Identification of two positive transcriptional elements within the 91-base pair promoter for mouse testis angiotensin...

Testis angiotensin-converting enzyme (testis ACE) is an isozyme of ACE only expressed by male germ cells during spermiogenesis. It is the result of a strong sperm-specific promoter found within the 12th intron of the somatic ACE gene. Previous studies have localized the boundaries of the mouse testis ACE promoter as being from -91 to -9, relative to the transcriptional start site, and have suggested two important DNA regulatory elements starting at positions -55 and -32. DNA constructs were made in which these motifs were either eliminated or substituted. Each construct was tested for its ability to promote transcription in vitro, using a rat testis nuclear extract. Disruption of either motif reduced in vitro transcription to about 30% of control levels, while mutations of both elements abolished transcription. Two sites were selected inside each motif and altered by point mutation. Each of four constructs, containing a mutation at -51, -48, -30, or -28, transcribed at 29% or less the efficiency of the parent construct. The DNA element at -55, TGAGGTCA, is homologous to a consensus cyclic AMP response element. The motif at -32, TCTTAT, is located at a position analogous to a TATA box. Substitution of the -32 motif with a consensus TATA box sequence, TATAAA, stimulated transcriptional activity about 3-fold. As measured by gel mobility shift, oligonucleotides encompassing the -32 motif and the consensus TATA box formed different DNA-protein complexes. However, the -32 motif oligonucleotide was recognized by nuclear proteins prepared from either liver or testis nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)