J Korean Soc Microbiol.  1998 Jun;33(3):295-306.

Isolation of a Partial Human cDNA Encoding a Factor Binding to the Perfect Palindrome of Enhancer A of HLA Class I Promoter, Homologous to NF-kB2

• Park JH
• Lee TJ
• Kim SJ

Abstract

Genes encoding sequence-specific DNA binding proteins have been isolated by screening cDNA libraries constructed in rgt11 expression vector with recognition site DNAs. We isolated a rgt11 recombinant human cDNA clone, designated to C2, using a DNA probe consisted of heptamer of the perfect palindrome (PP; GGGGATTCCCC) of enhancer A (Enh A) of HLA dass I promoter. Sequencing analysis showed that this clone contained a partial cDNA homologous to NF-kB2. Lysogenic E. coli containing the C2 was generated and crude cell extract was prepared. Immunoblot using anti-B-galactosidase antibody showed that this lysogenic E. coli expressed B-galactosidase fusion protein. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay were done using crude cell extract and their patterns were compared with nuclear protein extracted from an EBV transformed B lymphoblastoid cell line (BLCL). EMSA showed that crude cell extract prepared from E. coli lysogen speci5cally bound to the PP of Enh A region of HLA class I gene. DNase I footprinting assay showed that the binding sequence of this recombinant B-galactosidase fusion protein was identical to that of nuclear protein extracted from a BLCL. Our data indicate that a Agt11 recombinant cDNA clone was isolated from a human cDNA library using the PP of Enh A of the HLA class I promoter and this clone encoded a B-galactosidase fusion protein capable of binding to the PP and belongs to a NF-xB subunit.

MeSH Terms

Cell Line
Clone Cells
Deoxyribonuclease I
DNA
DNA, Complementary*
DNA-Binding Proteins
Electrophoretic Mobility Shift Assay
Gene Library
Genes, MHC Class I
Herpesvirus 4, Human
Humans*
Mass Screening
Nuclear Proteins
DNA
DNA, Complementary
DNA-Binding Proteins
Deoxyribonuclease I
Nuclear Proteins