A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

19F nuclear magnetic resonance studies of the coat protein of bacteriophage M13 in synthetic phospholipid vesicles and deoxycholate micelles.

The nonlytic, filamentous coliphage M13 offers an excellent model system for the study of membrane-protein interactions. We prepare derivatives of the protein containing fluorine-labeled amino acids and use 19F nuclear magnetic resonance (NMR) to study the protein in both deoxycholate micelles and phospholipid vesicles. We have previously described the in vivo preparation of an m-fluorotyrosyl derivative of M13 coat protein and also a method for incorporation of high levels of this protein into small, uniformly sized phospholipid vesicles of defined composition. Herein we describe the in vivo preparation and the characterization of an m-fluorophenylalanine derivative. We simultaneously compare the environment and mobility of the tyrosine and phenylalanine residues (the former in the hydrophobic region of the protein and the latter in the hydrophilic regions) as influenced by bile salt detergent or lipid interactions.     

Chick embryo fibroblasts produce two forms of hyaluronidase

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.     

A multilocus genealogical approach to phylogenetic species recognition in the model eukaryote Neurospora

To critically examine the relationship between species recognized by phylogenetic and reproductive compatibility criteria, we applied phylogenetic species recognition (PSR) to the fungus in which biological species recognition (BSR) has been most comprehensively applied, the well-studied genus Neurospora. Four independent anonymous nuclear loci were characterized and sequenced from 147 individuals that were representative of all described outbreeding species of Neurospora. We developed a consensus-tree approach that identified monophyletic genealogical groups that were concordantly supported by the majority of the loci, or were well supported by at least one locus but not contradicted by any other locus. We recognized a total of eight phylogenetic species, five of which corresponded with the five traditional biological species, and three of which were newly discovered. Not only were phylogenetic criteria superior to traditional reproductive compatibility criteria in revealing the full species diversity of Neurospora, but also significant phylogenetic subdivisions were detected within some species. Despite previous suggestions of hybridization between N. crassa and N. intermedia in nature, and the fact that several putative hybrid individuals were included in this study, no molecular evidence in support of recent interspecific gene flow or the existence of true hybrids was observed. The sequence data from the four loci were combined and used to clarify how the species discovered by PSR were related. Although species-level clades were strongly supported, the phylogenetic relationships among species remained difficult to resolve, perhaps due to conflicting signals resulting from differential lineage sorting.     

An isotopic perspective on the transport of Byzantine mining camp laborers into southwestern Jordan

The Byzantine Empire managed a complex administrative network that controlled the mining and processing of natural resources from within its boundaries. Scholars relying upon archeological and textual evidence debate the level of imperial involvement in these ventures, particularly in the provinces. Ancient sources note that many mining camps, for instance, purportedly contained criminal laborers and elite administrators transported from distant locales, indicating significant organization and expenditures by the imperial administration to run the mines. This analysis explores the presence of these nonlocal individuals in a cemetery associated with the third to seventh century A.D. mining camp of Phaeno (Faynan), located in modern Jordan. Strontium isotope analysis of 31 burials indicates that most spent their childhood in a similar geological region as Phaeno, implying that they were locally born. The δ¹⁸O results mirror the homogeneous ⁸⁷Sr/⁸⁶Sr values, confirming a local origin for most of the sample. Isotopic evidence therefore suggests that the Phaeno mining camp was largely a local operation, contrary to the picture presented in textual sources, although the profits surely padded imperial coffers. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Mitochondrial Profiling of Acute Myeloid Leukemia in the Assessment of Response to Apoptosis Modulating Drugs

BH3 profiling measures the propensity of transformed cells to undergo intrinsic apoptosis and is determined by exposing cells to BH3-mimicking peptides. We hypothesized that basal levels of prosurvival BCL-2 family proteins may modulate the predictive power of BH3 profiling and termed it mitochondrial profiling. We investigated the correlation between cell sensitivity to apoptogenic agents and mitochondrial profiling, using a panel of acute myeloid leukemias induced to undergo apoptosis by exposure to cytarabine, the BH3 mimetic ABT-199, the MDM2 inhibitor Nutlin-3a, or the CRM1 inhibitor KPT-330. We found that the apoptogenic efficacies of ABT-199 and cytarabine correlated well with BH3 profiling reflecting BCL2, but not BCL-XL or MCL-1 dependence. Baseline BCL-2 protein expression analysis increased the ability of BH3 profiling to predict resistance mediated by MCL-1. By utilizing engineered cells with overexpression or knockdown of BCL-2 family proteins, Ara-C was found to be independent, while ABT-199 was dependent on BCL-XL. BCL-2 and BCL-XL overexpression mediated resistance to KPT-330 which was not reflected in the BH3 profiling assay, or in baseline BCL-2 protein levels. In conclusion, mitochondrial profiling, the combination of BH3 profiling and prosurvival BCL-2 family protein analysis, represents an improved approach to predict efficacy of diverse agents in AML and may have utility in the design of more effective drug combinations.     

GENOME‐WIDE INVESTIGATION OF REPRODUCTIVE ISOLATION IN EXPERIMENTAL LINEAGES AND NATURAL SPECIES OF NEUROSPORA: IDENTIFYING CANDIDATE REGIONS BY MICROARRAY‐BASED GENOTYPING AND MAPPING

Inherent incompatibilities between genetic components from genomes of different species may cause intrinsic reproductive isolation. In evolution experiments designed to instigate speciation in laboratory populations of the filamentous fungus Neurospora, we previously discovered a pair of incompatibility loci (dfe and dma) that interact negatively to cause severe defects in sexual reproduction. Here we show that the dfe‐dma incompatibility also is a significant cause of genetic isolation between two naturally occurring species of Neurospora (N. crassa and N. intermedia). The strong incompatibility interaction has a simple genetic basis (two biallelic loci) and antagonistic epistasis occurs between heterospecific alleles only, consistent with the Dobzhansky–Muller model of genic incompatibility. We developed microarray‐based, restriction‐site associated DNA (RAD) markers that identified ～1500 polymorphisms between the genomes of the two species, and constructed the first interspecific physical map of Neurospora. With this new mapping resource, the approximate genomic locations of the incompatibility loci were determined using three different approaches: genome scanning, bulk‐segregant analyses, and introgression. These population, quantitative, and classical genetics methods concordantly identified two candidate regions, narrowing the search for each incompatibility locus to only ～2% of the nuclear genome. This study demonstrates how advances in high‐throughput, genome‐wide genotyping can be applied to mapping reproductive isolation genes and speciation research.