Cloning and sequence analysis of the gene and cDNA encoding mouse spermidine/spermine N1-acetyltransferase — a gene uniquely regulated by polyamines and their analogs

Abstract

The polyamine catabolizing enzyme, spermidine/spermine N 1-acetyltransferase (SSAT), has been implicated as a critical determinant of polyamine pool maintenance. SSAT has recently been shown to be positively regulated in human cell lines by polyamines and their analogs at the level of mRNA accumulation. Mouse LA-4 lung adenoma cells treated with either spermine or the spermine analog, N 1, N 12-bis(ethyl)spermine, produced a 2.3 and 6.5-fold increase, respectively, in SSAT mRNA. Prior evidence for transcriptional control of the enzyme prompted investigation of SSAT gene structure and its regulatory elements. The mouse SSAT gene was isolated as a 3650 bp EcoRI fragment from a λ-J1 Mus saxicola genomic library by hybridization with human SSAT cDNA. An additional 431 bp downstream from the 3′ EcoRI site were sequenced from a BamHI fragment (total gene sequence, 4066 bp). The gene contains six exons and five introns. Sequence analysis of the 774 bp of the 5′ non-coding region revealed the absence of TATAA or CCAAT sequence motifs and the presence of a number of binding motifs in the 5′ region of the gene with consensus binding sequences for transcription factors SP1, AP1, E2F, AP2, PEA-3 and others. The deduced amino acid sequence of the coding region differs from that of the human SSAT cDNA by five amino acids. The 527 bp of the 3′ non-coding region contains four possible polyadenylation signal sites of which only one displays a typical consensus sequence. A 940 bp SSAT cDNA was isolated from Mus domesticus (BALB-C) liver λgt11 cDNA library. It contains a 5′ untranslated region 89 bp in length and a 3′ untranslated region 376 bp in length. The amino acid sequence deduced from Mus domesticus differs from that of Mus saxicola by one amino acid, from the hamster cDNA, by four amino acids and from the human cDNA by six amino acids. Further elucidation of the structural features of the SSAT gene may reveal how it is positively regulated by polyamines and their analogs.