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

A lambda EMBL3 rat genomic library was screened to clone a phage that conta ined the promoter region of the kidney-type mitochondrial glutaminase gene. The resulting lambda GA1 phage contained 13.7 kb of genomic DNA that was m apped by Southern blotting and restriction analysis. The 2.22 kb and 0.83 k b SacI fragments of lambda GA1 were sequenced and the transcription initiat ion site was identified by RNase mapping. The reported sequence contains 22 87 bp of the promoter, the entire exon 1 (542 bp), and 223 bp of the initia l intron of the glutaminase gene. The initial exon contains 141 bp of 5'-no ntranslated sequence and 401 bp of coding sequence that encodes the 72-amin o acid mitochondrial targeting presequence and 61 amino acids from the N-te rminus of the mature 66 kDa glutaminase subunit. Various segments of the GA promoter were cloned into a chloramphenicol acetyltransferase (CAT) expres sion vector. The resulting GA-CAT constructs were transfected into LLC-PK1- F+ kidney cells to assess the promoter function of the isolated genomic DNA . The GA(-402)CAT construct produced a 10-fold greater CAT activity than th e promoter-less pCAT vector. Analysis of various deletion constructs indica ted 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 a nd two Sp1 sites. (C) 2001 Elsevier Science B.V. All rights reserved.