Using archaeal histones for precise DNA fragmentation

The fragmentation of DNA is a useful procedure for many molecular biology procedures. However, most methods used to fragment DNA are poorly controllable, and cannot be used to create small fragments. We describe a method to generate random DNA fragments of a predictable size to be cloned in expression vectors for the construction of display libraries. The DNA is allowed to form complexes with archaeal histones from Methanothermus fervidus (HMf) and the HMf/DNA core complex is naturally protected from nuclease DNaseI activity, giving rise to DNA fragments of approximately 60 bp and multiples thereof. We found that by varying the wt/wt ratio between DNA and HMf, the concentration of DNA and the incubation time with DNaseI, DNA fragments of desired size can be obtained. This approach should be applicable to the efficient fragmentation of DNA for the construction of phage display polypeptide libraries, as well as any other molecular biology procedures in which small DNA fragments of defined size are required.     

RELATIONSHIP BETWEEN M-A CONSTITUENT AND CLEAVAGE FRACTURE BEHAVIOUR OF GRANULAR BAINITIC WELD METAL

The cleavage fracture mechanism of granular bainitie weld metal in 15MnVN steel has beeninvestigated.SEM observation of a special specimen for revealing simultaneously fracture sur-face and microstructure shows that the cleavage of the weld is initiated by M-A constituent.The 95th percentile equivalent diameter of M-A constituent controls the cleavage fracturestress of the weld metal.     

Perspectives on molecular computing

In 1996, we began a research project on molecular computers under the new program “Research for the Future” funded by the Japan Society for the Promotion of Science. In this paper, we first summarize the research that has been completed in the field of DNA computing and the research problems that must be overcome. We also report some achievements of our research project in the first two years. We then propose a new direction in research towardsautonomous molecular computers, and describe the author’s work on the implementation of state machines using DNA molecules. We finally discuss the future perspectives on molecular computing based on our experiences. Masami Hagiya, Ph.D.: He received M.Sc. from University of Tokyo in 1982, and D.Sc. from Kyoto University in 1988. After the years in Research Institute for Mathematical Sciences, Kyoto University, he returned to University of Tokyo in 1992. He has been working on programming languages, verification and synthesis of programs, and automated deduction. In addition, he is interested in bio-computing since he was involved in the human genome project of Japan. He is currently organizing a project on molecular computing under the “Research for the Future” program of JSPS.     

Role of DNA-Dependent Protein Kinase in Mediating Cyst Growth in Autosomal Dominant Polycystic Kidney Disease

DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein involved in DNA damage response (DDR) signaling that may mediate kidney cyst growth in autosomal dominant polycystic kidney disease (ADPKD) due to its pleiotropic effects on proliferation and survival. To test this hypothesis, the expression of DNA-PK in human ADPKD and the in vitro effects of DNA-PK inhibition in a three-dimensional model of Madin-Darby Canine Kidney (MDCK) cyst growth and human ADPKD cells were assessed. In human ADPKD, the mRNA expression for all three subunits of the DNA-PK complex was increased, and using immunohistochemistry, the catalytic subunit (DNA-PKcs) was detected in the cyst lining epithelia of human ADPKD, in a focal manner. In vitro, NU7441 (a DNA-PK kinase inhibitor) reduced MDCK cyst growth by up to 52% after long-term treatment over 6–12 days. Although human ADPKD cell lines (WT9-7/WT9-12) did not exhibit synthetic lethality in response to DNA-PK kinase inhibition compared to normal human kidney cells (HK-2), the combination of low-dose NU7441 enhanced the anti-proliferative effects of sirolimus in WT9-7 and WT9-12 cells by 17 ± 10% and 11 ± 7%, respectively. In conclusion, these preliminary data suggest that DNA-PK mediates kidney cyst growth in vivo without a synthetically lethal interaction, conferring cell-specificity in human ADPKD cells. NU7441 enhanced the anti-proliferative effects of rapamycin complex 1 inhibitors, but the effect was modest.