PstI digestion also results in a 75 bp fragment not visible under standard conditions. Restriction digests of the clone give the following sizes (kb): EcoRI--3.0, 2.18; BamHI--4.55, 0.58; KpnI--uncut; HindIII--5.2; PstI--3.5, 1.08, 0.64. Insert encodes a 12 bp 5' untranslated region followed by a 1269 bp coding region. When the insert was cloned into pSVL and transfected into COS cells, acid phosphatase activity was expressed and 40% was membrane-associated. The EcoRI insert of pCT29-8 was inserted into pBEH and transfected into BHK cells. Studies of processing in this system suggest that the enzyme is synthesized and transported to lysosomes as a transmembrane protein with a short cytoplasmic tail. The insert detects a 2.3 kb mRNA in human liver and a 21 kb EcoRI fragment in human DNA. It detects a 5.0 kb EcoRI fragment in mouse DNA. |
