Pilot-scale production of recombinant plasmid pUDK-HGF / 生物工程学报

pUDK-HGF, the recombinant plasmid DNA encoding human hepatocyte growth factor (HGF), can treat ischaemic disease. A great quantity of pharmaceutical pUDK-HGF is needed. A pilot-scale production process of pUDK-HGF was established based on a new chromatographic media (plasmidselect), including fermentation, cell harvesting, alkaline lysis, ultrafiltration, RNA removing and buffer exchanging on Sephacryl S-1000, capturing supercoiled plasmid DNA with plasmidselect, and removing the salt with Sepharose 6BFF. The process does not use RNase enzyme and toxic solvents.

Three model systems measure oxidation/nitration damage caused by peroxynitrite.

Diseases activate the innate immune response which causes ancillary damage to the human body. Peroxynitrite (OONO-) or its carbon dioxide derivatives cause oxidation/nitration and hence mutation to various body polymers e.g. DNA, RNA, protein, lipids and sugars. The control of the ancillary damage can come from antioxidants which inhibit control the amount of peroxynitrite available for damage. In this paper we have developed three different levels of antioxidant screening: (i) Peroxynitrite or SIN-1 reaction with luminol to produce light, and the inhibition of light by substances therefore represents antioxidation. (ii) Nicking of plasmid DNA occurs via oxidants: and is prevented by antioxidants. (iii) Detection of plasmid luciferase activity post-oxidation and infection indicates either prevention or repair of damage: via antioxidants. We found green tea and a number of its polyphenolic constituents effective only at the first level of antioxidation, while extracts of various fruit help at all levels antioxidation. In the final analysis, a combination of green tea extracts and fruits is suggested to produce more complete antioxidant protection.

Axial distortion as a sensor of supercoil changes: a molecular model for the homeostatic regulation of DNA gyrase.

Negative supercoiling stimulates transcription of many genes. In contrast, transcription of the genes coding for DNA gyrase is subject to a novel mechanism of autoregulation, wherein relaxation of the template DNA stimulates their transcription. Since DNA gyrase is the sole supercoiling activity in the eubacterial cell, relaxation-stimulated transcription (RST) could reflect an autoregulatory mechanism to maintain supercoil levels within the cell. Extensive deletion and mutational analyses of Escherichia coli gyrA promoter have shown that the -10 region is essential for RST; however, a molecular model has proved to be elusive. We find a strong bend centre immediately downstream of the -10 region in the gyrA promoter. On the basis of analysis of various mutants in the -10 region, we propose a model where axial distortion acts as a sensor of topological changes in DNA. Our model is consistent with earlier data with E. coli gyrA anmd gyrB promoters. We also extrapolate the model to explain the phenomenon of RST of gyr promoters in other organisms and contrast it with promoters induced by supercoiling.

Construction and Application of Painting probe for Chromosome band 5q14-q15, 6q23, 16p11.2 and 22q12

PURPOSE: Chromosome microdissection has become a very powerful approach to generate chromosome band-specific library and painting probes for physical mapping or cytogenetic analysis. We have constructed here band-specific painting probes for human chromosomes by microdissection and polymerase chain reaction(PCR). METHODS: We pretreated the microdissected fragments with Topoisomerase I(Topo I) which catalyzes the relaxation of supercoiled DNA, and performed two initial rounds of DNA synthesis with T7 DNA polymerase followed by conventional PCR to enable the reliable preparation of fluorescent in situ hybridization(FISH) probe. RESULTS: It was possible to construct region-specific painting probes for 5q14-q15, 6q23, 16p11.2 and 22q12. The painting probes were successful in determining the corresponding chromosome bands in SNU-484 cell line, IMR-32 cell line. CONCLUSION: These painting probes can be used to detect the related marker chromosomes in congenital malformations and other solid tumors, and the band specific probe pools may be used effectively to analyse the genes.

Construction of Painting probe for Chromosome Band 18p11.1-p11.2 / 대한해부학회지

Chromosome microdissection has become a very powerful approach to generate chromosome band-specific library and painting probes for physical mapping or cytogenetic analysis. Here we have constructed the band-specific painting probe for human chromosome 18p11.1-p11.2 by microdissec-tion and polymerase chain reaction (PCR). We pretreated the microdissected fragments with Topoisomerase I (Topo I) which catalyzes the relaxation of supercoiled DNA, and performed two initial rounds of DNA synthesis with T7 DNA polymerase followed by conventional PCR to enable the reliable preparation of fluorescent in situ hybridization (FISH) probe from a single microdissected chromosome. With this method, it was possible to construct region-specific painting probe for 18p11.1-p11.2. The probe has been used successfully to determine the trisomy 18. Thus this painting probe can be applied to detect the related marker chromosomes in various solid tumors, and the band specific probe pools may be used to analyse the genes.

Construction and Analysis of Painting Probe for Homogeneously Staining Regions in Human Neuroblastoma Cell line IMR-32

Painting probe for HSRs in IMR-32 Neuroblastoma, a pediatric malignant neoplasm of neural crest origin, has a wide range of clinical virulence. The mechanisms contributing to the development of neuroblastomas are largely unclear, but non-random chromosomal changes identified over the past years suggest the involvement of genetic alterations. Amplification of the human N-myc proto-oncogene is frequently seen either in extrachromosomal double minutes or in homogeneously staining regions(HSRs) of aggressively growing neuroblastomas. N-myc maps to chromosome 2 band 24, but HSR have never been observed at this band, suggesting transposition of N-myc during amplification. We have constructed and analyzed the region-specific painting probe for HSR in neuroblastoma IMR-32 to determine the derivative chromosomes. Microdissection was performed on HSR using an inverted microscope with the help of microglass needles and an micromanipulator. We pretreated the microdissected fragments with Topoisomerase I which catalyzes the relaxation of supercoiled DNA, and performed two initial rounds of DNA synthesis with T7 DNA polymerase followed by conventional PCR to enable the reliable preparation of Fluorescent in situ hybridization probe from a single microdissected chromosome. With this method, it was possible to construct the region-specific painting probe for HSR. The probe hybridized specifically to the HSRs of IMR-32, and to 2p24, 2p13 of normal chromosome. Our results suggest there was coamplification of N-myc together with DNA of the chromosome 2p24 and 2p13. Moreover, the fluorescent signals for the amplified chromosomal regions in IMR-32 cells were also easily recognized at a Thus this painting probe can be applied to detect the similar amplification of N-myc in neuroblastoma tissue, and the probe pool for HSR may be used to identify the cancer-relevant genes.

Molecular mechanics studies of homopolymeric and mixed sequence Py.Pu*Pu triple helices.

DNA triple helices containing two purine strands and one pyrimidine strand (C.G*G and T.A*A) have been studied, using model building followed by energy minimisation, for different orientations of the third strand resulting from variation in the hydrogen bonding between the Watson-Crick duplex and the third strand and the glycosidic torsion angle in the third strand. Our results show that in the C.G*G case the structure with a parallel orientation of the third strand, resulting from Hoogsteen hydrogen bonds between the third strand and the Watson-Crick duplex, is energetically the most favourable while in the T.A*A case the antiparallel orientation of the third strand, resulting from reverse Hoogsteen hydrogen bonds, is energetically the most favourable. These studies when extended to the mixed sequence triplexes, in which the second strand is a mixture of G and A, correspondingly the third strand is a mixture of G and A/T, show that though the parallel orientation is still energetically more favourable, the antiparallel orientation becomes energetically comparable with an increasing number of thymines in the third strand. Structurally, for the mixed triplexes containing G and T in the third strand, it is seen that the basepair non-isomorphism between the C.G*G and the T.A*T triplets can be overcome with some changes in the base pair parameters without much distortion of either the backbone or the hydrogen bonds.

Superhelical density of goat mitochondrial DNA: fluorimetric studies.

EtBr binding to DNA has been used to determine the superhelical density of Goat mtDNA. The differential binding of closed circular and nicked circular DNA is estimated by simple fluorescence measurements, which is a function of state of DNA supercoiling. The method is simple and quick over the cesium chloride density gradient centrifugation studies.

Denatured supercoiled DNA--structural and biological activity.

Supercoiled DNA on treatment with NaOH followed by neutralization produces a condensed structure (form Id). This structure does not split into topoisomers when run on long gel in presence of intercalating agents and the migration of this form does not change appreciably in presence or absence of ethidium bromide. Relaxation of form Id by topoisomerase I from pea chloroplast is facilitated more than form I. Single-stranded binding (SSB) protein binds more to form Id as evidenced from gel retardation study. Hydroxyl radical nicking is facilitated in this form. Compared to form I, this form produces half the number of transformants, but adsorption and penetration remain almost same in both the forms. Post-transformational growth using 32P labelled form I and form Id showed greater amount of degradation in form Id.