Transposon excision from an atypical site: a mechanism of evolution of novel transposable elements

The role of transposable elements in sculpting the genome is well appreciated but remains poorly understood. Some organisms, such as humans, do not have active transposons; however, transposable elements were presumably active in their ancestral genomes. Of specific interest is whether the DNA surrounding the sites of transposon excision become recombinogenic, thus bringing about homologous recombination. Previous studies in maize and Drosophila have provided conflicting evidence on whether transposon excision is correlated with homologous recombination. Here we take advantage of an atypical Dissociation (Ds) element, a maize transposon that can be mobilized by the Ac transposase gene in Arabidopsis thaliana, to address questions on the mechanism of Ds excision. This atypical Ds element contains an adjacent 598 base pairs (bp) inverted repeat; the element was allowed to excise by the introduction of an unlinked Ac transposase source through mating. Footprints at the excision site suggest a micro-homology mediated non-homologous end joining reminiscent of V(D)J recombination involving the formation of intra-helix 3' to 5' trans-esterification as an intermediate, a mechanism consistent with previous observations in maize, Antirrhinum and in certain insects. The proposed mechanism suggests that the broken chromosome at the excision site should not allow recombinational interaction with the homologous chromosome, and that the linked inverted repeat should also be mobilizable. To test the first prediction, we measured recombination of flanking chromosomal arms selected for the excision of Ds. In congruence with the model, Ds excision did not influence crossover recombination. Furthermore, evidence for correlated movement of the adjacent inverted repeat sequence is presented; its origin and movement suggest a novel mechanism for the evolution of repeated elements. Taken together these results suggest that the movement of transposable elements themselves may not directly influence linkage. Possibility remains, however, for novel repeated DNA sequences produced as a consequence of transposon movement to influence crossover in subsequent generations.     

The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface

Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z-domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small-angle X-ray scattering data of the antibody–protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three-dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects.     

Molecular dynamics simulations of the chemokine CCL2 in complex with pull down-derived heparan sulfate hexasaccharides

Background

Binding of chemokines to glycosaminoglycans (GAGs) is a crucial step in leukocyte recruitment to inflamed tissues.

Cell-derived apolipoprotein E (ApoE) particles inhibit vascular cell adhesion molecule-1 (VCAM-1) expression in human endothelial cells

Sub-endothelial infiltration of monocytes occurs early in atherogenesis and is facilitated by cell adhesion molecules that are up-regulated on activated endothelium. Apolipoprotein E (apoE) helps protect against atherosclerosis, in part, because apoE particles secreted by macrophages have local beneficial effects at lesion sites. Here, we hypothesize that such protection includes anti-inflammatory actions and investigate whether cell-derived apoE can inhibit tumor necrosis factor-alpha-mediated up-regulation of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Two models were used to mimic endothelial exposure to macrophage-derived apoE. In the first, HUVECs were transiently transfected to secrete apoE; VCAM-1 induction inversely correlated with secretion of apoE into the media (r = -0.76, p < 0.001). In the second, incubation of HUVECs with media from recombinant Chinese hamster ovary (CHO) cells expressing apoE (CHO(apoE)) also reduced VCAM-1 in a dose-dependent manner (r = -0.70, p < 0.001). Characterization of CHO(apoE) cell-derived apoE revealed several similarities to apoE particles secreted by human blood monocyte-derived macrophages. The suppression of endothelial activation by apoE most likely occurs via stimulation of endothelial nitric oxide synthase; apoE increased levels of intracellular nitric oxide and its surrogate marker, cyclic guanosine monophosphate, while the nitric oxide synthase inhibitor, ethyl-isothiourea, blocked its effect. We propose that apoE secreted locally at lesion sites by macrophages may be anti-inflammatory by stimulating endothelium to release NO and suppress VCAM-1 expression.     

Adaptation and conservation of physiological systems in the evolution of human hypoxia tolerance

Analysis of human responses to hypobaric hypoxia in different lineages (lowlanders, Andean natives, Himalayan natives, and East Africans) indicates 'conservative' and 'adaptable' physiological characters involved in human responses to hypoxia. Conservative characters, derived by common descent, dominate and indeed define human physiology, but in five hypoxia response systems analyzed, we also found evidence for 'adaptable' characters at all levels of organization in all three high altitude lineages. Since Andeans and Himalayans have not shared common ancestry with East Africans for most of our species history, we suggest that their similar hypoxia physiology may represent the 'ancestral' condition for humans--an interpretation consistent with recent evidence indicating that our species evolved under 'colder, drier, and higher' conditions in East Africa where the phenotype would be simultaneously advantageous for endurance performance and for high altitude hypoxia. It is presumed that the phenotype was retained in low capacity form in highlanders and in higher capacity form in most lowland lineages (where it would be recognized by most physiologists as an endurance performance phenotype). Interestingly, it is easier for modern molecular evolution theory to account for the origin of 'adaptable' characters through positive selection than for conserved traits. Many conserved physiological systems are composed of so many gene products that it seems difficult to account for their unchanging state (for unchanging structure and function of hundreds of proteins linked in sequence to form the physiological system) by simple models of stabilizing selection.     

Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair

Previous in vitro studies indicated that hepatic stellate cells (HSC) and rat liver myofibroblasts (rMF) have to be regarded as different cell populations of the myofibroblastic lineage with fibrogenic potential. Employing the discrimination features defined by these studies the localization of HSC and rMF was analyzed in diseased livers. Normal and acutely as well as chronically carbon tetrachloride-injured livers were analyzed by immunohistochemistry and by in situ hybridization. In normal livers HSC [desmin/glial fibrillary acid protein (GFAP)-positive cells] were distributed in the hepatic parenchyma, while rMF (desmin/smooth muscle alpha actin-positive, GFAP-negative cells colocalized with fibulin-2) were located in the portal field, the walls of central veins, and only occasionally in the parenchyma. Acute liver injury was characterized almost exclusively by an increase in the number of HSC, while the amount of rMF was nearly unchanged. In early stages of fibrosis, HSC and rMF were detected within the developing scars. In advanced stages of fibrosis, HSC were mainly present at the scar–parenchymal interface, while rMF accounted for the majority of the cells located within the scar. At every stage of fibrogenesis, rMF, in contrast to HSC, were only occasionally detected in the hepatic parenchyma. HSC and rMF are present in normal and diseased livers in distinct compartments and respond differentially to tissue injury. Acute liver injury is followed by an almost exclusive increase in the number of HSC, while in chronically injured livers not only HSC but also rMF are involved in scar formation. Accepted: 16 September 1999     

Constitutional Mutations of the hSNF5/INI1 Gene Predispose to a Variety of Cancers

Biallelic, truncating mutations of the hSNF5/INI1 gene have recently been documented in malignant rhabdoid tumor (MRT), one of the most aggressive human cancers. This finding suggests that hSNF5/INI1 is a new tumor-suppressor gene for which germline mutations might predispose to cancer. We now report the presence of loss-of-function mutations of this gene in the constitutional DNA from affected members but not from healthy relatives in cancer-prone families. Furthermore, a constitutional mutation is documented in a patient with two successive primary cancers. In agreement with the two-hit model, the wild-type hSNF5/INI1 allele is deleted in the tumor DNA from mutation carriers. In all tested cases, DNA from parents demonstrated normal hSNF5/INI1 sequences, therefore indicating the de novo occurrence of the mutation, which was shown to involve the maternal allele in one case and the paternal allele in two other cases. These data indicate that constitutional mutation of the hSNF5/INI1 gene defines a new hereditary syndrome predisposing to renal or extrarenal MRT and to a variety of tumors of the CNS, including choroid plexus carcinoma, medulloblastoma, and central primitive neuroectodermal tumor. This condition, which we propose to term "rhabdoid predisposition syndrome," may account for previous observations of familial and multifocal cases of the aforementioned tumor types. It could also provide the molecular basis for cases of Li-Fraumeni syndrome without p53 germline mutations.     

The Ku protein complex interacts with YY1, is up-regulated in human heart failure, and represses alpha myosin heavy-chain gene expression

Human heart failure is accompanied by repression of genes such as alpha myosin heavy chain (alphaMyHC) and SERCA2A and the induction of fetal genes such as betaMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human alphaMyHC promoter. We have now identified a region of the alphaMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human alphaMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases alphaMyHC mRNA expression and increases skeletal alpha-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the alphaMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human alphaMyHC promoter during heart failure.