Cloning of an origin of DNA replication of Xenopus laevis

Journal Article · Sun Aug 31 20:00:00 EDT 1980 · Proc. Natl. Acad. Sci. U.S.A.; (United States)

DOI:https://doi.org/10.1073/pnas.77.9.5292· OSTI ID:7099115

Watanabe, S; Taylor, J H

DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Eschrichia coli plasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X. laevis. After measurement of the (/sup 3/H)-thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (550 base pairs) and several kilobases of frog DNA, were selected for more extensive analysis. In spite of the small size of th segment in pSW14, it incorporates in 2 hr at least 3 times as much labeled thymidine as either pSW9 or the vector alone. To determine the number of replications of pSW14, a novel method was employed. The results showed that about 50% of the labeled, supercoiled DNA recovered from eggs after 4 hr was sensitive to EcoRI digestion, which indicates that most of the DNA that incorporated (/sup 3/H)thymidine had replicated twice during the 4 hr in the unfertilized eggs of X. laevis. We conclude the pSW14 has a functional origin in the Xenopus DNA segment.

• Research Organization: Florida State Univ., Tallahassee
• OSTI ID: 7099115
• Journal Information: Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 77:9; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMPHIBIANS
ANIMALS
AQUATIC ORGANISMS
BACTERIA
CELL CONSTITUENTS
CLONING
DNA
DNA REPLICATION
DNA-CLONING
ESCHERICHIA COLI
EVALUATION
FROGS
MICROORGANISMS
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PLASMIDS
VERTEBRATES