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 FIM 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 ZnC12 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.

The expression of the PHOS gene of Saccharomyces cerevisiae is transcriptionally regulated in response to the level of inorganic phosphate present in the growth medium. We have identified, by DNA deletion analysis, the sequences (upstream activator sequences) that mediate this response. The sequence 5' CTGCACAATG 3' is present in two copies located within a 60-base-pair region. The presence of a single copy of the sequence is sufficient for the phosphatemediated transcriptional response. In addition, a DNA fragment that contains two copies of this sequence will act to repress transcription of a CYCI-lacZ fusion when placed either upstream or downstream of the CYCI activator sequence.The transcriptional regulation of genes in Saccharomyces cerevisiae is mediated through sequences located one hundred to several hundred bases upstream to the coding sequences (1)(2)(3)(4)(5) (12,13). Sequences at the 5' end determine a precise nucleosome positioning along the inactive gene sequence (14). In this study, using DNA deletions, we localize the sequences responsible for the transcriptional activation of PHOS transcription in response to the concentration of Pi in the growth medium. Also, using CYCI-PHOSlacZ gene fusions, we have demonstrated the presence of a "negative" factor involved in the repression of PHOS gene expression. MATERIALS AND METHODSStrains and Media. Strains of S. cerevisiae utilized in this study are listed in Table 1. Strains were grown in YCAD Fig. 1 for the structure of the wild-type plasmid p19UTT2) contains the E. coli plasmid pUC19, the yeast URA3 selectable marker, the 2-,um circle autonomously replicating segment, the CYCI transcription terminator, and a 1.2-kilobase Sal I/Pst I fragment containing the remainder of the PH05 gene. Thus, the plasmid contains unique BamHI and Sal I sites, allowing the analysis of deletions created within pBS-627 and its derivatives. pLG669Z, a CYCI-lacZ vector was obtained from L. Guarente (19). Derivatives either lacking the 430-bp Xho I fragment (p669-AXho) or with unique Xho I sites (p669-XP or p669-XD) were constructed in the laboratory.DNA Deletion Analysis and DNA Sequencing. DNA deletions within pBS-627 or its derivatives were created by either of two methods. (i) pBS-627 was cleaved with appropriate restriction enzymes, the single-stranded ends were filled-in with DNA polymerase I, and the plasmid was recircularized with T4 DNA ligase. (it) pBS-627 was cleaved with either TthlllI or Cla I and treated with BAL-31 for various time intervals. The purified DNA was subsequently treated with T4 DNA polymerase and recircularized with T4 DNA ligase in the presence of eitherXho I linker (for the TthlllI-cleaved sample) or Cla I linker (for the Cla I-cleaved sample). Individual E. coli transformants were screened by restriction analysis and subsequently the deletion fragment was purified by polyacrylamide gel electrophoresis. The fragment was Abbreviation: bp, base pair(s). 6070The publication costs of this article were defrayed in part by page charge payment...

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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.