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PubMed2000

Polyadenylation of three classes of chloroplast RNA in Chlamydomonas reinhadtii.

Three classes of RNA, represented by atpB and petD mRNAs, Arg and Glu tRNAs, and 5S rRNA, were found to exist in polyadenylated form in Chlamydomonas reinhardtii chloroplasts. Sequence analysis of cDNA clones derived from reverse transcriptase-polymerase chain reaction protocols used to select polyadenylated RNAs revealed that, at least for the mRNAs and tRNAs, there are three apparent types of polyadenylation. In the first case, the poly(A) tail is added at or near the mature 3' end, even when this follows a strong secondary structure. In the second case, the tail is added to pre-mRNA or pre-tRNA, suggesting a possible competition between polyadenylation and RNA-processing pathways. Finally, in all cases, the poly(A) tail can be added internally, possibly as a part of an RNA-decay pathway. The tails found in Chlamydomonas chloroplasts differ from those of spinach chloroplasts in adenine content, being nearly homopolymeric (>98% adenine) versus 70% in spinach, and are similar in length to those of Escherichia coli, being mostly between 20 and 50 nt. In vitro assays using a Chlamydomonas chloroplast protein extract showed that a 3' end A25 tail was sufficient to stimulate rapid degradation of atpB RNA in vitro, with a lesser effect for petD, and only minor effects on trnE. We therefore propose that polyadenylation contributes to mRNA degradation in Chlamydomonas chloroplasts, but that its effect may vary.

Animals↗

PubMed2004

Comparison of transgene expression mediated by several Fiber-modified adenovirus vectors in trophoblast cells.

The transfer of genes of interest is a useful method for studying placental biology. Recombinant adenovirus (Ad) vector is an efficient vector for transgene expression. An interaction between the fiber of Ad and the coxsackievirus and adenovirus receptor on the cell membrane is the first step in infection. We previously developed fiber-modified Ad vectors and showed that they improved transgene activity in several cell lines when compared to wild-type vector. In the present study, we examined the ability of three fiber-modified Ad vectors to transduce human choriocarcinoma cell lines (JEG-3, JAR and BeWo) and rat trophoblast cell lines (Rcho-1, TR-TBT 18d-1 and TR-TBT 18d-2). We compared the transgene efficacy of wild-type Ad-L2 vector, Ad-RGD(HI)-L2 vector containing an Arg-Gly-Asp motif, Ad-K7(C)-L2 vector containing a 7-tandem lysine motif, and Ad-RGD(HI)K7(C)-L2 vector containing both motifs in the fiber. We used the luciferase gene as a reporter gene. In the human and rodent trophoblast cell lines, Ad-RGD(HI)-L2 had the greatest infectious potential, followed by Ad-RGD(HI)K7(C)-L2, Ad-K7(C)-L2 and Ad-L2. Compared to the amount of luciferase produced by wild-type vector, Ad-RGD(HI)-L2 mediated 8.1-fold the amount of luciferase in JEG-3 cells, 13.5-fold in JAR cells, 76.8-fold in BeWo cells, 5.0-fold in Rcho-1, 19.4-fold in TR-TBT 18d-1 and 15.0-fold in TR-TBT 18d-2. These results indicate that Ad-RGD(HI) is a potential recombinant Ad vector for transgene expression in some trophoblast cell lines.

Adenoviridae↗

PubMed2006

Experimental study of recombinant eukaryotic expression vector of human eNOS in ECV304.

BACKGROUND AND PURPOSE: Gene transfer with recombinant non-viral vectors encoding vasodilator proteins, such as endothelial nitric oxide synthase (eNOS), maybe a preferential choice in gene therapy of artery restenosis following angioplasty, stent or anastomosis. However, the transfection rate of a non-viral vector, the harmful effects of eNOS transfection on endothelial cells (EC) and the control release of nitric oxide (NO) have been controversial. We designed the eukaryotic expression vector pcDNA3.1-eNOS to study the regulated expression of eNOS (in the presence of various chemical agents) and to evaluate the exogenous NO effect on EC proliferation in vitro. METHODS: The full-length human eNOS cDNA was inserted into the EcoRI cloning site of the pcDNA3.1 expression plasmid and the eNOS direction was tested by restriction enzyme digestion with XhoI to construct recombinant pcDNA3.1-eNOS. After co-transfection of pcDNA3.1-eNOS with pcDNA3.0-EGFP mediated by cationic liposomes into Human umbilical vein endothelial cells (ECV304), the transfection rate and the effect on ECV304 proliferation were calculated by fluorescence microscopy and flowcytometry. eNOS mRNA and protein were detected by reverse transcription-PCR (RT-PCR) and immunofluorescence, respectively. The eNOS activity, NO release and changes of the relevant cells growth curve were assessed after treating the transfected cells with four independent factors including ie Ca2+, L-arginine (L-Arg), Ethylene Diamine Tetraacetic Acid (EDTA) and N-nitro-L-arginine methylester (L-NAME). In addition, we examined the non-transfected cells status by isolated sodium nitroprusside (SNP) treatment. RESULT: eNOS cDNA was inserted into pcDNA3.1 in the proper direction. RT-PCR analysis showed that pcDNA3.1-eNOS transfected cells could express eNOS mRNA. The rate of eNOS transfection was 39.6 +/- 3.4%. Immunofluorescence staining displayed that subcellular localisation of eNOS was most prominent in plasma membrane and perinuclear regions of the cell. The eNOS activity of eNOS transfected cells had not increased significantly, whereas, in the presence of Ca2+, L-Arg, EDTA, and L-NAME, the eNOS activity was 96.98 +/- 13.47, 32.57 +/- 6.39, 11.63 +/- 3.02, 15.56 L 7.34 U/ml respectively and the NO level was 55.34 +/- 11.19, 9.43 +/- 4.51, 2.63 +/- 1.41, 3.73 +/- 1.65 mmol/L, respectively. Meanwhile, the growth curves of EC shifted. SNP also had obvious growth-inhibiting effects on the cells. Together, the ECV304 growth curve went downward in a NO concentration-dependent manner. CONCLUSION: Eukaryotic expression vector pcDNA3.1-eNOS was constructed successfully with the ability to express human eNOS mRNA and protein in EC effectively. The activity of eNOS in EC could be regulated by certain exogenous factors. Ca2+ was an important factor promoting NO release and excess NO had a cytotoxic effect on EC in vitro. Controlled release of NO in vivo and polygenic measurements might be considered in more clinical gene therapy studies.

Cell Line↗