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We have previously shown that transcription of the rat serine: pyruvate/alanine: glyoxylate aminotransferase (SPT/AGT) gene occurs from two start sites. cAMP-responsive transcription from the upstream start site (+1 bp) generates mRNA for the precursor of mitochondrial SPT/AGT containing a mitochondrial targeting N-terminal signal sequence. Transcription from the downstream site (+66 bp) generates mRNA which encodes peroxisomal SPT/ AGT (17). It is possible that the upstream and downstream transcription start sites are specified by different promoters, allowing different regulation of transcription from the two sites. In order to examine the existence of a proximal promoter associated with the downstream transcription initiation, transient expression analysis was carried out using HepG2 cells. A region of the SPT/AGT gene surrounding the downstream start site (+21 to + 106 bp) promoted the transcription of the fused chloramphenicol acetyltransferase (CAT) gene. Ligation of upstream sequences did not augment the promoter activity of the +21 to + 106 fragment, and the +64 to +106 fragment caused only very low expression of CAT, suggesting that a proximal promoter is located between +21 and +106 bp. The +21 to +106 region does not contain known promoter sequences, such as TATA, CCAAT and GC boxes, and does not exhibit sequence similarity to known classes of TATA-less promoters. There is a sequence which bears some similarity to the housekeeping initiator protein 1 (HIP 1) binding sequence, but gel shift competition analysis showed that nuclear protein binding to the HIP1 recognition sequence is not effectively competed for by the +21 to +106 fragment. Unexpectedly, the expression of the CAT plasmids bearing only the putative proximal promoter region was augmented by N6,2’-O-dibutyryl cAMP, although the mechanism and physiological significance of this enhancement have not been clear. These results altogether indicate that transcription from the downstream start site of the rat SPT/AGT gene is promoted by its own promoter which is still to be delimited and characterized further. 3Present address: Department of Internal Medicine, The tor protein 1 Branch Hospital, Nagoya University School of Medi*Since 'serine:pyruvate aminotransferase (SPT, EC cine, Higashiku, Nagoya 461, Japan 2.6.1.51) and alanine: glyoxylate aminotransferase—I 4T0 whom correspondence should be addressed (AGT, EC 2.6.1.44) are the same enzyme and both the Abbreviations.' SPT/AGT, serine:pyruvate/alanine: glyserinezpyruvate and alaninezglyoxylate aminotransfeoxylate aminotransferase; CAT, chloramphenicol acety1rase activities are believed to be of physiological importransferase; (Bt)2cAMP,N6,2’-O-dibutyry1cAMP, CRE, tance, this enzyme is called serine:pyruvate/alanine: cAMP-responsive element; HIP1, housekeeping initiaglyoxylate aminotransferase (SPT/AGT) in this paper. l

biomedical research

&lt;b&gt;CHARACTERIZATION OF TRANSCRIPTION FROM THE DOWNSTREAM START SITE OF THE RAT SERINE: PYRUVATE/ALANINE: GLYOXYLATE AMINOTRANSFERASE &lt;/b&gt;&lt;b&gt;GENE &lt;/b&gt;