Using a pericentromeric interspersed repeat to recapitulate the phylogeny and expansion of human centromeric segmental duplications

Despite considerable advances in sequencing of the human genome over the past few years, the organization and evolution of human pericentromeric regions have been difficult to resolve. This is due, in part, to the presence of large, complex blocks of duplicated genomic sequence at the boundary between centromeric satellite and unique euchromatic DNA. Here, we report the identification and characterization of an approximately 49-kb repeat sequence that exists in more than 40 copies within the human genome. This repeat is specific to highly duplicated pericentromeric regions with multiple copies distributed in an interspersed fashion among a subset of human chromosomes. Using this interspersed repeat (termed PIR4) as a marker of pericentromeric DNA, we recovered and sequence-tagged 3 Mb of pericentromeric DNA from a variety of human chromosomes as well as nonhuman primate genomes. A global evolutionary reconstruction of the dispersal of PIR4 sequence and analysis of flanking sequence supports a model in which pericentromeric duplications initiated before the separation of the great ape species (>12 MYA). Further, analyses of this duplication and associated flanking duplications narrow the major burst of pericentromeric duplication activity to a time just before the divergence of the African great ape and human species (5 to 7 MYA). These recent duplication exchange events substantially restructured the pericentromeric regions of hominoid chromosomes and created an architecture where large blocks of sequence are shared among nonhomologous chromosomes. This report provides the first global view of the series of historical events that have reshaped human pericentromeric regions over recent evolutionary time.     

Miniaturizable homogenous time-resolved fluorescence assay for carboxypeptidase B activity

An epitope-unmasking, homogeneous time-resolved fluorescence (HTRF) assay has been developed for measuring carboxypeptidase B (CPB) activity in a miniaturized high-throughput screening format. The enzyme substrate (biotin-RYRGLMVGGVVR-OH) is cleaved by CPB at the C terminus, causing release of the C-terminal Arg residue. The product (biotin-RYRGLMVGGVV-OH) is recognized specifically by a monoclonal antibody (G2-10) which is labeled with Eu(3+)-cryptate ([Eu(3+)]G2-10 mAb), and the complex is detected by fluorescence resonance energy transfer using streptavidin labeled with allophycocyanin ([XL665]SA). The CPB HTRF assay is readily adapted from 96- to 1536-well format as a robust (Z(')>0.5) assay for high-throughput screening.     

Lysyl oxidase is required for vascular and diaphragmatic development in mice

Lysyl oxidase (LOX) is an enzyme responsible for the cross-linking of collagen and elastin both in vitro and in vivo. The unique functions of the individual members of this multigene family have been difficult to ascertain because of highly conserved catalytic domains and overlapping tissue expression patterns. To address this problem of functional and structural redundancy and to determine the role of LOX in the development of tissue integrity, Lox gene expression was deleted by targeted mutagenesis in mice. Lox-targeted mice (LOX(-/-)) died soon after parturition, exhibiting cardiovascular instability with ruptured arterial aneurysms and diaphragmatic rupture. Microscopic analysis of the aorta demonstrated fragmented elastic fiber architecture in homozygous mutant null mice. LOX activity, as assessed by desmosine (elastin cross-link) analysis, was reduced by approximately 60% in the aorta and lungs of homozygous mutant animals compared with wild type mice. Immature collagen cross-links were decreased but to a lesser degree than elastin cross-links in LOX(-/-) mice. Thus, lysyl oxidase appears critical during embryogenesis for structural stability of the aorta and diaphragm and connective tissue development.     

Receptor activator of NF-kappaB ligand induction via Jak2 and Stat5a in mammary epithelial cells

Prolactin (PRL) is the primary hormone that, in conjunction with local factors, leads to lobuloalveolar development during pregnancy. Recently, receptor activator of NF-kappaB ligand (RANKL) has been identified as one of the effector molecules essential for lobuloalveolar development. The molecular mechanisms by which PRL may induce RANKL expression have not been carefully examined. Here we report that RANKL expression in the mammary gland is developmentally regulated and dependent on PRL and progesterone, whereas its receptor RANK (receptor activator of NF-kappaB) and decoy receptor osteoprotegerin (OPG) are constitutively expressed at all stages in both normal (PRL+/-) and prolactin knockout (PRL-/-) mice. In vitro, PRL markedly increased RANKL expression in primary mammary epithelial cells and RANKL-luciferase reporter activity in CHOD6 cells, which constitutively express the PRL receptor. We identified a gamma-interferon activation sequence (GAS) in the region between residues -965 to -725 of the RANKL promoter, which conferred a PRL response. Using dominant negative mutants of recombinant Jak2 and Stat5 in CHOD6 cells, and by reconstituting the Jak2/Stat5 pathway in COS7 cells, we determined that Jak2 and Stat5a are essential for the PRL-induced RANKL expression in mammary gland.     

The role of loop 5 in acetylcholine receptor channel gating

Nicotinic acetylcholine receptor channel (AChR) gating is an organized sequence of molecular motions that couples a change in the affinity for ligands at the two transmitter binding sites with a change in the ionic conductance of the pore. Loop 5 (L5) is a nine-residue segment (mouse alpha-subunit 92-100) that links the beta4 and beta5 strands of the extracellular domain and that (in the alpha-subunit) contains binding segment A. Based on the structure of the acetylcholine binding protein, we speculate that in AChRs L5 projects from the transmitter binding site toward the membrane along a subunit interface. We used single-channel kinetics to quantify the effects of mutations to alphaD97 and other L5 residues with respect to agonist binding (to both open and closed AChRs), channel gating (for both unliganded and fully-liganded AChRs), and desensitization. Most alphaD97 mutations increase gating (up to 168-fold) but have little or no effect on ligand binding or desensitization. Rate-equilibrium free energy relationship analysis indicates that alphaD97 moves early in the gating reaction, in synchrony with the movement of the transmitter binding site (Phi = 0.93, which implies an open-like character at the transition state). alphaD97 mutations in the two alpha-subunits have unequal energetic consequences for gating, but their contributions are independent. We conclude that the key, underlying functional consequence of alphaD97 perturbations is to increase the unliganded gating equilibrium constant. L5 emerges as an important and early link in the AChR gating reaction which, in the absence of agonist, serves to increase the relative stability of the closed conformation of the protein.     

Human CD4+ T cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of IL-12 to kill tumors in situ by indirect effects of IFN-gamma

By implanting nondisrupted pieces of human lung tumor biopsy tissues into SCID mice, it has been possible to establish viable grafts of the tumor, as well as the tumor-associated microenvironment, including inflammatory cells, fibroblasts, tumor vasculature, and the extracellular matrix. Using this xenograft model, we have evaluated and characterized the effects of a local and sustained release of human rIL-12 (rhIL-12) from biodegradable microspheres. In response to rhIL-12, the human CD45+ inflammatory cells present within the xenograft mediate the suppression or the complete arrest of tumor growth in SCID mice. Analysis of the cellular events reveals that human CD4+ and CD8+ T cells are induced by rhIL-12 to produce and secrete IFN-gamma. Serum levels of human IFN-gamma in mice bearing rhIL-12-treated tumor xenografts correlate directly with the degree of tumor suppression, while neutralizing Abs to human IFN-gamma abrogate the IL-12-mediated tumor suppression. Gene expression profiling of tumors responding to intratumoral rhIL-12 demonstrates an up-regulation of IFN-gamma and IFN-gamma-dependent genes not observed in control-treated tumors. Genes encoding a number of proinflammatory cytokines, chemokines (and their receptors), adhesion molecules, activation markers, and the inducible NO synthase are up-regulated following the introduction of rhIL-12, while genes associated with tumor growth, angiogenesis, and metastasis are decreased in expression. NO contributes to the tumor killing because an inhibitor of inducible NO synthase prevents IL-12-induced tumor suppression. Cell depletion studies reveal that the IL-12-induced tumor suppression, IFN-gamma production, and the associated changes in gene expression are all dependent upon CD4+ T cells.     

Phylogeny and development of marine model species: strongylocentrotid sea urchins

The phylogenetic relationships of ten strongy-locentrotid sea urchin species were determined using mitochondrial DNA sequences. This phylogeny provides a backdrop for the evolutionary history of one of the most studied groups of sea urchins. Our phylogeny indicates that a major revision of this group is in order. All else remaining unchanged, it supports the inclusion of three additional species into the genus Strongylocentrotus (Hemicentrotus pulcherrimus, Allocentrotus fragilis, and Pseudocentrotus depressus). All were once thought to be closely related to this genus, but subsequent revisions separated them into other taxonomic groupings. Most strongylocentrotid species are the result of a recent burst of speciation in the North Pacific that resulted in an ecological diversification. There has been a steady reduction in the complexity of larval skeletons during the expansion of this group. Gamete attributes like egg size, on the other hand, are not correlated with phylogenetic position. In addition, our results indicate that the rate of replacement substitutions is highly variable among phylogenetic lineages. The branches leading to S. purpuratus and S. franciscanus were three to six times longer than those leading to closely related species.     

Characterization of angiosperm nrDNA polymorphism, paralogy, and pseudogenes

Many early reports of ITS region (ITS 1, 5.8S, and ITS 2) variation in flowering plants indicated that nrDNA arrays within individuals are homogeneous. However, both older and more recent studies have found intra-individual nrDNA polymorphism across a range of plant taxa including presumed non-hybrid diploids. In addition, polymorphic individuals often contain potentially non-functional nrDNA copies (pseudogenes). These findings suggest that complete concerted evolution should not be assumed when embarking on phylogenetic studies using nrDNA sequences. Here we (1). discuss paralogy in relation to species tree reconstruction and conclude that a priori determinations of orthology and paralogy of nrDNA sequences should not be made based on the functionality or lack of functionality of those sequences; (2). discuss why systematists might be particularly interested in identifying and including pseudogene sequences as a test of gene tree sampling; (3). examine the various definitions and characterizations of nrDNA pseudogenes as well as the relative merits and limitations of a subset of pseudogene detection methods and conclude that nucleotide substitution patterns are particularly appropriate for the identification of putative nrDNA pseudogenes; and (4). present and discuss the advantages of a tree-based approach to identifying pseudogenes based on comparisons of sequence substitution patterns from putatively conserved (e.g., 5.8S) and less constrained (e.g., ITS 1 and ITS 2) regions. Application of this approach, through a method employing bootstrap hypothesis testing, and the issues discussed in the paper are illustrated through reanalysis of two previously published matrices. Given the apparent robustness of the test developed and the ease of carrying out percentile bootstrap hypothesis tests, we urge researchers to employ this statistical tool. While our discussion and examples concern the literature on plant systematics, the issues addressed are relevant to studies of nrDNA and other multicopy genes in other taxa.