Expression of thermotolerance following microinjection of poly(A)RNA isolated from thermotolerant CHO cells.
Poly(A)RNA was isolated from thermotolerant cells and microinjected into recipient non-tolerant Chinese hamster ovary (CHO) cells. The injected cells expressed thermotolerance to a subsequent test heat treatment both in terms of the end-points of colony formation (cell survival) and resumption of protein synthesis after test heating (translational labelling). The magnitude of thermotolerance expression was dependent on the experimental end-point (increase up to 3.8-fold for translational labelling and approximately 2-fold for survival) and on the time between microinjection and the test heat treatment. Control experiments showed that poly(A)RNA from non-tolerant cells did not alter the heat response of microinjected cells. Proteins corresponding to the poly(A)RNA from thermotolerant cells were analysed by in vitro translation and by labelling of microinjected cells, followed by SDS-PAGE. In vitro translations showed high levels of transcripts for classical heat-shock proteins (HSP 70/72, 89, 110) in poly(A)RNA from thermotolerant versus control cells. However, proteins synthesized in intact cells showed no detectable differences when cells were microinjected with poly(A)RNA from thermotolerant versus control cells, or not injected at all. In principle the data show that microinjection of specific poly(A)RNA fractions can be used for defining the contribution of individual gene products to the cellular heat response.
Henle
K J
KJ
Department of Medicine, University of Arkansas for Medical Sciences, Little Rock 72205.
Lumpkin
C K
CK
Norris
J S
JS
Nolen
G T
GT
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D006358:Hot Temperature; D008845:Microinjections; D011061:Poly A / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger
D006360:Heat-Shock Proteins; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA