Isolation and Characterization of a Novel Gene Up-Regulated by Granulocyte Colony-Stimulation Factor in a Murine Progenitor Cell Line • 1409

Abstract

Hematopoietic stem cells give rise to progenitor cells with progressively more restricted fates. This process is directed, in part, by a network of stimulating factors. Elucidation of the genetic events of hematopoiesis will provide insight into diseases of this system and novel strategies that may be employed to manipulate lineage commitment in a clinical setting. Granulocyte Colony-Stimulating Factor (G-CSF) is a hematopoietic growth factor which induces differentiation and maturation of the interleukin-3 (IL-3) dependent murine progenitor cell line 32D C13. In order to identify genes involved in commitment of myeloid progenitor cells to a differentiation program, 32D cells were withdrawn from IL-3 and cultured with G-CSF at a concentration of 400 U/ml. The cells were analyzed by napthol chloroesterase and myeloperoxidase staining at 24 hour time periods. At 96 hours after addition of G-CSF, 86% of the cells were napthol chloroesterase positive while 77% were myeloperoxidase positive. mRNA was extracted from a population of cells cultured for 96 hours with G-CSF, reverse transcribed to constitute the “tester” cDNA population, and used in a subtractive hybridization procedure with“driver” IL-3 cDNA. After two rounds of subtractive hybridization, the products were cloned into the Bluescript plasmid and screened for duplicates and true subtraction. DNA sequencing revealed the identity of a 164 base pair clone having 100% homology to the Tag-7 gene isolated in a differential display analysis of metastatic mouse tumors (Kustikova, et al; Genetika, 1996). Isolation of the full length cDNA sequence from a newborn murine brain library confirmed close homology to the Tag-7 gene. The deduced amino acid sequence encodes a novel protein that lacks known functional motifs. Both IL-3 and G-CSF induced 32D mRNA were probed for presence of the Tag-7 transcript by northern blot analysis. Signal quantitation showed a three-fold increase of Tag-7 message in the 96 hour G-CSF induced cells. Examination by RT-PCR indicated differential expression of Tag-7 in hematopoietic organs derived from adult and newborn mice. The authors conclude that the Tag-7 gene plays some yet undefined regulatory role in cell proliferation and differentiation. Further efforts are underway to isolate a corresponding human gene and to define the role of the Tag-7 gene in hematopoiesis.