| Project/Area Number |
63480510
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
分子遺伝学・分子生理学
|
|---|
| Research Institution | Osaka University |
|---|
Principal Investigator |
ITOH Tateo Osaka Univ., Biology Dept., Associate Professor, 理学部, 助教授 (40051817)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
HORII Toshihiro Osaka Univ., Biology Dept., Research associate, 理学部, 助手 (80142305)
|
|---|
| Project Period (FY) |
1988 – 1990
|
| Project Status |
Completed (Fiscal Year 1990)
|
| Budget Amount *help |
¥6,800,000 (Direct Cost: ¥6,800,000)
Fiscal Year 1990: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1989: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1988: ¥4,300,000 (Direct Cost: ¥4,300,000)
|
|---|
| Keywords | Colicin E2 / Colicin E3 / Replication initiator protein / Replication origin / Plasmid-specificity / Primer RNA / Primase / Reconstituted system / 一方向複製 / プライマーRNA |
|---|
| Research Abstract |
The basic replicons of ColE2 and ColE3 consist of three functional portions : A gene for the plasmid-specific initiator protein (Rep ; 33-35 Kda) ; an origin (32 and 33 bp) capable of initiating replication in the presence of the Rep protein ; a gene for an antigens RNA (RNA I ; 115 n) complementary to the 5' untranslated region of the Rep mRNA and negatively regulating initiation of replication.
Specificity of interaction of the Rep proteins and the origins is determined by the 20 amino acid-region near the carboxy terminal portion of the Rep protein of each plasmid and a single base pair deletion (ColE2) or insertion (ColE3) in the origin.
The partially purified ColE2 Rep protein was shown to bind specifically to the ColE2 origin by using the filter-binding method.
In vitro replication of ColE2 and ColE3 DNA in crude E. Coli cell extracts requires the Rep proteins and host DNA polymerase I but not RNA polymerase and DnaG primase. Replication starts from a region containing the cloned origin and proceeds unidirectionally. The 5' end of the newly synthesized leading strand was localized at a fixed position within the origin. The 3' end of the newly synthesized lagging strand was localized at the downstream end of the origin. This seems to be the basic mechanism for the unidirectional replication. The newly synthesized leading strand contains three ribonucleotide residues (ppApGpA) at its 5' end.
The ColE2 Rep protein in conjunction with the host DNA polymerase I and single-strand DNA binding protein specifically starts the leading strand synthesis at the identical position with the identical primer RNA. The reaction specifically requires ADP in addition to ATP and GTP and incorporates ADP into the primer RNA. The Rep protein seems to be a novel sequence-specific primase capable of functioning on double-strand DNA.
|
