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PMID: 1721704 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

In vitro self-splicing reactions of the chloroplast group I intron Cr.LSU from Chlamydomonas reinhardtii and in vivo manipulation via gene-replacement.

Nucleic acids research ·Vol. 19 ·No. 23 ·1991-12-11 ·Pages 6611-8

Thompson AJ, Herrin DL

Abstract

The group I intron from the chloroplast rRNA large subunit of Chlamydomonas reinhardtii (Cr.LSU) undergoes autocatalytic splicing in vitro. Cr.LSU displays a range of reactions typical of other group I introns. Under optimal conditions, the 5' cleavage step proceeds rapidly, but the exon-ligation step is relatively slow, and no pH dependent hydrolysis of the 3' splice site occurs. A requirement for high temperature and high [Mg2+] suggests involvement of additional splicing factors in vivo. The positions of three cyclization sites of the free intron have been mapped; two of these sites represent reactions analogous to 5'-splice site cleavage, whereas the third is an example of G-exchange. Cr.LSU contains an open reading frame (ORF) potentially encoding an 163 amino acid polypeptide. ORF function has been investigated by using chloroplast gene replacement via particle bombardment. We have shown that the ORF can be deleted from Cr.LSU without affecting splicing in vivo and it thus does not encode an essential splicing factor.

MeSH Terms
Adenosine Triphosphate/metabolism Animals Base Sequence Blotting, Southern Chlamydomonas reinhardtii/genetics,ultrastructure Chloroplasts/metabolism Cloning, Molecular Endoribonucleases/genetics Hydrogen-Ion Concentration Introns Kinetics Molecular Sequence Data Mutation Nucleic Acid Conformation Nucleotidyltransferases/genetics Open Reading Frames Plasmids RNA/metabolism RNA Splicing/genetics RNA, Circular RNA, Ribosomal/genetics Restriction Mapping Temperature Transformation, Genetic
Chemicals
RNA, Circular RNA, Ribosomal RNA Adenosine Triphosphate Nucleotidyltransferases mRNA maturase Endoribonucleases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Thompson A J
University of Texas, Department of Botany, Austin 78713.
Herrin D L
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/19.23.6611
Published
1991-12-11
Pages
6611-8
Language
English
Region
England
NLM ID
0411011
PMCID
PMC329230
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/19.23.6611 PMC Full Text View journal details
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