Plant Genome I Conference
Town & Country Conference Center, San Diego, CA, November, 1992.
PG-I: 28pg1
LOCALIZATION AND CHARACTERIZATION OF REPLICATION ORIGINS IN
TOBACCO CHLOROPLAST DNA.
Zhun Lu and Brent L. Nielsen, Dept. of Botany and Microbiology,
Auburn University, Auburn, AL 36849.
Chloroplast DNA (ctDNA) replicates by a double displacement
loop (D-loop) mechanism, with the two D-loops generally located
about 6-7 kbp apart. This has been confirmed in several species,
including Chlamydomonas reinhardii, pea, and Oenothera. In order
to characterize ctDNA replication origins in tobacco, ctDNA was
gently isolated from young leaves by sucrose gradient isolation
of chloroplasts, lysis by sarkosyl, and CsCl gradient
purification. Nicked molecules were repaired by treatment with
Klenow enzyme and T4 DNA ligase, and for some samples RNA was
removed by incubation with RNase A and RNase H. The 51 ends of
nascent replication D-loop molecules in this preparation were
then labeled with [6 32P]- ATP by polynucleotide kinase. The
ctDNA was restricted with PstI, and ctDNA molecules containing
D-loops were enriched by BND- cellulose chromatography. The high
salt-caffeine eluate was used to probe Southern blots of total
tobacco ctDNA as well as specific tobacco ctDNA clones, in order
to localize the D-loops. Significant hybridization was observed
only to the Sal 1 and 4 fragments in the inverted repeat region
near the rRNA genes, similar to the location which has been
reported in other chloroplast genomes. This location agrees with
the single D-loop in each inverted repeat recently reported for
tobacco proplastid DNA from cultured cells [Y. Takeda, H.
Hirokawa and T. Nagata, Mol. Gen. Genet. 232:191-198 (1992)],
except that we have identified a larger region (8-10 kbp),
of hybridization, which may indicate the presence of two D-loops
in each inverted repeat. In order to characterize the number and
location of replication origins, subclones of the inverted repeat
and adjacent regions are being prepared for in vitro DNA
replication reactions using a partially purified tobacco
chloroplast enzyme fraction. These results, as well as
2-dimensional agarose gel electrophoresis analysis of in vivo and
in vitro replication intermediates, will be presented.
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