Isolation and characterization of the promoter region of the rat kidney-type glutaminase gene

Abstract

A λEMBL3 rat genomic library was screened to clone a phage that contained the promoter region of the kidney-type mitochondrial glutaminase gene. The resulting λGA1 phage contained 13.7 kb of genomic DNA that was mapped by Southern blotting and restriction analysis. The 2.22 kb and 0.83 kb SacI fragments of λGA1 were sequenced and the transcription initiation site was identified by RNase mapping. The reported sequence contains 2287 bp of the promoter, the entire exon 1 (542 bp), and 223 bp of the initial intron of the glutaminase gene. The initial exon contains 141 bp of 5′-nontranslated sequence and 401 bp of coding sequence that encodes the 72-amino acid mitochondrial targeting presequence and 61 amino acids from the N-terminus of the mature 66 kDa glutaminase subunit. Various segments of the GA promoter were cloned into a chloramphenicol acetyltransferase (CAT) expression vector. The resulting GA-CAT constructs were transfected into LLC-PK 1-F + kidney cells to assess the promoter function of the isolated genomic DNA. The GA −402CAT construct produced a 10-fold greater CAT activity than the promoter-less pCAT vector. Analysis of various deletion constructs indicated that elements located between −402 and −63 bp must act in synergy with more proximal elements to create a functional promoter. The initial 402 bp segment lacks a TATA sequence but is GC-rich and contains two CCAAT boxes and two Sp1 sites.