Allozyme genotypes of Dyckia Brevifolia

Allozyme genotypes of reproductive rosettes and mating system of Dyckia brevifolia Baker

The Contributions of Wobbling and Superwobbling to the Reading of the Genetic Code

<div><p>Reduced bacterial genomes and most genomes of cell organelles (chloroplasts and mitochondria) do not encode the full set of 32 tRNA species required to read all triplets of the genetic code according to the conventional wobble rules. Superwobbling, in which a single tRNA species that contains a uridine in the wobble position of the anticodon reads an entire four-fold degenerate codon box, has been suggested as a possible mechanism for how tRNA sets can be reduced. However, the general feasibility of superwobbling and its efficiency in the various codon boxes have remained unknown. Here we report a complete experimental assessment of the decoding rules in a typical prokaryotic genetic system, the plastid genome. By constructing a large set of transplastomic knock-out mutants for pairs of isoaccepting tRNA species, we show that superwobbling occurs in all codon boxes where it is theoretically possible. Phenotypic characterization of the transplastomic mutant plants revealed that the efficiency of superwobbling varies in a codon box-dependent manner, but—contrary to previous suggestions—it is independent of the number of hydrogen bonds engaged in codon-anticodon interaction. Finally, our data provide experimental evidence of the minimum tRNA set comprising 25 tRNA species, a number lower than previously suggested. Our results demonstrate that all triplets with pyrimidines in third codon position are dually decoded: by a tRNA species utilizing standard base pairing or wobbling and by a second tRNA species employing superwobbling. This has important implications for the interpretation of the genetic code and will aid the construction of synthetic genomes with a minimum-size translational apparatus.</p> </div

Targeted inactivation of the two plastid <i>trnT</i> genes.

<p>(A) Physical map of the <i>trnT-UGU</i> containing region in the tobacco plastid genome (ptDNA; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Shinozaki1" target="_blank">[44]</a>). Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. Selected restriction sites used for cloning or RFLP analysis are indicated. The hybridization probe and the expected sizes of detected DNA fragments are also shown. Introns are represented by open boxes. (B) Map of the transformed plastid genome in <i>ΔtrnT-UGU</i> transplastomic plants. The selectable marker gene <i>aadA</i> (grey box) is inserted into the <i>trnT-UGU</i> gene in the same transcriptional orientation. (C) Physical map of the <i>trnT-GGU</i> containing region in the tobacco ptDNA. (D) Map of the transformed plastid genome in Δ<i>trnT-GGU</i> plants. (E) RFLP analysis of Δ<i>trnT-UGU</i> transplastomic lines. Independently generated transplastomic lines are designated by Arabic numerals following the tRNA gene name. All transplastomic lines remain heteroplasmic and show both the 1.9 kb wild type-specific hybridization band and the 3.1 kb band diagnostic of the transformed plastid genome. Wt: wild type. (F) RFLP analysis of Δ<i>trnT-GGU</i> transplastomic plants. All lines are homoplasmic and show exclusively the 3.7 kb band diagnostic of the transgenic ptDNA. (G) Seed assays to confirm heteroplasmy of Δ<i>trnT-UGU</i> plants and homoplasmy of Δ<i>trnT-GGU</i> plants. Seeds were germinated in the absence or in the presence of spectinomycin. Δ<i>trnT-UGU</i> plants produce mostly antibiotic-sensitive seedlings and a few antibiotic-resistant seedlings, as expected for a heteroplasmic situation. Moreover, many of the resistant seedlings are variegated indicating their composition of tissues possessing and tissues lacking the transgenic plastid genome. In contrast, the Δ<i>trnT-GGU</i> lines produce homogeneous antibiotic-resistant progeny, confirming their homoplasmic status. (H) Analysis of tRNA-Thr(GGU) accumulation in the wild type, a heteroplasmic Δ<i>trnT-UGU</i> line and a homoplasmic Δ<i>trnT-GGU</i> line by northern blotting. Hybridization of electrophoretically separated RNA isolated from purified chloroplasts to a plastid <i>trnT-GGU</i> probe confirms complete lack of mature tRNA-Thr(GGU) in the Δ<i>trnT-GGU</i> homoplasmic knock-out line, whereas its accumulation is unaltered in the heteroplasmic Δ<i>trnT-UGU</i> line. Note accumulation of a ∼1.5 kb hybridizing RNA species in the Δ<i>trnT-GGU</i> line, which corresponds to the tRNA-Thr(GGU) disrupted with the <i>aadA</i> cassette. To control for RNA loading, part of the ethidium bromide-stained gel (containing the two largest 23S rRNA hidden break products) prior to blotting is also shown.</p

Analysis of plastid protein synthesis and photosynthetic parameters in Δ<i>trnL-CAA</i>, Δ<i>trnS-GGA</i>, Δ<i>trnT-GGU</i>, and Δ<i>trnV-GAC</i> plants.

<p>(A) Assessment of RbcL protein accumulation by western blotting using a specific anti-RbcL antibody. For semiquantitative analysis, a dilution series of wild-type protein was loaded. Consistent with the differences in the severity of the growth phenotypes (cf. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen-1003076-g005" target="_blank">Figure 5</a>), the Δ<i>trnV-GAC</i> and Δ<i>trnL-CAA</i> mutants show the smallest reduction in RbcL accumulation, whereas the Δ<i>trnS-GGA</i> mutant and especially the Δ<i>trnT-GGU</i> mutant are more strongly affected, with RbcL levels being in the range of the 25% dilution of the wild-type sample in the Δ<i>trnT-GGU</i> mutant. Reduced synthesis of chloroplast proteins is also apparent, when total plant protein samples are separated by gel electrophoresis and stained with Coomassie (lower panel). The two most abundant proteins (representing the large and small subunits of Rubisco, RbcL and RbcS) are indicated. Reduced abundance of chloroplast proteins in the Δ<i>trnS-GGA</i> and Δ<i>trnT-GGU</i> mutants also becomes evident by a stronger background staining (coming from a large number of lower abundant nuclear-encoded proteins). (B) Analysis of chlorophyll content, chlorophyll a∶b ratio and the maximum quantum efficiency of photosystem II (F_V/F_M) in wild-type plants and homoplasmic transplastomic tRNA knock-out mutants. Datasets are shown for plants grown under ∼80 µE m⁻² s⁻¹ light intensity. Young Δ<i>trnL-CAA</i>, Δ<i>trnS-GGA</i> and Δ<i>trnV-GAC</i> plants were measured after 7 weeks of growth, Δ<i>trnT-GGU</i> plants after 30 weeks (when they had reached a similar size as the other lines after 7 weeks). Mature Δ<i>trnL-CAA</i>, Δ<i>trnS-GGA</i> and Δ<i>trnV-GAC</i> plants were measured after 20 weeks of growth, Δ<i>trnT-GGU</i> plants were raised at ∼20 µE m⁻² s⁻¹ for 40 weeks and then grown for 4 weeks at ∼80 µE m⁻² s⁻¹. The fourth leaf from the top was analyzed. For each plant line, three different plants were measured. F_V/F_M represents the maximum quantum efficiency of PSII in the dark adapted state. The error bars indicate the standard deviation, statistically significant differences from the wild type (p<0.05; Student's t-test) are indicated by asterisks.</p

Targeted deletion of the plastid <i>trnS-GGA</i> gene.

<p>(A) Physical map of the region in the tobacco plastid genome harboring the <i>trnS-GGA</i> gene. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. The bent arrows indicate the borders of the transformation plasmid. Restriction sites used for RFLP analysis are indicated. The hybridization probe and the expected sizes of detected DNA fragments are also shown. Introns are represented by open boxes. (B) Map of the transformed plastid genome in Δ<i>trnS-GGA</i> transplastomic lines. The <i>aadA</i> cassette is shown as grey box. (C) RFLP analysis of Δ<i>trnS-GGA</i> plastid transformants. All lines are homoplasmic and show exclusively the 2.9 kb band diagnostic of the transplastome. Independently generated transplastomic lines are designated by Arabic numerals following the tRNA gene name, the following capital letter indicates an individual plant. Wt: wild type. (D) tRNA-Ser(GGA) accumulation in wild-type plants and Δ<i>trnS-GGA</i> transplastomic lines. Hybridization to a plastid <i>trnS-GGA</i> probe reveals weak signals in all transplastomic plants, which are presumably caused by cross-hybridization to the mitochondrial <i>trnS-GGA</i>. To control for RNA loading, part of the ethidium bromide-stained gel (showing the two largest 23S rRNA hidden break products) prior to blotting is also shown. (E) tRNA-Ser(GGA) accumulation in isolated chloroplasts of wild type plants and a Δ<i>trnS-GGA</i> knock-out line. Hybridization to the plastid <i>trnS-GGA</i> probe confirms complete absence of the tRNA from the transplastomic line. (F) Confirmation of homoplasmy of the Δ<i>trnS-GGA</i> lines by inheritance assays. Germination of seeds harvested from transplastomic plants on spectinomycin-containing medium results in a homogeneous population of green antibiotic-resistant seedlings. (G) Comparison with spectinomycin-sensitive wild-type seedlings. Antibiotic sensitivity is evidenced by the white phenotype of all seedlings.</p

Decoding of the 64 triplets of the genetic code in plastids.

<p>Codon recognition by standard Watson-Crick base pairing, wobbling and/or superwobbling is indicated by the nucleotide in the wobble position of the anticodon of the tRNA species that can decode the triplet. Essential tRNA species are indicated in bold, non-essential tRNAs in normal font. The codon usage in plastids of <i>Nicotiana tabacum</i> is shown on a greyscale. Superscript numbers and indices indicate nucleoside modifications in the wobble position (N₃₄) of the anticodon of the tRNA species. ¹: 2′-O-methyluridine <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Pillay1" target="_blank">[26]</a>; ²: 2′-O-methylcytidine <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Pillay1" target="_blank">[26]</a>; ³: unknown modification <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Pfitzinger1" target="_blank">[23]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Sprouse1" target="_blank">[45]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Francis1" target="_blank">[46]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Francis2" target="_blank">[47]</a>; ⁴: inosine <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Karcher1" target="_blank">[28]</a>; ⁵: lysidine <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Francis3" target="_blank">[48]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Muramatsu1" target="_blank">[49]</a>; ⁶: 5-carboxymethylaminomethyl uridine (cmnm⁵U; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Schn1" target="_blank">[50]</a>); ⁷: 5-methylaminomethyl-2-thiouridine (mam⁵s²U; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Schn2" target="_blank">[51]</a>; <a href="http://trnadb.bioinf.uni-leipzig.de/" target="_blank">http://trnadb.bioinf.uni-leipzig.de/</a>); ^*: modification status of the wobble uridine unknown (RNA sequence not determined); -: stop codon.</p

Targeted deletion of the plastid <i>trnL-CAA</i> gene.

<p>(A) Physical map of the region in the tobacco plastid genome containing the gene for <i>trnL-CAA</i>. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. Selected restriction sites used for cloning and RFLP analysis are indicated. The hybridization probe and the expected sizes of detected DNA fragments are also shown. Introns are represented by open boxes. (B) Map of the transformed plastid genome in Δ<i>trnL-CAA</i> transplastomic plants. The <i>aadA</i> cassette replacing <i>trnL-CAA</i> is shown as grey box. (C) RFLP analysis of Δ<i>trnL-CAA</i> plastid transformants. All lines are homoplasmic and show exclusively the 3.1-kb band diagnostic of the transplastome. Wt: wild type. (D) tRNA-Leu(CAA) accumulation in the wild type and Δ<i>trnL-CAA</i> lines assessed by northern blotting. Hybridization to a plastid <i>trnL-CAA</i> probe confirms complete absence of the tRNA from homoplasmic knock-out lines. The ethidium bromide-stained agarose gel prior to blotting is also shown. (E) Confirmation of the homoplasmic state of the Δ<i>trnL-CAA</i> lines by inheritance assays. Germination of seeds from transplastomic plants on spectinomycin-containing medium results in a homogeneous population of green antibiotic-resistant seedlings. (F) Wild-type seedlings are sensitive to spectinomycin and bleach out in the presence of the antibiotic.</p

Reference graph track showing observed coverage values.

<p>Different colors show the minimum (light blue), mean (blue), and maximum (dark blue) observed coverage values for all genomic regions (data aggregation above 100 bp). <i>Araucaria angustifolia</i>, <i>Araucaria bidwilli</i>, and <i>Podocarpus lambertii</i> sequence reads were mapped on <i>Podocarpus totara</i>; <i>Pinus patula</i> sequence reads were mapped on <i>Pinus thunbergii</i>.</p

Chloroplast DNA visualization of <i>Araucaria angustifolia</i> in 0.7% agarose gel stained with ethidium bromide.

<p>(A) Ladder 1 kb and cpDNA isolated with modified high salt method; (B) Ladder 1 kb and cpDNA isolated with sucrose method; (C) Ladder 1 kb and cpDNA isolated with high salt plus saline Percoll method.</p

Flowchart showing the major steps for chloroplast isolation according to high salt plus saline Percoll.

<p>Flowchart showing the major steps for chloroplast isolation according to high salt plus saline Percoll.</p