Proc. Natl. Acad. Sci. USA Vol. 83, pp. 7633-7637, October 1986 Biochemistry Isolation and characterization of the cDNA for murine granulocyte colony-stimulating factor (murine fibrosarcoma NFSA cells/cDNA sequence/protein homology/growth factor/differentiation) MASAYUKI TSUCHIYA, SHIGETAKA ASANO, YOSHITo KAZIRO, AND SHIGEKAZU NAGATA Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan Communicated by Charles Yanofsky, June 26, 1986 ABSTRACT A cDNA sequence coding for murine granu- In this report, we describe the isolation of the murine locyte colony-stimulating factor (G-CSF) has been isolated G-CSF cDNA from a recombinant X phage library prepared from a cDNA library prepared with mRNA derived from from the murine fibrosarcoma NFSA cell line, which pro- murine fibrosarcoma NFSA cells, which produce G-CSF con- duces G-CSF constitutively. The murine G-CSF cDNA was stitutively. Identification of murine G-CSF cDNA was based on identified by using cross-hybridization with human G-CSF the cross-hybridization with human G-CSF cDNA under a cDNA under a low-stringency condition. The cDNA was low-stringency condition. The cDNA can encode a polypeptide expressed in monkey COS cells under the simian virus 40 consisting of a 30-amino acid signal sequence, followed by a (SV40) early promoter. The protein produced by COS cells mature G-CSF sequence of 178 amino acids with a calculated had an ability to stimulate the granulocyte colony formation Mr of 19,061. The nucleotide sequence and the deduced amino in bone marrow cells and to support the proliferation of acid sequence of murine G-CSF cDNA were 69.3% and 72.6% murine NFS-60 myeloid leukemic cells. homologous, respectively, to the corresponding sequences of human G-CSF cDNA. The murine G-CSF cDNA, when intro- MATERIALS AND METHODS duced into monkey COS cells under the simian virus 40 promoter, could direct the synthesis of a protein that can Cell Lines and Isolation ofmRNA. The murine fibrosarcoma stimulate the granulocyte colony formation from mouse bone cell line NFSA was kindly provided by Mikio Shikita (Na- marrow cells and support the proliferation of murine NFS-60 tional Institute of Radiological Sciences). The cells were myeloid leukemia cells. grown in Dulbecco's minimal essential medium (Nissui Seiyaku, Tokyo). Total cellular RNA was extracted from about 4 x 107 cells by the guanidine thiocyanate method (11), Colony stimulating factors (CSFs) have been identified as and poly(A)+ RNA was selected by oligo(dT)-cellulose col- factors that can allow proliferation and differentiation of umn chromatography (12). hematopoietic progenitor cells from bone marrow on semi- Construction of the cDNA Library. Double-stranded DNA solid culture systems (1, 2). In the murine system, four complementary to NFSA mRNA was synthesized as de- different CSFs-i.e., granulocyte-macrophage CSF (GM- scribed (13), methylated with EcoRI methylase (New En- CSF), granulocyte CSF (G-CSF), macrophage CSF (M- gland Biolabs), and ligated with the EcoRI linker. The CSF), and interleukin 3 (IL-3)-have been highly purified double-stranded DNA was then digested with EcoRI and and characterized (2), and the gene structures for two ofthem size-fractionated on 1.2% agarose gel (Low Gel Tempura- (GM-CSF, IL-3) have been determined (3-5). ture, Bio-Rad). DNA ranging from 1,200 to 1,800 bp was Among these CSFs, murine G-CSF has been purified recovered, and ligated with an EcoRI-digested Xgtl0 vector initially from the medium conditioned by lung cells from mice (14). The hybrid DNA was packaged in vitro, and a cDNA injected with bacterial endotoxin (6). The purified murine library consisting of 1.0 x 106 plaque-forming units (pfu) was G-CSF has a Mr of 24,000-25,000 (6) and is distinguished constructed on Escherichia coli C600 high-frequency from other CSFs by its ability to stimulate exclusively lysogeny cells. neutrophilic granulocyte colony formation from bone mar- Hybridization and DNA Sequence Analysis. Blot (15) and row cells and to induce the terminal differentiation ofmyeloid plaque hybridization (16) were carried out as described (17) leukemia cells such as WEHI-3B D+ in vitro (6). except that the hybridization temperature was lowered to Recently, we (7, 8) and others (9) have reported the 37°C and the filter was washed at 42°C in 15 mM NaCl/1.5 isolation and expression ofthe cDNA for human G-CSF from mM sodium citrate, pH 7.0/0.1% NaDodSO4. The nucleotide cDNA libraries constructed with mRNA prepared from sequence of cDNA was determined by the chain-termination method after subcloning into M13 phage derivatives (18). human carcinoma cells that produce G-CSF constitutively. It Transfection of COS Cells and in Vitro Colony-Formation was found that there are two different G-CSF mRNAs Assay. COS cells (2 x 106) in 10 ml of Dulbecco's minimal (G-CSFa and G-CSFb mRNAs) for human G-CSF (8). G- essential medium were transfected with 20 ,Ag of plasmid CSFa and G-CSFb mRNAs can code for polypeptides con- DNA. At 72 hr after transfection, the medium was collected sisting of 207 and 204 amino acids, respectively, both of and assayed for G-CSF activity as described (19). In brief, 5 which are functionally active. Although G-CSF has no x 104 bone marrow cells from a C3H/He mouse were apparent species specificity between the murine and human cultured with or without 0.1 ml of test sample in 1 ml of system (10), the availability of the purified murine G-CSF by McCoy's 5A medium containing 40% horse serum and 0.3% recombinant DNA technology might prove valuable in the agar. After incubation at 37°C in humidified 5% CO2 in air for study of the in vivo and in vitro functions of G-CSF in the 5 days, colonies consisting of >50 cells were counted. murine model system. Abbreviations: CSF, colony-stimulating factor; G-CSF, M-CSF, and The publication costs of this article were defrayed in part by page charge GM-CSF, CSFs that can stimulate colony formation ofgranulocytes, payment. This article must therefore be hereby marked "advertisement" macrophages, and granulocytes/macrophages, respectively; IL-3, in accordance with 18 U.S.C. §1734 solely to indicate this fact. interleukin 3; SV40, simian virus 40; bp, base pairs. 7633 Downloaded by guest on September 29, 2021 7634 Biochemistry: Tsuchiya et al. Proc. Natl. Acad. Sci. USA 83 (1986) Cell-Proliferation Assay. The NFS-60 cell line (20) obtained \1 1 2 3 from James N. Ihie (National Cancer Institute-Frederick Cancer Research Facility, Frederick, MD) was routinely ( WI' - maintained in RPMI 1640 medium (GIBCO) containing 10% fetal calf serum (Microbiological Associates), 50 puM 2- mercaptoethanol, and 4% ofthe COS cells' supernatant (a gift from Ken-ichi Arai, DNAX Institute) transfected with mouse 4.9 3 4. 92' - IL-3 expression plasmid (3) or 10 ng of the purified human 3. - 0 G-CSF per ml (19). The proliferation assay was carried out in 96-well microtiter plates as described (21) with a minor modification. Samples to be examined were serially diluted 1:2 in 50 tkl ofRPMI medium and mixed with 50 ,ul ofNFS-60 cells (1 x 106 cells per ml), which had been washed exten- sively with the medium without IL-3 or G-CSF. The cultures were incubated for 24 hr at 370C, after which 0.25 ACi (1 Ci = 37 GBq) of [3H]thymidine (specific activity, 2 Ci/mmol; Amersham) was added per well and further incubated for 6 hr at 37TC. The cells were subsequently harvested with an automated cell harvester unit (Titertek, Flow Laboratories) onto filter paper and were assayed for [3H]thymidine incor- poration. FIG. 1. Blot-hybridization analysis of mRNA derived from the RESULTS human CHU-2 and murine NFSA cell lines. Poly(A)+ RNAs were electrophoresed through 1.2% agarose gel containing formaldehyde Presence of mRNA Homologous to Human G-CSF mRNA in and blotted onto a nitrocellulose filter. The EcoRI fragment ofhuman Murine Fibrosarcoma NFSA Cells. Murine fibrosarcoma G-CSF cDNA (pBRG-4 in ref. 7) was labeled by nick-translation (15) NFSA cells constitutively produce proteins having CSF with [32P]dATP (specific activity, 3,000 Ci/mmol) and was hybrid- activity (22). CSF proteins produced by NFSA cells were ized as described. Lanes: 1, 2 ug of poly(A)+ RNA from human separated into two fractions. One stimulates colony forma- CHU-2 cells; 2 and 3, 5 and 10 ,ug of poly(A)+ RNA from murine tion of mainly granulocytes and is active on human as well as NFSA cells, respectively; M, size markers (32P-labeled DNA frag- mouse bone marrow cells, while the other stimulates colony ments) run in parallel. Sizes are given in kb. Ori, origin of formation of macrophages from only mouse bone marrow electrophoresis. cells (23). Since murine G-CSF is known to work on human cells (10), the former fraction was thought to be the murine eukaryote mRNA (24), the first ATG codon was tentatively equivalent of human G-CSF. To examine this possibility, assigned as the initiation codon. The initiation codon is mRNA from NFSA cells was analyzed by blot hybridization followed by 207 codons before a TAG termination codon at using human G-CSF cDNA as probe. Under a low-stringency nucleotide positions 692-694 is encountered. The 3' noncod- hybridization condition, a single band of about 1.5 kb could ing region of 669 nucleotides contains the AATAAA be detected in mRNA from NFSA cells with human G-CSF polyadenylylation signal (25) at positions 1,346-1,351, al- cDNA (pBRG-4 cDNA in ref.