Sequence analysis of the 5' flanking sequence of ovine lipoprotein lipase (LPL).

Lipoprotein lipase is found in many species and Ussue types 11) and is the pivotal enzyme in controlling lipid flux between plasma triacylglycerol rich lipoprotein particles and the tissues. Because tissue LPL activities alter with the physiological state of an organism, it is of great commercial and economic importance, and an understanding of the way factors interact to regulate its expression may, eventually, allow the remote manipulation of deposition of tissue lipids in v i m The 5 flanking sequence of Human I2.31. Mouse 141 and Chicken [5] LPL have already been determined and although chicken, in general, shares a lower level of sequence homology with human and mouse there is a remarkable level of conservation of cisacting elements across the species. Therefore this study was devised to investigate the 5 flanking sequence of ovine LPL to investigate whether these control elements were also conserved in sheep, a ruminant, with a very different metabolism to the species already investigated. High molecular weight ovine genomic DNA was isolated, from liver, and used to construct a sheep genomic library. This library consisted of 1.2 million independent clones with an average insert size of 14Kbk4Kb. A 1.7% ovine LPL cDNA (R1660) had already been isolated and sequenced previously[6] and was used to screen approx. 200.000 plaques of the sheep genomic library. From this screen one putative plaque was isolated, purified to homogeneity and designated LPLl. During the sequencing of R1660 a fragment corresponding to 225bp of the 5 end of R1660 had been generated using Pst I. This fragment was cloned into pGEM 3Zf and was designated LPL225c. LPLl was characterised by single and double digests with a range of restriction enzymes (HindIII. Sacl. Kpnl. BamHI, EcoFU and PstI). I t was determined from these digests that LPLl contained an insert of approx. 18Kb. The above digests were transferred to a nylon membrane and probed with 32P-labelled LPL225c. A single low molecular weight band of approx. 700bp was observed in the Pst I digest. This fragment was then subcloned into a vector suitable for sequencing. The vector chosen was pGEM 3Zf. Ligations were performed, transformed into JM109 and recombinants selected by blue/white screening. Plasmids were extracted from recombinant colonies and digested with Pst I. Digests were separated on an agarose gel, the DNA was transferred to a nylon membrane and probed again with 32P labelled LPL225c. A positive clone was identified and designated LPL7OOg. LPL7OOg was used as the template for sequencing reactions using the M 1 3 universal forward and reverse sequencing primers. Using the sequence already obtained, oligonucleotides were synthesised to internal sites in LPL7OOg in an attempt to "walk" through the clone. The sequence obtained from this Pst I sub-clone of LPLl included part of exon 1 and in addition 430bp of the 5 flanking sequence of LPL. This sequence was searched for known cis-elements using FINDPA'ITERNS from the Genetics Computer Group (GCG) suite of programs using the transcription factors database as the dictionary of ciselements and additionally by REGULON the authors own programme. The results of this analysis is shown in Table 1.