Nucleotide sequence of cDNA clones of the murine myb proto-oncogene.

We have isolated cDNA clones of murine c-myb mRNA which contain approximately 2.8 kb of the 3.9-kb mRNA sequence. Nucleotide sequencing has shown that these clones extend both 5' and 3' to sequences homologous to the v-myb oncogenes of avian myeloblastosis virus and avian leukemia virus E26. The sequence contains an open reading frame of 1944 nucleotides, and could encode a protein which is both highly homologous, and of similar size (71 kd), to the chicken c-myb protein. Examination of the deduced amino acid sequence of the murine c-myb protein revealed the presence of a 3-fold tandem repeat of 52 residues near the N terminus of the protein, and has enabled prediction of some of the likely structural features of the protein. These include a high alpha-helix content, a basic region toward the N terminus of the protein and an overall globular configuration. The arrangement of genomic c-myb sequences, detected using the cDNA clones as probes, was compared with the reported structure of rearranged c-myb in certain tumour cells. This comparison suggested that the rearranged c-myb gene may encode a protein which, like the v-myb protein, lacks the N-terminal region of c-myb.     

Differentiation of mouse erythroleukemia cells is blocked by late up-regulation of a c-myb transgene.

During chemically induced differentiation of Friend virus-infected mouse erythroleukemia (MEL) cell lines, there is a biphasic down-regulation of the c-myb proto-oncogene. A plasmid containing a murine c-myb cDNA controlled by a mouse metallothionein I promoter was transfected into the C19 MEL cell line. For six transfected clones, it was found that expression of the exogenous c-myb mRNA could be up-regulated by the addition of 120 microM ZnCl2 and that the N,N'-hexamethylenebisacetamide-induced differentiation of these transfectants was inhibited in proportion to the level of exogenous c-myb mRNA expression. By adding or removing ZnCl2 at different times during the induction process, it was possible to show that up-regulation of exogenous c-myb limited to the first 2 days of induction had little or no effect on differentiation. In contrast, continuous expression of exogenous c-myb beginning at any time during the period of induction blocked further differentiation. These results suggest that during HMBA induction of MEL cells, the early down-regulation of c-myb mRNA is not necessary for terminal differentiation, whereas the down-regulation of c-myb at a later time is necessary.     

Molecular cloning of a differentiation-related mRNA in the adipogenic cell line 1246.

The 1246 cell line is a C3H mouse teratoma-derived adipogenic cell line that can proliferate and differentiate in defined medium. We have constructed a recombinant phage library containing complementary DNAs (cDNAs) prepared from mRNA of differentiated 1246 cells. This library was screened using a differential hybridization technique. We have isolated five different cDNA clones corresponding to mRNAs that are induced during adipogenesis of 1246 cells and one cDNA clone corresponding to mRNA that is decreased during adipogenesis. Among the mRNAs expressed during adipose differentiation, some are not expressed in undifferentiated cells, whereas some are expressed at very low levels under these conditions. Moreover, the level of induction during differentiation and the temporal expression of the mRNAs corresponding to these cDNAs varied. Our results indicate that one of the cDNA clones isolated, called 154, which selects a 2.2-kilobase mRNA, was induced 100-fold at a very early time during the onset of the differentiation program in 1246 cells and also in adipocyte precursors in primary culture. Direct sequencing of 154 cDNA insert revealed no homology with sequences in GenBank and PIR protein databases. The expression of 154 mRNA was stimulated by accelerators of differentiation such as dexamethasone and isobutylmethylxanthine and inhibited by tumor necrosis factor alpha, transforming growth factor beta, and epidermal growth factor, which are known inhibitors of 1246 cell differentiation. In addition, 154 mRNA level in adipocytes was down-regulated by tumor necrosis factor alpha, but not by transforming growth factor beta or epidermal growth factor. These results suggest that the increase in 154 mRNA expression is related to the onset of adipose differentiation. Further analysis of this clone should allow characterization of a novel protein induced early during the process of differentiation.     

Isolation of differentially expressed human cDNA clones: similarities between mouse and human embryonal carcinoma cell differentiation

The study of early human development is of great importance but has been limited by the lack of suitable reagents. Recently, however, the human embryonal carcinoma (EC) cell line NT2D1 has been isolated. This cell line will differentiate upon exposure to retinoic acid (RA). A cDNA library was constructed from poly(A)+ RNA derived from NT2D1 cells treated with 10(-5) M-RA for 7 days (delta NT2D1 cells). By differential cDNA screening, it was found that 1.12% of delta NT2D1 cDNA recombinants screened detected an increase in signal with 32P-cDNAs derived from delta NT2D1 as compared with NT2D1. To compare RA-induced differentiation of mouse and human EC cells, the delta NT2D1 cDNA library was rescreened with 32P-cDNAs derived from the mouse EC cell line F9 and the result compared with 32P-cDNA derived from F9 differentiated to parietalendoderm (F9PE)-like cells and visceral-endoderm (F9VE)-like cells. Approximately 1.2% of the delta NT2D1 cDNA recombinants detected a differential increase in signal following differentiation of mouse EC cells to F9VE and/or F9PE. Of these homologous regulated sequences, 0.3% were common to both mouse and human EC cell RA-induced differentiation. Five different cDNA clones were isolated that detect a marked increase (5- to 75-fold) in mRNA abundance following RA-induced differentiation of NT2D1. Of these five clones, three detect homologous mRNAs which also increase in abundance following differentiation of the mouse EC cell line F9 to PE- and/or VE-like cells; the other two clones do not detect sequences in the mouse mRNAs tested. One clone shows homology to SPARC, a gene known to be regulated during mouse embryonic development. While another clone, SO5A, has a limited range of expression, being detected in F9VE and in a human parietal-endoderm-like cell, but not in F9PE and a human visceral-endoderm-like cell. This work shows that there are both similarities and differences in mouse and human EC cell differentiation, and these cDNA clones provide some of the first reagents for studying the molecular biology of human development.