Unusual DNA binding characteristics of an in vitro translation product of the CCAAT binding protein mYB-1.

We have isolated a cDNA that encodes the murine CCAAT-binding protein mYB-1. The deduced amino acid sequence shows 95% identity with its presumed human homologue (hYB-1A) which was originally isolated as a protein that binds to the Y box of MHC class II genes. In vitro translated mYB-1 binds to CCAAT boxes of the MHCIIE alpha, HSVTK and mouse PCNA promoters but not to alpha-globin or human thymidine kinase CCAAT boxes. Interestingly, complexes formed between the in vitro translated protein and the various CCAAT boxes display the property of being competed more efficiently with self competitor DNA, regardless of the CCAAT box initially used as a probe. A similar phenomenon was observed in a cell extract of Con-A stimulated murine splenocytes when the same competition assays were performed. These results may reflect the generation of multiple forms of a particular CCAAT-binding protein, such as mYB-1, that display distinct, yet overlapping, DNA binding specificities.

Regulation of c-myc mRNA levels by insulin or platelet-poor plasma.

Cell lines have been permanently established from BALB/c3T3 cells that constitutively express either the murine p53, the human IGF-1 gene, or both (Gai et al., 1988). The derivative cell lines grow well in platelet-poor plasma or in serum-free medium supplemented with the appropriate growth factors, while BALB/c3T3 cells do not grow in platelet-poor plasma, nor do they grow in serum-free medium unless supplemented with both platelet-derived growth factor and insulin (or IGF-1). In BALB/c3T3 cells, steady-state levels of c-myc mRNA decrease promptly and sharply once the cells are transferred to platelet-poor plasma. In the derivative cell lines, constitutively expressing p53, IGF-1, or both, c-myc mRNA levels remain elevated and actually increase when the cells are transferred to platelet-poor plasma. In serum-free medium, the c-myc mRNA levels decreased in BALB/c3T3 cells, as well as in the derivative cell lines. However, in the latter cell lines, but not in BALB/c3T3, the addition of platelet-poor plasma or insulin again increased the expression of c-myc. The increase in c-myc mRNA levels could be partially explained by an increase in transcription. These results indicate that in certain cell lines the expression of c-myc mRNA can be induced by insulin or platelet-poor plasma.

Abrogation of the requirements for added growth factors in 3T3 cells constitutively expressing the p53 and IGF-1 genes.

Two constructs (co-transfected with selectable markers) were used to establish cell lines from BALB/c3T3 cells: a p53 mini-gene under the control of an LTR promoter and a synthetic IGF-1 coding sequence driven by the SV40 early promoter. BALB/c3T3 cells do not grow in plasma or in 1% serum or in soft agar and in defined media require both platelet-derived growth factor (PDGF) and insulin (or IGF-1). Cells carrying only the LTR/p53 mini-gene grew well in plasma (but not in 1% serum), in soft agar and in serum-free medium containing only insulin. Cells carrying only the SV40/IGF-1 gene grew (but not too vigorously) in soft agar and grew well in serum-free medium containing PDGF but no insulin. Cells carrying both constructs grew very well in plasma, in soft agar and in serum-free medium without PDGF nor insulin. The results indicate that the requirements for growth of BALB/c3T3 cells can be reduced to two genes: IGF-1 and a gene replacing PDGF (p53 in our case). An unexpected, but interesting observation was that cells carrying the LTR/p53 gene grew slower in 10% serum than in plasma.