Abstract
BackgroundIn this report we evaluate the use of Xenopus laevis oocytes as a matched germ cell system for characterizing the organization and transcriptional activity of a germ cell-specific X. laevis promoter.Principal FindingsThe promoter from the ALF transcription factor gene was cloned from X. laevis genomic DNA using a PCR-based genomic walking approach. The endogenous ALF gene was characterized by RACE and RT-PCR for transcription start site usage, and by sodium bisulfite sequencing to determine its methylation status in somatic and oocyte tissues. Homology between the X. laevis ALF promoter sequence and those from human, chimpanzee, macaque, mouse, rat, cow, pig, horse, dog, chicken and X. tropicalis was relatively low, making it difficult to use such comparisons to identify putative regulatory elements. However, microinjected promoter constructs were very active in oocytes and the minimal promoter could be narrowed by PCR-mediated deletion to a region as short as 63 base pairs. Additional experiments using a series of site-specific promoter mutants identified two cis-elements within the 63 base pair minimal promoter that were critical for activity. Both elements (A and B) were specifically recognized by proteins present in crude oocyte extracts based on oligonucleotide competition assays. The activity of promoter constructs in oocytes and in transfected somatic Xenopus XLK-WG kidney epithelial cells was quite different, indicating that the two cell types are not functionally equivalent and are not interchangeable as assay systems.ConclusionsOverall the results provide the first detailed characterization of the organization of a germ cell-specific Xenopus promoter and demonstrate the feasibility of using immature frog oocytes as an assay system for dissecting the biochemistry of germ cell gene regulation.
Highlights
The factors and mechanisms that control transcriptional regulation in spermatocytes and oocytes of higher organisms have not been as well characterized as those in somatic cells [1,2,3]. This is due in part to the fact that germ cells cannot be propagated in cell culture and because cell-free extracts from complex tissues such as the testis are composed of contaminating subpopulations of germ cells and somatic cells
Studies on the mechanisms of mammalian germ cell gene expression have tended to rely on somatic cell culture systems—where germ cell genes should normally be off—or on cell-free extracts derived from mixed somatic and germ cell populations from whole tissue sources
Identification of the Xenopus laevis ALF promoter Since the genome sequence of X. laevis is not available, the first step in this project was to isolate the promoter of the ALF gene from purified genomic DNA
Summary
The factors and mechanisms that control transcriptional regulation in spermatocytes and oocytes of higher organisms have not been as well characterized as those in somatic cells [1,2,3] This is due in part to the fact that germ cells cannot be propagated in cell culture and because cell-free extracts from complex tissues such as the testis are composed of contaminating subpopulations of germ cells and somatic cells. Studies on the mechanisms of mammalian germ cell gene expression have tended to rely on somatic cell culture systems—where germ cell genes should normally be off—or on cell-free extracts derived from mixed somatic and germ cell populations from whole tissue sources Despite these issues, many regulatory factors have been proposed as regulators of germ cell gene expression. In this report we evaluate the use of Xenopus laevis oocytes as a matched germ cell system for characterizing the organization and transcriptional activity of a germ cell-specific X. laevis promoter
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