Regulation of ALF promoter activity in Xenopus oocytes.
BACKGROUND
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.


RESULTS
The 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.


CONCLUSIONS
Overall 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.
Li
Dan
D
Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX, USA.
Raza
Abbas
A
DeJong
Jeff
J
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D019175:DNA Methylation; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D014157:Transcription Factors / Q000235:genetics*; D029867:Xenopus Proteins / Q000235:genetics*; D014982:Xenopus laevis
D017931:DNA Primers; C485620:GTF2A1L protein, Xenopus; D014157:Transcription Factors; D029867:Xenopus Proteins
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