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Stable Transformants (stable + transformant)
Selected AbstractsA designed curved DNA segment that is a remarkable activator of eukaryotic transcriptionFEBS JOURNAL, Issue 24 2006Noriyuki Sumida To identify artificial DNA segments that can stably express transgenes in the genome of host cells, we built a series of curved DNA segments that mimic a left-handed superhelical structure. Curved DNA segments of 288 bp (T32) and 180 bp (T20) were able to activate transcription from the herpes simplex virus thymidine kinase (tk) promoter by approximately 150-fold and 70-fold, respectively, compared to a control in a transient transfection assay in COS-7 cells. The T20 segment was also able to activate transcription from the human adenovirus type 2 E1A promoter with an 18-fold increase in the same assay system, and also activated transcription from the tk promoter on episomes in COS-7 cells. We also established five HeLa cell lines with genomes containing T20 upstream of the transgene promoter and control cell lines with T20 deleted from the transgene locus. Interestingly, T20 was found to activate transcription in all the stable transformants, irrespective of the locus. This suggests that the T20 segment may allow stable expression of transgenes, which is of importance in many fields, and may also be useful for the construction of nonviral vectors for gene therapy. [source] Use of , -Glucuronidase Activity to Quantify the Growth of Fusarium oxysporum f. sp. radicis-lycopersici during Infection of TomatoJOURNAL OF PHYTOPATHOLOGY, Issue 6 2005K. K. Papadopoulou Abstract The , -glucuronidase (gus) reporter gene was integrated into the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici (FORL) in a co-transformation experiment using the hygromycin B resistance (hph) gene as selective marker, which resulted in the generation of 10 mitotically stable transformants. One transformant, F30, was selected based on the results of prior detailed characterization of the 10 transformants for growth rate, conidia production and pathogenicity in comparison with the wild-type strain. A strong positive correlation was found between GUS activity and accumulated biomass of in vitro -grown fungus and therefore GUS activity was used to study fungal growth quantitatively in two tomato lines. Although a parallel increase in lesion development and GUS activity was noted for both tomato lines, a correlation between the GUS activity and disease progression was not always possible. Interestingly, the levels of GUS activity obtained for the more resistant line were higher than those obtained for the susceptible line, indicating that disease progression in tomato caused by FORL may not be related only to the amount of fungal biomass within the root tissue. [source] Continuous expression in tobacco leaves of a Brassica napus PEND homologue blocks differentiation of plastids and development of palisade cellsTHE PLANT JOURNAL, Issue 1 2005Paul Wycliffe Summary Brassica napus complementary deoxyribonucleic acid (cDNA) clones encoding a DNA-binding protein, BnPEND, were isolated by Southwestern screening. A distinctive feature of the protein was a bZIP-like sequence in the amino-terminal portion, which, after expression in Escherichia coli, bound DNA. BnPEND transcripts were present in B. napus roots and flower buds, and to a lesser extent in stems, flowers and young leaves. Treatment in the dark for 72 h markedly increased the amount of BnPEND transcript in leaves of all ages. Sequence comparison showed that BnPEND was similar to a presumed transcription factor from B. napus, GSBF1, a protein deduced from an Arabidopsis thaliana cDNA (BX825084) and the PEND protein from Pisum sativum, believed to anchor the plastid DNA to the envelope early during plastid development. Homology to expressed sequence tag (EST) sequences from additional species suggested that BnPEND homologues are widespread among the angiosperms. Transient expression of BnPEND fused with green fluorescent protein (GFP) in Nicotiana benthamiana epidermal cells showed that BnPEND is a plastid protein, and that the 15 amino acids at the amino-terminal contain information about plastid targeting. Expression of BnPEND in Nicotiana tabacum from the Cauliflower Mosaic Virus 35S promoter gave stable transformants with different extents of white to light-green areas in the leaves, and even albino plants. In the white areas, but not in adjacent green tissue, the development of palisade cells and chloroplasts was disrupted. Our data demonstrate that the BnPEND protein, when over-expressed at an inappropriate stage, functionally blocks the development of plastids and leads to altered leaf anatomy, possibly by preventing the release of plastid DNA from the envelope. [source] Genetic Manipulation of Rubisco: Chromatium vinosum rbcL is expressed in Nicotiana tabacum but does not form a functional proteinANNALS OF APPLIED BIOLOGY, Issue 1 2002P J MADGWICK Summary N. tabacum lines that lacked functional Rubisco were transformed with plasmids encoding a chloroplast transit peptide in frame with C. vinosum rbcL and stable transformants generated. However, the transgene was transcribed at a low level and no Rubisco activity or C. vinosum large subunits were detectable in any line. [source] Entamoeba histolytica sirtuin EhSir2a deacetylates tubulin and regulates the number of microtubular assemblies during the cell cycleCELLULAR MICROBIOLOGY, Issue 7 2010Somasri Dam Summary We have discovered four sirtuin genes in Entamoeba histolytica, two of which are similar to eukaryotic sirtuins and two to bacterial and archaeal sirtuins. The eukaryotic sirtuin homologue, EhSir2a, showed NAD+ -dependent deacetylase activity and was sensitive to class III HDAC inhibitors. Localization of EhSir2a at different cellular sites suggested that this deacetylase could have multiple targets. Using an E. histolytica cDNA library in the yeast two-hybrid genetic screen, we identified several proteins that bound to EhSir2a. These proteins included Eh ,-tubulin, whose interaction with EhSir2a was validated in E. histolytica. We have shown that EhSir2a deacetylated tubulin and localized with microtubules in E. histolytica. Increased expression levels of EhSir2a in stable transformants led to reduced number of microtubular assemblies in serum synchronized cells. This effect was abrogated by mutations in the deacetylase domain of EhSir2a, showing that EhSir2a deacetylase activity affected the stability and number of microtubular assemblies during the cell cycle of E. histolytica. Our results suggest that epigenetic modification of tubulin by EhSir2a is one of the mechanisms that regulates microtubular assembly in E. histolytica. [source] | ||||||||||