The sulfatase gene family: cross-species PCR cloning using the MOPAC technique

Abstract

Several human sulfatase cDNAs have recently been cloned, revealing highly conserved domains of protein similarity. We have used this information for the isolation of sulfatase genes in different species using the polymerase chain reaction (PCR). Degenerate oligonucleotide primers corresponding to these regions of identity among human arylsulfatases A, B, and steroid sulfatase (ARSA, ARSB, and STS) were designed. The primers were used in the PCR amplification of reverse transcribed RNA (RT-PCR) from multiple tissues in human and mouse. Amplification products were obtained from mouse liver and from human liver, lymphoblasts, kidney, intestine, heart, muscle, and brain cDNA samples. Each of the PCR products was subcloned into a plasmid vector, and several subclones were characterized by colony hybridization and DNA sequencing. All the previously identified human ARSA, ARSB, and STS were found among our clones, indicating the power of the technique. Sequence analysis of two mouse clones showed high degrees of homology with the human ARSA and ARSB sequences, respectively, and likely represent the murine homologues of these enzymes. These are the first sulfatase genes isolated in the mouse. A murine equivalent for STS could not be identified, suggesting its strong diversity from the human homologue.