Expression cloning of genes for GPI-anchor biosynthesis.

T. Kinoshita, M. Takahashi, N. Inoue, T. Miyata, J. Takeda

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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)

Original languageEnglish
Pages (from-to)127-132
Number of pages6
JournalBrazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.]
Volume27
Issue number2
Publication statusPublished - 1994 Feb

ASJC Scopus subject areas

  • Biophysics
  • Neuroscience(all)
  • Biochemistry
  • Physiology
  • Immunology
  • Pharmacology, Toxicology and Pharmaceutics(all)
  • Cell Biology

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