IDENTIFICATION OF ALTERNATIVELY SPLICED TRANSCRIPTS ENCODING MURINE MACROPHAGE-COLONY-STIMULATING FACTOR

We have isolated a novel cDNA encoding macrophage colony-stimulating f actor (M-CSF) from a murine stromal cell line, ST2. The cDNA included an entire coding sequence of the M-CSF gene but contained an additiona l sequence of 140 base pairs (bp). Northern blot analysis demonstrated that other murine cell lines such as a fibroblastic cell line (L) and a stromal cell line (PA6) also expressed the transcripts correspondin g to the clone, The nucleotide sequence analyses of the cDNA and the c loned M-CSF genome revealed that the 140-bp insertion sequence was par t of intron 1 which separated exon 1 and exon 2: the former contained part of the amino acid residues of the signal sequence and the latter the rest of the signal sequence and the first 22 amino acid residues o f the mature protein. The insertion of the 140-bp intron sequence not only changed the amino acid sequence of the signal peptide but also ge nerated an in-frame termination codon. However, instead of the dysfunc tion of the original initiation codon, the 140-bp insertion sequence c ontained a putative ATG; initiation codon that preserved the original open reading frame. Finally, we found that the cDNA directed the expre ssion of a secreted and biologically active M-CSF protein when it was introduced into COS7 cells and M-CSF activity in the culture supernata nts was measured using an M-CSF-dependent cell line. These results ind icate the presence of an alternatively spliced M-CSF transcript which utilizes an alternate initiation codon in order to specify active RI-C SF protein. (C) 1998 Academic Press.