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.     

Functional analysis of the regulatory region adjacent to the cargB gene of Saccharomyces cerevisiae. Nucleotide sequence, gene fusion experiments and cis-dominant regulatory mutation analysis

In Saccharomyces cerevisiae the expression of the cargB gene (coding for ornithine aminotransferase) is submitted to dual regulation: an induction by allophanate and a specific induction process by arginine. We have determined the nucleotide sequence of the cargB gene along with its 5' region. The coding portion of the gene encodes a protein of 423 amino acid residues with a calculated Mr value of 46049. To characterize further the regulatory mechanisms modulating the expression of the gene we have analyzed fusions of several fragments of the 5' non-coding region to lacZ, compared the 5' sequences of the cargA (coding for arginase) and cargB coregulated genes and determined the nature of two constitutive cis-dominant mutations affecting the arginine control. These approaches allowed us to define three domains in the 5' non-coding region. The upstream one is implicated in the induction by allophanate. The two other domains are involved in the specific control by arginine; the target of the ARGR gene products, that mediate a positive regulation by arginine, lies upstream of another site where a repression by the CARGRI molecule occurs. The constitutive cargB+O- mutations are located in this repressor domain. The 5' non-coding region of cargA presents the same two-domain organization. These two domains contain three sequences homologous to the cargA and cargB 5' regions.     

Nucleotide sequence of the 5' flanking region responsible for the enhancement of the expression of yeast enolase 1 gene

Several plasmids carrying different length of the 5' flanking region of yeast (Saccharomyces cerevisiae) enolase 1 gene (ENO1) which is fused in frame to the Escherichia coli lacZ gene were constructed by recombination in vitro. Promoter activity of ENO1 was assayed by measuring beta-galactosidase activity of the fused gene product. Comparison of the promoter activity of these plasmids suggests that the sequences required for a strong promoter activity lie within the DNA segment -724 to -353 base pairs (bp) upstream from the start of ENO1 coding sequence. The nucleotide sequence of this region was determined.     

Light-regulated expression of the pea plastocyanin gene is mediated by elements within the transcribed region of the gene

Expression of the pea plastocyanin gene ( PetE ) is regulated by light in both pea and transgenic tobacco plants. However, the PetE promoter with the 5' untranslated leader region does not direct light-regulated expression of the GUS reporter gene in transgenic tobacco. This suggested that sequences downstream of the translation start of the PetE gene are required for light-regulated expression. To investigate this possibility the expression of a series of chimeric gene constructs in transgenic tobacco plants was examined to assess the contributions of the promoter, the 5' untranslated leader region, the coding region and the 3' region of the PetE gene to light-regulated expression. Both the coding region and the 5' untranslated leader region of the PetE gene were found to be required for full light regulation. Full light regulation of chimeric gene constructs containing the cauliflower mosaic virus (CaMV) 35S promoter required the deletion of CaMV 5' leader and polylinker sequences from the constructs. The presence of CaMV and polylinker sequences at the 5' end of the PetE leader masked the light regulation directed by the transcribed region of the pea PetE gene.     

Deletion analysis of the mouse alpha 1(III) collagen promoter.

A chimeric gene was constructed by fusing the DNA sequences containing the 5' flanking region of the mouse alpha 1(III) collagen gene to the coding sequence of the bacterial chloramphenicol acetyltransferase (CAT) gene. Transient transfection experiments indicated that the alpha 1(III) promoter is active in NIH 3T3 fibroblasts and BC3H1 smooth muscle cells. The activity of the alpha 1(III) collagen promoter-CAT plasmid is stimulated approximately ten fold by the presence of the SV40 enhancer element. Removing sequences upstream of -200 stimulates the activity of the chimeric gene eight fold. Further deletion analysis identified sequences located between -350 and -300 that were instrumental in repressing the activity of the promoter. This 50 bp region contains a direct repeat sequence that may be involved in the regulation of the mouse alpha 1(III) collagen gene. Truncating the alpha 1(III) promoter to -80 further stimulated expression. We propose that the positive regulatory elements of this gene appear to be located within the first 80 bp of the promoter, whereas elements located further upstream exert a negative effect on the expression of the gene. Regulation of the alpha 1(III) gene contrasts with that of the alpha 2(I) collagen gene, which appears to be regulated by several positive elements located in various regions of the promoter.