Quantitative EEG in subcortical neglect

We have analyzed the regulatory roles of the first intron (intron-1) of the bovine beta-casein gene in the bovine beta-casein/CAT expression system using a mouse mammary epithelial cell line, HC11. After a combined treatment of HC11 cells with insulin, dexamethasone and prolactin, the induced expression of p beta c1.8/+ICAT vector including 2 kb intron-1 and 1.8 kb promoter was greatly increased to 23.5 folds, while that of p beta ca.8CAT basic vector with 1.8 kb promoter only, was 6.5. A classical enhancer activity was shown in the 2 kb intron fragment from the experiment in which the orientation and the position of the intron-1 on the vectors were changed. The enhancer activity was largely dependent on the lactogenic hormones, especially prolactin. A stepwise reduction of the inducibility in the 5' to 3' deletion analysis of the intron-1 indicates the existence of several functional elements in the region. In particular, an internal fragment (+1071 to +1490) was important for the prolactin-dependent enhancing activity of the intron-1. These results suggest that several elements in the intron-1 of the bovine beta-casein gene cooperatively interact not only with each other but also with its promoter for hormonal induction.     

Structural and functional characterization of the human alpha3 nicotinic subunit gene promoter

We describe the structural and functional features of the human alpha3 nicotinic receptor subunit promoter. A 0.35-kb region immediately upstream of the start codon was identified that when transfected in human neuroblastoma cells was able to drive the expression of the luciferase reporter gene with a strength comparable to that of the well-characterized simian virus 40 promoter/enhancer. This region displayed the features of a multistart-site, GC-rich, TATA-less, and CAAT-less promoter, containing many overlapping Sp1 and AP-2 putative binding sites. Further dissections of the 0.35-kb fragment revealed that its 3' region, specifying the 5' UT of the mRNA, plays a relevant positive effect in determining the strength of the promoter. This region contains putative cis-acting elements for AP-2, nuclear factor-kappaB, and the recently described multiple-start site element downstream-1. By mutation analysis, we showed that these sites are functional and when combined increase the promoter activity by 4-fold. The 0.35-kb promoter was found to be under the negative control of upstream sequences that include a modern Alu repeat. The alpha3 Alu repeat works as a composite region, containing both positive and negative elements that control the activity of the downstream promoter. Finally, we investigated the tissue-specific activity of the human alpha3 gene 5' regulatory sequences, showing that they are able to drive the expression of the reporter gene preferentially in neuronal cells.     

Multiple control elements are required for expression of the human CD34 gene

Two cis regulatory elements of the human CD34 gene, the promoter and a 3' enhancer, have previously been described. In transient transfection assays, the promoter was not sufficient to direct cell type specific expression. In contrast, the 3' enhancer was active only in CD34+ cell lines, suggesting that this element might be responsible for stem cell-restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive sites, we detected a strong CD34+ cell type-specific site in intron 4. This region, however, did not work as an enhancer by itself. By analyzing stable transfectants and transgenic animals, we demonstrated that the 3' enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression.     

Preattentive visual search and perceptual grouping in schizophrenia

Differentiated retinal pigment epithelial (RPE) cells in vivo express basal levels of FGF-5, a secreted member of the FGF gene family. RPE cells proliferate in response to pathological events, resulting in a transient increase in FGF-5 gene expression. The goal of this study is to identify cis-acting sequences in the FGF-5 gene promoter which upregulate FGF-5 gene expression when differentiated RPE cells enter the cell cycle and proliferate. In vitro cultures of RPE cells were transfected with various FGF-5 promoter/luciferase deletion constructs, using methods specifically optimized for proliferating and differentiated RPE cells. A proximal promoter/enhancer whose activity is not cell-context dependent was identified between FGF-5 sequences -314 and +48. In addition, a silencer element (-1256/-883) was identified in the distal region which is active only in differentiated RPE cells. When tested in a heterologous system, the same element had silencer activity in differentiated cells. Two small regions in the distal FGF-5 gene promoter, -1195/-1173 and -984/-967 were able to specifically bind to nuclear proteins from differentiated RPE cells but not from proliferating RPE cells as evidenced by gel mobility shift assays. Therefore, FGF-5 gene expression in the RPE may be regulated by the formation of differentiation-specific complexes.