Restriction sites as identification tags for lymphocyte cDNAs.
A cDNA library was prepared from BW 5147 murine lymphoma cells in lambda ecc 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 cutting. Aliquots of DNA preparations from this sector were treated with XhoI, SmaI, NcoI, PvuII, PstI, HindIII, EcoRI, BamHI, and ApaLI before being used as templates in a cell-free expression system. The polypeptide products were separated by two-dimensional (2-D) gel electrophoresis and radiofluorographs of the gels were submitted to computer-aided image analysis. The matched patterns were inspected for the presence or absence of spots upon individual endonuclease treatments. Thereafter the results were integrated in a data matrix which served as a basis to construct "restriction tags" for all spots. These (restriction) tags are binary numbers termed "cut numbers" and are a representation 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 ranged 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 numbers, 136 were assigned to the 493 spots. Restriction tags are available to facilitate retrieval of cDNA clones from the (partitioned) cDNA library.
Frey
J R
JR
Basel Institute for Immunology, Switzerland. frey@bii.ch
Fol
B
B
Lefkovits
I
I
D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D018076:DNA, Complementary / Q000097:blood*; D015180:Electrophoresis, Gel, Two-Dimensional; D015723:Gene Library; D007091:Image Processing, Computer-Assisted; D008214:Lymphocytes / Q000378:metabolism*; D051379:Mice; D015183:Restriction Mapping
D018076:DNA, Complementary; D004262:DNA Restriction Enzymes