Michael D. Cole

Novel myc oncogene RNA from abortive immunoglobulin-gene recombination in mouse plasmacytomas

We have found that the myc oncogene has been modified by abortive recombination with the alpha heavy-chain immunoglobulin constant-region (C alpha) gene in five different mouse plasmacytoma lines. Recombination occurred approximately 0.8-2.0 kb to the 5 side of two distinct coding regions, defined by sequence homology between the chicken cellular and plasmacytoma myc genes. The myc and C alpha genes were always in opposite transcriptional orientation, with the recombination site within the C alpha switch region sequences. DNA recombination was found to correlate with the production of a novel 2.1 kb species of myc RNA that was 0.4 kb shorter than the normal cellular transcript. No elevated levels of myc RNA were evident, suggesting that DNA rearrangements have altered the myc oncogene product. This oncogene activation corresponds to the chromosomal translocations found in nearly all plasmacytomas.

Fibroblast lines expressing activated c-myc oncogenes are tumorigenic in nude mice and syngeneic animals

The influence of c-myc expression on fibroblast growth and morphology was investigated by transfection of c-myc genes linked to viral promoters. No foci were observed after transfection of either NIH/3T3 or Rat 2 cells. Cell lines containing activated c-myc genes were established using SV2-neo coselection and several growth parameters of the cells were studied. The cells showed a slight increase in refractility and formed colonies in soft agar with an efficiency of only 1%-2%. The c-myc-transfected cells grew well in 0.5% serum while the controls did not. The major difference in cell growth noted was that c-myc-transfected cells were tumorigenic when inoculated into nude mice or syngeneic rats. Analysis of RNA from the tumorigenic cells showed a level of c-myc expression from the transfected genes that was 2 to 6 fold higher than that from the endogenous gene. The level of c-myc RNA in the fibroblast tumors was similar to that found in mouse plasmacytomas. Expression of the endogenous c-myc gene was unaffected by the transfected genes for subconfluent cells in culture, but the gene was shut off in the nude mouse tumors. These results demonstrate that constitutive c-myc expression leads to tumorigenicity in immortalized cell lines.

Characterization of a novel plasmid DNA found in mitochondria of N. crassa

We have identified a plasmid DNA that is found within mitochondria of wild-type N. crassa strain Mauriceville-1c (FGSC #2225), but that shows no detectable sequence homology with mitochondrial DNA. The plasmid DNA consists of an oligomeric series of circular molecules of monomer length 3.6 kb. There are two unusual clusters of restriction enzyme sites, one consisting of six Eco RI sites in a 1 kb region, and the other of five or more Pst I sites in a 0.4 kb region. RNA gel transfer hybridization experiments show a major transcript 3.3 to 3.4 kb long, close to the monomer length of the plasmid. The latter finding implies that the plasmid DNA contains specific sites for the initiation and termination of transcription.

Transcriptional activation of the translocated c-myc oncogene in mouse plasmacytomas: Similar RNA levels in tumor and proliferating normal cells

We examine the influence of the immunoglobulin locus on the expression of the translocated c-myc oncogene in mouse plasmacytomas. The level of c-myc RNA was 30- 35-fold greater in tumor cells than in normal, quiescent B cells. Mitogen stimulation of the lymphocytes with lipopolysaccharide induced a 15-fold increase in c-myc expression per cell to a level that was similar to that in the transcription of the translocated c-myc gene involved initiation from sequences in the first c-myc intron. Abundant RNA transcripts were also found from the noncoding strand of the c-myc intron in most tumor lines. S1 nuclease mapping was used to locate the intronic sequences that are used to initiate the tumor-specific c-myc RNAs. Six different initiation sites within the intron were mapped, none of which have the TATA sequence usually associated with eucaryotic RNA polymerase II promoters. The noncoding strand transcripts were also found to initiate in the c-myc intron. Transcription of the c-myc coding strand was independent of the position of the translocation breakpoint, even when the heavy chain switch and constant regions were deleted.

Myc meets its max

c-myc, the earliest discovered and perhaps most prominent nuclear oncogene, has been implicated in the control of normal cell proliferation and the transformation and differentiation of many cell lineages (reviewed in Cole, 1936; Luscher and Eisenman, 1999). c-Myc represents the paradigm for two broad classes of transcription factors that contain either the basic/helix-loop-helix (HLH; Murre et al., 1939a) or the basic/leucine repeat structure (LR; Landschulz et al., 19BB). Thus, it is’particularly ironic that the direct demonstration of a function for the c-Myc protein has only recently been achieved with two reports of the cloning of a heterodimeric partner of c-Myc, called Max, that facilitates sequence-specific DNA-binding activity. A partner was predicted because c-Myc, like c-Fos, contains a leucine repeat with many charged residues at the coiledcoil interface that seem to prevent homodimer formation (O’Shea et al., 1989). If Max is the one and true partner of c-Myc, it opens new avenues of investigation that will finally address the key issue of how c-Myc can exert such a diverse range of biological activities. Myc Functions in DNA Binding as a Heterodlmer The clear demonstration of a DNA-binding function for Myc came first from the elegant use of a potentially powerful approach to the study of protein dimers, that of direct screening of a bacterial expression library with labeled protein. Blackwood and Eisenman (1991) used a labeled fusion protein containing the C-terminus of c-Myc to isolate a clone encoding a novel protein, Max. Like Myc itself, Max contains adjacent basic, HLH, and LR domains. Max forms heterodimers with all three Myc proteins (c-, N-, and L-Myc) but does not dimerize wlth other HLH proteins, even those that also contain combined HLHlLR domains such as TFE?t (Beckmann et al., 1999) USF (Gregor et al., 1990) and AP-4 (Hu et al., 1990). The Myc-Max heterodimer binds to the sequence CACGTG, which has been shown to bind weakly to Myc homodimers that form at very high protein concentrations (Blackwell et al., 1990). An alternative PCR approach that led to the cloning of max employed primers chosen to match conserved domains of Myc (Prendergast et al., 1991). One of the amplified cDNA molecules matched the sequence of max reported by Blackwood and Eisenman (1991). Analogous experiments demonstrated heterodimer formation and sequence-specific DNA binding. However, major differences were reported in the two studies in the migration of the Myc-Max complex, and in homodimer binding activity; these discrepancies are unresolved but seem difficult to attribute to reagents or constructs. The structure of Max appears to be extremely simple; it is comprised of only 169 amino acids, 99 of which constitute the DNA-bindingldimerization domain (see figure). Minireview

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