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In the small intestine and in HTC hepatoma cells, the gamma-glutamyl transpeptidase (GGT) single-copy gene is transcribed into a 2.5 kb and a 2.2 kb mRNA. Cloning of the GGT cDNA sequences from HTC cells demonstrates that the 2.5 kb mRNA (mRNA(IV-1)) differs from the other rat GGT transcripts by a 371-base unique leader sequence which maps in the gene as 2 separate exons upstream of the 3 promoters which have been previously characterized. We established that the transcription of these two mRNAs is initiated on a new promoter (promoter IV) and occurs in the small intestine, in the epididymis, and in some hepatoma cells. The primary transcript initiated on GGT promoter IV is then alternatively spliced into the 2.5 kb mRNA(IV-1) or the 2.2 kb mRNA(IV-2) which is shorter in its 5'-untranslated sequence. The rat GGT gene exhibits a complex transcriptional organization leading to the transcription of five mRNAs from four independent promoters in a tissue-specific manner. The expression of the GGT promoter IV in the HTC hepatoma cells as well as in the small intestine could reveal that the HTC-transformed cells originate from liver precursor cells which still have the capacity to evolve toward different lineages. Thus, the GGT promoter IV will be valuable to isolate factors involved in the differentiation and carcinogenic processes.

In rat, gamma-glutamyl transpeptidase (GGT) is encoded by multiple mRNAs (mRNAI, mRNAII, mRNAIII, and mRNAIV) that differ only in their 5' untranslated regions and are transcribed from a single-copy gene. Using oligonucleotides designed from the 5' untranslated sequences of the GGT mRNAII and mRNAIII, we amplified a 3.4-kb genomic sequence which contains the promoter region for mRNAII. The sequence flanking the two initiation start sites for mRNAII contains consensus motifs for several potential regulatory proteins and a TATA-like element at the expected position 26 bp upstream from the predominant start site. The sequence from positions -528 to +72 associated with the chloramphenicol acetyltransferase (CAT) reporter gene drives a promoter activity in LLC-PK1, a pig kidney cell line. Deletion analysis revealed that the region from nucleotides -528 to -322 mediates an activation of the promoter activity, whereas the sequence from -322 to -114 has a negative effect. Furthermore, the structural organization of the 5' end of the GGT gene reveals that the GGT mRNAIII is transcribed from a third promoter located upstream from the promoter II on the GGT gene. By Northern blot analysis, the promoter II was found to be expressed only in the kidney and in the epididymis. We also identified two new mRNA species which are expressed in the H5 hepatoma cells. Therefore, the GGT gene expression reveals a strong tissue- or cell-specific pattern which is based on the transcription of several mRNA species from multiple promoters.

Two gamma-glutamyl transpeptidase mRNAs (mRNAI and mRNAII), with alternate 5'-untranslated regions, are expressed in the rat kidney. Oligonucleotides were designed based upon these two alternate 5' sequences and used as primers to amplify GGT genomic DNA sequences. The genomic organization of the mRNAI and mRNAII 5'-untranslated sequences reveals that the mRNAs are encoded from two separate promoters which are 2.1 kbp apart on the single GGT gene. A 2775 base pair genomic sequence, which contains the proximal GGT promoter, was cloned from two overlapping amplified fragments. S1 mapping analysis shows that the kidney GGT mRNAI is transcribed from several start sites on this promoter which displays neither a classical TATA box nor Sp1 binding sites. Chimeric plasmids, including the GGT promoter region for mRNAI, associated with the chloramphenicol acetyltransferase (CAT) reporter gene, were transiently expressed in a kidney (LLCPK) and in a hepatoma (HTC) cell line. A sequence extending 308 bases upstream from the major GGT mRNAI start site drives a promoter activity which is 5-fold higher in LLCPK than in HTC cells and is sufficient to confer cell specificity to the GGT proximal promoter.