Sequence requirements for the stimulation of gene amplification by a mammalian genomic element.
HSAG-1 is a 3.4-kb genomic element from a human chronic lymphocytic leukemia--Chinese hamster ovary (CHO) hybrid cell line shown to stimulate the amplification of expression vectors in cis when transfected into a variety of cell lines [McArthur and Stanners, J. Biol. Chem. 266 (1991) 6000-6005]. Subfragments of HSAG-1 were tested for amplification activity by insertion into the vector, pSV2DHFR. The results suggest that multiple positive- and negative-acting elements were present that influenced amplification activity. The deletion of regions believed to contain positive-acting elements decreased or abolished the amplification stimulatory activity of the most active 1.45-kb fragment, supporting this hypothesis. The construction of composite sequences containing multiple positive elements and lacking negative elements, however, failed to enhance the activity; maximum activity was obtained only with the original intact configuration of elements. Two of two CHO HSAG-1-like elements tested had an activity equivalent to HSAG-1, while one of 24 random CHO genomic fragments tested had an activity as high as HSAG-1. The combination of sequence and structural features needed to affect the frequency of gene amplification may therefore be quite common in the mammalian genome.
Beitel
L K
LK
Department of Biochemistry and Cancer Centre, McGill University, Montreal, Quebec, Canada.
McArthur
J G
JG
Stanners
C P
CP
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005784:Gene Amplification; D005822:Genetic Vectors; D006801:Humans; D006822:Hybrid Cells; D015451:Leukemia, Lymphocytic, Chronic, B-Cell; D008969:Molecular Sequence Data; D015183:Restriction Mapping
D004247:DNA; D015246:Deoxyribonuclease EcoRI; C025580:endodeoxyribonuclease XBAI; D015252:Deoxyribonucleases, Type II Site-Specific