Identification of regulatory elements in the human renin gene promoter has been hindered by the lack of suitable renin-expressing cell lines. In this paper, the SV40 viral enhancer is coupled to a human renin promoter/chloramphenicol acetyltransferase (CAT) reporter gene construct, to determine whether promoter regulatory elements can be identified in the context of enhanced transcription in the normally non-renin-expressing HeLa cell line. The present results indicate that the SV40 enhancer can overcome tissue-specificity of the human renin promoter, and confer correctly initiated transcriptional activity to HeLa cells. Analysis of a series of 5’-end deletions of the human renin promoter linked to the CAT gene and SV40 enhancer identified negative regulatory elements between positions-275 and -225, and between -142 and -102 of the human renin promoter, as well as a positive regulatory element between -225 and -142. Thus, studies of renin promoter regulatory elements need not be limited to renin-expressing cells, but can be performed in non-renin-expressing cells with the addition of an enhancer. This strategy can be generally applicable to the study of tissue-specific gene regulation in cases where there are no specific cell lines available.
In a series of studies to investigate the structural features of the biological crystals such as the tooth and bone, following the previous observations of the tooth crystal using an electron microscope, we examined the ultrastructure of the human bone crystals at near atomic resolution through the cross and longitudinal sections of the crystals.
The materials used for this study were the normal bone tissue obtained from the buccal alveolar compact bone of the human mandibule in the portion of the lower first molar. The small cubes of the bone tissue were fixed in glutaraldehyde and osmium tetroxide and embedded in epoxy resin using the routine methods. The ultrathin sections were cut with a diamond knife without decalcification. The sections were examined with the HITACHI H-800 type transmission electron microscope operated at 200 kV. Each crystal was observed at the initial magnification of 300,000 times and at the final magnification of 10,000,000 times and over.
Using this approach, we showed the configuration of the hydroxyapatite structure in the cross and longitudinal sections of the bone crystals deposited within and between the collagen fibrils (intrafibrillar and interfibrillar crystals) in the bone tissue.
Furthermore, using the same approach, we observed the crystal lattices of the hydroxyapatite structure appearing in the cross and longitudinal sections. We sincerely believe that the electron micrographs shown in this report are the first atomic images from the section obtained from the hydroxyapatite crystal from the human alveolar bone.