BMC Genomics 2007, 8:99 http://www.biomedcentral.com/1471-2164/8/99 FAilgigunrmee1nt of the 5'-flanking region of four mammalian HL genes Alignment of the 5'-flanking region of four mammalian HL genes . From the rat, mouse, macaque and human HL genes, exon-1 and 30 kb of upstream sequence was aligned by the MLAGAN algorithm of the mVista program. The sequences of rat (A), mouse (B) and macaque (C) are aligned to the human HL sequence (x-axis); numbering is relative to the transcrip- tional start site. Conserved regions (> 70% homology over 100 bp window) are shaded. The boxes indicate conserved regions among the four sequences, as determined by RankVista (P ≤ 10-5), with the P-values given above. ent from promoter-less pCAT-Basic, suggesting that there is weak enhancer activity between nucleotides -1697 and -2287. Further deletion to position -754 slightly increased promoter activity to levels significantly above back- ground. Shortening the insert from -754 to -446 resulted in a 5-fold increase in promoter activity, suggesting the presence of a strong negative regulatory element in this region of the rat HL gene. CAT expression was not signifi- cantly affected by deleting the insert from -446 to -211. The presence of the weak enhancer element between - 2287 and -1697, and the negative element between -754 and -446 corresponds to positive and negative elements in the human HL upstream regulatory region observed by Oka et al. [23]. We assume, therefore, that both these ele- ments are present in homologous parts of the rat and human gene. Indeed, the global alignment of the four spe- cies by mVista detected homology at these parts of the gene (Fig. 1), but homology did not exceed the 70 % over 100 bp mark used as threshold in this analysis. Appar- ently, potentially important elements may be missed due to the high stringency of the conservation rule in the mVista program. To test the importance of the conserved sequence mod- ules within the -220 to +9 region, further 5'-deletions in the rat HL promoter region were made (Fig. 5). Transcrip- tional activity of the rHL-127 construct, in which module A has been removed, was not significantly different from that of the rHL-446 or rHL-221 constructs. Similarly, removal of the highly conserved intervening sequence Page 4 of 13 (page number not for citation purposes)