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Molecular cloning of spinach chloroplast DNA isolated by alkaline lysisDrager, Robert Gray 01 January 1987 (has links)
Chloroplast genomes of land plants show conservation of structure and gene arrangement. The spinach chloroplast genome is comprised of a covalently closed. circular DNA molecule of 150 kilobases and is typical of these plants. Approximately 20% of the proteins found in the spinach chloroplast are encoded by the chloroplast genome and translated on chloroplast ribosomes. The remainder are encoded on chromosomes in the nucleus, translated on cytoplasmic ribosomes and transported into the chloroplast.
Spinach chloroplast DNA was isolated from crude 2 chloroplast preparations by a new method. Chloroplasts were lysed with alkaline sodium dodecyl sulfate, contaminating macromolecules precipitated with acidified potassium acetate and plastid DNA was purified by phenol:chloroform extraction and ethanol:ammonium acetate precipitation. The yield was approximately 50 ug chloroplast DNA per 100 grams leaf material. The DNA consisted of 10% circular molecules and 90% linear molecules.
The chloroplast DNA was digested with restriction enzyme PstI and the fragments were cloned into the plasmid vector pUC9. Several recombinant plasmids were isolated and the chloroplast DNA inserts identified. The recombinant plasmid pRD105 containing the PstI #5 fragment was subjected to further investigation. The ClaI restriction sites of the PstI #5 fragment were mapped and the insert was subcloned into the plasmid vector pGEM4, which bears bacteriophage SP6 and T7 RNA polymerase promoter sequences.
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Maintenance Of Mammary Epithelial Phenotype By Transcription Factor Runx1 Through Mitotic Gene BookmarkingRose, Joshua 01 January 2019 (has links)
Breast cancer arises from a series of acquired mutations that disrupt normal mammary epithelial homeostasis and create multi-potent cancer stem cells that can differentiate into clinically distinct breast cancer subtypes. Despite improved therapies and advances in early detection, breast cancer remains the leading diagnosed cancer in women.
A predominant mechanism initiating invasion and migration for a variety of cancers including breast, is epithelial-to-mesenchymal transition (EMT). EMT— a trans-differentiation process through which mammary epithelial cells acquire a more aggressive mesenchymal phenotype—is a regulated process during early mammary gland development and involves many transcription factors involved in cell lineage commitment, proliferation, and growth. Despite accumulating evidence for a broad understanding of EMT regulation, the mechanism(s) by which mammary epithelial cells maintain their phenotype is unknown.
Mitotic gene bookmarking, i.e., transcription factor binding to target genes during mitosis for post mitotic regulation, is a key epigenetic mechanism to convey regulatory information for cell proliferation, growth, and identity through successive cell divisions. Many phenotypic transcription factors, including the hematopoietic Runt Related Transcription Factor 1 (RUNX1/AML1), bookmark target genes during mitosis. Despite growing evidence, a role for mitotic gene bookmarking in maintaining mammary epithelial phenotype has not been investigated.
RUNX1 has been recently identified to play key roles in breast cancer development and progression. Importantly, RUNX1 stabilizes the normal breast epithelial phenotype and prevents EMT through repression of EMT-initiating pathways. Findings reported in this thesis demonstrate that RUNX1 mitotically bookmarks both RNA Pol I and II transcribed genes involved in proliferation, growth, and mammary epithelial phenotype maintenance. Inhibition of RUNX1 DNA binding by a specific small molecule inhibitor led to phenotypic changes, apoptosis, differences in global protein synthesis, and differential expression of ribosomal RNA as well as protein coding genes and long non-coding RNA genes involved in cellular phenotype. Together these findings reveal a novel epigenetic regulatory role of RUNX1 in normal-like breast epithelial cells and strongly suggest that mitotic bookmarking of target genes by RUNX1 is required to maintain breast epithelial phenotype. Disruption of RUNX1 bookmarking results in initiation of epithelial to mesenchymal transition, an essential first step in the onset of breast cancer.
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Characterization Of Epigenetic Plasticity And Chromatin Dynamics In Cancer Cell ModelsGerrard, Diana Lea 01 January 2019 (has links)
Cancer progression is driven by cumulative changes that promote and maintain the malignant phenotype. Epigenetic alterations are central to malignant transformation and to the development of therapy resistance. Changes in DNA methylation, histone acetylation and methylation, noncoding RNA expression and higher-order chromatin structures are epigenetic features of cancer, which are independent of changes in the DNA sequence. Despite the knowledge that these epigenetic alterations disrupt essential pathways that protect cells from uncontrolled growth, how these modifications collectively coordinate cancer gene expression programs remains poorly understood. In this dissertation, I utilize molecular and informatic approaches to define and characterize the genome-wide epigenetic patterns of two important human cancer cell models. I further explore the dynamic alterations of chromatin structure and its interplay with gene regulation in response to therapeutic agents.
In the first part of this dissertation, pancreatic ductal adenocarcinoma (PDAC) cell models were used to characterize genome-wide patterns of chromatin structure. The effects of histone acetyltransferase (HAT) inhibitors on chromatin structure patterns were investigated to understand how these potential therapeutics influence the epigenome and gene regulation. Accordingly, HAT inhibitors globally target histone modifications and also impacted specific gene pathways and regulatory domains such as super-enhancers. Overall, the results from this study uncover potential roles for specific epigenomic domains in PDAC cells and demonstrate epigenomic plasticity to HAT inhibitors.
In the second part of this dissertation, I investigate the dynamic changes of chromatin structure in response to estrogen signaling over a time-course using Estrogen Receptor (ER) positive breast cancer cell models. Accordingly, I generated genome-wide chromatin contact maps, ER, CTCF and regulatory histone modification profiles and compared and integrated these profiles to determine the temporal patterns of regulatory chromatin compartments. The results reveal that the majority of alterations occur in regions that correspond to active chromatin states, and that dynamic chromatin is linked to genes associated with specific cancer growth and metabolic signaling pathways. To distinguish ER-regulated processes in tamoxifen-sensitive and in tamoxifen-resistant (TAMR) cell models, we determined the corresponding chromatin and gene expression profiles using ER-positive TAMR cancer cell derivatives. Comparison of the patterns revealed characteristic features of estrogen responsiveness and show a global reprogramming of chromatin structure in breast cancer cells with acquired tamoxifen resistance.
Taken together, this dissertation reveals novel insight into dynamic epigenomic alterations that occur with extrinsic stimuli and provides insight into mechanisms underlying the therapeutic responses in cancer cells.
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Genetic and Environmental Influences of Bullying Involvement: A Longitudinal Twin StudyDunbar, Ellyn 01 January 2018 (has links)
Introduction—Bullying involvement is associated with many long-term adverse outcomes. Bullied children are at risk for internalizing disorders including anxiety, depression and suicidal behavior in childhood and adulthood. Bullies are also at risk for psychiatric disorders, specifically externalizing disorders. Bully victims—children who are both bullied and bullies—have a particularly poor prognosis, with a higher risk for internalizing and externalizing disorders. The purpose of this study is to study the epidemiology, risk of psychiatric disorders, and genetic and environmental influences of being bullied, a bully, and a bully victim—in the sample and individually in males and females.
Methods—Twins (N=2,844, aged 8-17) from the Virginia Twin Study of Adolescent Behavioral Development and the Young Adult Follow-Up were used to study bullying involvement. Child and mother responses from three waves of data collection were used to determine bullying involvement status and to diagnose internalizing and externalizing disorders. The epidemiology of bullying involvement was examined. The odds ratios (OR) of being involved in bullying and having a psychiatric disorder were calculated. The twin methodology was used to estimate the genetic and environmental influences of bullying involvement.
Results—In the sample, 14.56% were bullied, 17.33% were bullies, and 10.69% were bully victims. Males are more often involved in bullying, but females are more severely affected by their involvement. Bullied children are at a higher risk for internalizing disorders, especially young adult depression (OR 1.29). Bullies are at a higher risk for externalizing disorders, and depression (OR 1.72). Bully victims are at a higher risk for nearly every disorder tested. Bullying involvement is heritable, and being bullied has a dominance genetic component. The heritability of being bullied, a bully, and a bully victim is 48.12%, 54.81%, and 62.62% respectively.
Conclusion—Individuals involved in bullying are at risk for serious and long-lasting psychiatric disorders. Interventions need to be developed that target each category of bullying involvement, and the specific disorders that these children are at risk for, while keeping in mind that their involvement is heritable.
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Evidence-Based Practice Self-Study Education Program for Staff Nurses on GenomicsNorman-Marzella, Nancy L 01 January 2019 (has links)
Nurses routinely obtain genomic data when collecting family health histories. However, they report low confidence in their knowledge and understanding of genomics and the genetically engineered medications prescribed for their patients. The purpose of this project was the development and implementation of an evidence-based online education program about genetics and genomics to increase the nurses' understanding and ability to provide competent care for their patients receiving treatments based on the science of genomics. Knowles's principles of adult learning theory guided the development and delivery of the online education project to 12 medical-surgical registered nurses employed in a hospital in the northeastern United States. The Johns Hopkins nursing evidence-based practice model provided a guideline for organizing and evaluating the level and quality of evidence. A 2-tailed paired t test showed that the nurses' knowledge and understanding about genetics and genomics increased after participating in the evidence-based education program. The increase in nurses' knowledge on genomics has the potential to provide nurses with the competence and confidence to collaborate with physicians and pharmacists regarding treatment plans incorporating genomics, resulting in effective team collaboration and a positive social change that could improve patient outcomes.
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Determining the Influence of the Extracellular Proteinase from <em>Brevibacterium linens</em> on the Metabolism of <em>Lactococcus lactis</em> spp. <em>lactis</em> Using Functional GenomicsXie, Yi 01 May 2003 (has links)
Since the catabolism of amino acids in cheese results in the formation of most volatile flavor compounds, a proper intracellular pool of amino acids must be established in order to produce a desirable flavor production in cheese. Generation of this pool of amino acids requires complex interactions among casein and its derivatives, proteolytic enzymes, and transport systems in the associated bacteria, including lactococci. In this project, we hypothesized that casein hydrolysis by the extracellular proteinases of Brevibacterium linens BL2 modulates the expression profile of proteolytic related genes in Lactococcus lactis spp. lactis IL1403.
In order to monitor the global gene regulation patterns in L. lactis ssp. lactis IL1403, a high-throughput gene expression tool was needed to study the gene expression profiles on a genomic scale. In this project, we developed a novel oligonucleotide-based filter DNA array protocol for this purpose. The success of this oligonucleotide-based DNA array was dependent on technical innovations including polyI tailing, indirect high density biotin labeling, careful probe design, and integrated computational data analysis. The utility and validity of this protocol were demonstrated by profiling the expression of 375 metabolically related genes in L. lactis ssp. lactis IL1403 during heat, acid, and osmotic stresses.
Subsequently the DNA macroarray was used to profile the gene expression changes of L. lactis spp. lactis IL1403 growing in a peptide-limited medium, in a casitone-based peptide-rich medium, and in a casein hydrolyte by B. linens BL2 proteolytic enzymes. L. lactis ssp. lactis IL1403 experienced nitrogen starvation even with an abundance of peptide resources because of lack of expression of peptide transporter genes. Conversely, a peptide pool generated by B. linens BL2 proteolytic activities was sufficient to sustain the growth of L. lactis ssp. lactis IL1403. The repression of the peptide transporter and other peptidase genes of L. lactis ssp. lactis IL1403 was relieved in this medium. Interestingly, the Opt system, a di-tripeptide transporter, was used as a primary peptide transporter, instead of the Opp system whose genes were not actively transcripted in IL1403.
We also conducted additional experiments to further describe the protease in B. linens BL2 responsible for the peptide pool generation. This enzyme was secreted as a non-active zymogen and matured into the active protease. Both proteolysis and maturation processes were regulated. Collectively, this work demonstrated that a unique protease of B. linens BL2 generated a pool of pep tides transportable by L. lactis IL1403 and induced changes in gene expression in L. lactis IL1403. Consequently, this body of work demonstrated the hypothesis to be true.
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Arquitetura genética do consumo alimentar residual em bovinos Nelore /Silva, Beatriz Pressi Molina da January 2018 (has links)
Orientador: Josineudson Augusto II Vasconcelos Silva / Banca: Rusbel Raul Aspilcueta Borquis / Banca: Henrique Nunes de Oliveira / Resumo: A alimentação é o componente mais dispendioso e relevante na produção de bovinos de corte. Com a necessidade de tornar a bovinocultura de corte mais rentável e sustentável, características de eficiência alimentar ganharam importância, entretanto, apresentam dificuldades pela onerosa obtenção da informação. A característica relacionada a eficiência alimentar e com mais estudos na última década, consumo alimentar residual (CAR), possui independência fenotípica de características de crescimento e de produção. O avanço na utilização da tecnologia de marcadores moleculares, como os SNPs, possibilitou o uso de novas ferramentas genômicas. Neste sentindo, é de interesse econômico e científico investigar mecanismos genéticos e biológicos que influenciam o CAR. O objetivo do estudo foi identificar regiões genômicas associadas ao CAR, avaliar diferenças genômicas em grupos contrastantes de animais, além de estimar os efeitos da substituição alélica de SNP que apresentem diferentes frequências alélicas em bovinos Nelore (Bos indicus). Foram utilizadas informações fenotípicas de 946 animais participantes do teste de eficiência alimentar nos anos de 2010 a 2017, com informação de genótipos de 956 animais. O modelo abordado para predição dos valores genéticos foi o WssGBLUP/S2, e a cada iteração foram reestimados efeitos de SNPs a partir de valores genético genômico (GEBVs). Foram encontradas oito regiões explicando variância genética total acima de 1%, totalizando 12,25% da variância gené... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Feeding is the most expensive and relevant component in the production of beef cattle. With the need to make beef cattle production more profitable and sustainable, feed efficiency traits have gained importance, however, they present difficulties due to the onerous obtaining of the information. The trait related to feed efficiency and with more studies in the last decade, residual feed intake (RFI), has phenotypic independence of growth and production traits. Advances in the use of molecular marker technology, such as SNPs, provide new genomic tools. In this sense, it is of economic and scientific interest to investigate genetic and biological mechanisms that influence RFI. The objective of the study was to identify genomic regions associated with RFI, and in contrasting groups of animals, to evaluate genomic differences and to estimate the effects of allelic substitution of SNPs that present different allelic frequencies in Nelore (Bos indicus) cattle. Phenotypic information from 946 animals participating in the feed efficiency test was used from 2010 to 2017, with information on 956 animals genotypes. The model addressed for prediction of genetic values was the WssGBLUP/S2, and at each iteration were re-estimated effects of SNPs from genomic genetic values (GEBVs). Eight regions were found explaining over 1% of the total genetic variance, and accounting for 12.25% of the total genetic variance of the RFI, located on chromosomes 5, 6, 9, 11, 14, 17 and 27, with 71 genes iden... (Complete abstract click electronic access below) / Mestre
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The LASSO linear mixed model for mapping quantitative trait lociFoster, Scott David January 2006 (has links)
This thesis concerns the identification of quantitative trait loci (QTL) for important traits in cattle line crosses. One of these traits is birth weight of calves, which affects both animal production and welfare through correlated effects on parturition and subsequent growth. Birth weight was one of the traits measured in the Davies' Gene Mapping Project. These data form the motivation for the methods presented in this thesis. Multiple QTL models have been previously proposed and are likely to be superior to single QTL models. The multiple QTL models can be loosely divided into two categories : 1 ) model building methods that aim to generate good models that contain only a subset of all the potential QTL ; and 2 ) methods that consider all the observed marker explanatory variables. The first set of methods can be misleading if an incorrect model is chosen. The second set of methods does not have this limitation. However, a full fixed effect analysis is generally not possible as the number of marker explanatory variables is typically large with respect to the number of observations. This can be overcome by using constrained estimation methods or by making the marker effects random. One method of constrained estimation is the least absolute selection and shrinkage operator (LASSO). This method has the appealing ability to produce predictions of effects that are identically zero. The LASSO can also be specified as a random model where the effects follow a double exponential distribution. In this thesis, the LASSO is investigated from a random effects model perspective. Two methods to approximate the marginal likelihood are presented. The first uses the standard form for the double exponential distribution and requires adjustment of the score equations for unbiased estimation. The second is based on an alternative probability model for the double exponential distribution. It was developed late in the candidature and gives similar dispersion parameter estimates to the first approximation, but does so in a more direct manner. The alternative LASSO model suggests some novel types of predictors. Methods for a number of different types of predictors are specified and are compared for statistical efficiency. Initially, inference for the LASSO effects is performed using simulation. Essentially, this treats the random effects as fixed effects and tests the null hypothesis that the effect is zero. In simulation studies, it is shown to be a useful method to identify important effects. However, the effects are random, so such a test is not strictly appropriate. After the specification of the alternative LASSO model, a method for making probability statements about the random effects being above or below zero is developed. This method is based on the predictive distribution of the random effects (posterior in Bayesian terminology). The random LASSO model is not sufficiently flexible to model most QTL mapping data. Typically, these data arise from large experiments and require models containing terms for experimental design. For example, the Davies' Gene Mapping experiment requires fixed effects for different sires, a covariate for birthdate within season and random normal effects for management group. To accommodate these sources of variation a mixed model is employed. The marker effects are included into this model as random LASSO effects. Estimation of the dispersion parameters is based on an approximate restricted likelihood (an extension of the first method of estimation for the simple random effects model). Prediction of the random effects is performed using a generalisation of Henderson's mixed model equations. The performance of the LASSO linear mixed model for QTL identification is assessed via simulation. It performs well against other commonly used methods but it may lack power for lowly heritable traits in small experiments. However, the rate of false positives in such situations is much lower. Also, the LASSO method is more precise in locating the correct marker rather than a marker in its vicinity. Analysis of the Davies' Gene Mapping Data using the methods described in this thesis identified five non-zero marker-within-sire effects ( there were 570 such effects). This analysis clearly shows that most of the genome does not affect the trait of interest. The simulation results and the analysis of the Davies' Gene Mapping Project Data show that the LASSO linear mixed model is a competitive method for QTL identification. It provides a flexible method to model the genetic and experimental effects simultaneously. / Thesis (Ph.D.)--School of Agriculture, Food and Wine, 2006.
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Structural studies of three cell signaling proteins : crystal structures of EphB1, PTPA, and YegSBakali, Amin January 2007 (has links)
<p>Kinases and phosphatases are key regulatory proteins in the cell. The disruption of their activities leads ultimately to the abolishment of the homeostasis of the cell, and is frequently correlated with cancer. EphB1 is a member of the largest family of receptor tyrosine kinases. It is associated with neurogenesis, angiogenesis, and cancer. The cytosolic part of the human EphB1 receptor is composed of two domains. Successful generation of soluble constructs, using a novel random construct screening approach, led to the structure determination of the kinase domain of this receptor. The native structure and the complex structure with an ATP analogue revealed novel features in the regulation of the Eph family of kinases.</p><p>The structure of PTPA, an activator of protein phosphatase 2 A, a tumor suppressor and a key phosphatase in the cell was solved. The structure revealed a novel fold containing a conserved cleft predicted to be involved in interaction with PP2A.</p><p>Finally, the structure of YegS, an <i>Escherichia coli</i> protein annotated as a putative diacylglycerol kinase, has been determined. Beside the elucidation of its atomic structure, a phosphatidylglycerol (PG) kinase activity, never seen before, has been assigned to YegS based on biochemical studies. The YegS structure shows resemblance to the fold previously seen in NAD kinases. The structure also revealed the existence of a novel metal site that could potentially play a regulatory role. The YegS structure has important implications for understanding related proteins in pathogenic organisms and is the first homologue of a human lipid kinase for which the structure has been elucidated.</p>
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Endocytic trafficking is required for neuron cell death through regulating TGF-beta signaling in <i>Drosophila melanogaster</i>Wang, Zixing 01 August 2011 (has links)
Programmed cell death (PCD) is an essential feature during the development of the central nervous system in Drosophila as well as in mammals. During metamorphosis, a group of peptidergic neurons (vCrz) are eliminated from the larval central nervous system (CNS) via PCD within 6-7 h after puparium formation. To better understand this process, we first characterized the development of the vCrz neurons including their lineages and birth windows using the MARCM (Mosaic Analysis with a Repressible Cell Marker) assay. Further genetic and MARCM analyses showed that not only Myoglianin (Myo) and its type I receptor Baboon is required for neuron cell death, but also this death signal is extensively regulated by endocytic trafficking in Drosophila melanogaster. We found that clathrin-mediated membrane receptor internalization and subsequent endocytic events involved in Rab5-dependent early endosome and Rab11-dependent recycling endosome differentially participate in TGF-β [beta] signaling. Two early endosome-enriched proteins, SARA and Hrs, are found to act as a cytosolic retention factor of Smad2, indicating that endocytosis mediates TGF-β [beta] signaling through regulating the dissociation of Smad2 and its cytosolic retention factor.
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