Current investigations are directed at cloning the gene coding for human leukoregulin, a 32-kD glycoprotein with the unique ability to induce permeability of tumor cell plasma membranes. mRNA from leukoregulin secreting human peripheral blood lymphocytes and a cDNA library constructed from the mRNA are being used to isolate the leukoregulin gene. Two gene cloning strategies are being pursued. In the first approach, mRNA is microinjected directly into Xenopus oocytes which are subsequently examined for expression of membrane-permeability activity. Approximately 4.5% of injected oocytes secrete membrane-permeability activity. The frequency and level of expression are increased approximately 10-fold following injection of size-fractionated mRNA isolated from sucrose- density gradients. In the second approach, the cDNA library has been probed with a 17 mer oligonucleotide consensus sequence probe which has been designed based upon highly conserved sequences in endogenous lectin genes. The rationale for this approach is that the properties of leukoregulin are consistent with the cytokine being an endogenous lectin. Probing of the cDNA library with a consensus endogenous lectin sequence probe, if homologous with a portion of the leukoregulin gene, could greatly facilitate identification and isolation of the gene. A colony from the cDNA library hybridizing with the oligonucleotide probe and containing a unique sequence of greater than 700 base pairs has been isolated. The cDNA insert has been hybridized to a human genomic DNA library and a 16-kb DNA genomic insert isolated. A 5-kb portion of the genomic insert has been biotinylated and mapped to band q24.2 of human chromosome 12 by fluorescent in situ hybridization. The size of the 63C1 gene is estimated to be 3 kb. Investigations are in progress to isolate a full-length cDNA sequence which in turn will be evaluated for leukoregulin activity in Xenopus oocytes.
