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Nucleic Acids Research, 1994, Vol. 22, No. 7 1194-1201
© 1994

MOLECULAR BIOLOGY

Identification of positive and negative transcriptional regulatory elements of the rabbit angiotensin-converting enzyme gene

Tauqir Y. Goraya1,2, Sean P. Kessler1, Ravi S. Kumar1,+, Janice Douglas2 and Ganes C. Sen1,2,*

1Department of Molecular Biology, The Cleveland Clinic Foundation 9500 Euclid Avenue, Cleveland, OH 44195 2The Department of Physiology and Biophysics, Case Western Reserve University Cleveland, OH 44106, USA

*To whom correspondence should be addressed

Received December 29, 1993. Revised March 2, 1994. Accepted March 2, 1994.

The two tissue-specific mRNAs encoding the isozymes of rabbit angiotensin-converting enzyme (ACE) are generated from the same gene by alternative choice of two transcription initiation sites 5.7 kb apart. In the current study, we have characterized the regulatory sites controlling the transcription of the larger pulmonary isozyme mRNA. For this purpose, reporter genes driven by varying lengths of upstream region of the ACE gene were transfected into ACE-producIng cells. Our results demonstrated that the transcription of this gene is primarily driven by positive elements within the first 274 bp DNA upstream of the transcription initiation site. The reporter gene driven by this region was expressed in two ACE-producing cells but not in two ACE-non-producing cells thereby establishing its tissue specificity. Our experiments also revealed the existence of a strong negative element located between –692 and –610 positions. This element suppressed the expression of the reporter gene in a dose-dependent and position and orientation-independent fashion thus suggesting that it is a true silencer element. It could also repress the expression of a reporter gene driven by the heterologous strong promoter of the ß-actln gene. The repressing effects of the negative element could be partially overcome by cotransfectlng the isolated negative element along with the reporter gene containing the negative element. This result was possibly due to the functional removal of a limiting frans-actlng factor which binds to this element. Electrophoretlc mobility shift assays revealed that the negative element can form several complexes with proteins present in the nuclear extract of an ACE-produclng cell line. At least part of the negative element is strongly conserved In the upstream regions of the human and mouse ACE genes.

+ Present address: Oncogene Science 106 Charles Lindberg Blvd. Union Dale, NY 11553-3649, USA


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