Search results for "Cloning"
showing 10 items of 498 documents
Two heterologously expressed Planobispora rosea proteins cooperatively induce Streptomyces lividans thiostrepton uptake and storage from the extracel…
2010
Abstract Background A bacterial artificial chromosomal library of Planobispora rosea, a genetically intractable actinomycete strain, was constructed using Escherichia coli-Streptomyces artificial chromosome (ESAC) and screened for the presence of genes known to be involved in the biosynthesis of antibiotics. Results One clone with a 40 kb insert showed antimicrobial activity against Gram positive bacteria. Insert sequence analysis and subcloning experiments revealed that the bioactivity was due to a 3.5 kb DNA fragment containing two open reading frames. These orfs encode two proteins with high similarity to a putative membrane protein of Streptomyces coelicolor and to the nogalamycin resis…
Cloning, purification, and nucleotide-binding traits of the catalytic subunit A of the V1VO ATPase from Aedes albopictus.
2007
The Asian tiger mosquito, Aedes albopictus, is commonly infected by the gregarine parasite Ascogregarina taiwanensis, which develops extracellularly in the midgut of infected larvae. The intracellular trophozoites are usually confined within a parasitophorous vacuole, whose acidification is generated and controlled by the V(1)V(O) ATPase. This proton pump is driven by ATP hydrolysis, catalyzed inside the major subunit A. The subunit A encoding gene of the Aedes albopictus V(1)V(O) ATPase was cloned in pET9d1-His(3) and the recombinant protein, expressed in the Escherichia coli Rosetta 2 (DE3) strain, purified by immobilized metal affinity- and ion-exchange chromatography. The purified prote…
Evaluating Bug-Fixing in Software Product Lines
2016
[Background] Bug-fixing could be complex in industrial practice since thousands of products share features in their configuration. Despite the importance and complexity of bug-fixing, there is still a lack of empirical data about the difficulties found in industrial Software Product Lines (SPLs). [Aims] This paper aims to evaluate engineers' performance fixing errors and propagating the fixes to other configured products in the context of an industrial SPL. [Method] We designed and conducted an empirical study to collect data with regard to bug-fixing tasks within the context of a Induction Hob SPL in the BSH group, the largest manufacturer of home appliances in Europe. [Results] We found t…
Introduction of Cytochrome P-450 Genes into V79 Chinese Hamster Cells to Generate New Mutagenicity Test Systems
1989
Usually, cultivated cells have poor capabilities to metabolize promutagens and procarcinogens. This is particularly true for cells that grow fast and have a high cloning efficiency, as is the case with V79 Chinese hamster cells. For this reason, these cells are being extensively used in mutagenicity tests. But, due to the fact that particularly these cells lack cytochrome P-450 activities, promutagens and procarcinogens have to be incubated with an exogenous metabolizing system, e.g. liver homogenate preparations, in order to generate reactive metabolites. These extracellularly generated metabolites are then given to V79 cells in order to check for their potency to mutate the chromosomal DN…
Cloning and characterization of new orphan nuclear receptors and their developmental profiles duringTenebriometamorphosis
1999
Five PCR fragments corresponding to a part of the DNA-binding domain of different hormone nuclear receptors were isolated from Tenebrio molitor mRNAs. The sequence identity of three of them with known Drosophila nuclear receptors strongly suggests that they are the Tenebrio orthologs of seven-up, DHR3 and β-FTZ-F1, and thus named Tmsvp, TmHR3 and TmFTZ-F1. The full-length sequences of the other two were established. TmHR78 is either a new receptor of the DHR78 family or the same gene which has evolved rapidly, particularly in the E domain. TmGRF belongs to the GCNF1 family and its in vitro translated product binds to the extended half site TCAAGGTCA with high affinity. The periods of expres…
Cloning and expression of the sponge longevity gene SDLAGL.
2000
Porifera show a characteristic Bauplan in spite of the fact that (almost) all cells are telomerase-positive and presumably provided with an unlimited potency for cell proliferation. One gene, SDLAGL, was identified in the marine sponge Suberites domuncula whose deduced polypeptide showed high sequence similarity to the longevity assurance genes from other Metazoa. While in single cells no transcripts of SDLAGL could be identified, high expression was seen after re-aggregation of single cells and in proliferating cells of primmorphs.
High Expression of Human CYP2C in Immortalized Human Liver Epithelial Cells
2010
Cell lines stably expressing high levels of single isozymes of human CYP2C genes (CYP2C8, CYP2C9, CYP2C18 and CYP2C19) have been successfully generated by transfecting liver epithelial human cells (THLE) with an appropriate expression vector. To this aim, cDNAs encoding for each CYP2C gene were inserted by blunt-ended cloning into the unique insertion site of the singular expression vector pCMVneo. The recombinant pCMV2C8, pCMV2C9, pCMV2C18 and pCMV2C19 vectors were liposome-mediated transfected into THLE cells. The resulting transgenic cells, designated as T5-2C8, T5-2C9, T5-2C18 and T5-2C19, were cloned and the expression of the ectopic gene, mRNA and protein, was investigated by RT-PCR a…
Interrogation of genomes by molecular copy-number counting (MCC)
2006
Human cancers and some congenital traits are characterized by cytogenetic aberrations including translocations, amplifications, duplications or deletions that can involve gain or loss of genetic material. We have developed a simple method to precisely delineate such regions with known or cryptic genomic alterations. Molecular copy-number counting (MCC) uses PCR to interrogate miniscule amounts of genomic DNA and allows progressive delineation of DNA content to within a few hundred base pairs of a genomic alteration. As an example, we have located the junctions of a recurrent nonreciprocal translocation between chromosomes 3 and 5 in human renal cell carcinoma, facilitating cloning of the br…
Cloning and sequencing of the chicken egg-white avidin-encoding gene and its relationship with the avidin-related genes Avrl-Avr5
1995
Abstract The gene encoding chicken egg-white avidin (Avd) was amplified from chromosomal DNA, cloned and sequenced. The entire coding region of preavidin (pre-Avd) containing four exons was identified by comparing the Avd gene (1119 bp) with the cDNA. It had a high identity percentage (91–95%) with the previously isolated Avd-related genes 1–5 (Avrl–Avr5) . Interestingly, comparison of Avd with the Avr genes showed that the introns were better conserved (on average 97%) than the exons (90%). The Avd gene, as well as the cDNA, encodes a Gln residue at position 53 of the mature protein, which is in contrast to the previously determined amino-acid sequence.
Evolutionary analysis of G-proteins in early metazoans: Cloning of α- and β-subunits from the sponge Geodia cydonium1The sequences reported here have…
1998
G-protein-coupled (seven-transmembrane segment)-receptors represent a major group of metazoan receptors, involved in transduction of extracellular signals. The G-proteins, which are made up of Galpha/beta/gamma-subunits, link the receptors to the effector system(s). To analyze the phylogenetic relationships among the metazoan alpha-subunits of G-proteins, cDNAs of alpha-subunits were isolated from Geodia cydonium, a marine sponge belonging to the lowest metazoan phylum, Porifera. One encodes a putative isotype of a stimulator of the adenylyl cyclase (Galpha s), another one a putative inhibitor of the adenylyl cyclase (Galpha i/o) and the third one a putative activator of phospholipase C (Ga…