title: Localization of replication origins in pea chloroplast DNA.
abstract: The locations of the two replication origins in pea chloroplast DNA (ctDNA) have been mapped by electron microscopic analysis of restriction digests of supercoiled ctDNA cross-linked with trioxalen. Both origins of replication, identified as displacement loops (D-loops), were present in the 44-kilobase-pair (kbp) SalI A fragment. The first D-loop was located at 9.0 kbp from the closest SalI restriction site. The average size of this D-loop was about 0.7 kbp. The second D-loop started 14.2 kbp in from the same restriction site and ended at about 15.5 kbp, giving it a size of about 1.3 kbp. The orientation of these two D-loops on the restriction map of pea ctDNA was determined by analyzing SmaI, PstI, and SalI-SmaI restriction digests of pea ctDNA. One D-loop has been mapped in the spacer region between the 16S and 23S rRNA genes. The second D-loop was located downstream of the 23S rRNA gene. Denaturation mapping of recombinants pCP 12-7 and pCB 1-12, which contain both D-loops, confirmed the location of the D-loops in the restriction map of pea ctDNA. Denaturation-mapping studies also showed that the two D-loops had different base compositions; the one closest to a SalI restriction site denatured readily compared with the other D-loop. The recombinants pCP 12-7 and pCB 1-12 were found to be highly active in DNA synthesis when used as templates in a partially purified replication system from pea chloroplasts. Analysis of in vitro-synthesized DNA with either of these recombinants showed that full-length template DNA was synthesized. Recombinants from other regions of the pea chloroplast genome showed no significant DNA synthesis activity in vitro.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
authors.forename: R
authors.initials: R
authors.lastname: Meeker
authors.forename: B
authors.initials: B
authors.lastname: Nielsen
authors.forename: K K
authors.initials: KK
authors.lastname: Tewari
mesh_terms: D002736:Chloroplasts / Q000648:ultrastructure*; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004278:DNA, Superhelical / Q000096:biosynthesis / Q000648:ultrastructure*; D004259:DNA-Directed DNA Polymerase; D015252:Deoxyribonucleases, Type II Site-Specific; D007887:Fabaceae; D008854:Microscopy, Electron; D010946:Plants, Medicinal
chemical_list: D004278:DNA, Superhelical; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes; C025651:CCCGGG-specific type II deoxyribonucleases; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C029792:GTCGAC-specific type II deoxyribonucleases