In the present study, we have constructed a subtraction cDNA library to identify novel genes induced by IFN-γ in GM-CSF-derived bone marrow macrophages (mø). Mø were treated with 50 U/ml IFN-γ for 40, 70 and 140 min to induce expression of early genes regulated by IFN-γ, and the mø were pooled. Poly(A)+RNA was prepared from both unactivated and IFN-γ-stimulated mø, and cDNA libraries were constructed in λZAP. Genes expressed in common by both mø populations were removed by subtraction using biotin-avidin precipitation of hybrid complexes. Further selection was performed by differential screening using cDNA prepared from mRNA of unactivated mø as a probe, followed by colony hybridization to remove sister clones. Of 17 clones from which sequence information was obtained, two appeared to be identical with the murine genes, C10 (clone GM2B1) and Mac-2 (clone GM2C4) and an additional two clones had high similarity to human cDNAs encoding proteins of unknown function. cDNAs containing sequences which did not match published sequences were used to probe Northern blots prepared from both unstimulated and IFN-γ-activated GM-CSF- and CSF-1-derived mø. Five clones (GM1A2, GM1B4, GM1F2, GM2A12 and GM2B8) showed enhanced transcript levels following IFN-γ treatment of GM-CSF-derived mø, but demonstrated high constitutive transcript levels in CSF-1-derived mø. In addition, C10 transcripts were constitutively expressed by GM-CSF-derived mø, but not by CSF-1-derived mø, even after activation by IFN-γ. These data suggest that much of the functional heterogeneity of GM-CSF- and CSF-1-derived mø resides in the differential expression of early genes specifically induced by IFN-γ.
- subtraction cDNA library