We have shown previously that an increase in the level of accumulated pS2 mRNA is first detectable in MCF-7 cells after 3 hr of estradiol treatment. Using in vitro nuclear run-on transcription with nuclei prepared from MCF-7 cells grown in the presence of estradiol or in estradiolstripped medium, we demonstrate here that expression of the pS2 gene is controlled by estrogen at the transcriptional level, Induction of transcription is a very early event that is already apparent within 15 min after addition of estradiol to the culture medium. In addition, pretreatment of the cells with the protein synthesis inhibitor cycloheximide does not prevent induction of pS2 gene transcription, indicating that it corresponds to a primary effect of estrogen. The pS2 gene in MCF-7 cells represents a unique example of a human gene whose transcription is directly controlled by estrogen.

The translational positions of nucleosomes in the promoter region of the mouse mammary tumor virus (MMTV) were defined at high resolution. Nucleosome boundaries were determined in primer extension assays using full-length single-stranded mononucleosomal DNA prepared from cells treated with formaldehyde, a reversible protein-DNA cross-linking agent. Multiple boundaries were observed in both the nucleosome A (Nuc-A) and Nuc-B region of the promoter, indicating multiple nucleosome translational frames. The different nucleosome frames in both the Nuc-A and Nuc-B regions were occupied unequally. The most frequently occupied frames were found clustered within 50-60 bases of each other, resulting in a distribution centered in the positions defined previously at low resolution for Nuc-A and Nuc-B. The most abundant 5' ends of the frames in the B region were found between -235 and -187, and the 3' ends between -86 and -36, whereas in the A region the most abundant 5' ends were between -22 and +42, and the 3' ends between + 121 and +186. Although frames in the Nuc-B region of the LTR extend at a low frequency in the 5' direction toward the Nuc-C region, there is a sharp discontinuity in the 3' direction toward Nuc-A, suggesting the presence of a boundary constraint in the A-B linker. The positions and relative occupancies of nucleosome frames, in either the B or the A region, did not change when the promoter was activated with dexamethasone.

copies of a consensus response element commonly referred to as the 29,33,51,63), plus, in some cases, an overlapping consensus octamer element, ATGCTAAT (39; C. apRhys, D. M. Ciufo, E. A. O'Neill, T. J. Kelly, and G. S. Hayward, submitted for publication). In addition, 11 CCCGCCC elements, which appear to be associated with basal expression properties of the IE175-IE68 region, have been shown by DNA footprinting studies to bind to the purified cellular Sp-1 factor (25).During HSV infection, the IE175 gene product is essential for progression of the lytic cycle (8, 50, 60) and for activation of the transcription of the delayed-early (DE) class of viral genes (30,31,46,50). The isolated viral DE and late promoters, whether associated with their own genes (11,55) or as hybrid HSV promoter-driven reporter genes, also respond to virus superinfection in an IE175-dependent fashion, both when in an integrated state in permanent DNAtransfected cell lines (6,9,11,12,35,53) and in transientexpression assays with superinfecting virus (14,40). In DNA cotransfection assays, the isolated IE175 gene encodes a trans-acting factor that stimulates expression of HSV DE promoter targets (13,21,41,52) and inhibits expression from its own promoter in IE175- CAT (9,22,42,43). Although the HSV IE110 gene product was shown to stimulate expression 4307 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from with polynucleotide kinase and [-y-32P]ATP. The two com-J. VIROL.on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from DNA TARGET SITES FOR IE175 BINDING AND AUTOREGULATION plementary oligonucleotides were annealed by heating to 65°C in 50 mM NaCI-10 mM MgCl2-25 mM Tris hydrochloride (pH 8.0)-100 ,ug of bovine serum albumin per ml-2 mM P-mercaptoethanol followed by slow cooling. For cloning, the double-stranded oligonucleotides were ligated after phosphorylation with polynucleotide kinase to BamHI-plus-BglII-cleaved pGH59 DNA. The resulting plasmids, pGH123a and pGH123b, contained sequences from -8 to

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We previously described a two-plasmid system for the identification of promoters recognized by Salmonella enterica serovar Typhimurium (S. Typhimurium) sigmaE. The S. Typhimurium sigmaE-dependent rpoEp3 promoter was active in the E. coli two-plasmid system only after arabinose-induced expression of S. Typhimurium rpoE. In the present study, we have exploited this two-plasmid system for the identification of nucleotides critical for activity of the rpoEp3 promoter. A library of randomly mutated DNA fragments containing the rpoEp3 promoter was cloned upstream of a lacZalpha reporter gene and screened for activity in the presence of S. Typhimurium sigmaE. The clones exhibiting reduced LacZ activity were sequenced to identify the mutations. The activity of the mutated rpoEp3 promoters were studied further using a luciferase-based promoter-probe plasmid. All of the important nucleotides of the rpoEp3 promoter (in capital) were located in the -35 (ggAActt) and -10 (TctaA) regions. The critical nucleotides were also the most conserved in known sigmaE-dependent promoters. The study also revealed the importance of the 16-bp spacing between -10 and -35 region, as reducing the spacing to 15-bp greatly reduced activity of the promoter. This method should be generally applicable for the identification of important nucleotides in the cognate promoters of other sigma factors.

The control of beta-galactosidase specified by the lactose transposon Tn951 (inserted into RP1 to give pGC9114) has been studied in Escherichia coli K12, Proteus mirabilis, Pseudomonas aeruginosa and Pseudomonas putida; in the first two species comparison could be made with Flac. In E. coli K12, the Tn951 and chromosomally encoded enzymes showed marked qualitative differences in regulatio, the former giving a substantially lower maximum induced level and induction ratio. Several parameters were slightly affected by strain background. In P. mirabilis, beta-galactosidase control determined by both Flac (in accord with earlier work) and pGC9114 was markedly different from E. coli in that maximal induced levels were about an order of magnitude lower and the induction ratio was reduced to 3 to 5. In Ps. aeruginosa and Ps. putida, Tn951-specified lac expression was qualitatively similar to that in P. mirabilis. Possible reasons for anomalous expression in Proteus and Pseudomonas are discussed.

Deregulated expression of v-abl and BCR/abl genes has been associated with myeloproliferative syndromes and myelodysplasia, both of which can progress to acute leukemia. These studies identify the localization of the oncogenic form of the abl gene product encoded by the Abelson murine leukemia virus in the nuclei of myeloid cells and the association of the v-Abl protein with the transcriptional regulator cyclic AMP response elementbinding protein (CREB). We have mapped the specific domains within each of the proteins responsible for this interaction. We have shown that complex formation is a prerequisite for transcriptional potentiation of CREB. Transient overexpression of the homologous cellular protein c-Abl also results in the activation of promoters containing an intact CRE. These observations identify a novel function for v-Abl, that of a transcriptional activator that physically interacts with a transcription factor.

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