Overexpression of uvomorulin in a compaction-negative F9 mutant cell line.
The mutant F9 cell line F9att-5.51 synthesizes reduced amounts of uvomorulin (UM) protein and we hypothesized earlier (Adamson, Baribault, and Kemler, Dev. Biol. (1990), 138, 338) that this may account for its inability to compact into tightly aggregated balls of cells. Subsequently, when 5.51 cells are treated with retinoic acid to stimulate their differentiation, they are unable to form embryoid bodies as do wild-type cells which form an outer epithelial layer of visceral endoderm cells. We have now examined the possibility that the UM protein made in the mutant line is defective, but find that it is normal in structure and stability. The gene coding for UM appears to be normal as does the mRNA which is synthesized at a normal rate but is severely reduced in steady-state measurements of mutant cells. A rescue experiment was performed by increasing levels of UM in mutant cells by means of transfection with a UM expression vector. The resulting cells expressed abundant UM mRNA and protein but were still unable to form compacted aggregates and did not differentiate into embryoid bodies. Interestingly, the stability of endogenous UM mRNA was improved in the presence of exogenous UM; therefore, a positive feedback mechanism contributes to low mRNA levels in mutant cells. The accumulated data suggest that UM in 5.51 cells is unable to mount a compaction activity because a distal connecting link in the multicomponent process initiated by UM is missing or or aberrant. The missing component is likely to connect UM to actin and the cytoskeleton of the cell.
Calogero
A
A
La Jolla Cancer Research Foundation, California 92037.
Samuels
M
M
Darland
T
T
Edwards
S A
SA
Kemler
R
R
Adamson
E D
ED
D000818:Animals; D001483:Base Sequence; D015820:Cadherins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D004707:Endoderm / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D010449:Peptide Mapping; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism; D013724:Teratoma / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D015820:Cadherins; D012333:RNA, Messenger