Rapid access to genetic information is central to the revolution taking place in molecular genetics. The simultaneous analysis of the entire human mitochondrial genome is described here. DNA arrays containing up to 135,000 probes complementary to the 16.6-kilobase human mitochondrial genome were generated by light-directed chemical synthesis. A two-color labeling scheme was developed that allows simultaneous comparison of a polymorphic target to a reference DNA or RNA. Complete hybridization patterns were revealed in a matter of minutes. Sequence polymorphisms were detected with single-base resolution and unprecedented efficiency. The methods described are generic and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability.

The mitochondrial genomes of seed plants are unusually large and vary in size by at least an order of magnitude. Much of this variation occurs within a single family, the Cucurbitaceae, whose genomes range from an estimated 390 to 2,900 kb in size. We sequenced the mitochondrial genomes of Citrullus lanatus (watermelon: 379,236 nt) and Cucurbita pepo (zucchini: 982,833 nt)--the two smallest characterized cucurbit mitochondrial genomes--and determined their RNA editing content. The relatively compact Citrullus mitochondrial genome actually contains more and longer genes and introns, longer segmental duplications, and more discernibly nuclear-derived DNA. The large size of the Cucurbita mitochondrial genome reflects the accumulation of unprecedented amounts of both chloroplast sequences (>113 kb) and short repeated sequences (>370 kb). A low mutation rate has been hypothesized to underlie increases in both genome size and RNA editing frequency in plant mitochondria. However, despite its much larger genome, Cucurbita has a significantly higher synonymous substitution rate (and presumably mutation rate) than Citrullus but comparable levels of RNA editing. The evolution of mutation rate, genome size, and RNA editing are apparently decoupled in Cucurbitaceae, reflecting either simple stochastic variation or governance by different factors.

The chloroplast genome encodes proteins required for photosynthesis, gene expression, and other essential organellar functions. Derived from a cyanobacterial ancestor, the chloroplast combines prokaryotic and eukaryotic features of gene expression and is regulated by many nucleus-encoded proteins. This review covers four major chloroplast posttranscriptional processes: RNA processing, editing, splicing, and turnover. RNA processing includes the generation of transcript 5' and 3' termini, as well as the cleavage of polycistronic transcripts. Editing converts specific C residues to U and often changes the amino acid that is specified by the edited codon. Chloroplasts feature introns of groups I and II, which undergo protein-facilitated cis- or trans-splicing in vivo. Each of these RNA-based processes involves proteins of the pentatricopeptide motif-containing family, which does not occur in prokaryotes. Plant-specific RNA-binding proteins may underpin the adaptation of the chloroplast to the eukaryotic context.

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Chlamydomonas reinhardtii is a unicellular eukaryotic alga possessing a single chloroplast that is widely used as a model system for the study of photosynthetic processes. This report analyzes the surprising structural and evolutionary features of the completely sequenced 203,395-bp plastid chromosome. The genome is divided by 21.2-kb inverted repeats into two single-copy regions of approximately 80 kb and contains only 99 genes, including a full complement of tRNAs and atypical genes encoding the RNA polymerase. A remarkable feature is that >20% of the genome is repetitive DNA: the majority of intergenic regions consist of numerous classes of short dispersed repeats (SDRs), which may have structural or evolutionary significance. Among other sequenced chlorophyte plastid genomes, only that of the green alga Chlorella vulgaris appears to share this feature. The program MultiPipMaker was used to compare the genic complement of Chlamydomonas with those of other chloroplast genomes and to scan the genomes for sequence similarities and repetitive DNAs. Among the results was evidence that the SDRs were not derived from extant coding sequences, although some SDRs may have arisen from other genomic fragments. Phylogenetic reconstruction of changes in plastid genome content revealed that an accelerated rate of gene loss also characterized the Chlamydomonas/Chlorella lineage, a phenomenon that might be independent of the proliferation of SDRs. Together, our results reveal a dynamic and unusual plastid genome whose existence in a model organism will allow its features to be tested functionally.

Arabidopsis thaliana chloroplasts contain at least two 3′ to 5′ exoribonucleases, polynucleotide phosphorylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 3′ processing. However, the observed maturation defects do not affect chloroplast translation, suggesting that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose-containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast-encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unnecessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 5′ and 3′ ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 3′ extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloroplast ribosomal RNAs leads to a reduction in the number of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development.

We identify and functionally characterize MRL1, a conserved nuclear-encoded regulator of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. The nonphotosynthetic mrl1 mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase/oxygenase, and the resulting block in electron transfer is partially compensated by redirecting electrons toward molecular oxygen via the Mehler reaction. This allows continued electron flow and constitutive nonphotochemical quenching, enhancing cell survival during illumination in spite of photosystem II and photosystem I photoinhibition. The mrl1 mutant transcribes rbcL normally, but the mRNA is unstable. The molecular target of MRL1 is the 59 untranslated region of rbcL. MRL1 is located in the chloroplast stroma, in a high molecular mass complex. Treatment with RNase or deletion of the rbcL gene induces a shift of the complex toward lower molecular mass fractions. MRL1 is well conserved throughout the green lineage, much more so than the 10 other pentatricopeptide repeat proteins found in Chlamydomonas. Depending upon the organism, MRL1 contains 11 to 14 pentatricopeptide repeats followed by a novel MRL1-C domain. In Arabidopsis thaliana, MRL1 also acts on rbcL and is necessary for the production/stabilization of the processed transcript, presumably because it acts as a barrier to 59>39 degradation. The Arabidopsis mrl1 mutant retains normal levels of the primary transcript and full photosynthetic capacity.

The Chlamydomonas reinhardtii chloroplast atpB mRNA contains sequences at its 3' end that can form a complex stem/loop structure. Deletions of part or all of this sequence in transformed C. reinhardtii cells led to decreased atpB mRNA accumulation, whereas transcription rates were unaffected. The reduction of mRNA to 20% to 35% of wild-type levels in transformants without 3' stem/loops was correlated with the accumulation of atpB mRNA that was heterogeneous in size. These results indicated that RNA secondary structures function both in mRNA stabilization and in 3' end formation in C. reinhardtii chloroplasts. Furthermore, deletion of the stem/loop resulted in a decrease in the steady-state level of the ATPase beta-subunit to approximately 60% of wild-type levels, suggesting that translational and/or post-translational mechanisms may influence the steady-state level of the atpB gene product.