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A specific cis-hairpin ribozyme facilitates infection of a TMV-based DNA vector in tobacco protoplasts.
- Source :
-
Journal of virological methods [J Virol Methods] 2003 Aug; Vol. 111 (2), pp. 101-9. - Publication Year :
- 2003
-
Abstract
- The effect of a specific cis-hairpin ribozyme on TMV-based vectors in the infection of tobacco protoplasts was studied. Vectors contained full-length TMV genome cDNA linked to a T7 promoter or a CaMV 35S promoter at the 5'-end and an NOS gene polyadenylation signal at the 3'-end. The coat protein (CP) gene was replaced with the green fluorescent protein (GFPuv) gene allowing quantification of protoplast infection. In plasmids pTMVGFPRIB (T7-driven) and pSTMVGFPRIB (CaMV 35S-driven), the cDNA fragment of the cis-hairpin ribozyme (designed to specifically cleave the transcripts immediately downstream of the 3'-terminus of TMV RNA) was inserted between the 3'-terminus of TMV genome and NOS sequence. The in vitro transcript TMVGFPRIB was three- to fivefold more infectious than the control TMVGFPNOS. Northern blot analysis indicated that the 3'-terminal non-viral sequence had been cleaved from the in vitro transcripts by the cis-hairpin ribozyme soon after in vitro transcription. pSTMVGFPRIB and pSTMVGFPNOS plasmid DNAs were, as expected, less infectious than their in vitro transcript counterparts. However, pSTMVGFPRIB was somewhat more infectious than pSTMVGFPNOS. Northern blot analysis indicated that pSTMVGFPRIB synthesized more genomic and sub-genomic RNAs in the protoplasts. The significant increase in infectivity and viral RNA synthesis is due to the specific activity of the cis-hairpin ribozyme in vivo. Therefore, the cis-hairpin ribozyme described here may improve TMV-based vectors in the expression of foreign protein in plants.
- Subjects :
- Base Sequence
Green Fluorescent Proteins
Luminescent Proteins genetics
Luminescent Proteins metabolism
Molecular Sequence Data
Plasmids
RNA, Catalytic chemistry
RNA, Catalytic genetics
RNA, Viral biosynthesis
RNA, Viral genetics
Tobacco Mosaic Virus physiology
Transcription, Genetic
Genetic Vectors
Protoplasts virology
RNA, Catalytic metabolism
Nicotiana virology
Tobacco Mosaic Virus genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0166-0934
- Volume :
- 111
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Journal of virological methods
- Publication Type :
- Academic Journal
- Accession number :
- 12880925
- Full Text :
- https://doi.org/10.1016/s0166-0934(03)00153-8