Aurintricarboxylic acid (ATA) is a general inhibitor of nucleases. ATA has been shown to inhibit the following enzymes in vitro: DNAse I, RNAse A, S1 nuclease, exonuclease III, and restriction endonucleases Sal I, Bam HI, Pst I and Sma I. The observed inhibition is consistent with the proposal by Blumenthal and Landers (BBRC 55, 680, 1973) that most nucleic acid binding proteins will be sensitive to ATA. The action of ATA as a nuclease inhibitor can be used to advantage in the isolation of cellular nucleic acids.

We have compared therbcL andatpB transcription units from spinach, maize, and pea. In most cases multiple transcripts were found for a given chloroplast gene. The 5' termini of these transcripts were determined by S1 nuclease protection and primer extension analyses. TherbcL transcripts have 5' termini 178-179 and 64 nucleotides (spinach), 300 and 59-63 nucleotides (maize), and 178 and 65 nucleotides (pea) upstream from their respective protein coding regions. TheatpB transcripts have 5' termini (453-454, 272-273, 179, and 99 nucleotides (spinach), 298-302 nucleotides (maize), and 351-355 nucleotides (pea) upstream from their respective protein coding regions. The intergenic distance between therbcL andatpB genes is relatively constant (152 to 157 base pairs) among the three chloroplast genomes. In spinach, maize, and pea, the 80 base pairs surrounding the 5' end of therbcL gene (±40 base pairs) have 85% sequence homology. Similarly, the 60 base pairs preceding theatpB gene have 48% sequence homology. Both genes have '-10' and '-35' regions that resemble the prokaryotic consensus promoter sequence. The larger, but not smaller,rbcL transcripts from spinach and pea can be labeled with alpha-(32)P-GTP by guanylyltransferase. These data suggest that DNA sequences 178-179 (spinach), 300 (maize), and 178 (pea) base pairs before therbcL protein coding regions represent sites of transcription initiation. The sequences 59-65 base pairs before therbcL protein coding regions may correspond to sites of RNA cleavage.

We have developed an homologous in vitro system from spinach chloroplasts that correctly initiates transcription of the plastid genes for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) and the beta subunit of the plastid ATPase (atpB). The transcriptionally active extracts from spinach chloroplasts require circular DNA templates for specific initiation. The RNA polymerase activity is insensitive to rifampicin. The extent of transcription in vitro is a function of the extract:template ratio. The efficiency of the rbcL transcription in vitro is more than one transcript per one hundred templates per hour.

To determine whether chloroplast RNA polymerase will accurately terminate transcription in vitro, we have fused the spinach chloroplast rbcL promoter to the 3' end of the rbcL gene as well as to various factor independent transcription terminators from E. coli. Transcription of the rbcL minigene did not result in production of the expected 265 nucleotide RNA. However, the spinach chloroplast RNA polymerase did terminate transcription with varying efficiency at the thra, rrnB, rrnC and gene 32 terminators. The most efficient transcription termination was observed for the threonine attenuator. For each of the prokaryotic terminators, the chloroplast enzyme ceased transcription at essentially the same position as the E. coli RNA polymerase. These data indicate that the transcription termination process in chloroplasts has some features in common with the mechanism used in prokaryotes.

We present a new approach to edit both mitochondrial and chloroplast genomes. Organelles have been considered off-limits to CRISPR due to their impermeability to most RNA and DNA. This has prevented applications of Cas9/gRNA-mediated genome editing in organelles while the tool has been widely used for engineering of nuclear DNA in a number of organisms in the last several years. To overcome the hurdle, we designed a new approach to enable organelle genome editing. The plasmids, designated “Edit Plasmids,” were constructed with two expression cassettes, one for the expression of Cas9, codon-optimized for each organelle, under promoters specific to each organelle, and the other cassette for the expression of guide RNAs under another set of promoters specific to each organelle. In addition, Edit Plasmids were designed to carry the donor DNA for integration between two double-strand break sites induced by Cas9/gRNAs. Each donor DNA was flanked by the regions homologous to both ends of the integration site that were short enough to minimize spontaneous recombination events. Furthermore, the donor DNA was so modified that it did not carry functional gRNA target sites, allowing the stability of the integrated DNA without being excised by further Cas9/gRNAs activity. Edit Plasmids were introduced into organelles through microprojectile transformation. We confirmed donor DNA insertion at the target sites facilitated by homologous recombination only in the presence of Cas9/gRNA activity in yeast mitochondria and Chlamydomonas chloroplasts. We also showed that Edit Plasmids persist and replicate in mitochondria autonomously for several dozens of generations in the presence of the wild-type genomes. Finally, we did not find insertions and/or deletions at one of the Cas9 cleavage sites in Chloroplasts, which are otherwise hallmarks of Cas9/gRNA-mediated non-homologous end joining (NHEJ) repair events in nuclear DNA. This is consistent with previous reports of the lack of NHEJ repair system in most bacteria, which are believed to be ancestors of organelles. This is the first demonstration of CRISPR-mediated genome editing in both mitochondria and chloroplasts in two distantly related organisms. The Edit Plasmid approach is expected to open the door to engineer organelle genomes of a wide range of organisms in a precise fashion.

No abstract

No abstract

The large subunit gene (rbcL) of ribulose 1,5-bisphosphate carboxylase was transcribed in vitro by using maize and pea chloroplast extracts and a cloned plastid DNA template containing 172 base pairs (bp) of the maize rbcL protein-coding region and 791 bp of upstream sequences. Three major in vitro RNA species were synthesized which correspond to in vivo maize rbcL RNAs with 5' termini positioned 300, 100 to 105, and 63 nucleotides upstream of the protein-coding region. A deletion of 109 bp, including the "-300" 5' end (the 5' end at position -300), depressed all rbcL transcription in vitro. A plasmid DNA containing this 109-bp fragment was sufficient to direct correct transcription initiation in vitro. A cloned template, containing 191 bp of plastid DNA which includes the -105 and -63 rbcL termini, did not support transcription in vitro. Exogenously added -300 RNA could be converted to the -63 transcript by maize chloroplast extract. These results established that the -300 RNA is the primary maize rbcL transcript, the -63 RNA is a processed form of the -300 transcript, and synthesis of the -105 RNA is dependent on the -300 region. The promoter for the maize rbcL gene is located within the 109 bp flanking the -300 site. Mutagenesis of the 109-bp chloroplast sequence 11 bp upstream of the -300 transcription initiation site reduced rbcL promoter activity in vitro.Ribulose 1,5-bisphosphate carboxylase (RUBISCO) catalyzes CO2 fixation, the first step of the Calvin cycle (40). In higher plants, the chloroplast enzyme is composed of eight identical, catalytic polypeptides of 50,000 to 60,000 kilodaltons and eight smaller polypeptides of 12,000 to 20,000 kilodaltons (2). The small subunit, whose function is unknown, is encoded in the nucleus by a small multigene family (7,14,19). The large subunit gene (rbcL) is present as a single copy on the multicopy chloroplast genome (5). Under most conditions, the synthesis of the two RUBISCO subunits is coordinated (55) and regulated by light (15, 50) and cytokinins (23). In maize and other C4 plants, RUBISCO expression is leaf cell-type specific (30). The maize mRNAs encoding the two subunits are present in bundle sheath cells but not in mesophyll cells, which assimilate CO2 via ribulose 1,5-bisphosphate and C4 dicarboxylic acids, respectively (11,35).In chloroplast genomes of higher plants, the genes for the large subunit of RUBISCO and the beta subunit of ATP synthetase (atpB) are adjacent and transcribed divergently (31, 60). The 5' ends of the two genes are separated by approximately 150 base pairs (bp) (52). Northern hybridization analyses of transcripts from a broad spectrum of angiosperms indicate that the rbcL gene is generally transcribed as a 1.6-kilobase mRNA, although larger transcripts are also observed (46). Si nuclease protection experiments have revealed the presence of two 5' termini for the rbcL RNAs of maize, barley, spinach, and peas (16,21,41,49,62.) Tobacco has a single rbcL RNA with its 5' end at position -180 relative to the coding region (53). In maize ...