AN AMINO-ACID CHANGE IN NOVEL PROTEIN D123 IS RESPONSIBLE FOR TEMPERATURE-SENSITIVE G1-PHASE ARREST IN A MUTANT OF RAT FIBROBLAST LINE 3Y1

A temperature-sensitive mutant of rat fibroblast line 3Y1, 3Y1tsD123, is arrested in the G1 phase of the cell cycle at restrictive temperatu re. Using a human cDNA expression library, we succeeded in the molecul ar cloning of a cDNA (designated as D123 cDNA) that functionally compl emented the temperature-sensitive mutation. We then isolated rat D123 cDNA from a 3Y1 cDNA library using the human D123 cDNA probe. Both hum an and rat D123 cDNA contained a long open-reading-frame coding for a novel protein (designated as D123 protein) of 336 amino acids. A compa rison of the nucleotide sequences of rat D123 cDNA and the correspondi ng cDNA of 3Y1tsD123 - obtained by polymerase chain reaction in conjun ction with reverse transcription - showed that there was a difference in a single nucleotide that must cause a single amino acid change in D 123 protein. Antibody raised against the recombinant protein of human D123 cDNA detected a 44-kDa D123 protein in human embryo lung fibrobla sts and 3Y1. The level of D123 protein was much lower in 3Y1tsD123 tha n in 3Y1 at both permissive (33.8 degrees C) and restrictive (39.8 deg rees C) temperatures. We suppose that quantitative and qualitative cha nges of D123 protein due to the amino acid change cause the temperatur e-sensitive arrest of 3Y1tsD123. On the other hand, the level of D123 protein in 3Y1 did not change significantly whether cells were prolife rating or arrested at the saturation cell density. This indicates that the G1 arrest of 3Y1 at the saturation cell density does not involve the control of D123 protein level. (C) 1996 Academic Press, Inc.