Identification and isolation of a BTB-POZ-containing gene expressed in oocytes and early embryos of the zebrafish Danio rerio.

In this report, we describe the cloning of a cDNA from the zebrafish Danio rerio encoding a protein containing a BTB-POZ domain closely resembling the BTBD1 and BTBD2 proteins previously identified in mammals. However, unlike other BTB-POZ-containing genes, expression of this gene in adults is most abundant in oocytes, where the RNA can be detected at all stages of oogenesis examined. The presence of the RNA persists through early cleavage, but is decreased significantly by gastrulation. Although the function of this gene has yet to be determined, its resemblance to the BTB-POZ family of genes coupled with its expression pattern suggests that it may have an important function in oogenesis and (or) early zebrafish development.

The affinity and activity of the multiple hormone response element in the proximal promoter of the human oxytocin gene.

In vivo there appears to be a marked association between oestrogen levels and the expression of the oxytocin (OT) gene in most tissues. Transfection and DNA-protein binding experiments using high levels of either oestrogen receptor (ER)alpha or ERbeta imply a direct interaction of these transcription factors with the multiple hormone response element (HRE) at approximately -160 from the transcription start site of the OT gene in most species. In an extensive set of experiments, we show, using both transfection and protein-DNA binding, that low to moderate amounts of either oestrogen receptor, while being able to interact directly with a classic oestrogen response element (ERE) fail to interact with the human OT -160 HRE. Instead, this element, similar to its bovine counterpart, has a high affinity for the orphan receptors steroidogenic factor 1 and chicken ovalbumin upstream promoter transcription factor. Second, the human and bovine OT promoter can be made artificially responsive towards oestrogen in a cotransfection system over-expressing ERalpha or ERbeta, but not in cells expressing natural levels of these steroid receptors. Interestingly, nuclear extracts from both ERalpha-positive MCF7 cells and ERalpha-negative MDA-MB231 cells both contain a transcription factor which binds specifically to both the hOT-HRE element and to a classic ERE, and which has orphan receptor-like binding properties rather than those of an oestrogen receptor. Together, these and other results suggest that oestrogen action in vivo on the OT gene in all species is more likely to involve a DNA-independent mechanism than classic direct interactions with dimeric oestrogen receptors.

The role of SF-1/Ad4BP in the control of the bovine gene for the steroidogenic acute regulatory (StAR) protein.

The bovine gene for the steroidogenic acute regulatory protein (StAR) was cloned and sequenced, including 2 kb of the upstream control region of the gene. The gene comprises seven exons arranged similarly to those of the human and mouse gene sequences. The sequence analysis identified three cis elements corresponding to the binding motif for the transcription factor SF-1/Ad4BP, at - 100, - 240 and - 1190 from the transcription start site. Electrophoretic mobility shift analysis (EMSA) using nuclear proteins from bovine corpus luteum and bovine adrenal as well as in vitro transcribed/translated SF-1/Ad4BP consistently showed that only the site at -1190 bound the transcription factor significantly. Very weak binding was detectable also at the - 240 site, but none at the -100 site. Heterologous transfection of StAR promoter deletion-reporter constructs into Hela cells cotransfected with an expression vector for bovine SF-1/Ad4BP, showed that this transcription factor can specifically act on the bovine StAR gene promoter, but preferentially in regions corresponding to the two proximal SF-1/Ad4BP elements at - 100 and - 240, though with only low relative effect. Furthermore, additional cotransfection of a construct expressing a constitutive protein kinase A catalytic subunit to mimic the effects of cAMP stimulation, led to a small SF-1/Ad4BP-dependent increase in reporter activity mediated only by the same proximal sites. Since the bovine StAR gene promoter does not appear to have a functional cAMP responsive element (CRE), either this effect is mediated in this system directly by SF-1/Ad4BP, or by other factors interacting with this transcription factor, but which do not involve CRE-mediated gene activation. Taken together, the results show that there is a discrepancy between the results of the EMSA experiments and those using transfection of promoter-reporter constructs, which needs to be resolved before a clear understanding of SF-1/Ad4BP-mediated regulation of the StAR gene is attained.