Constitutive and inducible expression of a transgene directed by heterologous promoters in a trout liver cell line

Activities of heterologous promoters and enhancers in cultured rainbow trout liver cells were examined employing the bacterial chloramphenicol acetyltransferase gene as the reporter. SV40 promoter-enhancer and Rous sarcoma virus long terminal repeat directed constitutive expression at high levels. Moloney murine leukemia virus long terminal repeat and SV40 promoter combined with Adenovirus type 2 enhancer were also constitutively expressed. Drosophila Hsp70 promoter was activated when the transformed cells were cultured at 25 degrees C, a higher temperature than the temperature normally used, in faithful response to heat shock.     

Sequence Requirements for Myosin Gene Expression and Regulation in Caenorhabditis Elegans

Four Caenorhabditis elegans genes encode muscle-type specific myosin heavy chain isoforms: myo-1 and myo-2 are expressed in the pharyngeal muscles; unc-54 and myo-3 are expressed in body wall muscles. We have used transformation-rescue and lacZ fusion assays to determine sequence requirements for regulated myosin gene expression during development. Multiple tissue-specific activation elements are present for all four genes. For each of the four genes, sequences upstream of the coding region are tissue-specific promoters, as shown by their ability to drive expression of a reporter gene (lacZ) in the appropriate muscle type. Each gene contains at least one additional tissue-specific regulatory element, as defined by the ability to enhance expression of a heterologous promoter in the appropriate muscle type. In rescue experiments with unc-54, two further requirements apparently independent of tissue specificity were found: sequences within the 3' non-coding region are essential for activity while an intron near the 5' end augments expression levels. The general intron stimulation is apparently independent of intron sequence, indicating a mechanistic effect of splicing. To further characterize the myosin gene promoters and to examine the types of enhancer sequences in the genome, we have initiated a screen of C. elegans genomic DNA for fragments capable of enhancing the myo-2 promoter. The properties of enhancers recovered from this screen suggest that the promoter is limited to muscle cells in its ability to respond to enhancers.     

Promoter and enhancer elements from the rat elastase I gene function independently of each other and of heterologous enhancers.

An elastase-human growth hormone (hGH) fusion gene containing 205 base pairs of elastase 5' flanking region is expressed exclusively in pancreatic acinar cells of transgenic mice. This paper shows that the promoter region (-72 to +8) and the enhancer (-205 to -73) function independently of each other. The elastase enhancer can activate the heterologous mouse metallothionein gene and the hGH gene promoters; conversely, enhancers from the thymocyte-specific murine leukemia virus MCF13 and the metal regulatory elements from the metallothionein gene can activate the elastase promoter in a variety of cell types. Combinations of immunoglobulin and elastase enhancers with a heterologous promoter and the hGH gene result in expression in all of the tissues predicted by the sum of each enhancer acting alone. Thus these enhancer elements act independently of each other, suggesting that they do not have silencing activity in cells in which they are normally inactive.     

Regulation of gene expression in preimplantation mouse embryos: effects of the zygotic clock and the first mitosis on promoter and enhancer activities

Previous studies have reported that promoters requiring enhancers for full activity in mammalian somatic cells also require enhancers when injected into mouse two-cell embryos, whereas the same promoters can be expressed just as efficiently in the absence of an enhancer when injected into arrested one-cell embryos. Experiments were designed to determine whether this phenomenon reflected normal developmental changes at the beginning of mammalian development, or simply differences in the physiological states of these cells under the experimental conditions employed. The activity of three different promoters that function in a wide variety of mammalian cells was measured both in embryos whose morphological development was arrested and in embryos that continued development in vitro. Expression of the injected gene was related to the onset of zygotic gene expression ("zygotic clock"), the phase of the cell proliferation cycle, the use of aphidicolin to arrest cell proliferation, and formation of two-cell embryos in vitro and in vivo. The results demonstrated that promoter activity was tightly linked to zygotic gene expression, while the need for enhancers to stimulate promoter activity depended only on formation of a two-cell embryo. These results further support the hypothesis that the first mitosis induces a general repression of promoters prior to initiation of zygotic gene expression that is relieved specifically by enhancers.     

Expression of reporter genes introduced by microinjection and electroporation in fish embryos and fry.

The technique for foreign gene transfer in fish is becoming a novel method for genetic engineers to produce useful transgenic fish as well as for experimental purposes. Our studies of transgenic fish have been focused on methods of gene transfer and regulatory elements for transgene expression. I describe the characteristics of 3 gene transfer methods we have established (i.e., microinjection into fertilized eggs, microinjection into oocytes, and electroporation). Also described is a series of experiments to estimate activities of several promoters and enhancers in a fish cell line. Finally, experiments to examine activities of the elements shown to be active in the cell line in fish embryos and fry are described. SV2, miw, and metallothioneine promoters of trout and mouse were shown to be active in these experiments.     

Porcine CD4 promoters and enhancers can direct foreign gene expression in human cells

Porcine CD4 proximal promoter and enhancer sequences were cloned and aligned with the corresponding human and murine sequences. The alignment showed nucleotide homology between porcine and human sequences was 62.4 % for the CD4 promoter and 56.6 % for the CD4 enhancer. The nucleotide homology between porcine and murine sequences was 42.5 % for the CD4 promoter and 25.4 % for the CD4 enhancer. The proximal enhancer and promoter regions of the CD4 gene from porcine, murine and human cells were compared for their ability to direct foreign gene expression in transiently transfected human cell lines. The results indicated the porcine CD4 promoters and enhancers could effectively direct expression of a foreign gene in human cells. The porcine promoter was equally efficient as CMV and EF-1α in directing gene expression.     

Analysis of the Maize Polyubiquitin-1 Promoter Heat Shock Elements and Generation of Promoter Variants with Modified Expression Characteristics

The maize polyubiquitin-1 (Ubi-1) promoter is one of a few select promoters used to express foreign genes in monocots, such that recombinant proteins can be produced at commercially viable levels. Modifying the activity, specificity and responsiveness of such promoters provides a means to achieve desired levels and patterns of expression of genes encoding target products. Ubi-1 is constitutively expressed but is further induced by heat shock. The promoter contains two overlapping sequences with similarity to defined heat shock elements and we show that these sequences are also present upstream of the Ubi-1 homologue isolated from teosinte. Both the maize and teosinte promoters can mediate a heat shock response in transgenic maize. We have dissected the overlapping maize Ubi-1 promoter heat shock elements and demonstrate that the 3' element is required to mediate a heat shock response. The Ubi-1 promoter is particularly active in tissues consisting of rapidly dividing cells, and within the seed it is strongly biased towards driving expression in the embryo. However, replacement of the heat shock elements with a trimer of a basic domain/leucine zipper factor binding site of a pea lectin promoter shifts the balance in seed expression towards the endosperm. The Ubi-1 variants described here differ in their overall activity in the seed, but they all show potential for driving high levels of heterologous gene expression in maize.     

Cytotoxicities of cytosine deaminase and herpes simplex virus thymidine kinase genes in melanoma cells are not affected by the promoter strength

The right choice of regulatory elements (promoters and enhancers) is essential for the preparation within cancer cells of the therapeutic genetic constructs aimed at the gene-programmed enzymatic transformation of non-toxic prodrugs into toxins. It is generally accepted that the efficiency of gene therapy constructions is dependent, in particular, on the strength of promoters regulating the expression of therapeutic genes. Using melanoma-specific promoters and gene enhancers of human melanoma inhibitory activity and mouse tyrosinase and therapeutic genes, namely, HSVtk encoding herpes simplex virus thymidine kinase and FCU1 encoding cytosine deaminase/uracil phosphoribosyltransferase hybrid protein gene, we demonstrated that the promoter strength was not critical for the development of cytotoxic effects. The cytotoxic activity of these genes was shown to be influenced by the concentration of the prodrug added.     

Enhancer and promoter element interactions dictate cyclic adenosine monophosphate mediated and cell-specific expression of the glycoprotein hormone alpha-gene

The glycoprotein hormone alpha-gene is preferentially expressed in placental cell lines, but it is also expressed in several other cell lines indicating that the differential activity of the alpha-gene regulatory elements in various cell types is more quantitative than qualitative. The 5'-flanking region of the alpha-gene contains several distinct DNA regulatory sequences including an upstream regulatory element [(URE) -181 to -150 base pairs (bp)] that stimulates basal expression and an 18 bp twice-repeated cAMP-responsive element [(CRE) -146 to -111 bp]. We constructed an array of fusion genes containing the URE and/or the CRE linked to different truncated promoters [alpha-gene, somatostatin (SRIF), glucagon, Simian Virus 40]. These constructions were transiently expressed in placental, fibroblast, or islet cell lines to identify regulatory sequences involved in cell-specific expression as well as interactions between the URE, the CRE, and different promoter elements. The URE, CRE, and alpha-promoter elements contribute approximately 3-, 6-, and 5-fold, respectively, to preferential expression in JEG-3 cells. In JEG-3 cells, the URE is strictly dependent on the CRE for activity, but it functions in a promoter-independent manner. In contrast, the CRE is markedly promoter dependent. When linked to heterologous enhancers, the alpha-promoter is more active in JEG-3 cells than in other cell lines, thereby contributing substantially to preferential expression in placental cells. Although the CREs derived from the alpha and SRIF genes both activate expression of the alpha promoter, only the alpha CRE activates the SRIF promoter in JEG-3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)