PlasmaDNA: a free, cross-platform plasmid manipulation program for molecular biology laboratories

<p>Abstract</p> <p>Background</p> <p>Most molecular biology experiments, and the techniques associated with this field of study, involve a great deal of engineering in the form of molecular cloning. Like all forms of engineering, perfect information about the starting material is crucial for successful completion of design and strategies.</p> <p>Results</p> <p>We have generated a program that allows complete <it>in silico </it>simulation of the cloning experiment. Starting with a primary DNA sequence, PlasmaDNA looks for restriction sites, open reading frames, primer annealing sequences, and various common domains. The databases are easily expandable by the user to fit his most common cloning needs. PlasmaDNA can manage and graphically represent multiple sequences at the same time, and keeps in memory the overhangs at the end of the sequences if any. This means that it is possible to virtually digest fragments, to add the digestion products to the project, and to ligate together fragments with compatible ends to generate the new sequences. Polymerase Chain Reaction (PCR) fragments can also be virtually generated using the primer database, automatically adding to the fragments any 5' extra sequences present in the primers.</p> <p>Conclusion</p> <p>PlasmaDNA is a program available both on Windows and Apple operating systems, designed to facilitate molecular cloning experiments by building a visual map of the DNA. It then allows the complete planning and simulation of the cloning experiment. It also automatically updates the new sequences generated in the process, which is an important help in practice. The capacity to maintain multiple sequences in the same file can also be used to archive the various steps and strategies involved in the cloning of each construct. The program is freely available for download without charge or restriction.</p

GDNF promotes tubulogenesis of GFRα1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase

Glial cell line–derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor α1 (GFRα1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRα1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRα1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRα1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRα1-positive and GFRα1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling

Roles of c-Src and hDRR1 in glioma cell invasion

Malignant glioma is the major brain tumor in adults, and has a poor prognosis. The failure to control invasive cell subpopulations may be the key reason for local glioma recurrence after radical tumor resection, and may contribute substantially to the failure of the other treatment modalities, such as radiation therapy and chemotherapy. As a model for this invasion, we have implanted spheroids from a human glioma cell line (U251) in three-dimensional collagen type I matrices which these cells readily invade. First, we observed that the Src family kinase specific pharmacological inhibitors PP2 and SU6656 significantly inhibited the invasion of the cells in this assay, which was then confirmed by expression of two inhibitors of Src family function, dominant inhibitory Src and CSK. Fluorescent time-lapse microscopy on U251 cells stably expressing a YFP-actin construct shows that PP2 caused the disappearance of peripheral membrane ruffles within minutes in monolayer cultures, and induced the loss of actin bursting at the leading tip of the invadopodium in three-dimensions. The inhibition of Src family activity is thus a potential therapeutic approach to treating highly invasive malignant glioma. In the second part, we analyze the role of a novel protein, hDRR1, which was cloned from a functional screen of genes involved in glioma cell hyperinvasion. We show that hDRR1 localizes endogenously and when overexpressed to the actin cytoskeleton, via two independent but homologous actin-localization domains. We also show that the C-terminus of hDRR1 can bind to the light chain of MAP1A and MAP1B, and that hDRR1 overexpression in invading glioma cells results in a decrease in the percentage of cells adopting a polarized morphology. These results suggest that hDRR1 represents a novel actin-tubulin bridging protein that plays important roles in cytoskeletal events

Phenotypic analyses of PTP-PEST null cells and gene targeting of its substrate, p130cas

To understand the physiological role of the protein tyrosine phosphatase (PTP) PEST, the phenotypes of PTP-PEST (--/--) mouse fibroblasts have been studied PTP-PEST was shown to translocate to the membrane periphery following stimulation by the extracellular matrix protein fibronectin, and to play a role in cell migration, spreading, and cytokinesis. For this action, PTP-PEST was shown to regulate the phosphorylation levels of p130cas, paxillin, FAK and PSTPIP, and their association with SH2 domain-containing proteins like Src and Crk. From these proteins, p130cas is known to be a direct substrate. Experiments for the obtention of p130cas (--/--) embryos and cell lines have also been initiated, up to the generation of adult chimeric animals. In the process, a genomic fragment of p130cas containing a C-terminal exon has been cloned and analysed

PlasmaDNA: a free, cross-platform plasmid manipulation program for molecular biology laboratories-2

<p><b>Copyright information:</b></p><p>Taken from "PlasmaDNA: a free, cross-platform plasmid manipulation program for molecular biology laboratories"</p><p>http://www.biomedcentral.com/1471-2199/8/77</p><p>BMC Molecular Biology 2007;8():77-77.</p><p>Published online 17 Sep 2007</p><p>PMCID:PMC2075515.</p><p></p>inding sites in an orientation favorable for a PCR. B) PCR window, selecting Plasmid X as the template, and the two primers. The resulting fragment can be added to the project to be further analyzed and manipulated

PlasmaDNA: a free, cross-platform plasmid manipulation program for molecular biology laboratories-0

<p><b>Copyright information:</b></p><p>Taken from "PlasmaDNA: a free, cross-platform plasmid manipulation program for molecular biology laboratories"</p><p>http://www.biomedcentral.com/1471-2199/8/77</p><p>BMC Molecular Biology 2007;8():77-77.</p><p>Published online 17 Sep 2007</p><p>PMCID:PMC2075515.</p><p></p>window selecting NotI as an enzyme. C) After adding both digestion products to the project, the project contains three fragments, including the original plasmid. Each of the new fragments can then be further analyzed and manipulated. The fragments can be visualized together and to scale, as shown, or one by one for greater resolution

article ID bav101; V C The Author(s) Database

These authors contributed equally to this work. Abstract The DNA target sequence is the key element in designing detection methods for genetically modified organisms (GMOs). Unfortunately this information is frequently lacking, especially for unauthorized GMOs. In addition, patent sequences are generally poorly annotated, buried in complex and extensive documentation and hard to link to the corresponding GM event. Here, we present the JRC GMO-Amplicons, a database of amplicons collected by screening public nucleotide sequence databanks by in silico determination of PCR amplification with reference methods for GMO analysis. The European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) provides these methods in the GMOMETHODS database to support enforcement of EU legislation and GM food/feed control. The JRC GMO-Amplicons database is composed of more than 240 000 amplicons, which can be easily accessed and screened through a web interface. To our knowledge, this is the first attempt at pooling and collecting publicly available sequences related to GMOs in food and feed. The JRC GMOAmplicons supports control laboratories in the design and assessment of GMO methods, providing inter-alia in silico prediction of primers specificity and GM targets coverage. The new tool can assist the laboratories in the analysis of complex issues, such as the detection and identification of unauthorized GMOs. Notably, the JRC GMO-Amplicons database allows the retrieval and characterization of GMO-related sequences included in patents documentation. Finally, it can help annotating poorly described GM sequences and identifying new relevant GMO-related sequences in public databases. The JRC GMO-Amplicons is freely accessible through a web-based portal that is hosted on the EU-RL GMFF website