On the mechanism of homologous recombination of DNA in plant cells

1992 Fiscal Year Final Research Report Summary

• Project/Area Number: 03660097
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: 応用生物化学・栄養化学
• Research Institution: Sophia University (1992); The Institute of Physical and Chemical Research (1991)
• Principal Investigator: MAKINO Osamu Sophia University, Faculty of Science and Technology, Associate Professor, 理工学部, 助教授 (70231587)
• Project Period (FY): 1991 – 1992
• Keywords: homologous recombination / Tobacco / gene conversion / yeast / PCR / non-reciprocal recombination

Research Abstract

Homologous recombination of DNA is one of the essential metabolism for living organisms, and the investigations on this important system would be quite helpful for artificial manipulation of the genome. Therefore, I have started the work on the homologous recombination using higher plant and yeast both of them are important in basic science and applied biotechnology.
First cooperation with Max-Planck-Institut of FRG, I tried to detect the homologous recombination between DNA molecules introduced into Tobacco cells. I used two independent deletion mutants of NPTII(Kanamycin resistant gene of Tn5 of E.coli)as reporter and found that the recombination between the two single-stranded DNAs occurred very efficiently only when the two single-stranded allelic DNAs have opposite direction to each other. The possible partial double-stranded form with relatively long loops was suggested to be key for this recombination. Therefore, I have carried out PCR experiments to make the mechanism of this ef ficient recombination clearer. When the whole DNAs from Km resistant callus were analyzed, all of them contained full length NPTII gene, and some of them contained shorter DNA which corresponds to the introduced mutant NPTII gene with one of the deletions. But, shortest DNA which has both deletions simultaneously was not detected in any callus DNA used. Additional experiments using fission yeast and same reporter gene with deletions have been carried out using electroporation. Even when DNAs were extracted transiently after introduction of single-stranded DNAs were changed to complete length only when they have opposite orientation like the case for Tobacco. But, shortest NPTII gene with double deletions was not detected after the analysis using PCR.
In both organisms, DNA of NPTII gene with both deletion was not detected. These results would suggest the non reciprocal homologous recombination between the two single-stranded DNA with opposite orientation and also the existence of similar mechanism with gene conversion. The results also showed that the single-stranded DNA introduced into cells are processed by the machinery of recombination or repair before replicating the complementary strand to form double-stranded DNA, i.e. the observed recombination is not the recombination between double-stranded DNAs. Partially double-stranded hetero-duplex DNA formed from the two DNAs introduced into cells was suggested to be an essential intermediate in both organisms used in this study.