Identification of alternatively spliced transcripts encoding murine macrophage colony-stimulating factor.
We have isolated a novel cDNA encoding macrophage colony-stimulating factor (M-CSF) from a murine stromal cell line, ST2. The cDNA included an entire coding sequence of the M-CSF gene but contained an additional 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 corresponding to the clone. The nucleotide sequence analyses of the cDNA and the cloned M-CSF genome revealed that the 140-bp insertion sequence was part 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 of the mature protein. The insertion of the 140-bp intron sequence not only changed the amino acid sequence of the signal peptide but also generated an in-frame termination codon. However, instead of the dysfunction of the original initiation codon, the 140-bp insertion sequence contained a putative ATG initiation codon that preserved the original open reading frame. Finally, we found that the cDNA directed the expression of a secreted and biologically active M-CSF protein when it was introduced into COS7 cells and M-CSF activity in the culture supernatants was measured using an M-CSF-dependent cell line. These results indicate the presence of an alternatively spliced M-CSF transcript which utilizes an alternate initiation codon in order to specify active M-CSF protein.
Suzu
S
S
Biochemical Research Laboratory, Morinaga Milk Industry Co. Ltd., Higashihara 5-1-83, Kanagawa, Zama, 228, Japan.
Hatake
K
K
Ota
J
J
Mishima
Y
Y
Yamada
M
M
Shimamura
S
S
Kimura
F
F
Motoyoshi
K
K
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D017422:Sequence Analysis, DNA; D014162:Transfection / Q000235:genetics
D017077:Culture Media, Conditioned; D012333:RNA, Messenger; D016173:Macrophage Colony-Stimulating Factor