RESTRICTION SITES AS IDENTIFICATION TAGS FOR LYMPHOCYTE CDNAS

A cDNA library was prepared from BW 5147 murine lymphoma cells in lamb da ece III phage and randomly partitioned into 291 sectors, each with 800-1000 recombinant phage plaques. One sector was chosen for further characterization in terms of sensitivity to restriction endonuclease c utting. Aliquots of DNA preparations from this sector were treated wit h XhoI, SmaI, NcoI, PvuII, PstI, HindIII, EcoRI, BamHI, and ApaLI befo re being used as templates in a cell-free expression system. The polyp eptide products were separated by two-dimensional (2-D) gel electropho resis and radiofluorographs of the gels were submitted to computer-aid ed image analysis. The matched patterns were inspected for the presenc e or absence of spots upon individual endonuclease treatments. Thereaf ter the results were integrated in a data matrix which served as a bas is to construct ''restriction tags'' for all spots. These (restriction ) tags are binary numbers termed ''cut numbers'' and are a representat ion of the set of recognition sequences which are (or are not) part of the coding sequence. From 493 sequences (visualized as 2-D gel spots) , 12 were not cut by any of the nine enzymes, while 45 were cut by all of them. The percentages of sequences resistant to enzyme treatment r anged between 17% and 77% for NcoI and XhoI, respectively. The enzyme treatments led to the appearance of a certain portion of ''new spots'' , probably products from truncated sequences. From 512 possible cut nu mbers, 136 were assigned to the 493 spots. Restriction tags are availa ble to facilitate retrieval of cDNA clones from the (partitioned) cDNA library.