Pure Wild Type Research Articles

Genetic diversity, linkage disequilibrium, and population structure analysis of the tea plant (Camellia sinensis) from an origin center, Guizhou plateau, using genome-wide SNPs developed by genotyping-by-sequencing

BackgroundTo efficiently protect and exploit germplasm resources for marker development and breeding purposes, we must accurately depict the features of the tea populations. This study focuses on the Camellia sinensis (C. sinensis) population and aims to (i) identify single nucleotide polymorphisms (SNPs) on the genome level, (ii) investigate the genetic diversity and population structure, and (iii) characterize the linkage disequilibrium (LD) pattern to facilitate next genome-wide association mapping and marker-assisted selection.ResultsWe collected 415 tea accessions from the Origin Center and analyzed the genetic diversity, population structure and LD pattern using the genotyping-by-sequencing (GBS) approach. A total of 79,016 high-quality SNPs were identified; the polymorphism information content (PIC) and genetic diversity (GD) based on these SNPs showed a higher level of genetic diversity in cultivated type than in wild type. The 415 accessions were clustered into three groups by STRUCTURE software and confirmed using principal component analyses (PCA)—wild type, cultivated type, and admixed wild type. However, unweighted pair group method with arithmetic mean (UPGMA) trees indicated the accessions should be grouped into more clusters. Further analyses identified four groups, the Pure Wild Type, Admixed Wild Type, ancient landraces and modern landraces using STRUCTURE, and the results were confirmed by PCA and UPGMA tree method. A higher level of genetic diversity was detected in ancient landraces and Admixed Wild Type than that in the Pure Wild Type and modern landraces. The highest differentiation was between the Pure Wild Type and modern landraces. A relatively fast LD decay with a short range (kb) was observed, and the LD decays of four inferred populations were different.ConclusionsThis study is, to our knowledge, the first population genetic analysis of tea germplasm from the Origin Center, Guizhou Plateau, using GBS. The LD pattern, population structure and genetic differentiation of the tea population revealed by our study will benefit further genetic studies, germplasm protection, and breeding.

791 PROSTATE CANCER PROMOTING MUTATIONS OF THE COI GENE ASSOCIATED WITH G PROTEIN DYSREGULATION

You have accessJournal of UrologyProstate Cancer: Basic Research (IV)1 Apr 2013791 PROSTATE CANCER PROMOTING MUTATIONS OF THE COI GENE ASSOCIATED WITH G PROTEIN DYSREGULATION Qian Sun, Rebecca S. Arnold, and John A. Petros Qian SunQian Sun Atlanta, GA More articles by this author , Rebecca S. ArnoldRebecca S. Arnold Atlanta, GA More articles by this author , and John A. PetrosJohn A. Petros Atlanta, GA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.355AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Inherited missense mutations of the mitochondrially encoded cytochrome oxidase 1 (COI) gene are present in 25% of prostate cancer patients. Laboratory analysis of one such prostate cancer ûassociated mutation (T6124C; Met74Thr) demonstrated that the mutation caused increased cell proliferation and in vivo tumor growth. To better understand the cell signaling events that this mutation produces we performed whole genome expression profiling of mutant and wild type cancer cell lines. METHODS The T6124C mutation of the COI gene was transferred from a prostate cancer patient's peripheral blood cells to a strong cancer nucleus (134B cells) that were devoid of their native mtDNA in a cytoplasmic transfer protocol to make cytoplasmic hybrid (cybrid) cell lines that contained either pure mutant or pure wild type mitochondria. RNA was prepared followed by fluorescent RT-PCR and hybridization to Agilent whole genome expression chips. Differences in gene expression were determined by averaging signal intensity for the two wild type cell lines and comparing to the average of the two mutant cell lines. D-score, q-value and fold change were used to determine significant findings. RESULTS The most highly induced gene was RGS1 (regulator of G-protein signaling 1) that was three logs higher in mutant cells. RGS1 is highly expressed in a broad variety of cancers including cervical, melanoma, renal cell and T-cell leukemias. Also upregulated (55 fold) was P2RY8 (purinergic receptor P2Y, G protein coupled 8) a single gene transforming agent overexpressed in leukemia. Amongst the most significantly downregulated (26-fold) transcripts was GPSM 2 (G protein signaling modulator 2) which is essential to maintenance of cell polarity. CONCLUSIONS Inherited missense mutations of the COI gene increase the risk for the development of prostate cancer and are present in 25% of all men with prostate cancer. One such prostate cancer patient-derived mutation has been studied extensively and found to increase cell proliferation and in vivo tumor growth and induces the RGS1 gene known to be overexpressed in a wide variety of human cancers as well as P2RY8, another G protein coupled protein that is potent in transformation. The loss of the G protein signal modulator GPSM2 suggest disruption of cell polarity, an essential characteristic of malignant transformation. We therefore propose that inherited mtDNA mutations predispose individuals to the development of prostate cancer in part by dysregulation of G-protein signaling thereby promoting transformation while disrupting cell polarity. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e326 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Qian Sun Atlanta, GA More articles by this author Rebecca S. Arnold Atlanta, GA More articles by this author John A. Petros Atlanta, GA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Detection and Clinical Significance of Intratumoral EGFR Mutational Heterogeneity in Chinese Patients with Advanced Non-Small Cell Lung Cancer

PurposeThis study evaluated occurrence and potential clinical significance of intratumoral EGFR mutational heterogeneity in Chinese patients with non-small cell lung cancer (NSCLC).Materials and MethodsEighty-five stage IIIa-IV NSCLC patients who had undergone palliative surgical resection were included in this study. Of these, 45 patients carried EGFR mutations (group-M) and 40 patients were wild-type (group-W). Each tumor sample was microdissected to yield 28–34 tumor foci and Intratumoral EGFR mutation were determined using Denaturing High Performance Liquid Chromatography (DHPLC) and Amplification Refractory Mutation System (ARMS). EGFR copy numbers were measured using fluorescence in situ hybridization (FISH).ResultsMicrodissection yielded 1,431 tumor foci from EGFR mutant patients (group-M) and 1,238 foci from wild-type patients (group-W). The EGFR mutant frequencies in group-M were 80.6% (1,154/1,431) and 87.1% (1,247/1,431) using DHPLC and ARMS, respectively. A combination of EGFR-mutated and wild-type cells was detected in 32.9% (28/85) of samples by DHPLC and 28.2% (24/85) by ARMS, supporting the occurrence of intratumoral heterogeneity. Thirty-one patients (36.5%) were identified as EGFR FISH-positive. Patients harboring intratumoral mutational heterogeneity possessed lower EGFR copy numbers than those tumors contained mutant cells alone (16.7% vs. 71.0%, P<0.05). Among 26 patients who had received EGFR-TKIs, the mean EGFR mutation content was higher in patients showing partial response (86.1%) or stable disease (48.7%) compared with patients experiencing progressive disease (6.0%) (P = 0.001). There also showed relationship between progression-free survival (PFS) and different content of EGFR mutation groups (pure wild type EGFR, EGFR mutation with heterogeneity and pure mutated EGFR) (P = 0.001).ConclusionApproximately 30% of patients presented intratumoral EGFR mutational heterogeneity, accompanying with relatively low EGFR copy number. EGFR mutant content was correlated with the response and prognosis of EGFR-TKIs.

NMDA Receptors Mediate Synaptic Competition in Culture

BackgroundActivity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo, where activity of inputs can be differentially regulated, but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. Here, we develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons.Methodology/Principal FindingsGluN1 -/- (KO) mouse hippocampal neurons lacking the essential NMDA receptor subunit were cultured alone or cultured in defined ratios with wild type (WT) neurons. The absence of functional NMDA receptors did not alter neuron survival. Synapse development was assessed by immunofluorescence for postsynaptic PSD-95 family scaffold and apposed presynaptic vesicular glutamate transporter VGlut1. Synapse density was specifically enhanced onto minority wild type neurons co-cultured with a majority of GluN1 -/- neighbour neurons, both relative to the GluN1 -/- neighbours and relative to sister pure wild type cultures. This form of synaptic competition was dependent on NMDA receptor activity and not conferred by the mere physical presence of GluN1. In contrast to these results in 10% WT and 90% KO co-cultures, synapse density did not differ by genotype in 50% WT and 50% KO co-cultures or in 90% WT and 10% KO co-cultures.Conclusions/SignificanceThe enhanced synaptic density onto NMDA receptor-competent neurons in minority coculture with GluN1 -/- neurons represents a cell culture paradigm for studying synaptic competition. Mechanisms involved may include a retrograde ‘reward’ signal generated by WT neurons, although in this paradigm there was no ‘punishment’ signal against GluN1 -/- neurons. Cell culture assays involving such defined circuits may help uncover the rules and mechanisms of activity-dependent synaptic competition in the developing nervous system.

Juxtaposition of the Steroid Binding Domain-like I and II Regions Constitutes a Ligand Binding Site in the σ-1 Receptor

sigma-1 receptors represent unique binding sites that are capable of interacting with a wide range of compounds to mediate different cellular events. The composition of the ligand binding site of this receptor is unclear, since no NMR or crystal structures are available. Recent studies in our laboratory using radiolabeled photoreactive ligands suggested that the steroid binding domain-like I (SBDLI) (amino acids 91-109) and the steroid binding domain-like II (SBDLII) (amino acids 176-194) regions are involved in forming the ligand binding site(s) ( Chen, Y., Hajipour, A. R., Sievert, M. K., Arbabian, M., and Ruoho, A. E. (2007) Biochemistry 46, 3532-3542 ; Pal, A., Hajipour, A. R., Fontanilla, D., Ramachandran, S., Chu, U. B., Mavlyutov, T., and Ruoho, A. E. (2007) Mol. Pharmacol. 72, 921-933 ). In this report, we have further addressed this issue by utilizing our previously developed sulfhydryl-reactive, cleavable, radioiodinated photocross-linking reagent: methanesulfonothioic acid, S-((4-(4-amino-3-[125I]iodobenzoyl) phenyl)methyl) ester (Guo, L. W., Hajipour, A. R., Gavala, M. L., Arbabian, M., Martemyanov, K. A., Arshavsky, V. Y., and Ruoho, A. E. (2005) Bioconjugate Chem. 16, 685-693). This photoprobe was shown to derivatize the single cysteine residues as mixed disulfides at position 94 in the SBDLI region of the wild type guinea pig sigma-1 receptor (Cys94) and at position 190 in the SBDLII region of a mutant guinea pig sigma-1 receptor (C94A,V190C), both in a sigma-ligand (haloperidol or (+)-pentazocine)-sensitive manner. Significantly, photocross-linking followed by Endo Lys-C cleavage under reducing conditions and intramolecular radiolabel transfer from the SBDLI to the SBDLII region in the wild type receptor and, conversely, from the SBDLII to the SBDLI region in the mutant receptor were observed. These data support a model in which the SBDLI and SBDLII regions are juxtaposed to form, at least in part, a ligand binding site of the sigma-1 receptor.

Modulation of the Viral ATPase Activity by the Portal Protein Correlates with DNA Packaging Efficiency

DNA packaging in tailed bacteriophages and herpesviruses requires assembly of a complex molecular machine at a specific vertex of a preformed procapsid. As in all these viruses, the DNA translocation motor of bacteriophage SPP1 is composed of the portal protein (gp6) that provides a tunnel for DNA entry into the procapsid and of the viral ATPase (gp1-gp2 complex) that fuels DNA translocation. Here we studied the cross-talk between the components of the motor to control its ATP consumption and DNA encapsidation. We showed that gp6 embedded in the procapsid structure stimulated more than 10-fold the gp2 ATPase activity. This stimulation, which was significantly higher than the one conferred by isolated gp6, depended on the presence of gp1. Mutations in different regions of gp6 abolished or decreased the gp6-induced stimulation of the ATPase. This effect on gp2 activity was observed both in the presence and in the absence of DNA and showed a strict correlation with the efficiency of DNA packaging into procapsids containing the mutant portals. Our results demonstrated that the portal protein has an active control over the viral ATPase activity that correlates with the performance of the DNA packaging motor.

Endoglin Null Endothelial Cells Proliferate Faster and Are More Responsive to Transforming Growth Factor β1 with Higher Affinity Receptors and an Activated Alk1 Pathway

Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.

Assessment of Helicobacter pylori clarithromycin resistance mutations in archival gastric biopsy samples.

First, to assess the clarithromycin resistance (Cla(R)) rate 1) in patients with persistent Helicobacter pylori (H. pylori) infection after eradication,2) in patients with untreated infection and 3) in patients with successful status post eradication. Second, to evaluate the techniques sequencing and line probe hybridisation INNO-LipA for resolution of uniform and mixed populations in archival gastric biopsy samples. The genomic 2142/43 23s rRNA mutations of the 50S ribosomal subunit conferring Cla(R) were detected by PCR-based assays. A total of 130 patients were investigated. Out of 21 patients of a first series with persistent infection after eradication, 19 (90%; CI (95%): 67-99%) exhibited point mutations at position 2142/43. In the second series of untreated patients, primary resistance was observed in 8 out of 93 patients (9%; CI: 4-16%). In a third series of 16 successfully eradicated patients, pure wild type populations (WT; for loci 2142/43) without any minimal mutated part were found (resistance rate 0%; CI: 0-21%). Further, in all 24 biopsies with uniform mutated and in 8 of 11 biopsies with mixed populations the two molecular biological methods yielded concordant results (100%; CI: 86-100% and 73%; CI: 39-94%, respectively). In the Baden region of Switzerland, most clarithromycin resistant H. pylori strains harbour mutations at position 2142/43. The primary resistance rate is below 10%. Mixed populations, even with minor mutated part, cannot become successfully eradicated. The two investigated techniques are equally valid for resolution of uniform mutated or mixed H. pylori populations in archival biopsy material.

Assessment of Helicobacter pylori clarithromycin resistance mutations in archival gastric biopsy samples.

First, to assess the clarithromycin resistance (Cla(R)) rate 1) in patients with persistent Helicobacter pylori (H. pylori) infection after eradication,2) in patients with untreated infection and 3) in patients with successful status post eradication. Second, to evaluate the techniques sequencing and line probe hybridisation INNO-LipA for resolution of uniform and mixed populations in archival gastric biopsy samples. The genomic 2142/43 23s rRNA mutations of the 50S ribosomal subunit conferring Cla(R) were detected by PCR-based assays. A total of 130 patients were investigated. Out of 21 patients of a first series with persistent infection after eradication, 19 (90%; CI (95%): 67-99%) exhibited point mutations at position 2142/43. In the second series of untreated patients, primary resistance was observed in 8 out of 93 patients (9%; CI: 4-16%). In a third series of 16 successfully eradicated patients, pure wild type populations (WT; for loci 2142/43) without any minimal mutated part were found (resistance rate 0%; CI: 0-21%). Further, in all 24 biopsies with uniform mutated and in 8 of 11 biopsies with mixed populations the two molecular biological methods yielded concordant results (100%; CI: 86-100% and 73%; CI: 39-94%, respectively). In the Baden region of Switzerland, most clarithromycin resistant H. pylori strains harbour mutations at position 2142/43. The primary resistance rate is below 10%. Mixed populations, even with minor mutated part, cannot become successfully eradicated. The two investigated techniques are equally valid for resolution of uniform mutated or mixed H. pylori populations in archival biopsy material.

A Sulfenic Acid Enzyme Intermediate Is Involved in the Catalytic Mechanism of Peptide Methionine Sulfoxide Reductase fromEscherichia coli

Methionine oxidation into methionine sulfoxide is known to be involved in many pathologies and to exert regulatory effects on proteins. This oxidation can be reversed by a ubiquitous monomeric enzyme, the peptide methionine sulfoxide reductase (MsrA), whose activity in vivo requires the thioredoxin-regenerating system. The proposed chemical mechanism of Escherichia coli MsrA involves three Cys residues (positions 51, 198, and 206). A fourth Cys (position 86) is not important for catalysis. In the absence of a reducing system, 2 mol of methionine are formed per mole of enzyme for wild type and Cys-86 --> Ser mutant MsrA, whereas only 1 mol is formed for mutants in which either Cys-198 or Cys-206 is mutated. Reduction of methionine sulfoxide is shown to proceed through the formation of a sulfenic acid intermediate. This intermediate has been characterized by chemical probes and mass spectrometry analyses. Together, the results support a three-step chemical mechanism in vivo: 1) Cys-51 attacks the sulfur atom of the sulfoxide substrate leading, via a rearrangement, to the formation of a sulfenic acid intermediate on Cys-51 and release of 1 mol of methionine/mol of enzyme; 2) the sulfenic acid is then reduced via a double displacement mechanism involving formation of a disulfide bond between Cys-51 and Cys-198, followed by formation of a disulfide bond between Cys-198 and Cys-206, which liberates Cys-51, and 3) the disulfide bond between Cys-198 and Cys-206 is reduced by thioredoxin-dependent recycling system process.

DEMOGRAPHIC AND GENETIC RESPONSES OF TWO STRAINS OF TRIBOLIUM CASTANEUM TO A NOVEL ENVIRONMENT.

This is a study of the demographic and genetic consequences of long term exposure to a novel husbandry regime in laboratory populations of two strains of the flour beetle Tribolium castaneum. An alteration of the husbandry represents an environmental change, and a resulting shift in the demographic characteristics of populations exposed to this new environment for long periods of time could be attributed to an evolutionary response if the shift can be shown to be genetic. Certainly the demographic traits of populations have a genetic basis and are subject to natural selection. The effect of selection on demography has not been studied extensively though there have been some theoretical and empirical considerations. Some of these conclude that the intrinsic rate of natural increase r should be maximized either by an increase in fecundity or a decrease in developmental time (Dobzhansky, 1968; Stern, 1970). Other authors believe selection should maximize population size or biomass (Carson, 1957, 1961; Fisher, 1958; and Ayala, 1966). The empirical results presented here are contrary to these predictions and indicate some of the problems associated with making predictions at the population level based on the outcome of selection on individuals. This work has its antecedents in studies documented by Sokal and Sonleitner (1968) and Sokal and Fujii (1973). To follow changes in the frequency of the mutant allele black (b) subsequent to its introduction to wild type populations of the normally long-lived beetles, the above authors designed a scheme in which discrete generations were imposed on the experimental populations. Since laboratory stocks are maintained under conditions permitting overlapping generations, this novel regime radically altered the ecology of the experimental cultures. Competition among like life history stages was intensified and the reproductive period shortened by the process which will be described fully below. In their experiments, Sokal and his coworkers established many replicate populations with discrete generations for the pure wild type and pure bb (the Chicago Black strain marked by bb) as controls to their selection experiments. Experimental populations of both strains followed characteristic patterns of rapid increase in adult population size followed by a leveling off period. The pure bb populations then underwent a severe long term decline in adult numbers while the wild type populations declined only slightly, maintaining levels near their earlier peak. The time period involved is in excess of 40 generations. It is postulated that the altered population sizes have a genetic basis and are a result of adaptation to the novel environment. Differential responses in the Chicago Black and wild type strains result from genetic differences inherent in the two strains. 1 Current address: Department of Biology, University of Chicago, 1103 East 57th Street, Chicago, Illinois 60637. 2 Contribution Number 193 from the Program in Ecology and Evolution, State University of New York at Stony Brook.

Heterocytosis in Cellular Slime Molds

By isolating single spores it was found that five of the strains of Acrasiales maintained in this laboratory were heterogeneous and consisted of more than one cell type. In addition to spores producing wild type fruiting bodies, variants were found which deviated from the wild type in either the morphology of their fruiting bodies, or the morphogenetic process that gave rise to fruiting bodies. In one strain of Dictyostelium mucoroides two cell types persist in ratios of a limited range over a period of many serial subcultures. It was further shown in the case of pure wild type cultures of this strain, that a new cell type arose `spontaneously' during the course of serial subcultures and established itself firmly in the population. The different cell types of a given strain can cooperate to form a common pseudoplasmodium, which because of its cellular heterogeneity has been called a heterocyton. Mixing the amoebae of variants with even small amounts of wild type cells results in the formation of heterocytons that will produce a fruiting body with the wild type phenotype, that is, one which produces a normal stalk and sorus. Grafting experiments indicate that whenever wild type cells are involved, the final morphology of a fruiting body has the wild type phenotype.