A genomic approach to the study of Tribolium castaneum genetics, development & evolution

During the last decade, Tribolium castaneum has become the insect of choice for comparative genetics and developmental studies outside of drosophilids. Until recently, most molecular studies have focused on the comparative analysis of early development with a focus on segmentation and homeotic genes. In order to acquire independent knowledge on the genetic basis of insect development, a genomic approach consisting of EST and BAC-ends sequencing projects has been initiated in Tribolium. The EST project resulted in the production of 2,246 random sequences representing 488 non-redundant EST contigs. Of those, 280 sequences were selected, along with 86 independently cloned putative transcription factors, and further characterized by in situ hybridization. Expression analysis led to the identification of at least 25 novel genes putatively involved in diverse aspects of Tribolium embryonic development such as segmentation, appendage development, neurogenesis, myogenesis and terminal patterning. Comparative evolutionary analysis of the EST sequences verified that Tribolium is a slow evolving species when compared to dipterans. As predicted by the neutral theory, the data also revealed that evolutionary rates are a composite measure of both gene and species specific rates. To date, the BAC-ends sequencing project resulted in the production of 8,640 sequences covering 2.9% of the Tribolium genome. A functional analysis of a subset of these BAC-end sequences (BES) allowed the identification of 486 putative ORFs. It is estimated that of the 53,000 BES to be produced, 6,900 ORFs will be found, comprising 18% of the genome. Random sequencing of ESTs and production of BES are shown to be powerful ways to identify new genes, to help mapping the Tribolium genome and to identify coding regions in genomic sequences

Phylogenetic and functional analysis of the Cation Diffusion Facilitator (CDF) family: improved signature and prediction of substrate specificity

BACKGROUND The Cation Diffusion Facilitator (CDF) family is a ubiquitous family of heavy metal transporters. Much interest in this family has focused on implications for human health and bioremediation. In this work a broad phylogenetic study has been undertaken which, considered in the context of the functional characteristics of some fully characterised CDF transporters, has aimed at identifying molecular determinants of substrate selectivity and at suggesting metal specificity for newly identified CDF transporters. RESULTS Representative CDF members from all three kingdoms of life (Archaea, Eubacteria, Eukaryotes) were retrieved from genomic databases. Protein sequence alignment has allowed detection of a modified signature that can be used to identify new hypothetical CDF members. Phylogenetic reconstruction has classified the majority of CDF family members into three groups, each containing characterised members that share the same specificity towards the principally-transported metal, i.e. Zn, Fe/Zn or Mn. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The function of some conserved amino acids was assessed by site-directed mutagenesis in the poplar Zn2+ transporter PtdMTP1 and compared with similar experiments performed in prokaryotic members. An essential structural role can be assigned to a widely conserved glycine residue, while aspartate and histidine residues, highly conserved in putative transmembrane domains, might be involved in metal transport. The potential role of group-conserved amino acid residues in metal specificity is discussed. CONCLUSION In the present study phylogenetic and functional analyses have allowed the identification of three major substrate-specific CDF groups. The metal selectivity of newly identified CDF transporters can be inferred by their position in one of these groups. The modified signature sequence proposed in this work can be used to identify new hypothetical CDF members

β-Diketonate, β-Ketoiminate, and β-Diiminate Complexes of Difluoroboron

A series of β-diketonate, keto(aryl)iminato, and β-bis(aryl)iminato complexes of difluoroboron, twenty in total, have been prepared to assess the impact of chelate ring and aniline substitution on the structural, electrochemical, and photophysical properties of these ubiquitous chelates. DFT (B3LYP/6-31G*) calculations supplemented the experimental results and both demonstrated that replacing oxygen with the more electron-donating aniline groups serves to only fine-tune the electronic properties because both the HOMO and LUMO energies are affected by such substitution. The electronic properties of all compounds are most greatly influenced by the nature of the substituents bound to the carbon portion of the chelate ring. Each difluoroboron complex undergoes two ligand-based, one-electron reductions where the first reduction potential becomes less favorable with increasing aniline substitution. Similarly, replacing oxygen with the more electron-donating aniline groups gives rise to slightly red-shifted absorption and emission processes. Substitution on the aniline ring has little, if any, influence on the electronic properties of the resultant complexes