Construction and characterization of a selectable multidrug resistance-glucocerebrosidase fusion gene.
Gene fusions can be employed to ensure concomitant expression of two different proteins under the same transcriptional control elements. We have synthesized a retroviral expression vector (pHaMG1) containing a human multidrug resistance (MDR1)-glucocerebrosidase (GC) chimeric gene inserted between the long terminal repeats of the Harvey murine sarcoma virus. When introduced into psi-CRE mouse fibroblasts, pHaMG1 conferred the drug-selectable multidrug resistance phenotype, and drug-resistant clones produced active human GC of about 60 kDa. Percoll gradient fractionation of homogenates prepared from transfectants confirmed correct targeting of P-glycoprotein to the plasma membrane and of GC to lysosomes. Although this construction was designed as a translational fusion of the MDR1 gene product, P-glycoprotein, and human GC, no evidence for a fusion protein was found in transfected cells, and an analysis of the RNAs transcribed from the integrated pHaMG1 retroviral vector suggests that either P-glycoprotein and GC are translated from one mRNA and rapidly processed into two proteins or they are translated separately from different mRNAs. These results reveal the feasibility of using fusion genes, which are smaller than alternative constructions with two promoters or with an internal ribosome entry site, for coexpression of selectable and nonselectable cDNAs in retroviral vectors.
Aran
J M
JM
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Germann
U A
UA
Gottesman
M M
MM
Pastan
I
I
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D000818:Animals; D019677:Artificial Gene Fusion; D001483:Base Sequence; D056668:Cathepsin L; D002403:Cathepsins / Q000096:biosynthesis; D002460:Cell Line; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D019450:Genes, MDR; D005962:Glucosylceramidase / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D051379:Mice; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011993:Recombinant Fusion Proteins; D005962:Glucosylceramidase; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; C534278:Ctsl protein, mouse