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.     

Expression of muscle creatine kinase gene of mice and interaction of nuclear proteins with MEF-2, E boxes and A/T-rich elements during aging

The changes in the expression of muscle creatine kinase (MCK) gene in the heart and skeletal muscle of mice during aging were studied. Its expression declines as a function of age in the heart, however, no age-related change is observed in the skeletal muscle. The cis-acting elements, MEF-2, E boxes and A/T rich elements present in the enhancer region of the mouse MCK gene are known to regulate the expression of the gene. Hence, these elements were subcloned and electrophoretic mobility shift assay was carried out to investigate the changes in the binding of the nuclear trans-acting protein factors of the heart with these elements as a function of age. These factors showed specificity for the respective cis-acting elements. Furthermore, the binding of these factors was found to decrease during aging which may contribute to the age-related decline in the expression of the MCK gene and activity of the heart.     

Conserved signals in the 5' flanking region of eukaryotic nuclear tRNA genes.

The statistical analysis of 5' flanking regions of eukaryotic tRNA genes was done. The analysis of nucleotides in the sequence of fungi and invertebrates showed a high content of A and T in the flanking regions versus coding regions where G and C dominate. In contrast to these results in vertebrates sequences the preferences of any nucleotide in flanking regions was not observed. The analysis of tetrads showed five conserved signals: TTGT, (T/A)(T/A)ATA, A(C/T)(C/A)A in the tRNA genes of fungi, (A/T)TGA of invertebrates and (A/T)GAG of vertebrates. The analysis of 3' flanking regions did not show any conserved signals except well known poly-T tracks.     

Identification of nuclear factors which interact with the 5' flanking region of the EF-1 alpha O gene in Xenopus laevis.

The EF-1 alpha O gene of Xenopus laevis is a stage-specific gene, being transcribed in oogonia and oocytes, but not in postmeiotic germ cells and terminally differentiated cells. We found that two trans-acting factors from oocyte nuclear extract are able to interact with a DNA sequence in the 5'-upstream region of the EF-1 alpha O gene. Methylation interference experiments suggested that the two factors recognised the same DNA element. Gel retardation assays indicated that part of the protein binding site could be confined to a 21 bp sequence, located between -51 and -72, relative to the cap site. Interestingly, this region shares great homology to a negative regulatory segment in the promoter of the TFIIIA gene, another developmentally regulated gene.     

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.