Faculty Collaboration Database

Characterization of replication origins flanking the 23S rRNA gene in tobacco chloroplast DNA. Plant Mol Biol 1996 Nov;32(4):693-706

Date

11/01/1996

Pubmed ID

8980521

DOI

10.1007/BF00020210

Scopus ID

2-s2.0-0030292780 (requires institutional sign-in at Scopus site)   26 Citations

Abstract

Using 5' end-labeled nascent strands of tobacco chloroplast DNA (ctDNA) as a probe, replication displacement loop (D-loop) regions were identified. The strongest hybridization was observed with restriction fragments containing the rRNA genes from the inverted repeat region. Two-dimensional gel analysis of various digests of tobacco ctDNA suggested that a replication origin is located near each end of the 7.1 kb BamHI fragment containing part of the rRNA operon. Analysis of in vitro replication products indicated that templates from either of the origin regions supported replication, while the vector alone or ctDNA clones from other regions of the genome did not support in vitro replication. Sequences from both sides of the BamHI site in the rRNA spacer region were required for optimal in vitro DNA replication activity. Primer extension was used for the first time to identify the start site of DNA synthesis for the D-loop in the rRNA spacer region. The major 5' end of the D-loop was localized to the base of a stem-loop structure which contains the rRNA spacer BamHI site. Primer extension products were insensitive to both alkali and RNase treatment, suggesting that RNA primers had already been removed from the 5' end of nascent DNA. Location of an origin in the rRNA spacer region of ctDNA from tobacco, pea and Oenothera suggests that ctDNA replication origins may be conserved in higher plants.

Author List

Lu Z, Kunnimalaiyaan M, Nielsen BL

MESH terms used to index this publication - Major topics in bold

Base Composition
Base Sequence
DNA Replication
DNA, Chloroplast
Electrophoresis, Gel, Two-Dimensional
Molecular Sequence Data
Nucleic Acid Conformation
Plants, Toxic
RNA, Ribosomal, 23S
Replication Origin
Restriction Mapping
Sequence Analysis, DNA