Circular YAC vectors containing a small mammalian origin sequence can associate with the nuclear matrix.
Three different mammalian origins of DNA replication, 343, S3, and X24, have been cloned into a 15.8 kb circular yeast vector pYACneo. Subsequent transfection into HeLa cells resulted in the isolation of several stably maintained clones. Two cell lines, C343e2 and CS3e1, were found to have sequences maintained as episomes in long-term culture with a stability per generation of approximately 80%. Both episomes also contain matrix attachment region (MAR) sequences which mediate the binding of DNA to the nuclear skeleton and are thought to play a role in DNA replication. Using high salt extraction of the nucleus and fluorescent in situ hybridization, we were able to demonstrate an association of the 343 episome with the nuclear matrix, most probably through functional MAR sequences that allow an association with the nuclear matrix and associated regions containing essential replication proteins. The presence of functional MARs in small episomal sequences may facilitate the replication and maintenance of transfected DNA as an episome and improve their utility as small episomal constructs, potential microchromosomes.
Cossons
N
N
McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Nielsen
T O
TO
Dini
C
C
Tomilin
N
N
Young
D B
DB
Riabowol
K T
KT
Rattner
J B
JB
Johnston
R N
RN
Zannis-Hadjopoulos
M
M
Price
G B
GB
D000818:Animals; D018244:Chromosomes, Artificial, Yeast; D002999:Clone Cells; D004261:DNA Replication; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008322:Mammals; D015530:Nuclear Matrix / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection / Q000379:methods
D011994:Recombinant Proteins