A 6.1 kb 5' upstream region of the mouse tryptophan hydroxylase gene directs expression of E. coli lacZ to major serotonergic brain regions and pineal gland in transgenic mice

Tryptophan hydroxylase (TPH) catalyzes the first step of serotonin biosynthesis in serotonergic neurons and neuroendocrine cells. Serotonin influences diverse vital physiological functions and is thought to play an important role in several human psychiatric disorders. To localize DNA element(s) important for serotonergic tissue-specific expression of TPH, 6.1 kb of the 5' flanking region of the mouse TPH gene was fused to the coding region of the E. coli lacZ gene, and expression of the resulting fusion gene was analyzed in transgenic mice. The 6.1 kb of 5' flanking sequence was able to direct the expression of a lacZ reporter gene to serotonergic tissues in six lines of transgenic mice. A high level of lacZ expression in transgenic mice carrying the fusion gene was detected in the pineal gland as well as a moderate level of lacZ expression in serotonergic brain regions such as the median and dorsal raphe nuclei, the nuclei raphe magnus and raphe pallidus. In contrast, a smaller 5' flanking sequence of 1.1 kb directed no detectable serotonergic tissue-specific lacZ expression in five lines of transgenic mice. These results presented in this paper suggest first that DNA elements critical to serotonergic tissue-specific expression reside between -6.1 kb and -1.1 kb of 5' flanking region of the mouse TPH gene, but second that this region confers a restricted tissue-specific expression.     

Expression of rat bone sialoprotein promoter in transgenic mice

Bone sialoprotein (BSP) is a major protein of the mineralized bone extracellular matrix that has been implicated in the nucleation of hydroxyapatite crystals. Our previous studies have demonstrated that BSP mRNA is expressed by differentiated osteoblasts, odontoblasts, and cementoblasts involved in de novo mineralized tissue formation in a tissue-specific and developmentally regulated manner. To determine the basis of the selective expression of the BSP gene, we have generated four transgenic mouse lines in which 2.7 kb of the rat BSP promoter ligated to a luciferase reporter gene has been stably integrated into the mouse genome. Assays of luciferase activities in 5-day-old animals has revealed consistently high levels in bone tissues with negligible activities in various other organs including kidney, liver, stomach, intestine, and spleen. In some animals, variable expression was observed in brain and skin. Temporal analyses revealed the highest luciferase expression in neonatal bones, with expression decreasing markedly with subsequent growth and development, as observed previously for the endogenous gene in rats. Immunohistochemical analysis of luciferase activity and in situ hybridization of luciferase mRNA in bone tissues show that differentiated osteoblasts express the highest levels of luciferase, consistent with the induction of endogenous gene expression. These studies demonstrate that the regulation of the BSP gene during osteoblastic differentiation, together with its tissue-specific, developmentally regulated expression, is primarily mediated within the 2.7 kb region of the promoter.     

Tissue-specific activation of the osmotin gene by ABA, C2H4 and NaCl involves the same promoter region

The gene encoding the antifungal protein osmotin is induced by several hormonal and environmental signals. In this study, tissue-specific and inducer-mediated expression of the reporter gene beta-glucuronidase (uidA) fused to different fragment lengths of the osmotin promoter was evaluated in transgenic tobacco (Nicotiana tabacum). The region of the promoter between -248 to -108 (Fragment A) was found to be essential and sufficient for inducer (abscisic acid (ABA), C2H4 and NaCl)-mediated expression of the reporter gene. Expression of the reporter gene was developmentally regulated and increased with maturity of leaves, stem and flowers. Expression also was tissue-specific being most highly expressed in epidermis and vascular parenchyma of the stem. The regulators ABA, C2H4 and NaCl exhibited tissue-specific induction of this promoter. The promoter was specifically responsive to C2H4 in flowers at virtually all stages of development, but not responsive in these tissues to ABA or NaCl. Conversely, ABA and NaCl were able to induce reporter gene activity using promoter Fragment A in specific tissues of root where C2H4 was unable to induce activity. Further dissection of the promoter Fragment A into fragments containing either the conserved GCC element (PR); PR/AT; or G/AT sequences, and subsequent testing of these fragments fused to GUS in transgenic plants was performed. These experiments revealed that the promoter fragment containing PR element alone, although required, was barely able to allow responsiveness to C2H4. However, significant C2H4-induced activity was obtained with a promoter fragment containing the AT and PR elements together.     

Neuroblast pattern formation: regulatory DNA that confers the vnd/NK-2 homeobox gene pattern on a reporter gene in transgenic lines of Drosophila

DNA fragments -0.57, -2.2, -2.9, -5.3, and -8.4 kb in length from the upstream regulatory region of the vnd/NK-2 gene were cloned in the 5'-flanking region of a beta-galactosidase (beta-gal) reporter gene in the P-element pCaSpeR-AUG-beta-gal, and the effects of the DNA on the pattern and time of expression of beta-gal were determined in transgenic embryos. Embryos from 11 lines transformed with -8.4 kb of vnd/NK-2 regulatory DNA expressed beta-gal patterns that closely resemble those of vnd/NK-2. In embryos from four lines transformed with -5.3 kb of vnd/NK-2 DNA, beta-gal was found in the normal vnd/NK-2 pattern in the nerve cord but not in part of the cephalic region. beta-Gal patterns in embryos from transgenic lines containing -0.57, -2.2, or -2.9 kb of vnd/NK-2 DNA did not resemble vnd/NK-2. Null vnd/NK-2 mutant embryos containing the homozygous P-element p[-8.4 to +0.34 beta-gal] expressed little beta-gal in contrast to siblings with a wild-type vnd/NK-2 gene. We conclude that (i) the 8.4-kb DNA fragment from the vnd/NK-2 gene contains the nucleotide sequences required to generate the normal pattern of vnd/NK-2 gene expression, sequences that may be involved in the switch between neuroblast vs. epidermoblast pathways of development, (ii) the 5'-flanking region of the vnd/NK-2 gene between -5.3 and -8. 4 kb is required for vnd/NK-2 gene expression in the most dorsoanterior part of the cephalic region, and (iii) vnd/NK-2 protein is required, directly or indirectly, for maintenance of vnd/NK-2 gene expression.     

Glucocorticoid receptor-immunoreactivity in corticotrophin-releasing factor afferents to the locus coeruleus

Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using beta-galactosidase (LacZ) as a reporter gene. A 3,402-bp segment from the 5'-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5'-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.     

Myelin proteolipid protein intron 1 sequences do not appear to enhance myelin proteolipid protein gene transcription

Sequences from the first intron of the mouse myelin proteolipid protein (PLP) gene were examined for their ability to modulate PLP gene expression. Glial (N20.1) or nonglial (NIH 3T3) cells were transiently transfected with constructs that contained 1.4 kb of PLP promoter sequence driving luciferase reporter gene expression, as well as various portions of PLP intron 1 DNA. Although these same PLP intron 1 fragments enhanced reporter gene expression from a heterologous basal promoter in a previous study, the results reported here demonstrate that they do not augment PLP promoter activity. Thus, the regulation of PLP cell-type-specific expression, conferred by the first intron, appears to be mediated by an enhancer-independent mechanism.