Introduction and expression of the bacterial PaeR7 restriction endonuclease gene in mouse cells containing the PaeR7 methylase.
To study the factors essential for a functional restriction system, the PaeR7 restriction-modification system has been introduced and expressed in murine cells. Transfer of this system was accomplished in two steps. First, cells containing sufficient PaeR7 methylase to completely methylate the mouse genome were constructed. In the second step, the mouse metallothionein promoter-regulated, endonuclease expression vector linked to the hygromycin B resistance selection marker was used to transfect the high methylase-expressing cells. Sixty percent of the clones isolated contained PaeR7 endonuclease enzymatic activity. Transfected cells expressing both methylase and endonuclease were incapable of blocking infection by DNA viruses, and possible explanations are discussed.
Kwoh
T J
TJ
La Jolla Biological Laboratories, San Diego, CA 92138.
Obermiller
P S
PS
McCue
A W
AW
Kwoh
D Y
DY
Sullivan
S A
SA
Gingeras
T R
TR
D000256:Adenoviridae; D000818:Animals; D015257:DNA-Cytosine Methylases; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D014645:Herpesvirus 3, Human; D007739:L Cells / Q000201:enzymology* / Q000382:microbiology; D008668:Metallothionein; D051379:Mice; D018139:Simplexvirus; D014162:Transfection; D010948:Viral Plaque Assay
D008668:Metallothionein; C047392:DNA modification methylase PaeR7; D015257:DNA-Cytosine Methylases; C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific