and the stem-loop structures of the 3' UTRs are important for transcript 3'-end processing, and for accumulation of the mature mRNAs.

The 3' untranslated regions of chloroplast genes in Chlamydomonas reinhardtii do not serve as efficient transcriptional terminators.

A general characteristic of the 3' untranslated regions of plastid mRNAs is an inverted repeat sequence that can fold into a stem-loop structure. These stem-loops are superficially similar to structures involved in prokaryotic transcription termination, but were found instead to serve as RNA 3' end processing signals in spinach chloroplasts, and in the atpB mRNA of Chlamydomonas reinhardtii chloroplasts. In order to carry out a broad study of the efficiency of the untranslated sequences at the 3' ends of chloroplast genes in Chlamydomonas to function as transcription terminators, we performed in vivo run-on transcription experiments using Chlamydomonas chloroplast transformants in which different 3' ends were inserted into the chloroplast genome between a petD promoter and a reporter gene. The results showed that none of the 3' ends that were tested, in either sense or antisense orientation, prevented readthrough transcription, and thus were not highly efficient transcription terminators. Therefore, we suggest that most or all of the 3' ends of mature mRNAs in Chlamydomonas chloroplasts are formed by 3' end processing of longer precursors.

Selection on the codon bias of Chlamydomonas reinhardtii chloroplast genes and the plant psbA gene.

Plant chloroplast genes have a codon use that reflects the genome compositional bias of a high A+T content with the single exception of the highly translated psbA gene which codes for the photosystem II D1 protein. The codon usage of plant psbA corresponds more closely to the limited tRNA population of the chloroplast and is very similar to the codon use observed in the chloroplast genes of the green alga Chlamydomonas reinhardtii. This pattern of codon use may be an adaptation for increased translation efficiency. A correspondence between codon use of plant psbA and Chlamydomonas chloroplast genes and the tRNAs coded by the chloroplast genome, however, is not observed in all synonymous codon groups. It is shown here that the degree of correspondence between codon use and tRNA population in different synonymous groups is correlated with the second codon position composition. Synonymous groups with an A or T at the second codon position have a high representation of codons for which a complementary tRNA is coded by the chloroplast genome. Those with a G or C at the second position have an increased representation of codons that bind a chloroplast tRNA by wobble. It is proposed that the difference between synonymous groups in terms of codon adaptation to the tRNA population in plant psbA and Chlamydomonas chloroplast genes may be the result of differences in second position composition.

Function of 3' non-coding sequences and stop codon usage in expression of the chloroplast psaB gene in Chlamydomonas reinhardtii.

The rate of mRNA decay is an important step in the control of gene expression in prokaryotes, eukaryotes and cellular organelles. Factors that determine the rate of mRNA decay in chloroplasts are not well understood. Chloroplast mRNAs typically contain an inverted repeat sequence within the 3' untranslated region that can potentially fold into a stem-loop structure. These stem-loop structures have been suggested to stabilize the mRNA by preventing degradation by exonuclease activity, although such a function in vivo has not been clearly established. Secondary structures within the translation reading frame may also determine the inherent stability of an mRNA. To test the function of the inverted repeat structures in chloroplast mRNA stability mutants were constructed in the psaB gene that eliminated the 3' flanking sequences of psaB or extended the open reading frame into the 3' inverted repeat. The mutant psaB genes were introduced into the chloroplast genome of Chlamydomonas reinhardtii. Mutants lacking the 3' stem-loop exhibited a 75% reduction in the level of psaB mRNA. The accumulation of photosystem I complexes was also decreased by a corresponding amount indicating that the mRNA level is limiting to PsaB protein synthesis. Pulse-chase labeling of the mRNA showed that the decay rate of the psaB mRNA was significantly increased demonstrating that the stem-loop structure is required for psaB mRNA stability. When the translation reading frame was extended into the 3' inverted repeat the mRNA level was reduced to only 2% of wild-type indicating that ribosome interaction with stem-loop structures destabilizes chloroplast mRNAs. The non-photosynthetic phenotype of the mutant with an extended reading frame allowed us to test whether infrequently used stop codons (UAG and UGA) can terminate translation in vivo. Both UAG and UGA are able to effectively terminate PsaB synthesis although UGA is never used in any of the Chlamydomonas chloroplast genes that have been sequenced.

The salvage/turnover/repair (STOR) model for uniparental inheritance in Chlamydomonas: DNA as a source of sustenance.

The non-Mendelian inheritance of chloroplast genes in Chlamydomonas has engaged researchers for decades and has prompted numerous debates regarding molecular mechanisms and evolutionary significance. The hallmarks of chloroplast inheritance in Chlamydomonas are reviewed here, including observations on vegetative haploid cells, somatic hybrids, meiotic zygospores, and vegetative zygotes resulting from sexual reproduction. Models invoked to explain the typical uniparental maternal inheritance of chloroplast genes, and which center upon the presumed existence of sex-specific protectors and destroyers of chloroplast genomes, are briefly discussed. In an effort to bring together the diverse observations on chloroplast gene inheritance in somatic as well as sexual cells, a model is proposed that focuses on organelle DNA turnover as a source of sustenance for the cell during periods of starvation. The salvage/turnover/repair (STOR) model for chloroplast inheritance in Chlamydomonas proposes that as a consequence of the high ploidy of the chloroplast genome, many copies are dispensable; their degradation would provide nucleotides for recombination, repair, RNA synthesis and cell metabolism. The STOR model offers an alternative view of uniparental inheritance as a phenomenon of direct selective benefit to the organism rather than simply being of selfish benefit to the chloroplast genome. These concepts may also have application to other lower eukaryotes that have sexual reproduction coupled with an extended dormancy.

A suppressor of a mating-type limited zygotic lethal allele also suppresses uniparental chloroplast gene transmission in Chlamydomonas monoica.

Uniparental inheritance of Chlamydomonas chloroplast genes is thought to involve modification of maternal (mt+) chloroplast genomes to protect against a nuclease that is activated after gamete fusion. The mating-type limited mtl-1 mutant strain of Chlamydomonas monoica is unable to protect mt(+)-derived chloroplast DNA. Zygotes homozygous for mtl-1 lose all chloroplast DNA and fail to germinate. We have selected for suppression of this zygote-specific lethality, and have obtained 20 mutant strains that produce viable homozygotes despite the continued presence of the mtl-1 allele. Genetic analysis indicates that the suppressor mutations are all recessive alleles at a single locus (sup-1) which is unlinked to mtl-1. Crosses between sup-1 strains carrying distinctive chloroplast antibiotic resistance markers also show predominantly biparental chloroplast gene transmission. Chloroplast nucleoids of both parental origins (stained with the DNA-specific fluorochrome, DAPI) are retained in the zygotes homozygous for sup-1. The data are compatible with the idea that the sup-1 (suppressor of uniparental inheritance) locus may encode a chloroplast DNA nuclease that is expressed from both parental genomes.

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.

Evidence for Nuclear Control of the Expression of the atpA and atpB Chloroplast Genes in Chlamydomonas

Evidence for Nuclear Control of the Expression of the atpA and atpB Chloroplast Genes in Chlamydomonas

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.

Mapping of chloroplast mutations conferring resistance to antibiotics in Chlamydomonas: Evidence for a novel site of streptomycin resistance in the small subunit rRNA

A major obstacle to our understanding of the mechanisms governing the inheritance, recombination and segregation of chloroplast genes in Chlamydomonas is that the majority of antibiotic resistance mutations that have been used to gain insights into such mechanisms have not been physically localized on the chloroplast genome. We report here the physical mapping of two chloroplast antibiotic resistance mutations: one conferring cross-resistance to erythromycin and spiramycin in Chlamydomonas moewusii (er-nM1) and the other conferring resistance to streptomycin in the interfertile species C. eugametos (sr-2). The er-nM1 mutation results from a C to G transversion at a well-known site of macrolide resistance within the peptidyl transferase loop region of the large subunit rRNA gene. This locus, designated rib-2 in yeast mitochondrial DNA, corresponds to residue C-2611 in the 23 S rRNA of Escherichia coli. The sr-2 locus maps within the small subunit (SSU) rRNA gene at a site that has not been described previously. The mutation results from an A to C transversion at a position equivalent to residue A-523 in the E. coli 16 S rRNA. Although this region of the E. coli SSU rRNA has no binding affinity for streptomycin, it binds to ribosomal protein S4, a protein that has long been associated with the response of bacterial cells to this antibiotic. We propose that the sr-2 mutation indirectly affects the nearest streptomycin binding site through an altered interaction between a ribosomal protein and the SSU rRNA.

Mating type linked mutations which disrupt the uniparental transmission of chloroplast genes in chlamydomonas.

In Chlamydomonas reinhardtii, chloroplast genomes are normally transmitted by the mating type plus (mt+) parent and mitochondrial genomes by the mating type minus (mt-) parent. In this paper we describe three new nuclear mutations, designated mat-3-1 to -3, which are tightly linked to the mt+ allele and permit high transmission of chloroplast genomes from the mt- parent, but have no effect on transmission of mitochondrial genomes. We also show that mat-1, reported by others to be a nuclear mutation linked to mt- which promotes transmission of chloroplast genomes by the mt- parent, is probably a vegetative diploid since it contains both mt+ and mt- alleles. Vegetative diploids behave as if they are mt- with respect to mating, but possess a level of chloroplast gene transmission intermediate between that of haploid mt- and mt+ stocks.

Persistent heteroplasmic cells for mitochondrial genes in Saccharomyces cerevisiae

Genes in mitochondria and chloroplasts segregate rapidly during vegetative reproduction. Models to explain this vegetative segregation invoke either random segregation of organelle DNA molecules, or nonrandom segregation with random recombination events. All such models are basically stochastic. To look at vegetative segregation we took heteroplasmic (HET) cells containing mitochondrial mutations at the cap1, eryl and olil loci from several crosses. HETs were repeatedly selected and subcloned. Even after three to five successive subclonings (approximately 60-100 generations) some cells remained heteroplasmic. This confirms and extends previous observations of persistent HETs by Rank and Bech-Hansen (1972) and Forster and Kleese (1975), and by Bolen et al. (1980) for chloroplast genes in Chlamydomonas.

Extensive methylation of chloroplast DNA by a nuclear gene mutation does not affect chloroplast gene transmission in chlamydomonas

Based on analysis by high pressure liquid chromatography, greater than 35% of the cytosine residues in chloroplast DNA of vegetative cells were found to be methylated constitutively in the nuclear gene mutation ( me-1) of Chlamydomonas reinhardtii, which has an otherwise wild-type phenotype. Digestion of chloroplast DNA from vegetative cells and gametes of this mutant with restriction endonucleases Hpa II and Msp I reveals that in the 5′CCGG3′ sequence, CpG is methylated extensively, whereas CpC is only methylated occasionally. Hae III (5′GGCC3′) digestion of the mutant chloroplast DNA also shows extensive methylation of the GpC sequence. In contrast to the results of Sager and colleagues, which show a correlation between methylation of chloroplast DNA and transmission of chloroplast genes in crosses, our results with crosses of the me-1 mutant suggest that extensive chloroplast DNA methylation may be insufficient to account for the pattern of inheritance of chloroplast genes in Chlamydomonas.

The mat-1 gene in chlamydomonas regulates DNA methylation during gametogenesis

The inheritance of chloroplast genes in Chlamydomonas is regulated by methylation of chloroplast DNA during gametogenesis. The wild-type pattern of maternal inheritance results from the methylation of chloroplast DNA in female ( mt +) but not in male ( mt −) gametes, leading to preferential degradation of chloroplast DNA of male origin in zygotes. This paper describes the distribution of 5-methyl cytosine residues in restriction fragments of chloroplast DNA sampled during gametogenesis by two methods: ethidium bromide staining of agarose gels, and binding of antibody directed against 5-methyl cytosine onto restriction fragments blotted to nitrocellulose paper. Methylated cytosines are located in most if not all Eco RI and Msp I fragments, but the extent of methylation is not proportional to fragment size. The mat-1 mutation carried by males converts maternal inheritance of chloroplast genes to biparental inheritance. Chloroplast DNA of male gametes carrying the mat-1 mutation becomes methylated during gametogenesis. This methylation protects against restriction enzyme-promoted degradation in zygotes, as shown by physical data demonstrating the transmission to progeny of chloroplast genes carried on chloroplast DNA of the mat-1 male parent. Thus the mat-1 gene, which is linked to the mating-type locus, determines whether or not methylation of chloroplast DNA will occur in males during gametogenesis.

Early vegetative segregation of mitochondrial genes in Saccharomyces cerevisiae

The vegetative segregation of seven mitochondrial gene loci was studied in yeast. At various times after mating antibiotic resistant and sensitive strains, samples of the diploid progeny were examined to determine the segregation rates of the alleles at each locus in three- and four-factor crosses. The rate of segregation was approximately the same for the cap1, ery1, oli1, oli2, and par1 loci, which are scattered over about two-thirds of the mitochondrial DNA molecule. Differences in segregation rates were found but showed no consistent relationship to the map positions of the loci. This is in contrast to the segregation of chloroplast genes in Chlamydomonas, where loci segregate at rates proportional to their distance from an “attachment point” which appears to govern the partitioning of chloroplast DNA molecules between daughter chloroplasts when the chloroplast divides. Our data are compatible with a model in which the mitochondrial DNA molecules in a cell occur in a small number of groups corresponding to individual nucleoids or mitochondria. Most or all of the molecules in a group carry the same allele at any given locus. These genetically homogeneous groups of molecules may thus be the units of segregation, and may be partitioned randomly between mother cell and bud at each division.

Chloroplast gene inheritance studied by somatic fusion in Chlamydomonas reinhardtii

Somatic fusion between strains of Chlamydomonas containing complementing cell-wall and auxotrophic mutations, having the same mating-type (mt) and bearing chloroplast markers, have been performed to study the mode of chloroplast gene inheritance in the fusion products. About one third of the fusion products (mt (+)/mt (+) or mt (-)/mt (-)) transmitted chloroplast markers from both parents (= biparental fusion products). The rest of the population was equally distributed between fusion products transmitting the chloroplast marker of one parent or the other (uniparental fusion products) exclusively. Incubation of the fusion products in the dark for 48 hours, immediately after the fusion, decreases the frequency of biparental fusion products. The results indicate that the general process of elimination of chloroplast alleles is independent of the presence of both mt (+) and mt (-) alleles in the cell. In contrast, directional elimination (i.e. preferential elimination of paternal chloroplast alleles) does appear to depend upon heterozygosity at the mt locus. These results are discussed in relation to the models which have been proposed to explain the maternal inheritance of chloroplast genes in Chlamydomonas.

Chloroplast gene transmission in Chlamydomonas reinhardtii: A random choice model

Chloroplast genes in Chlamydomonas reinhardtii are inherited uniparentally. In a laboratory cross the majority (>95%) of the zygotes transmit to the meiotic progeny only those chloroplast genes donated by the maternal parent. This occurs even though the parents are isogamous and the chloroplasts from the two parents fuse shortly after mating. Uniparental inheritance of the chloroplast genes can be altered by several methods. If maternal gametes are irradiated with ultraviolet light prior to mating, the proportion of zygotes transmitting chloroplast genes from the paternal parent rises dramatically. In this paper I examine in detail the effects of uv irradiation on both maternal and paternal gametes and the effect of photore-activation following the uv irradiation. The effect of uv irradiation can be largely reversed by photoreactivation, and both the starting time and the intensity of the photoreactivating light used are found to be critical. Examination of the frequencies of the different zygote types obtained with respect to chloroplast gene transmission following uv treatment of the maternal gametes shows that they fit a hypergeometric distribution, in which choices are made from a population without replacement. By rearranging the basic hypergeometric equation I was able to estimate that the choice is made from a population of 27 maternal and 2 paternal units of gene transmission. These units probably contain more than one genome each, since their number is much lower than the estimated number of genomes per cell. My model explains both the observed distribution of zygote types and the bias in favor of maternal alleles found in the progeny of a biparental zygote, and may have a wider application to other organelle genetic systems. I also suggest that the extreme degree of uniparental inheritance of chloroplast genes in Chlamydomonas found in the laboratory may not be seen in nature.

Mutations that alter the transmission of chloroplast genes in Chlamydomonas.

Two mutations are described that alter the pattern of inheritance of chloroplast genes in Chlamydomonas. The mutant gene mat-1 linked to the mating type allele mt(-) greatly increases the frequency of exceptional zygotes, i.e., zygotes that transmit chloroplast genes from the mt(-) (male) parent. In some crosses, 80-90% of the zygotes are biparental, transmitting chloroplast genes from both parents. The mat-2 mutation, linked to mt(+), acts to decrease the frequency of exceptional zygotes below the spontaneous level. The mutant effects are discussed in terms of a DNA modification-restriction system, postulated to regulate the transmission of chloroplast DNA in zygotes.