Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants

A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa, showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.      

Solution structure of ApaG from Xanthomonas axonopodis pv. citri reveals a fibronectin-3 fold

ApaG proteins are found in a wide variety of bacterial genomes but their function is as yet unknown. Some eukaryotic proteins involved in protein-protein interactions, such as the human polymerase delta-interacting protein (PDIP38) and the F Box A (FBA) proteins, contain ApaG homology domains. We have used NMR to determine the solution structure of ApaG protein from the plant pathogen Xanthomonas axonopodis pv. citri (ApaG(Xac)) with the aim to shed some light on its molecular function. ApaG(Xac) is characterized by seven antiparallel beta strands forming two beta sheets, one containing three strands (ABE) and the other four strands (GFCC'). Relaxation measurements indicate that the protein has a quite rigid structure. In spite of the presence of a putative GXGXXG pyrophosphate binding motif ApaG(Xac) does not bind ATP or GTP, in vitro. On the other hand, ApaG(Xac) adopts a fibronectin type III (Fn3) fold, which is consistent with the hypothesis that it is involved in mediating protein-protein interactions. The fact that the proteins of ApaG family do not display significant sequence similarity with the Fn3 domains found in other eukaryotic or bacterial proteins suggests that Fn3 domain may have arisen earlier in evolution than previously estimated.     

Molecular and morphological diversity of on-farm hazelnut (Corylus avellana L.) landraces from southern Europe and their role in the origin and diffusion of cultivated germplasm

Hazelnut (Corylus avellana L.) is a traditional nut crop in southern Europe. Germplasm exploration conducted on-farm in five countries (Portugal, Spain, Italy, Slovenia, and Greece) identified 77 landraces. The present work describes phenotypic variation in nut and husk traits and investigates genetic relationships using ten simple sequence repeat (SSR) markers among these landraces, 57 well-known references cultivars, and 19 wild accessions. Among the 77 landraces, 42 had unique fingerprints while 35 showed a SSR profile identical to a known cultivar. Among the 42 unique landraces, morphological observations revealed high phenotypic diversity, and some had characteristics appreciated by the market such as nut round and caliber. Analysis of genetic relationships and population structure allowed investigation of the origin and spread of the cultivated germplasm in southern Europe. Our results indicate the existence of three primary centers of diversity in the Mediterranean basin: northwestern Spain (Tarragona) and southern Italy (Campania) in the West and Black Sea (Turkey) in the East. Moreover, the data suggest the existence of secondary gene pools in the Iberian (Asturias) and Italian (Liguria and Latium) Peninsulas, where local varieties were recently domesticated from wild forms and/or from introduced ancient domesticated varieties.     

Molecular imaging of epidermal growth factor receptor kinase activity

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is commonly altered in different tumor types, leading to abnormally regulated kinase activity and excessive activation of downstream signaling cascades, including cell proliferation, differentiation, and migration. To investigate the EGFR signaling events in real time and in living cells and animals, here we describe a multidomain chimeric reporter whose bioluminescence can be used as a surrogate for EGFR kinase activity. This luciferase-based reporter was developed in squamous cell carcinoma cells (UMSCC1) to generate a cancer therapy model for imaging EGFR. The reporter is designed to act as a phosphorylated substrate of EGFR and reconstitutes luciferase activity when it is not phosphorylated, thereby providing a robust indication of EGFR inhibition. We validated the reporter in vitro and demonstrated that its activity could be differentially modulated by EGFR tyrosine kinase inhibition with erlotonib or receptor activation with epidermal growth factor. Further experiments in vivo demonstrated quantitative and dynamic monitoring of EGFR tyrosine kinase activity in xenograft. Results obtained from these studies provide unique insight into pharmacokinetics and pharmacodynamics of agents that modulate EGFR activity, revealing the usefulness of this reporter in evaluating drug availability and cell targeting in both living cells and mouse models.