In:
Molecular and Cellular Biology, Informa UK Limited, Vol. 6, No. 2 ( 1986-02), p. 549-558
Kurzfassung:
A permanent human cell line, cell line LM205, was established by transforming primary human fibroblasts with a plasmid containing both simian virus 40 sequences with a defective origin of replication and a G418 resistance gene (neo) that lacked a eucaryotic transcriptional promoter. G418-resistant cells appeared spontaneously in clonal populations of LM205 cells at a frequency of approximately 10(-5) cell per cell plated in the presence of 400 micrograms of G418 per ml. G418 resistance was stable and correlated with the appearance of neo-specific RNA. Characterization of the neo gene in the G418-sensitive parental cell line by both a Southern blot analysis and a restriction map analysis of cloned sequences demonstrated that there was a stable integration site containing a single neo coding sequence. A Southern blot analysis of five G418-resistant subclones indicated that there were heterogeneous DNA rearrangements in the region of the neo gene that were unique in each subclone. Restriction mapping of a fragment containing the neo gene isolated from one of the resistant subclones demonstrated that the rearrangement was a tandem duplication that resulted in the relocation of the simian virus 40 bidirectional transcriptional promoter 5' to the neo gene. Tandem duplication was also consistent with the Southern blot polymorphisms observed in the other resistant subclones, suggesting that there were heterogeneous sites of recombination with respect to both the neo gene and the simian virus 40 promoter. Although these rearrangements resulted in an increase in neo gene copy number per cell, amplification showed no correlation quantitatively with the large increase in neo-specific RNA in these cells. Therefore, G418-resistant colony formation in cell line LM205 provides a method for studying both the mechanisms involved in this type of recombination and the factors influencing its frequency.
Materialart:
Online-Ressource
ISSN:
0270-7306
,
1098-5549
Sprache:
Englisch
Verlag:
Informa UK Limited
Publikationsdatum:
1986
ZDB Id:
1474919-1
SSG:
12
