GlcNAc phosphotransferase of the lysosomal targeting pathway

An isolated nucleic acid which hybridizes under stringent conditions to the complement of SEQ ID NO:5, wherein said stringent conditions comprise washing in 0.2×SSC and 0.1% SDS at 65° C., and wherein the nucleic acid encodes a biologically active γ-subunit of GlcNAc-phosphotransferase.

A method of producing a GlcNAc-phosphotransferase in a cell comprising

• transfecting into said cell a DNA construct comprising a targeting sequence homologous to a target site within or upstream of a endogenous GlcNAc-phosphotransferase gene contained in the cell, wherein said endogenous gene comprises the sequence in SEQ ID NO:4 or SEQ ID NO:5, an exogenous regulatory sequence, an exon, and an unpaired splice-donor site at the 3′ end of the exon, wherein said transfecting generates a homologously recombinant cell in which the splice-donor site is operatively linked to the second exon of the endogenous gene, and the exogenous regulatory sequence controls transcription of the construct driven, the endogenous gene, and any sequence lying between the construct-driven exon and the endogenous gene, to produce a RNA transcript that encodes the GlcNAc-phosphotransferase, so that the homologously recombinant cell produces the GlcNAc-phosphotransferase.

An isolated nucleic acid which encodes a protein comprising the amino acid sequence of SEQ ID NO:1.

An isolated nucleic acid which hybridizes under stringent conditions to the complement of nucleotides 165 to 2948 of SEQ ID NO:4, wherein said stringent conditions comprise washing in 0.2×SSC and 0.1% SDS at 65° C., and wherein the nucleic acid encodes a biologically active α-subunit of GlcNAc-phosphotransferase.

An isolated nucleic acid which encodes a protein comprising the amino acid sequence of SEQ ID NO:2.

An isolated nucleic acid which hybridizes under stringent conditions to the complement of nucleotide 2949 to 3932 of SEQ ID NO:4, wherein said stringent conditions comprise washing in 0.2×SSC and 0.1% SDS at 65° C., and wherein the nucleic acid encodes a biologically active β-subunit of GlcNAc-phosphotransferase.

An isolated nucleic acid which encodes a protein comprising the amino acid sequence of SEQ ID NO:3.

An isolated nucleic acid which hybridizes under stringent conditions to the complement of nucleotides 96 to 941 of SEQ ID NO:5, wherein said stringent conditions comprise washing in 0.2×SSC and 0.1% SDS at 65° C., and wherein the nucleic acid encodes a biologically active γ-subunit of GlcNAc-phosphotransferase.

A method of producing a GlcNAc-phosphotransferase in a cell comprising

• transfecting into said cell a DNA construct comprising a targeting sequence homologous to a target site within or upstream of a endogenous GlcNAc-phosphotransferase gene contained in the cell, wherein said endogenous gene comprises a sequence that hybridizes under stringent conditions to the complement of SEQ ID NO:4 or SEQ ID NO:5, which stringent conditions comprise washing in 0.2×SSC and 0.1% SDS at 65° C., an exogenous regulatory sequence, an exon, and an unpaired splice-donor site at the 3′ end of the exon, wherein said transfecting generates a homologously recombinant cell in which the splice-donor site is operatively linked to the second exon of the endogenous gene, and the exogenous regulatory sequence controls transcription of the construct driven, the endogenous gene, and any sequence lying between the construct-driven exon and the endogenous gene, to produce a RNA transcript that encodes the GlcNAc-phosphotransferase, so that the homologously recombinant cell produces the GlcNAc-phosphotransferase.