Hydrogen isotopic differences between C3 and C4 land plant lipids: consequences of compartmentation in C4 photosynthetic chemistry and C3 photorespiration

The ²H/¹H ratio of carbon‐bound H in biolipids holds potential for probing plant lipid biosynthesis and metabolism. The biochemical mechanism underlying the isotopic differences between lipids from C₃ and C₄ plants is still poorly understood. GC‐pyrolysis‐IRMS (gas chromatography‐pyrolysis‐isotope ratio mass spectrometry) measurement of the ²H/¹H ratio of leaf lipids from controlled and field grown plants indicates that the biochemical isotopic fractionation (ε²H_{lipid_biochem}) differed between C₃ and C₄ plants in a pathway‐dependent manner: ε²H_C4 > ε²H_C3 for the acetogenic pathway, ε²H_C4 < ε²H_C3 for the mevalonic acid pathway and the 1‐deoxy‐D‐xylulose 5‐phosphate pathway across all species examined. It is proposed that compartmentation of photosynthetic CO₂ fixation into C₄ mesophyll (M) and bundle sheath (BS) cells and suppression of photorespiration in C₄ M and BS cells both result in C₄ M chloroplastic pyruvate – the precursor for acetogenic pathway – being more depleted in ²H relative to pyruvate in C₃ cells. In addition, compartmentation in C₄ plants also results in (i) the transferable H of NADPH being enriched in ²H in C₄ M chloroplasts compared with that in C₃ chloroplasts for the 1‐deoxy‐D‐xylulose 5‐phosphate pathway pathway and (ii) pyruvate relatively ²H‐enriched being used for the mevalonic acid pathway in the cytosol of BS cells in comparison with that in C₃ cells.     

Molecular recognition of a branched peptide with HIV-1 Rev Response Element (RRE) RNA

Interaction of HIV-1 rev response element (RRE) RNA with its cognate protein, Rev, is critical for HIV-1 replication. Understanding the mode of interaction between RRE RNA and ligands at the binding site can facilitate RNA molecular recognition as well as provide a strategy for developing anti-HIV therapeutics. Our approach utilizes branched peptides as a scaffold for multivalent binding to RRE IIB (high affinity rev binding site) with incorporation of unnatural amino acids to increase affinity via non-canonical interactions with the RNA. Previous high throughput screening of a 46,656-member library revealed several hits that bound RRE IIB RNA in the sub-micromolar range. In particular, the lead compound, 4B3, displayed a K_d value of 410?nM and demonstrated selectivity towards RRE. A ribonuclease protection assay revealed that 4B3 binds to the stem-loop structure of RRE IIB RNA, which was confirmed by SHAPE analysis with 234 nt long NL4-3 RRE RNA. Our studies further indicated interaction of 4B3 with both primary and secondary Rev binding sites.     

Mutations in the ribonuclease H active site of HIV-RT reveal a role for this site in stabilizing enzyme-primer-template binding

The RNase H activity of HIV-RT is coordinated by a catalytic triad (E478, D443, D498) of acidic residues that bind divalent cations. We examined the effect of RNase H deficient E(478)-->Q and D(549)-->N mutations that do not alter polymerase activity on binding of enzyme to various nucleic acid substrates. Binding of the mutant and wild-type enzymes to various nucleic acid substrates was examined by determining dissociation rate constants (k(off)) by titrating both Mg(2+) and salt concentrations. In agreement with the unaltered polymerase activity of the mutant, the k(off) values for the wild-type and mutant enzymes were essentially identical using DNA-DNA templates in the presence of 6 mM Mg(2+). However, with lower concentrations of Mg(2+) and in the absence of Mg(2+), although both enzymes dissociated more rapidly, the mutant enzymes dissociated several-fold more slowly than the wild type. This was also observed on RNA-DNA templates. These results indicate that alterations in residues essential for Mg(2+) binding have a pronounced positive effect on enzyme-template stability and that the negative residues in the RNase H region of the enzyme have a negative influence on binding in the absence of Mg(2+). In this regard RT is similar to other nucleic acid cleaving enzymes that show enhanced binding upon mutation of active site residues.     

Synergistic Interactions between Drosophila Orthologues of Genes Spanned by De Novo Human CNVs Support Multiple-Hit Models of Autism

Autism spectrum disorders (ASDs) are highly heritable and characterised by deficits in social interaction and communication, as well as restricted and repetitive behaviours. Although a number of highly penetrant ASD gene variants have been identified, there is growing evidence to support a causal role for combinatorial effects arising from the contributions of multiple loci. By examining synaptic and circadian neurological phenotypes resulting from the dosage variants of unique human:fly orthologues in Drosophila, we observe numerous synergistic interactions between pairs of informatically-identified candidate genes whose orthologues are jointly affected by large de novo copy number variants (CNVs). These CNVs were found in the genomes of individuals with autism, including a patient carrying a 22q11.2 deletion. We first demonstrate that dosage alterations of the unique Drosophila orthologues of candidate genes from de novo CNVs that harbour only a single candidate gene display neurological defects similar to those previously reported in Drosophila models of ASD-associated variants. We then considered pairwise dosage changes within the set of orthologues of candidate genes that were affected by the same single human de novo CNV. For three of four CNVs with complete orthologous relationships, we observed significant synergistic effects following the simultaneous dosage change of gene pairs drawn from a single CNV. The phenotypic variation observed at the Drosophila synapse that results from these interacting genetic variants supports a concordant phenotypic outcome across all interacting gene pairs following the direction of human gene copy number change. We observe both specificity and transitivity between interactors, both within and between CNV candidate gene sets, supporting shared and distinct genetic aetiologies. We then show that different interactions affect divergent synaptic processes, demonstrating distinct molecular aetiologies. Our study illustrates mechanisms through which synergistic effects resulting from large structural variation can contribute to human disease.