Identification of estrogen-responsive DNA sequences by transient expression experiments in a human breast cancer cell line.

The expression of a hybrid gene formed by the promoter region of the Xenopus laevis vitellogenin gene B1 and the CAT coding region is regulated by estrogen when the gene is transfected into hormone-responsive MCF-7 cells. Furthermore, the 5' flanking region of the gene B1 alone can confer inducibility to heterologous promoters, although to a varying extent depending on the promoter used. Deletion mapping of he vitellogenin hormone-responsive sequences revealed that a 13 bp element 5'-AGTCACTGTGACC-3' at position -334 is essential for estrogen inducibility. We have shown previously that this 13 bp element is present upstream of several liver-specific estrogen-inducible genes.     

Structure and expression of the human gene encoding testicular H1 histone (H1t)

The gene coding for the human H1t histone, a testis-specific H1 subtype, was isolated from a genomic library using a human somatic H1 gene as a hybridization probe. The corresponding mRNA is not polyadenylated and encodes a 206-amino-acid protein. Sequence analysis and S1 nuclease mapping of the human H1t gene reveals that the 5' flanking region contains several consensus promoter elements, as described for somatic, i.e., S-phase-dependent H1 subtype genes. The 3' region includes the stem-and-loop structure necessary for mRNA processing of most histone mRNAs. Northern blot analysis with RNAs from different human tissues and cell lines revealed that only testicular RNA hybridized with this gene probe.     

Identification of an upstream repressor site controlling the expression of an anaerobic gene (ANB1) in Saccharomyces cerevisiae

The Saccharomyces cerevisiae anaerobic gene (ANB1) is negatively regulated both by oxygen and heme. A 299-base pair-long fragment from the 5'-flanking region of the ANB1 gene was found to confer oxygen-mediated negative regulation to an heterologous CYC1-LacZ hybrid gene. Studies with deletions of predefined length in this fragment demonstrated the presence of separate elements that comprise an upstream promoter that is active in the absence or presence of oxygen, and an upstream repressor site (URS) which confers strong repression upon the promoter element when oxygen is present. The promoter element is located 5' to the URS in the ANB1 gene. Mixed oligonucleotide-directed mutagenesis was used to obtain nucleotide substitutions in the URS which partially or completely inactivated this sequence without affecting the promoter activity. The URS region has three short direct repeats which seem to be important for function, as nucleotide substitutions within the repeats and not outside them, inactivated URS function. A model to explain the negative regulation of the ANB1 gene by oxygen and heme is proposed.     

Evidence that the respiratory syncytial virus polymerase is recruited to nucleotides 1 to 11 at the 3' end of the nucleocapsid and can scan to access internal signals

The 3'-terminal end of the respiratory syncytial virus genomic RNA contains a 44-nucleotide leader (Le) region adjoining the gene start signal of the first gene. Previous mapping studies demonstrated that there is a promoter located at the 3' end of Le, which can signal initiation of antigenome synthesis. The aim of this study was to investigate the role of the 3' terminus of the RNA template in (i) promoter recognition and (ii) determining the initiation site for antigenome synthesis. A panel of minigenomes containing additional sequence at the 3' end of the Le were analyzed for their ability to direct antigenome and mRNA synthesis. Minigenomes containing heterologous extensions of 6 nucleotides or more were unable to support efficient RNA synthesis. However, the activity of a minigenome with a 56-nucleotide extension could be restored by insertion of Le nucleotides 1 to 11 or 1 to 13 at the 3' end, indicating that these nucleotides, in conjunction with the 3' terminus, are sufficient to recruit polymerase to the template. Northern blot and 5' rapid amplification of cDNA ends analysis of antigenome RNA indicated that antigenome initiation occurred at the first position of Le, irrespective of the terminal extension. This finding demonstrates that the 3' terminus of the RNA is not necessary for determining the antigenome initiation site. Data are presented which suggest that following recruitment to a promoter at the 3' end of Le, the polymerase is able to scan and respond to a promoter signal embedded within the RNA template.     

Insulin-mediated inhibition of the induction of tyrosine aminotransferase by dexamethasone.

Insulin-mediated regulation of glucocorticoid-induced expression of the liver-specific gene tyrosine aminotransferase was studied in a clone of the Reuber rat hepatoma cells. Insulin inhibited dexamethasone-induced chloramphenicol acetyltransferase expression from approximately 4 kb of TAT 5' flanking sequence. The degree of this inhibition was comparable to the response of the endogenous gene. A construct of approximately 3 kbp of 5' flanking sequence exhibited no significant basal expression but retained sensitivity to glucocorticoids and to insulin inhibition of the glucocorticoid response. Results of further analysis of the insulin response in deletion constructs and constructs containing glucocorticoid responsive elements ligated to a heterologous promoter suggest that in addition to the glucocorticoid response elements a region close to the start site in the TAT promoter is necessary for insulin to inhibit glucocorticoid-mediated induction of expression.