Expression cloning of genes for GPI-anchor biosynthesis.
Cloning genes for glycosylphosphatidylinositol (GPI)-anchor biosynthesis is important to further understand its mechanisms and regulation. We have been using expression cloning methods in which a cDNA library was transfected into GPI-anchor-deficient mutant cells. The transfectants which restored surface expression of GPI-anchored proteins were isolated and the plasmids were rescued. In this way we previously cloned cDNAs of genes for complementation classes A and F, and named them PIG-A and PIG-F, respectively. In the present study we have cloned the gene for class B, termed PIG-B. In each case we used different methods. For cloning PIG-A cDNA we used a cDNA library made with an Epstein-Barr-virus-based vector and human class A mutant JY5 which expresses EBNA-1 protein. The EBNA-1 protein allows stable replication of oriP-containing plasmids in the episomal form. For cloning PIG-F cDNA we chose a transient expression method and cotransfected a human T-cell cDNA library made with a vector bearing an origin of replication of polyoma virus with a plasmid bearing polyoma virus large T into the class F murine thymoma mutant. This cotransfection strategy was unsuccessful for cloning PIG-B due to low transfection efficiency of the class B thymoma mutant SIA-b. Thus, we first established large T-expressing SIA-b cells and then transfected them with a cDNA library. PIG-B cDNA restored the surface expression of Thy-1 on SIA-b cells and also synthesis of mature type GPI-anchor precursors in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Kinoshita
T
T
Department of Immunoregulation, Osaka University, Japan.
Takahashi
M
M
Inoue
N
N
Miyata
T
T
Takeda
J
J
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D015723:Gene Library; D005822:Genetic Vectors; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis* / Q000172:deficiency / Q000235:genetics; D006457:Hemoglobinuria, Paroxysmal / Q000235:genetics / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D009154:Mutation / Q000235:genetics; D014162:Transfection
D018076:DNA, Complementary; D017261:Glycosylphosphatidylinositols