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OFAGE analysis of large DNA restriction fragments from multidrug resistant SEWA mouse cells
1990 (English)In: Hereditas, Wiley-Blackwell Publishing, Inc. , 1990, 9- p.Conference paper, (Refereed)
Abstract [en]

The aim of this study is to detect similarities and differences in the gene amplification process, when MDR cells develop after selection in different drugs. For this purpose, nine independent MDR sublines of the murine SEWA TC13K cell line were established by stepwise selection in actinomycinD( Al,A2,A4),colcemid(Cl,C2,C3),or vincristine (Vl, V3, V4). All MDR sublines displayed numerous DMs. DNA from all lines was digested with EcoRl and the infrequently cutting enzyme SfiI. DNA-fragments from the SfiI-digests were separated by orthogonal field alterating gel electrophoresis (OFAGE). Hybridizations were made with two probes: (1) cp28, a Chinese hamster P-glycoprotein cDNA sequence, kindly supplied by Dr Alexander Van der Bliek, The Netherlands Cancer Institute in Amsterdam; (2) Ie7, a genomic clone from a DM mini-library (Stihl et al., Hereditas 106:97, 1987). The hybridization patterns of the Sfildigests were very similar for both probes, while the patterns of the EcoRI-digests differed considerably. No drugspecific amplification pattern was revealed. The Ie7 probe did not cross-hybridize to the cp28 cDNA-sequence. However, low stringency hybridization of Ie7 to a 4.5 kb mRNA (hamster) has been reported (Diddens et al., Int. J. Cancer 40:635, 1987). Still, the similarities in the hybridization patterns suggest that Ie7 DNA-sequence is located close to the cp28 DNA-sequence, perhaps as a part of an intron. In the SfiI digest the cp28 probe hybridized to seven DNA-fragments (six of which were detected also with Ie7) that were amplified in most of the lines. The same DNAfragments were present but not amplified in the control line TC13K. The presence of several hybridizing fragments also in the control line is probably due to homology within the P-glycoprotein gene family. Other amplified sequences were unique to each cell line and are presumably a result of so-called novel joints. The large number of new hybridizing fragments thus reflects a recombinational process during amplicon formation. The presence of specific cytogenetic markers in some of the cell lines indicated that the MDR cells were of monoclonal origin. Therefore, it is possible that each line contains a homogeneous population of DMs, each contributing the same complex pattern of novel fragments in the SfiI analysis. Another explanation of this complexity would be thai different amplicons (with different novel joints) are located on different DMs in a heterogeneous DM population.

Place, publisher, year, edition, pages
Wiley-Blackwell Publishing, Inc. , 1990. 9- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:hb:diva-7499DOI: 10.1111/j.1601-5223.1990.tb00131.xLocal ID: 2320/7589OAI: oai:DiVA.org:hb-7499DiVA: diva2:888362
Conference
XIII Congress of Scandinavian Association of Geneticists, Gothenburg, Sweden, June 13-16
Available from: 2015-12-22 Created: 2015-12-22

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