Two N-myc polypeptides with distinct amino termini encoded by the second and third exons of the gene.

The N-myc and c-myc genes encode closely related nuclear phosphoproteins. We found that the N-myc protein from human tumor cell lines appears as four closely migrating polypeptide bands (p58 to p64) in sodium dodecyl sulfate-polyacrylamide gels. This and the recent finding that the c-myc protein is synthesized from two translational initiation sites located in the first and second exons of the gene (S. R. Hann, M. W. King, D. L. Bentley, C. W. Anderson, and R. N. Eisenman, Cell 52:185-195, 1988) prompted us to study the molecular basis of the N-myc protein heterogeneity. Dephosphorylation by alkaline phosphatase reduced the four polypeptide bands to a doublet with an electrophoretic mobility corresponding to the two faster-migrating N-myc polypeptides (p58 and p60). When expressed transiently in COS cells, an N-myc deletion construct lacking the first exon produced polypeptides similar to the wild-type N-myc protein, indicating that the first exon of the N-myc gene is noncoding. Furthermore, mutants deleted of up to two thirds of C-terminal coding domains still retained the capacity to produce a doublet of polypeptides, suggesting distinct amino termini for the two N-myc polypeptides. The amino-terminal primary structure of the N-myc protein was studied by site-specific point mutagenesis of the 5' end of the long open reading frame and by N-terminal radiosequencing of the two polypeptides. Our results show that the N-myc polypeptides are initiated from two alternative in-phase AUG codons located 24 base pairs apart at the 5' end of the second exon. Both of these polypeptides are phosphorylated and localized to the nucleus even when expressed separately. Interestingly, DNA rearrangements activating the c-myc gene are often found in the 1.7-kilobase-pair region between the two c-myc translational initiation sites and correlate with the loss of the longer c-myc polypeptide. Thus the close spacing of the two N-myc initiation codons could explain the relative resistance of the N-myc gene to similar modes of oncogenic activation.     

Nucleotide sequence of the coding region of the mouse N-myc gene.

A genomic clone for the mouse N-myc gene was isolated and the total nucleotide sequence (4807 bp) of the two coding exons and an intron located between them was determined. The amino acid sequence of the N-myc protein was deduced from the DNA sequence. This protein is composed of 462 amino acids, slightly larger than human and mouse c-myc proteins, and is rich in proline like the c-myc protein. Comparison of the amino acid sequences of the mouse N-myc and c-myc proteins showed that conserved sequences are located in eight regions: four regions are in the N-terminal half of the N-myc protein and are separated from each other by regions poorly homologous to those of the c-myc protein, and the four others are located in the C-terminal half, throughout which certain homology exists. A remarkable sequence containing 13 successive acidic amino acids is present in one of the conserved regions located in the middle of the N-myc protein.     

Amplification of the N-myc oncogene in an adenocarcinoma of the lung

c-myc oncogene is the most extensively studied member of the myc gene family, which now consists of three characterized members, namely the c-myc, N-myc, and L-myc genes. Deregulation owing to amplification and/or rearrangements of the c-myc gene have been described in a variety of human malignancies. Several neuroblastomas have amplifications of the N-myc genes. The c-myc, N-myc, or L-myc oncogenes are also found amplified in different cell lines from small cell carcinomas of the lung. In this study, we have examined the c-myc, N-myc, and c-erbB oncogenes in 34 clinical and autopsy tumor specimens representing various histopathological types of human lung cancer, including nine small cell lung cancers. A 30-fold amplification of the N-myc gene was found in a tumor histopathologically and histochemically verified as a typical adenocarcinoma. No amplifications of the c-myc or c-erbB oncogenes were seen in any of the tumors. In the DNA of one small cell carcinoma, an extra c-myc and N-myc cross-hybridizing restriction fragment was observed, possibly owing to an amplification of a yet uncharacterized myc-related gene.     

Structure and expression of canary myc family genes.

We found that the canary N-myc gene is highly related to mammalian N-myc genes in both the protein-coding region and the long 3' untranslated region. Examined coding regions of the canary c-myc gene were also highly related to their mammalian counterparts, but in contrast to N-myc, the canary and mammalian c-myc genes were quite divergent in their 3' untranslated regions. We readily detected N-myc and c-myc expression in the adult canary brain and found N-myc expression both at sites of proliferating neuronal precursors and in mature neurons.     

Hel-N1: an autoimmune RNA-binding protein with specificity for 3'' uridylate-rich untranslated regions of growth factor mRNAs.

We have investigated the RNA binding specificity of Hel-N1, a human neuron-specific RNA-binding protein, which contains three RNA recognition motifs. Hel-N1 is a human homolog of Drosophila melanogaster elav, which plays a vital role in the development of neurons. A random RNA selection procedure revealed that Hel-N1 prefers to bind RNAs containing short stretches of uridylates similar to those found in the 3' untranslated regions (3' UTRs) of oncoprotein and cytokine mRNAs such as c-myc, c-fos, and granulocyte macrophage colony-stimulating factor. Direct binding studies demonstrated that Hel-N1 bound and formed multimers with c-myc 3' UTR mRNA and required, as a minimum, a specific 29-nucleotide stretch containing AUUUG, AUUUA, and GUUUUU. Deletion analysis demonstrated that a fragment of Hel-N1 containing 87 amino acids, encompassing the third RNA recognition motif, forms an RNA binding domain for the c-myc 3' UTR. In addition, Hel-N1 was shown to be reactive with autoantibodies from patients with paraneoplastic encephalomyelitis both before and after binding to c-myc mRNA.     

Transrepression of the N-myc expression by c-myc protein

In the human neuroblastoma cell line IMR32 the N-myc gene happens to be amplified and actively expressed, whereas no stable c-myc RNA can be detected in the same cells. In this report, we show that in IMR32 cells the expression of the N-myc gene is repressed by introduction of a c-myc expression vector (c-myc cDNA conjugated with an SR promoter). Moreover, dose response experiments showed that the amount of endogenous c-myc protein present in HeLa cells (which express c-myc but not N-myc) is enough to repress the expression of N-myc in IMR32 cells.     

Specific phosphorylation of the acidic central region of the N-myc protein by casein kinase II.

The central region of the N-myc protein has a characteristic amino acid sequence EDTLSDSDDEDD, which is very similar to those of particular domains of adenovirus E1A, human papilloma virus E7, Simian virus 40 large T, c-myc and L-myc proteins. Domains of these three viral oncoproteins have recently been shown to be specific binding sites for the tumor-suppressor gene retinoblastoma protein. We have noted that the sequence of serine followed by a cluster of acidic amino acids is exactly the same as that of a typical substrate of casein kinase II (CKII). Therefore, we investigated whether these nuclear oncoproteins are phosphorylated by CKII. For this purpose, we fused the beta-galactosidase and N-myc genes including this domain and expressed it in Escherichia coli cells. Several mutant N-myc genes, containing single amino acid substitutions in this domain, were also used to produce fused proteins. Strong phosphorylation by CKII was detected with the fused protein of wild-type N-myc. However, no phosphorylation of beta-galactosidase itself was observed and the phosphorylations of fused mutant proteins were low. Another fused N-myc protein containing most of the C-terminal region downstream of this acidic region was not phosphorylated by CKII. Analysis of phosphorylation sites in synthetic peptides of this acidic region identified the major sites phosphorylated by CKII as Ser261 and Ser263. On two-dimensional tryptic mapping of phosphorylated N-myc proteins, major spots of in vitro-labeled and in-vivo-labeled N-myc proteins were detected in the same positions. These results suggest that two serine residues of the acidic central region of the N-myc protein are phosphorylated by CKII in vivo as well as in vitro. The functional significance of this acidic domain is discussed.     

Organization and expression of the chicken N-myc gene.

We cloned the chicken N-myc gene and analyzed its structure and expression. We found that it consisted of three exons with coding regions in exons 2 and 3. Comparison to mammalian N-myc genomic sequence indicated that nucleotide sequences of the 5'-flanking region, noncoding exon 1, and introns were not conserved, but coding and 3' noncoding sequences showed significant homology to mammalian N-myc. Alignment of deduced amino acid sequences of chicken and mammalian N-myc proteins revealed nine conserved domains interrupted by different lengths of nonhomologous sequences. Two of the domains were specific to N-myc proteins, and the other seven were common to c-myc proteins. Northern blot (immunoblot) and in situ hybridization analyses of 3.5-day-old chicken embryos revealed that high-level expression of the N-myc gene was confirmed to certain tissues, e.g., the central nervous system, neural crest derivatives, and mesenchyme of limb buds. In the beak and limb primordia, N-myc expression in the mesenchyme was higher toward the distal end, suggesting possible involvement in positional assignment of the tissue within the rudimentary structures.     

Neoplastic transformation by the human gene N-myc.

Amplification and abundant expression of a gene known as N-myc are found frequently in advanced stages of human neuroblastoma and may play a role in the genesis of several malignant human tumors. Previous studies have shown that N-myc can cooperate with a mutant allele of the proto-oncogene c-Ha-ras to transform embryonic rat cells in culture. Here we show that N-myc can also act alone to elicit neoplastic growth of an established line of rat fibroblasts (Rat-1). We used recombinant DNA vectors to express either N-myc or its kindred gene c-myc in transfected cells. Both genes caused morphological transformation, anchorage-independent growth, and tumorigenicity. We noticed two variables that appeared to influence the ability to isolate cells transformed by N-myc and c-myc: the abundance in which the genes were expressed and biological selection to eliminate untransformed cells from the cultures. Our findings sustain the belief that N-myc is an authentic proto-oncogene, lend further credibility to the role of N-myc in the genesis of human tumors, and establish a convenient assay that can be used to explore further the properties of both N-myc and c-myc.     

Nucleotide sequence of a transduced myc gene from a defective feline leukemia provirus.

The nucleotide sequence of a feline v-myc gene and feline leukemia virus (FeLV) flanking regions was determined. Both the nucleotide and predicted amino acid sequences are very similar to the murine and human c-myc genes (ca. 90% identity). The entire c-myc coding sequence is represented in feline v-myc and replaces portions of the gag and env genes and the entire pol gene. The coding sequence is in phase with the gag gene reading frame; v-myc, therefore, appears to be expressed as a gag-myc fusion protein. Viral sequences at the 3' myc-FeLV junction begin with the hexanucleotide CTCCTC, which is also found at the 3' fes-FeLV junction of both Gardner-Arnstein and Snyder-Theilen feline sarcoma viruses. These similarities suggest that some sequence specificity may exist for the transduction of cellular genes by FeLV. Feline v-myc lacks a potential phosphorylation site at amino acid 343 in the putative DNA-binding domain, whereas both human and murine c-myc have such sites. Avian v-myc has lost a potential phosphorylation site which is present in avian c-myc five amino acids from the potential mammalian site. If these sites are actually phosphorylated in normal c-myc proteins, their loss may alter the DNA-binding affinity of v-myc proteins.     

Contrasting patterns of c-myc and N-myc expression in proliferating, quiescent, and differentiating cells of the embryonic chicken lens.

The present study uses the polymerase chain reaction and in situ hybridization to examine c-myc and N-myc mRNA in the embryonic chicken lens at 6, 10, 14 and 19 days of development and compares the pattern of expression obtained with the developmental pattern of cell proliferation and differentiation. In the central epithelium, c-myc mRNA levels were proportional to the percentage of proliferating cells throughout development. N-myc mRNA expression in this region was relatively low and showed no correlation with cell proliferation. The ratio of N-myc to c-myc mRNA increased markedly with the onset of epithelial cell elongation and terminal fiber cell differentiation, although both c-myc and N-myc mRNAs continued to be expressed in postmitotic, elongating cells of the equatorial epithelium and in terminally differentiating lens fiber cells. Thus, increased expression of N-myc, a gene whose protein product may compete with c-myc protein for dimerization partners, accompanies the dissociation of c-myc expression and cell proliferation during terminal differentiation of lens fiber cells.     

Isolation and characterization of the human cellular myc gene product

Antibodies against the product of the human cellular myc gene (c-myc) were prepared against a bacterially expressed human c-myc protein by inserting the ClaI/BclI fragment of the human c-myc DNA clone in an expression vector derived from pPLc24. These antibodies cross-react with viral-coded myc (v-myc) proteins from MC29 and OK10 viruses. Furthermore, IgGs specific for synthetic peptides, corresponding to the 12 carboxy-terminal amino acids of the human c-myc gene and 16 internal amino acids, were isolated. By use of the various myc-specific antisera or IgGs, a protein of Mr 64 000 was detected in several human tumor cell lines including Colo320, small cell cancer of the lung (417d), HL60, Raji, and HeLa. This protein is larger than the corresponding v-myc or chicken c-myc proteins from avian virus transformed cells or avian bursa lymphoma cells (RP9), both of which are proteins of Mr 55 000. The human c-myc protein is located in the nucleus of Colo320 cells, exhibits a half-life of about 15 min, and is expressed at significantly lower levels than the viral protein. The human c-myc protein was enriched about 3000-fold from Colo320 cells using c-myc-specific IgG coupled to Sepharose beads. The protein binds to double-stranded DNA in vitro, a reaction that can be inhibited to more than 90% by c-myc specific IgG.