Promoter mapping of the mouse Tcp-10bt gene in transgenic mice identifies essential male germ cell regulatory sequences.
Transgenic mice were generated to localize essential promoter elements in the mouse testis-expressed Tcp-10 genes. These genes are expressed exclusively in male germ cells, and exhibit a diffuse range of transcriptional start sites, possibly due to the absence of a TATA box. A series of transgene constructs containing different amounts of 5' flanking DNA revealed that all sequences necessary for appropriate temporal and tissue-specific transcription of Tcp-10 reside between positions -1 to -973. All transgenic animals containing these sequences expressed a chimeric transgene at high levels, in a pattern that paralleled the endogenous genes. These experiments further defined a 227 bp fragment from -746 to -973 that was absolutely essential for expression. In a gel-shift assay, this 227-bp fragment bound nuclear protein from testis, but not other tissues, to yield two retarded bands. Sequence analysis of this fragment revealed a half-site for the AP-2 transcription factor recognition sequence. Gel shift assays using native or mutant oligonucleotides demonstrated that the putative AP-2 recognition sequence was essential for generating the retarded bands. Since the binding activity is testis-specific, but AP-2 expression is not exclusive to male germ cells, it is possible that transcription of Tcp-10 requires interaction between AP-2 and a germ cell-specific transcription factor.
Ewulonu
U K
UK
Jackson Laboratory, Bar Harbor, Maine 04609, USA.
Snyder
L
L
Silver
L M
LM
Schimenti
J C
JC
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus; D002874:Chromosome Mapping; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D005854:Germ Cells; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008869:Microtubule-Associated Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011506:Proteins; D012045:Regulatory Sequences, Nucleic Acid; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D044767:Ubiquitin-Protein Ligases; D056427:t-Complex Genome Region
D017931:DNA Primers; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D011506:Proteins; C533392:TCP10L3 protein, human; C533395:Tcp10 protein, mouse; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D004247:DNA; C116196:PPP1R11 protein, human; D044767:Ubiquitin-Protein Ligases