ABSTRACT
A gene coding for the Tac protein (interleukin-2 receptor alpha-subunit, IL-2R alpha) of the interleukin-2 receptor was constructed by chemoenzymatic gene synthesis. The gene designed for mutagenesis codes for a receptor protein where all 10 methionines are substituted by alanine, valine, leucine, and isoleucine. In addition, aspartate at position 6 is substituted by glutamate. This desmethionine IL-2R alpha and the wild-type IL-2R alpha genes were integrated into a eukaryotic expression vector and transferred into different cell lines. The recipient cell lines express both wild-type and mutant receptor proteins on their cell surfaces which are recognized equally by different monoclonal antibodies. It was possible to establish cell lines with high level IL-2R alpha chain expression by fluorescence-activated cell sorting. The wild-type IL-2R alpha expressed in LTK- cells is a glycoprotein with an apparent molecular size of about 60 kDa and a typical low interleukin-2 binding affinity of KD = 12 nM. Despite the fact that 11 amino acids are altered, no significant difference in the mutant IL-2R alpha is observed, exhibiting the same molecular size and a low interleukin-2 binding affinity of KD = 26 nM.
Subject(s)
Interleukin-2/metabolism , Receptors, Immunologic/genetics , Amino Acid Sequence , Base Sequence , Flow Cytometry , Methionine , Molecular Sequence Data , Molecular Weight , Mutation , Plasmids , Receptors, Immunologic/metabolism , Receptors, Interleukin-2 , TransfectionSubject(s)
DNA/isolation & purification , Egg Proteins/biosynthesis , Genes , Muramidase/biosynthesis , Ovomucin/biosynthesis , Protein Biosynthesis , Transcription, Genetic , Animals , Chickens , Cloning, Molecular , DNA/metabolism , DNA Restriction Enzymes , DNA, Recombinant/metabolism , Erythrocytes/metabolism , Microscopy, Electron , RNA, Messenger/metabolismABSTRACT
Hybrid phages that contain chicken lysozyme gene sequences have been isolated from a chicken DNA library. Two overlapping clones covering a region of 22 kilobase pairs around this gene have been studied by restriction mapping. Southern hybridization, and electron microscopic analysis of hybrids between lysozyme mRNA and the cloned cellular DNA. Three intervening sequences interrupt the lysozyme structural gene. The cellular gene is at least 3.9 kilobases long, about 6 times the length of the structural gene.
