- Biochemical Quantitation of PM2 Phage DNA as a Substrate for Endonuclease Assay
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Yoo Jin Joo , Hee-Ju Kim , Jae Yung Lee , Joon Kim
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J. Microbiol. 2004;42(2):99-102.
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DOI: https://doi.org/2038 [pii]
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 Abstract
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Bacteriophage PM2 has a closed circular form of double stranded DNA as a genome. This DNA from the phage is a useful source for nick-circle endonuclease assay in the fmol range. Due to difficulties in the maintenance of viral infectivity, storage conditions of the phage should be considered for the purification of PM2 DNA. The proper condition for a short-term storage of less than 2 months is to keep the PM2 phage at 4^oC; whereas the proper condition for a long-term storage of the PM2 phage for over 2 months is to keep it under liquid nitrogen in 7.5% glycerol. The optimal conditions for a high yield of phage progeny were also considered with the goal to achieve a successful PM2 DNA preparation. A MOI(Multiplicity Of Infection) of 0.03, in which the OD_600 of the host bacteria was between 0.3 and 0.5, turned out to be optimal for the mass production of PM2 phage with a burst size of about 214. Considerations of PM2 genome size, and the concentrations and radiospecific activities of purified PM2 DNA, are required to measure the endonuclease activity in the fmol range. This study reports the proper quantitation of radioactivity and the yield of purified DNA based on these conditions.
- Evaluation of Several Parameters of in situ Polymerase Chain Reaction (ISPCR) to Reduce the Leakage of Amplificants from Cells
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Jae Yung Lee , Chung-Kyoon Auh , George W. Jordan
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J. Microbiol. 2002;40(1):70-76.
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Proviral DNAs from HIV-1-infected CD4+ T cells (Molt/LAV cells) were amplified and detected in infected individual cells using polymerase chain reaction and in situ hybridization. In this in situ PCR, three parameters were considered to achieve effective amplification and retention of amplificants inside the cells by making high molecular weight PCR products intracellularly, forming agarose matrix against the cells, and maintaining the appropriate PCR temperature profile. Over the cycles of amplification, tailed primers with complementary overhanging sequences at their 5 sides manufactured high molecular weight products by using short primary products as a repeating unit. Agarose matrix could prevent the diffusion of the amplificants from the cells. Use of Thermanox coverslip inside the PCR tube offered target cells a similar temperature profile to that of conventional PCR in solution.
- Expression and Characterization of the Human rpS3 in a Methylotrophic Yeast Pichia pastoris
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Jae Yung Lee , Sang Oun Jung , BuHyun Youn , Oh Sik Kwon , Joon Kim
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J. Microbiol. 2000;38(2):88-92.
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A human ribosomal protein S3 (rpS3), which also functions as a DNA repair enzyme (UV endonuclease III), was expressed in a methylotrophic yeast, Pichia pastoris, and biochemically characterized. UV endonuclease activity was previously characterized, and this activity of mammalian rpS3 was found to be non-specific upon purification and storage. Under the Pichia expression system, the subcloned cDNA of the human rpS3 gene revealed a peptide of 42 kDa by SDS-PAGE and Western blot. The secreted form of human rpS3 rendered no endonuclease activity while the intracellular form showed UV specific endonuclease activity by the nick circle assay.
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