BMC Genomics 2007, 8:99 http://www.biomedcentral.com/1471-2164/8/99 comparative genomics in the identification of TFBS that are functional in gene expression. Methods Database analysis The annotated data of the mammalian genome sequence projects were accessed through the Ensembl genome server (e!42: Dec 2006) [30]. The exon-1 and 5'-upstream regulatory sequence of the hepatic lipase gene was availa- ble only for human (ENSG00000166035), rat (ENSRNOG00000015747), mouse (ENSMUSG00000032207), hedgehog (ENSETEG00000015177), chimpanzee (ENSPTRG00000007115) and rhesus macaque (ENSMMUG00000009566). Multiple sequence align- ment was performed with DNAMAN software package version 3.2 (Lynnon BioSoft, Quebec, Canada). Global sequence alignments were performed with the publicly available web-based tool mVista [12,31] using the MLA- GAN algorithm. A search for potential TFBS in the upstream regulatory region of a particular HL gene was performed online at Genomatix using the MatInspector program [2,32]. Clusters of TFBS that are conserved among the rat, mouse, human and macaque HL promoter regions were identified by the publicly available web-tool rVista [11,12,31]. Isolation of exon-1 and the 5'-flanking region of the rat HL gene A rat genomic library in λ DASH II (Stratagene, La Jolla, CA, USA) was used for isolation of the HL promoter region, using a HL cDNA probe corresponding to exons-1 and -2. The probe was generated by RT-PCR on 1 μg rat liver RNA using the oligonucleotides (5'-GGT AAG ACG AGA GAC ATG G-3', nt 1–19; numbering according to [33]) and (5'-CCC GTG GAT GAT CAT GAC AA-3', nt 285–266) as forward and reverse primers, respectively. The RT-PCR product was isolated by agarose gel electro- phoresis, and radiolabeled using [α32-P]dCTP and the Megaprime kit from Amersham (Amersham, UK). Filters containing 106 plaques were hybridized overnight at 42°C with 50 ng of the labeled cDNA probe in hybridiza- tion buffer (50 % (v/v) formamide, 0.5 % (w/v) SDS, 0.1 mg/ml denaturated herring sperm DNA and 2 × PIPES buffer; [34]). After washing in 0.2 × sodium chloride/ sodium citrate/0.5% SDS at 65°C for 5 min, the filters were exposed to autoradiography film. Two positive clones were identified, which were plaque-purified three times. One of these clones was selected for further analy- sis. Phage DNA was isolated and digested with EcoRI. A 6 kb fragment [35] was subcloned into pBluescript KS- (pBsE6) and its identity with the 5'-regulatory region of the rat HL gene was verified by sequence analysis. Construction of reporter plasmids The clone in pBluescript containing the 6 kb EcoRI frag- ment of the rat HL gene (pBsE6) was used to generate pro- moter-reporter constructs in pCAT-Basic (Promega, Madison, WI, USA). By digestion with PstI and XbaI, a 1.85-kb PstI/PstI, a 0.32-kb PstI/XbaI and a 0.15-kb XbaI/ XbaI fragment was isolated. First, the 0.32-kb PstI/XbaI (- 446/-127; numbering according to [35]) fragment was cloned into pCAT-Basic. From this construct, the rHL-446 CAT plasmid was generated by insertion of the 0.15-kb XbaI (-127/+9) fragment. Subsequently, rHL-2287 CAT was generated by insertion of the 1.85-kb PstI (-2287/- 446) fragment into rHL-446. The rHL-127 CAT construct was made by subcloning the 0.15-kb XbaI (-127/+9) frag- ment into pCAT-Basic. From the rHL-2287 CAT vector, the 5'-truncated rHL- 1697, rHL-1041 and rHL-747 constructs were generated by PCR using HindIII-restriction site-containing oligonu- cleotides 3F, 4F and 5F as upstream primer, respectively, and the CAT-gene specific oligonucleotide CATrev2 as downstream-primer (Table 2). After digestion of the PCR products with HindIII and PstI, the DNA fragments were purified by electrophoresis through agarose gel, and sub- sequently ligated into the rHL-446 CAT plasmid that had been digested with the same restriction enzymes. Simi- larly, the rHL-211 construct was generated from the rHL- 446 CAT by PCR using oligonucleotides 9F and CATrev2 as upstream and downstream primer, respectively, fol- lowed by ligation into the HindIII and XbaI sites of rHL- 446 CAT. Finally, the rHL-75, rHL-39 and rHL-23 con- structs were generated from pBsE6 using 7F, 6F and 11F as upstream, and T3Primer as downstream primer, respec- tively, followed by digestion and ligation into the HindIII and PstI sites of pCAT-Basic; subsequently, the resulting plasmids were digested with XbaI followed by self-liga- tion. Human HL promoter constructs were made in the pGL3- Basic luciferase reporter plasmid (Promega, Madison, WI, USA), starting from the hHL(-685/+13)-CAT plasmid described previously [36]. An upstream SacI restriction site was introduced by PCR using the HHL-685Sac primer (Table 2) and the downstream HHL+13Xba primer. After digestion with SacI and XbaI, the gel-purified DNA prod- ucts were ligated into the SacI and NheI sites of pGL3, thus generating the hHL-685Luc plasmid. Similarly, hHL- 306Luc, hHL-79Luc and hHL-36Luc plasmids were gener- ated by using HHL-306Nhe, HHL-79Kpn, and HHL- 36Kpn as upstream primers, respectively. To test the enhancer activity of conserved upstream sequences, the -14 kb, -22 kb and -10 kb elements were inserted into the enhancer site of the hHL-685Luc plasmid using the BamHI and SalI restriction sites. Human Page 11 of 13 (page number not for citation purposes)