Restriction Fragment Length Polymorphism Linkage Map for Arabidopsis thaliana

We have constructed a restriction fragment length polymorphism linkage map for the nuclear genome of the flowering plant Arabidopsis thaliana. The map, containing 90 randomly distributed molecular markers, is physically very dense; >50% of the genome is within 1.9 centimorgans, or approx 270 kilobase pairs, of the mapped DNA fragments. The map was based on the meiotic segregation of markers in two different crosses. The restriction fragment length polymorphism linkage groups were integrated with the five classically mapped linkage groups by virtue of mapped mutations included in these crosses. Markers consist of both cloned Arabidopsis genes and random low-copy-number genomic DNA clones that are able to detect polymorphisms with the restriction enzymes EcoRI, Bgl II, and/or Xba I. These cloned markers can serve as starting points for chromosome walking, allowing for the isolation of Arabidopsis genes of known map location. The restriction fragment length polymorphism map also can associate clones of unknown gene function with mutant phenotypes, and vice versa

Molecular Genetic Studies in Arabidopsis thaliana: I. Cloning and Characterization of the Alcohol Dehydrogenase Gene; II. Complementation of an Alcohol Dehydrogenase Mutant; III. A Restriction Fragment Length Polymorphism Map of the Genome

This thesis examines several genetic aspects of the flowering plant Arabidopsis thaliana which has previously been shown to possess several attributes for molecular genetic experiments such as a short life cycle, small size, fecundity, and a strong background of classical genetics. In particular, the nuclear genome is remarkably small and consists almost entirely of single copy sequences. Two approaches are established for the isolation and study of plant genes using Arabidopsis as a model system. One approach demonstrates complementation of a mutant phenotype using the Arabidopsis alcohol dehydrogenase (ADH) gene. The ADH gene was first isolated from a genomic DNA library by cross-hybridization with a maize ADH1 gene probe. The gene structure was studied by DNA sequence analysis and mapping of the transcript. The gene conferred wild-type ADH activity to an Arabidopsis ADH null mutant when introduced by Agrobacterium-mediated transformation. Transformed plants were subjected to genetic and molecular analyses. The ADH gene may have utility as a gene tag in transformation experiments. The second topic of this thesis is the construction of a genetic linkage map of restriction fragment length polymorphisms (RFLPs). The map provides an approach to cloning genes about which nothing more is known than a mutant phenotype and a map location because the RFLP markers can serve as starting points for the isolation of overlapping clones from a genomic DNA library. The map contains 90 RFLP markers distributed randomly throughout the nuclear genome. Since the genome consists of about 70,000 kilobase pairs, the markers are at an average physical spacing of approximately 780 kilobase pairs. The map is based on meiotic segregation of RFLPs in two different crosses detected with the restriction enzymes EcoRI, BglII, and XbaI. The RFLP linkage groups have been aligned with the standard genetic map of approximately 80 mutation markers.</p

To grow old: regulatory role of ethylene and jasmonic acid in senescence

Funding for Open Access provided by the UMD Libraries Open Access Publishing Fund.Senescence, the final stage in the development of an organ or whole plant, is a genetically programmed process controlled by developmental and environmental signals. Age-related signals underlie the onset of senescence in specific organs (leaf, flower, and fruit) as well as the whole plant (monocarpic senescence). Rudimentary to most senescence processes is the plant hormone ethylene, a small gaseous molecule critical to diverse processes throughout the life of the plant. The role of ethylene in senescence was discovered almost 100 years ago, but the molecular mechanisms by which ethylene regulates senescence have been deciphered more recently primarily through genetic and molecular studies in Arabidopsis. Jasmonic acid (JA), another plant hormone, is emerging as a key player in the control of senescence. The regulatory network of ethylene and JA involves the integration of transcription factors, microRNAs, and other hormones. In this review, we summarize the current understanding of ethylene’s role in senescence, and discuss the interplay of ethylene with JA in the regulation of senescence

Ferret: a sentence-based literature scanning system

The rapid pace of bioscience research makes it very challenging to track relevant articles in one’s area of interest. MEDLINE, a primary source for biomedical literature, offers access to more than 20 million citations with three-quarters of a million new ones added each year. Thus it is not surprising to see active research in building new document retrieval and sentence retrieval systems. We present Ferret, a prototype retrieval system, designed to retrieve and rank sentences (and their documents) conveying gene-centric relationships of interest to a scientist. The prototype has several features. For example, it is designed to handle gene name ambiguity and perform query expansion. Inputs can be a list of genes with an optional list of keywords. Sentences are retrieved across species but the species discussed in the records are identified. Results are presented in the form of a heat map and sentences corresponding to specific cells of the heat map may be selected for display. Ferret is designed to assist bio scientists at different stages of research from early idea exploration to advanced analysis of results from bench experiments. Three live case studies in the field of plant biology are presented related to Arabidopsis thaliana. The first is to discover genes that may relate to the phenotype of open immature flower in Arabidopsis. The second case is about finding associations reported between ethylene signaling and a set of 300+ Arabidopsis genes. The third case is on searching for potential gene targets of an Arabidopsis transcription factor hypothesized to be involved in plant stress responses. Ferret was successful in finding valuable information in all three cases. In the first case the bZIP family of genes was identified. In the second case sentences indicating relevant associations were found in other species such as potato and jasmine. In the third sentences led to new research questions about the plant hormone salicylic acid. Ferret successfully retrieved relevant gene-centric sentences from PubMed records. The three case studies demonstrate end user satisfaction with the system.https://doi.org/10.1186/s12859-015-0630-

Uncovering multiple axonal targeting pathways in hippocampal neurons

Neuronal polarity is, at least in part, mediated by the differential sorting of membrane proteins to distinct domains, such as axons and somata/dendrites. We investigated the pathways underlying the subcellular targeting of NgCAM, a cell adhesion molecule residing on the axonal plasma membrane. Following transport of NgCAM kinetically, surprisingly we observed a transient appearance of NgCAM on the somatodendritic plasma membrane. Down-regulation of endocytosis resulted in loss of axonal accumulation of NgCAM, indicating that the axonal localization of NgCAM was dependent on endocytosis. Our data suggest the existence of a dendrite-to-axon transcytotic pathway to achieve axonal accumulation. NgCAM mutants with a point mutation in a crucial cytoplasmic tail motif (YRSL) are unable to access the transcytotic route. Instead, they were found to travel to the axon on a direct route. Therefore, our results suggest that multiple distinct pathways operate in hippocampal neurons to achieve axonal accumulation of membrane proteins

Anti-Neuroinflammatory Effects of the Calcium Channel Blocker Nicardipine on Microglial Cells: Implications for Neuroprotection

Background/Objective Nicardipine is a calcium channel blocker that has been widely used to control blood pressure in severe hypertension following events such as ischemic stroke, traumatic brain injury, and intracerebral hemorrhage. However, accumulating evidence suggests that inflammatory processes in the central nervous system that are mediated by microglial activation play important roles in neurodegeneration, and the effect of nicardipine on microglial activation remains unresolved. Methodology/Principal Findings In the present study, using murine BV-2 microglia, we demonstrated that nicardipine significantly inhibits microglia-related neuroinflammatory responses. Treatment with nicardipine inhibited microglial cell migration. Nicardipine also significantly inhibited LPS plus IFN-γ-induced release of nitric oxide (NO), and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, nicardipine also inhibited microglial activation by peptidoglycan, the major component of the Gram-positive bacterium cell wall. Notably, nicardipine also showed significant anti-neuroinflammatory effects on microglial activation in mice in vivo. Conclusion/Significance The present study is the first to report a novel inhibitory role of nicardipine on neuroinflammation and provides a new candidate agent for the development of therapies for inflammation-related neurodegenerative diseases

Do proxies reflect patients' health concerns about urinary incontinence and gait problems?

BACKGROUND: While falls and urinary incontinence are prevalent among older patients, who sometimes rely on proxies to provide their health information, the validity of proxy reports of concern about falls and urinary incontinence remains unknown. METHODS: Telephone interviews with 43 consecutive patients with falls or fear of falling and/or bothersome urinary incontinence and their proxies chosen by patients as most knowledgeable about their health. The questionnaire included items derived from the Medical Outcomes Study Short Form 12 (SF-12), a scale assessing concerns about urinary incontinence (UI), and a measure of fear of falling, the Falls Efficacy Scale (FES). Scores were estimated using items asking the proxy perspective (6 items from the SF-12, 10 items from a UI scale, and all 10 FES items). Proxy and patient scores were compared using intraclass correlation coefficients (ICC, one-way model). Variables associated with absolute agreement between patients and proxies were explored. RESULTS: Patients had a mean age of 81 years (range 75–93) and 67% were female while proxies had a mean age of 70 (range 42–87) and 49% were female. ICCs were 0.63 for the SF-12, 0.52 for the UI scale, and 0.29 for the FES. Proxies tended to understate patients' general health and incontinence concern, but overstate patients' concern about falling. Proxies who lived with patients and those who more often see patients more closely reflected patient FES scores compared to those who lived apart or those who saw patients less often. Internal consistency reliability of proxy responses was 0.62 for the SF-12, 0.86 for the I-QOL, and 0.93 for the FES. In addition, construct validity of the proxy FES scale was supported by greater proxy-perceived fear of falling for patients who received medical care after a fall during the past 12 months (p < .05). CONCLUSION: Caution should be exercised when using proxies as a source of information about older patients' health perceptions. Questions asking about proxies' views yield suboptimal agreement with patient responses. However, proxy scales of UI and fall concern are internally consistent and may provide valid independent information

Modulators of Prostate Cancer Cell Proliferation and Viability Identified by Short-Hairpin RNA Library Screening

There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets