Restriction digests of the clone give the following sizes (kb): EcoRI--5.2; HindIII--5.2; BamHI--3.2, 1.8; PstI--3.2, 1.8; SalI--5.2; EcoRI/KpnI--2.9, 2.1. If the plasmid is linearized with HindIII, and transcribed from the SP6 promoter in the presence of m7-GpppG, mRNA for the secreted form of placental alkaline phosphatase is produced. mRNA prepared from this construct can be used as an internal standard for expression of mRNA injected into Xenopus oocytes. Linkage has been established between Waardenburg syndrome type 1 and ALPP in five families (LOD score 4.76 at a recombination fraction of 0.023. Waldenburg syndrome shows close linkage to FN1 and ALPP in one multigenerational family with 11 affected individuals. The described RsaI polymorphism was established using a 1.1 kb EcoRI/ApaI fragment of the ALPP cDNA (ALP-1). Constructed from pBC12/PLAP containing the complete cDNA for the membrane-anchored form by inserting a termination codon after the codon for amino acid 489. The EcoRI/KpnI fragment was then cloned into the vector. |
