Characterization of the 5' flanking region of rat glucokinase gene

The rat glucokinase (GK) gene containing the first exon was isolated and its 5' flanking region was characterized by the bacterial chloramphenicol acetyltransferase (CAT) assay. A transient expression assay with a series of 5' deletion constructs (-5.5 k to -48) of GK-CAT fusion genes indicated that the 5' flanking sequence up to nucleotide -87 was sufficient for promoter activity in adult rat hepatocytes, but its activity was much weaker than that of the SV40 enhancer/promoter. Similar promoter activity was also detected in dRLh-84 hepatoma cells, which do not express glucokinase. Insulin treatment caused no change in the CAT activity of hepatocytes transfected with the fusion genes. These results suggest that the 5' flanking region of the glucokinase gene up to -5.5 k does not contain enhancer elements responsible for tissue-specific expression or insulin regulation.     

Neuronal expression of enhanced green fluorescent protein directed by 5' flanking sequences of the rat aldolase C gene in transgenic mice

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a combination of distal and proximal 5' flanking sequences, the A+C+0.8 kilobase (kb) pairs fragments, ensured high brain-specific expression in vivo (Skala et al. 1998). We show here that the expression pattern conferred by these sequences, when placed in front of the chloramphenicol acetyltransferase (CAT) or the enhanced green fluorescent protein (EGFP) reporter genes in transgenic mice, is similar to the distribution of the endogenous mRNA and protein. Double immunostaining for neuronal or glial cell-specific markers and for the EGFP protein indicates that the A+C+0.8 kb genomic sequences from the rat aldolase C gene direct a predominant expression in neuronal cells of adult brain.     

Polymorphism within the 3' flanking region of the bovine growth hormone receptor gene

Growth hormone receptor (GHR) has a major role in the regulation of growth hormone action, and thus, is an obvious candidate gene associated with milk production traits in mammals. The present authors have sequenced 273 bp of the 3' flanking region of the bovine GHR , and found three length variants and one base substitution polymorphism in this region. Allele frequencies of the length variants differ between Finnish native and commercial dairy cattle breeds. The chromosomal localization of GHR was confirmed to bovine chromosome 20 by synteny mapping and linkage analysis.     

Glucocorticoids regulate peptidyl-glycine alpha-amidating monooxygenase gene expression in the rat hypothalamic paraventricular nucleus

Peptidyl-glycine alpha-amidating monooxygenase (PAM) is a posttranslational processing enzyme which catalyzes the formation of biologically active alpha-amidated peptides. The two major neuropeptides involved in the regulation of ACTH secretion [CRF and arginine vasopressin (AVP)], synthesized in the parvocellular part of the hypothalamic paraventricular nucleus (PVN), are amidated, and their synthesis and/or release is negatively regulated by glucocorticoids. In this study, using in situ hybridization, we have shown that PAM mRNA is abundantly expressed in the hypothalamic paraventricular and supraoptic nucleus. Surgical adrenalectomy (ADX) induced increases in PAM, CRF, and AVP mRNA in the parvocellular part of the PVN, while corticosterone treatment normalized these values. PAM and AVP gene expression were not changed in the magnocellular part of the PVN or in the supraoptic nucleus. These observations suggest that in addition to stimulation of CRF and AVP synthesis, ADX induces an increase in PAM synthesis in the PVN and, thus, support the hypothesis of increased secretion of both CRF and AVP after ADX.     

Isolation of a cytochrome P-450e gene variant and characterization of its 5' flanking sequences

A cytochrome P-450e gene variant has been isolated from the rat liver genomic library. It is a typical e gene clone but unique in having b-like single base substitutions at specific sites in the 5' flanking region. It also appears to have certain additional restriction sites in the introns. When compared with the cytochrome P-450b gene, the e gene has some of the repetitive motifs interrupted in the 5' flanking region. In addition, this region is characterized by the presence of alternating pyrimidine-purine stretch, steroid hormone regulatory elements, consensus eukaryotic enhancer sequence and sequences involved in general amino acid regulation.     

High-level tuber expression and sucrose inducibility of a potato Sus4 sucrose synthase gene require 5' and 3' flanking sequences and the leader intron.

The 3.6 kb of 5' flanking sequence, leader intron, and 0.7 kb of 3' sequence from the potato sucrose synthase gene Sus4-16 are sufficient to direct high-level expression in developing tubers, in basal tissues of axillary buds and shoots, and in meristems and caps of roots, and to confer sucrose inducibility in leaves. By examining a series of deletion and substitution constructs in transgenic potato plants, we found that this pattern of expression requires 5' flanking sequences both upstream and downstream of position -1500 and that sequences between positions -1500 and -267 are essential for sucrose induction. Replacement of the native 3' sequence with the nopaline synthase 3' sequence resulted in the loss of sucrose inducibility and of expression in basal tissues of axillary buds. A general decrease in expression in other tissues was also observed. Removal of the 1612-bp leader intron also had a dramatic effect on both the pattern and level of expression.     

Regulation of human interleukin-2 gene: functional DNA sequences in the 5' flanking region for the gene expression in activated T lymphocytes

Interleukin-2 (IL-2) is a lymphokine that plays a crucial role in the immune system, especially in the growth control of T lymphocytes. Expression of this lymphokine is restricted to activated T lymphocytes. Here we demonstrate the presence of unique DNA sequences in the 5' flanking region of the human IL-2 gene that control induced T-cell-specific gene expression. We also show that the DNA sequences function in an orientation-independent manner and activate a heterologous promoter which is otherwise inert in induced T cells. The DNA, which spans about 200 bp, contains regions with sequence homology to LTR sequences of HTLV-III (or LAV) and the 5' upstream region of the IL-2 receptor and interferon-gamma genes.     

Regulation of rat growth hormone receptor gene expression

A cDNA encoding the growth hormone (GH) receptor was cloned from rat liver. Both the nucleotide and translated amino acid sequence share greater than 70% similarity with the GH receptors from rabbit and human. An RNA probe was generated from this sequence for use in a solution hybridization assay to quantitate GH receptor mRNA expression in rat tissues. Expression was detected in 9/12 tissues examined, with the highest levels observed in the liver. Expression in liver, kidney, heart and muscle was developmentally regulated, being low at birth and rising to adult levels in 5 weeks. No difference was observed between hepatic expression in males and females, although livers from pregnant rats had elevated levels. Hypophysectomy and GH treatment did not affect hepatic GH receptor mRNA levels.