Evolutionary constraints on the plastid tRNA set decoding methionine and isoleucine

The plastid (chloroplast) genomes of seed plants typically encode 30 tRNAs. Employing wobble and superwobble mechanisms, most codon boxes are read by only one or two tRNA species. The reduced set of plastid tRNAs follows the evolutionary trend of organellar genomes to shrink in size and coding capacity. A notable exception is the AUN codon box specifying methionine and isoleucine, which is decoded by four tRNA species in nearly all seed plants. However, three of these four tRNA genes were lost from the genomes of some parasitic plastid-containing lineages, possibly suggesting that less than four tRNA species could be sufficient to decode the triplets in the AUN box. To test this hypothesis, we have performed knockout experiments for the four AUN-decoding tRNAs in tobacco (Nicotiana tabacum) plastids. We find that all four tRNA genes are essential under both autotrophic and heterotrophic growth conditions, possibly suggesting tRNA import into plastids of parasitic plastid-bearing species. Phylogenetic analysis of the four plastid tRNA genes reveals striking conservation of all those bacterial features that are involved in discrimination between the different tRNA species containing CAU anticodons

Can infants develop meningitis in the absence of bacteremia in the first ninety days of life? A retrospective chart review

The overall incidence of meningitis in infants 0-90 days is low; however, it remains a serious cause of morbidity and mortality among affected patients. It is standard of care to perform lumbar punctures as part of the work-up of fever in the first four weeks of life and sick-looking babies up to the age of 90 days. This particular procedure is often refused by parents, and physicians are left to predict the possibility of meningitis based on blood culture results.The aim of this study is to determine whether it would be safe to rule out meningitis based on a negative blood culture in this age group

Can infants develop meningitis in the absence of bacteremia in the first ninety days of life? A retrospective chart review

The overall incidence of meningitis in infants 0-90 days is low; however, it remains a serious cause of morbidity and mortality among affected patients. It is standard of care to perform lumbar punctures as part of the work-up of fever in the first four weeks of life and sick-looking babies up to the age of 90 days. This particular procedure is often refused by parents, and physicians are left to predict the possibility of meningitis based on blood culture results. The aim of this study is to determine whether it would be safe to rule out meningitis based on a negative blood culture in this age group.</jats:p

Nonessential Plastid-Encoded Ribosomal Proteins in Tobacco: A Developmental Role for Plastid Translation and Implications for Reductive Genome Evolution[W][OA]

Of seven plastid genome-encoded ribosomal proteins analyzed by reverse genetics in tobacco (Nicotiana tabacum), two were found to be nonessential: S15 and L36. Elimination of ribosomal protein S15 produced normal plants, but elimination of L36 resulted in mutants with reduced apical dominance and strikingly altered leaf morphology, uncovering a role for plastid translational activity in plant development

Phytochemical Composition, Antioxidant and Antiproliferative Activities of <i>Citrus hystrix</i>, <i>Citrus limon</i>, <i>Citrus pyriformis,</i> and <i>Citrus microcarpa</i> Leaf Essential Oils against Human Cervical Cancer Cell Line

The essential oil derived from Citrus plants has long been used for medicinal purposes, due to its broad spectrum of therapeutic characteristics. To date, approximately 162 Citrus species have been identified, and many investigational studies have been conducted to explore the pharmacological potential of Citrus spp. oils. This study investigated the volatile constituents of essential oil distilled from the leaves of C. hystrix, C. limon, C. pyriformis, and C. microcarpa, using gas chromatography–quadrupole mass spectrometry. A total of 80 secondary compounds were tentatively identified, representing 84.88–97.99% of the total ion count and mainly comprising monoterpene (5.20–76.15%) and sesquiterpene (1.36–27.14%) hydrocarbons, oxygenated monoterpenes (3.91–89.52%) and sesquiterpenes (0.21–38.87%), and other minor chemical classes (0.10–0.52%). In particular, 27 compounds (1.19–39.06%) were detected across all Citrus species. Principal component analysis of the identified phytoconstituents and their relative quantities enabled differentiation of the Citrus leaf oils according to their species, with the loading variables contributing to these metabolic differences being identified. The Citrus leaf oils were tested for their antioxidant and antiproliferative activities using 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The results indicated that C. limon displayed the highest DPPH radical scavenging ability (IC50 value of 29.14 ± 1.97 mg/mL), while C. hystrix exhibited the lowest activity (IC50 value of 279.03 ± 10.37 mg/mL). On the other hand, all the Citrus oils exhibit potent antiproliferative activities against the HeLa cervical cancer cell line, with IC50 values of 11.66 μg/mL (C. limon), 20.41 μg/mL (C. microcarpa), 25.91 μg/mL (C. hystrix), and 87.17 μg/mL (C. pyriformis)

Phytochemical Composition, Antioxidant and Antiproliferative Activities of Citrus hystrix, Citrus limon, Citrus pyriformis, and Citrus microcarpa Leaf Essential Oils against Human Cervical Cancer Cell Line

The essential oil derived from Citrus plants has long been used for medicinal purposes, due to its broad spectrum of therapeutic characteristics. To date, approximately 162 Citrus species have been identified, and many investigational studies have been conducted to explore the pharmacological potential of Citrus spp. oils. This study investigated the volatile constituents of essential oil distilled from the leaves of C. hystrix, C. limon, C. pyriformis, and C. microcarpa, using gas chromatography–quadrupole mass spectrometry. A total of 80 secondary compounds were tentatively identified, representing 84.88–97.99% of the total ion count and mainly comprising monoterpene (5.20–76.15%) and sesquiterpene (1.36–27.14%) hydrocarbons, oxygenated monoterpenes (3.91–89.52%) and sesquiterpenes (0.21–38.87%), and other minor chemical classes (0.10–0.52%). In particular, 27 compounds (1.19–39.06%) were detected across all Citrus species. Principal component analysis of the identified phytoconstituents and their relative quantities enabled differentiation of the Citrus leaf oils according to their species, with the loading variables contributing to these metabolic differences being identified. The Citrus leaf oils were tested for their antioxidant and antiproliferative activities using 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The results indicated that C. limon displayed the highest DPPH radical scavenging ability (IC50 value of 29.14 ± 1.97 mg/mL), while C. hystrix exhibited the lowest activity (IC50 value of 279.03 ± 10.37 mg/mL). On the other hand, all the Citrus oils exhibit potent antiproliferative activities against the HeLa cervical cancer cell line, with IC50 values of 11.66 μg/mL (C. limon), 20.41 μg/mL (C. microcarpa), 25.91 μg/mL (C. hystrix), and 87.17 μg/mL (C. pyriformis).</jats:p

Targeted deletion of the plastid <i>trnV-GAC</i> gene.

<p>(A) Physical map of the region in the tobacco plastid genome containing <i>trnV-GAC</i>. All genes shown transcribed from the left to the right. 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>trnV-GAC</i> lines. The <i>aadA</i> cassette is shown as grey box. (C) RFLP analysis of Δ<i>trnV-GAC</i> plastid transformants. Wt: wild type. (D) tRNA-Val(GAC) accumulation in the wild type and in Δ<i>trnV-GAC</i> lines determined by northern blotting. Hybridization to a plastid <i>trnV-GAC</i> probe confirms complete absence of the tRNA from homoplasmic transplastomic lines. To control for RNA loading, the 18S rRNA-containing part of the ethidium bromide-stained agarose gel prior to blotting is also shown. (E) Homoplasmy of Δ<i>trnV-GAC</i> lines as confirmed by inheritance assays. Transplastomic seeds germinated on spectinomycin-containing yield 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

Phenotypes of transplastomic plants generated with knock-out constructs for the tRNA genes <i>trnT-UGU</i>, <i>trnT-GGU</i>, <i>trnL-CAA</i>, <i>trnS-GGA</i>, and <i>trnV-GAC</i>.

<p>(A) Growth phenotype of a Δ<i>trnT-GGU</i> plant in comparison with a wild-type plant. Plants were grow from seeds in soil (with nitrogen-rich fertilizer) under ∼100 µE m⁻² s⁻¹ light intensity and photographed after 12 weeks. The red arrow points to the Δ<i>trnT-GGU</i> plant. (B) Flowering and seed set of a Δ<i>trnT-GGU</i> plant after 50 weeks of growth under ∼20 µE m⁻² s⁻¹ light intensity. (C) Growth of Δ<i>trnT-GGU</i> and Δ<i>trnT-UGU</i> plants in comparison with a wild-type plant after 6 weeks of growth on sucrose-containing synthetic medium (left pictures) and a subsequent 16 day growth period in soil (right pictures). Note the typical leaf-loss phenotype in the Δ<i>trnT-UGU</i> plant indicating essentiality of the tRNA gene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Rogalski2" target="_blank">[19]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003076#pgen.1003076-Rogalski3" target="_blank">[20]</a>. (D) Phenotypes of Δ<i>trnL-CAA</i>, Δ<i>trnS-GGA</i> and Δ<i>trnV-GAC</i> transplastomic plants in comparison with a wild-type plant after growth for 13 weeks in soil under standard greenhouse conditions (average light intensity: 200 µE m⁻² s⁻¹). (E) Phenotype of Δ<i>trnL-CAA</i>, Δ<i>trnS-GGA</i>, Δ<i>trnT-GGU</i> and Δ<i>trnV-GAC</i> transplastomic plants in comparison with a wild-type plant after growth for 20 weeks under low-light conditions (∼80 µE m⁻² s⁻¹).</p

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