Molecular cloning and regulated expression of the human c-myc gene in Escherichia coli and Saccharomyces cerevisiae: comparison of the protein products.
mRNA from human HL-60 cells was used to prepare a cDNA library, from which two full-length clones that encompass the complete c-myc coding region were isolated. One clone, pM1-11, contains all three exons of human c-myc. The second clone, pM4-10, represents a relatively rare transcript that initiated in the first intron and includes the coding exons 2 and 3. The cDNA insert in pM1-11 was used to express the human c-myc protein in both prokaryotic and eukaryotic cells. Insertion of the coding sequences in exons 2 and 3 into the appropriate expression vectors yielded detectable c-myc protein in Escherichia coli lacking the Lon protease and in Saccharomyces cerevisiae upon induction. The protein produced in E. coli has an apparent size of 60 kDa and appears to be unmodified, as it is identical in size to the protein synthesized in an in vitro system. In contrast, yeast cells synthesize two myc proteins, of 60 kDa and 62 kDa. The difference in apparent molecular mass between the two proteins appears to be due, in part, to phosphorylation. Subcellular fractionation of yeast cells showed that the c-myc phosphoprotein is located predominantly in the nuclear fraction.
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D007951:Leukemia, Myeloid / Q000235:genetics; D008297:Male; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics; D011519:Proto-Oncogenes; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013045:Species Specificity; D014158:Transcription, Genetic
D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D004247:DNA