Automatic microinjection system facilitates detection of growth inhibitory mRNA.
Naturally quiescent human lymphocytes, consisting predominantly of T cells, contain mRNA(s) that can inhibit DNA synthesis when injected into either human diploid fibroblasts (IMR-90) or transformed recipient cells (HeLa). By using an automated capillary microinjection system and a fluorescent coinjection marker (fluorescein isothiocyanate-dextran), individually injected cells can be retrieved and analyzed for DNA synthesis. mRNA isolated from resting T cells is able to block the cells from entering the S phase. The block is reversible and leads to a delay in DNA synthesis. The inhibitory effect is not observed if the injected mRNA is isolated from growth-activated T cells. The disappearance of the inhibition coincides with the approach of the G1/S boundary in both the donor T cells and the recipient human fibroblasts. The mRNA of resting T cells was size-fractionated and the peak inhibitory activity was recovered in a fraction approximately equal to 1.5 kilobases long.
Pepperkok
R
R
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Zanetti
M
M
King
R
R
Delia
D
D
Ansorge
W
W
Philipson
L
L
Schneider
C
C
D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004261:DNA Replication; D003911:Dextrans; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins; D006801:Humans; D008845:Microinjections; D012333:RNA, Messenger / Q000032:analysis* / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects
D003911:Dextrans; D005452:Fluoresceins; D012333:RNA, Messenger; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate