Sequence Determination and Bioinformatics Analyses of Collagenase (MMP1) Domains Cloned from Primate Genomic DNAs

The compelling evidence for the role of metalloproteases (MMP) in many physiological and pathological processes, such as cell-cell and cell-matrix adhesion and signaling, has made them attractive targets for therapeutic interventions.  We are interested in comparing the collagenase (MMP1) sequences comprising the fourth and fifth exons as well as the fourth intron in order to gain insights into the complex evolutionary relationships of this protease gene family in primates.  We hypothesize greater conservation between exons than introns in closely related primates.  Genomic DNAs isolated from Ateles geoffroyi and Pongo pygmeaus were PCR amplified using designed collagenase specific primers.  Collagenase PCR products were ligated to plasmid cloning vectors, transformed into Escherichia coli competent cells, and cultured.  Plasmid DNAs were subsequently purified, digested with restriction endonucleases, and visualized using agarose gel electrophoresis.  Following DNA sequence determination, Bioinformatics analyses were performed on two exons and three introns of the MMP1 segment cloned from A. geoffroyi and one exon and one intron of the MMP1 segment cloned from P. pygmaeaus.  Based upon preliminary results, there is a close evolutionary relationship of these two analyzed collagenase domains with those of previously characterized primate species including Callithrix jacchus, Macaca mulata, Nomascus leucogenys, Pan troglodytes, Pongo abelii, and humans.  Experiments in progress include the determination of the intron-exon structural boundaries, statistical analyses of sequence differences between species, and sequence submissions to the GenBank database.  We thank the Biology Department and the RISE (GM59429-13) and Honors Programs at UPR-Cayey.