Molecular cloning and regulated expression of the human c-myc gene in Escherichia coli and Saccharomyces cerevisiae: comparison of the protein products.

Abstract

ABSTRACTmRNA 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 farst intron and includes the coding exons 2 and 3. The cDNA insert in pMl-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.Activation of the cellular myc gene has been associated with neoplasia in a variety of species. In transformed cells, the c-myc gene can be altered by several mechanisms that result in abnormal or elevated expression of c-myc RNA (1, 2) and, presumably, in increased levels of c-myc protein. Analysis of genomic clones of the human c-myc gene shows that it contains three exons (3). Exon 1 represents a long 5' noncoding leader region, whereas all of the coding sequences are located in exons 2 and 3. The coding sequences are highly conserved among species and with v-myc, the viral oncogene homolog harbored by avian myelocytomatosis virus (4, 5). Immunoprecipitation studies show that myc-specific antisera recognize proteins of =60 kDa in human cells (6-8). The highly conserved nature of the c-myc protein in many vertebrate species suggests that c-myc must supply a crucial function in eukaryotic cells. Such a function has not been elucidated, although studies on v-myc and c-myc indicate that this protein is found predominantly in the nucleus and binds to double-stranded DNA (9-13).Studies on the structure and function of the c-myc gene product have been hampered by the difficulty in obtaining sufficient amounts of protein for biochemical analyses from human cells that express elevated levels of c-myc RNA (unpublished observations). In this report, we describe the isolation and characterization of cDNA clones containing the complete coding region of human c-myc. The cDNA clones were used to express the c-myc gene in heterologous cells under regulated conditions. The c-myc gene products synthesized in Escherichia coli and in Saccharomyces cerevisiae were compared and found to exhibit host-cell-specific modifications. MATERIALS AND METHODSCell Strains. E. coli strain KRR123 was derived from strain RR1 (14) by phage P1 transduction of the lon9 allele from str...

Poly(A) shortening and degradation of the 3' A+U-rich sequences of human c-myc mRNA in a cell-free system.

“…Cell-free mRNA decay reaction mixtures were incubated for various times (see Materials and Methods), after which RNA was purified, electrophoresed, transferred, and hybridized to a full-length c-myc cDNA probe (60 variable lengths (see below). Poly(A) was a major source of the heterogeneity, because discrete bands were observed when the RNA was treated with oligo(dT) and RNase H before electrophoresis (lane 8, P1 and P2).…”

Regulation of c-myc mRNA stability in vitro by a labile destabilizer with an essential nucleic acid component.

“…RNase protection mapping with RNases P1 and Ti, deoxyoligonucleotidedirected RNase H cleavage assays (H mapping), and blotting of c-myc mRNA were performed as previously described (6). The c-myc probes for these assays were derived from cDNA clone pMl-ll, kindly provided by Grace Ju and colleagues (42). In some P1-plus-Ti RNase-mapping experiments, the protected fragments were electrophoresed in a polyacrylamide minigel (7 by 9 cm; Bio-Rad Laboratories, Richmond, Calif.).…”

Characterization of three putative Lon proteases of Thermus thermophilus HB27 and use of their defective mutants as hosts for production of heterologous proteins

“…In E. coli, strains deficient in EcLon were used to direct overproduction of several heterologous gene products (Buell et al 1985;Miyamoto et al 1985;Brodin et al 1986;Walker et al 1990;Alexander et al 1992;Singh et al 1992;Hutter and Singh 1998;Philibert and Martineau 2004). In some cases, the production was observed only in Eclon mutants, not in wild type strains.…”

“…Physiologically, they are known to contribute to protein quality control in the cells, as well as regulating many cellular processes, by determining the fates of short-lived proteins (Goldberg 1992;Gottesman and Maurizi 1992;Gottesman 1996;Wicker et al 1999). The Lon protease was also reported to degrade heterologous gene-products in E. coli (Buell et al 1985;Miyamoto et al 1985;Brodin et al 1986;Walker et al 1990;Alexander et al 1992;Singh et al 1992;Hutter and Singh 1998;Philibert and Martineau 2004).…”