A positive regulatory sequence of the Saccharomyces cerevisiae ENO1 gene

ENO1-'lacZ fusions with various lengths of the ENO1 5'-flanking region were constructed on various types of yeast plasmid vectors. The fully expressed level of beta Gal directed by ENO1-'lacZ fusions differed depending on the type of vector, but on any type of vector, beta Gal activity was not greatly influenced by the carbon source in the medium. The 86-bp DNA region of ENO1 at position -487 to -402 upstream of the initiation codon, in which we had previously delimited the positive regulatory region of ENO1 (Uemura, H., Shiba, T., Paterson, M., Jigami, Y., & Tanaka, H. (1986) Gene 45, 67-75), exerted its function without requiring precise location with respect to the TATA box. The action of the positive regulatory region was not affected by its orientation. In addition, the substitution of the UASs of PHO5, encoding repressible acid phosphatase, with the regulatory region of ENO1 changed the expression of PHO5-'lacZ gene to constitutive, irrespective of the concentration of inorganic phosphate in the medium. Furthermore, the GCR1 gene cloned in a multicopy plasmid increased the expression of the ENO1-'lacZ fused genes.     

Lysozyme gene activity in chicken macrophages is controlled by positive and negative regulatory elements.

The chicken lysozyme gene is constitutively active in macrophages and under the control of steroid hormones in the oviduct. To investigate which DNA elements are involved in the control of its expression in macrophages we performed transient DNA transfer experiments with two different types of plasmids: 5'-deletion mutants of the upstream region of the chicken lysozyme gene and different fragments from this area in front of the thymidine kinase promoter (herpes simplex virus), each placed in front of the CAT (chloramphenicol acetyl transferase) coding sequence. Two enhancers (E-2.7 kb and E-0.2 kb) were characterized. They are active in macrophages, but not in chicken fibroblasts. Furthermore a negative element (N-2.4 kb) was identified, which is active in fibroblasts and promyelocytes, but not in mature macrophages. The combined action of all three elements contributes to the observed lysozyme gene activities: no activity in fibroblasts, moderate activity in promyelocytes and high activity in mature macrophages.     

Identification of a sequence containing the positive regulatory region of Saccharomyces cerevisiae gene ENO1

A DNA fragment which contains the 5'-flanking region of the Saccharomyces cerevisiae enolase 1 gene (ENO1) and a portion of the coding sequence was cloned in a plasmid pMC1587. This fragment was fused in frame to the lacZ gene of Escherichia coli. Many mutants which deleted a portion of the 5'-flanking region of ENO1 were isolated from this ENO1-lacZ fusion plasmid by in vitro recombination. Analysis of beta-galactosidase activity of these mutants indicated that the regulatory region responsible for an efficient expression of the ENO1-lacZ fused gene resides within an 86-bp sequence located at -487 to -402 upstream from the start codon of ENO1. We found that the segment encompassing the 86-bp region worked equally well in an inverted orientation, but the tandem duplication of the sequence did not enhance the expression of the fused gene.     

CACCC and GATA-1 sequences make the constitutively expressed alpha-globin gene erythroid-responsive in mouse erythroleukemia cells.

Although the human alpha-globin and beta-globin genes are co-regulated in adult life, they achieve the same end by very different mechanisms. For example, a transfected beta-globin gene is expressed in an inducible manner in mouse erythroleukemia (MEL) cells while a transfected alpha-globin gene is constitutively expressed at a high level in induced and uninduced MEL cells. Interestingly, when the alpha-globin gene is transferred into MEL cells as part of human chromosome 16, it is appropriately expressed in an inducible manner. We explored the basis for the lack of erythroid-responsiveness of the proximal regulatory elements of the human alpha-globin gene. Since the alpha-globin gene is the only functional human globin gene that lacks CACCC and GATA-1 motifs, we asked whether their addition to the alpha-globin promoter would make the gene erythroid-responsive in MEL cells. The addition of each of these binding sites to the alpha-globin promoter separately did not result in inducibility in MEL cells. However, when both sites were added together, the alpha-globin gene became inducible in MEL cells. This suggests that erythroid non-responsiveness of the alpha-globin gene results from the lack of erythroid binding sites and is not necessarily a function of the constitutively active, GC rich promoter.     

Secretion of foreign proteins mediated by chicken lysozyme gene regulatory sequences.

Exploitation of the insulating properties of the complete chicken lysozyme gene domain may facilitate the production of transgenic chicken bioreactors with the capacity to deposit valuable proteins in the egg white. Chimeric genes consisting of the chicken lysozyme gene regulatory sequences and sequences encoding foreign proteins could be inserted randomly into the chicken genome and retain appropriate expression levels. The research reported here established that chicken lysozyme gene regulatory sequences can be used to direct the production and secretion of green fluorescent protein (used as a reporter protein) in transiently transfected chicken blastodermal cells. Attempts to verify these findings in transgenic hens are currently in progress. To provide a rapid means of generating constructs encoding other foreign proteins under the control of lysozyme gene regulatory sequences that can facilitate the secretion of heterologous proteins in vivo, a generic lysozyme gene regulatory scaffold was created using a poxvirus-mediated gene targeting system.     

Effect of constitutively expressed phoP gene on the localization of Salmonella typhimurium within Mac-1 positive phagocytes

Intracellular localization of the wild-type (Spv+), the phoP-constitutively expressed strain (PhoPc), and the spv-deleted strain (Spv-) of Salmonella typhimurium was examined by the use of confocal laser scanning microscopy analysis of immunostained sections of mouse spleens after oral or subcutaneous inoculation. Only 40% of salmonellae of both the PhoPc and the Spv- strains were detected intracellularly within Mac-1 positive cells at day five after oral or day four after subcutaneous inoculation. In contrast, over 85% of salmonellae of the Spv+ strain were detected inside Mac-1 positive cells. In both inoculation trials, the splenic colony-forming unit values for the PhoPc and Spv- strains were significantly lower than the corresponding value for the Spv+ strain. These findings suggest that the constitutively expressed phoP gene of S. typhimurium attenuated virulence by limiting intracellular proliferation within mouse spleen phagocytes, and that the lack of spv genes had the same effect.