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 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 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 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)+ 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. 7) as a probe (Fig. 1). The size though the poly(A) tract is not included in this cDNA. ofthe hybridizing mRNA was slightly smaller than the human On the basis ofthe homology with the amino acid sequence G-CSF mRNA in human CHU-2 squamous carcinoma cells of human G-CSF (7), Val-1 was assigned to the NH2-terminal (7), which produce G-CSF constitutively. The result indi- amino acid of the mature, secreted murine G-CSF. The cates that NFSA cells synthesize an mRNA highly homolo- peptide containing amino acid residues -30 to -1 probably gous to human G-CSF mRNA. serves as a signal peptide for the secretion. The mature Identification of Murine G-CSF cDNA Clone. To isolate a murine G-CSF consists of 178 amino acids with a calculated cDNA clone containing the sequence coding for murine Mr of 19,061 and no potential N-glycosylation site (Asn-Xaa- G-CSF, a cDNA library was constructed with mRNA from murine NFSA cells. The double-stranded cDNA, ranging Thr) (26) is found. However, it is possible that murine G-CSF from 1.2 to 1.8 kb, was size-fractionated on an agarose gel is O-glycosylated because the natural G-CSF purified from electrophoresis, and a cDNA library was prepared with the the mouse lung-conditioned medium has a Mr of 24,000- XgtlO vector system (14). By screening 1 x 106 clones with 25,000 and has been thought to be glycosylated (6). Actually, human G-CSF cDNA, a total of 24 hybridizing clones was the common sequences for galactosamine attachment (27) obtained. Ten of the 24 clones were plaque-purified, and DNA was prepared from each clone. EcoRI digestion of the - I 0 -o _ _ _ z :z . recombinant DNAs released fragments of 1.3-1.8 kb, and ZZ :1 ;_: Z, Z, Qq CL. C. :z ll! Q, -,! : DNA from one clone having the =1.4-kb cDNA insert was I r subcloned to the EcoRI site of pBR327 (denoted as pMG2) L .L and further characterized by restriction analysis and nucleotide sequence determination. Nucleotide Sequence Analysis of Murine G-CSF cDNA. Fig. 2 shows the restriction map of pMG2 and the sequence 200 analysis strategy. The sequence was determined on both bp strands of the cDNA, crossing the restriction fragment The nucleotide is shown in FIG. 2. Restriction map of murine G-CSF cDNA (pMG2) and the junction. sequence (1,363 bp) Fig. strategy for nucleotide sequence determination. The untranslated 3, together with the deduced amino acid sequence. The sequences are represented by a line, while the coding sequences are pMG2 cDNA contains a single open reading frame containing boxed. The dark portion indicates the sequence coding for mature three potential initiation codons (nucleotide numbers 68-70, G-CSF protein, while the white region represents the sequence 95-97, and 104-106) like human G-CSF cDNA (7). Since the coding for the putative 30-residue signal sequence. Arrows show the nucleotide sequences surrounding the first methionine codon direction and the length of sequence determined by each independent are most optimal to the consensus sequence of higher- experiment. Downloaded by guest on September 29, 2021 Biochemistry: Tsuchiya et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7635

10 30 50 70 90 GTATAAAGGCCCCCTGGAGCTGGGCCCTGGCAGAGCCCAGAGCTGCAGCCCAGATCACCCAGAATCC ATG GCT CAA CTT TCT GCC CAG AGG Met Ala Gin Leu Ser Ala Gln Arg -30 110 130 150 170 CGC ATG AAG CTA ATG GCC CTG CAG CTG CTG CTG TGG CAA AGT GCA CTA TGG TCA GGA CGA GAG GCC GTT CCC CTG GTC ACT GTC AGC GCT Arg Met Lys Leu Met Ala Leu Gln Leu Leu Leu Trp Gin Ser Ala Leu Trp Ser Gly Arg Glu Ala Val Pro Leu Val Thr Val Ser Ala -20 -10 -1 1 190 210 230 250 270 CTG CCA CCA TCC CTG CCT CTG CCC CGA AGC TTC CTG CTT AAG TCC CTG GAG CAA GTG AGG AAG ATC CAG GCC AGC GGC TCG GTG CTG CTG Leu Pro Pro Ser Leu Pro Leu Pro Arg Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys lie Gln Ala Ser Gly Ser Val Leu Leu 10 20 30 290 310 330 350 GAG CAG TTG TGT GCC ACC TAC AAG CTG TGT CAC CCC GAG GAG CTG GTG TTG CTG GGC CAC TCT CTG GGG ATC CCG AAG GCT TCC CTG AGT Glu Gln Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Lys Ala Ser Leu Ser 40 50 60 370 390 410 430 450 GGC TGC TCT AGC CAG GCC CTG CAG CAG ACA CAG TGC CTA AGC CAG CTC CAC AGT GGG CTC TGC CTC TAC CAA GGT CTC CTG CAG GCT CTA Gly Cys Ser Ser Gln Ala Leu Gln Gin Thr Gln Cys Leu Ser Gin Leu His Ser Gly Leu Cys Leu Tyr Gln Gly Leu Leu Gin Ala Leu 70 80 90 470 490 510 530 TCG GGT ATT TCC CCT GCC CTG GCC CCC ACC TTG GAC TTG CTT CAG CTG GAT GTT GCC AAC TTT GCC ACC ACC ATC TGG CAG CAG ATG GAA Ser Gly Ile Ser Pro Ala Leu Ala Pro Thr Leu Asp Leu Leu Gln Leu Asp Val Ala Asn Phe Ala Thr Thr Ile Trp Gln Gln Met Glu 100 110 120 550 570 590 610 630 AAC CTA GGG GTG GCC CCT ACT GTG CAG CCC ACA CAG AGC GCC ATG CCA GCC TTC ACT TCT GCC TTC CAG CGC CGG GCA GGA GGT GTC CTG Asn Leu Gly Val Ala Pro Thr Val Gln Pro Thr Gln Ser Ala Met Pro Ala Phe Thr Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu 130 140 150 650 670 690 710 GCC ATT TCG TAC CTG CAG GGC TTC CTG GAG ACG GCT CGC CTT GCT CTG CAC CAC TTG GCC TAG ACCTGAGCAGAAAGCCCTTTCCAGATAGTTTATTT Ala Ile Ser Tyr Leu Gin Gly Phe Leu Glu Thr Ala Arg Leu Ala Leu His His Leu Ala End 160 170 750 770 790 810 830 ATCTCTATTTAATATTTATGCATATTTAAGCCTACTATTTAAAGACAAAGACGAGAAAATGGAGCTCTAAGCTTCTAGATCATTCTCTCCACTTCCGAGTTTTGTTCTCCTGCTTAGAG 870 890 910 930 950 CAGAGAGAGAAGGCTCTTGTGTCCTCCTGTGGAGGCCAGGGAAGGAGATGGGTAAATACCAAGTATTGATTCCTGCTGCTGCTCCAGGCACCCAGTTCTGTGGCAGTACCCCCAAAAAA 970 990 1010 1030 1050 1070 TCAGTGAGCCCTGCCGTGCTGAGGCACCATCTCAGGGGGGCCCAGGCAGCATCTGGTCTCCCTTCCGGGGGACAAGACATCCCTGTTTAATATTTAAACAGCAGTGTTCCCAAACTGGG 1090 1110 1130 1150 1170 1190 TTCTTATATCCCTTGCTCTGGTCAACCAGGTTGCAGGGTTTCCTGTCCTCACAGGAACGAAGTCCCTAAAGAAACAGTGGCAGCCAGGTTTAGCCCCGGAATTGACTGGATTCCTTTTT 1210 1230 1250 1270 1290 1310 TAGGGCCCTGCTGGCCTGGAAGTTGGAGTGGGGGGCAGAGGAGGCAGGAGGAAGCCTGGGGGGGGGGTTGGCATGGAGGGAGGCCTTCCCATCCACCCTCACCCTCCACCCCACCTGTC 1330 1350 ACTATAGCCAAGCTTGCGGATAATAAAGTGTGGTGTTCC

FIG. 3. Nucleotide sequence and the deduced amino acid sequence of pMG2 encoding murine G-CSF. Numbers above and below each line refer, respectively, to nucleotide position and amino acid position. Amino acids are numbered starting at Val-1 of the mature G-CSF sequence, which was postulated on the basis of homology with human G-CSF sequence (7, 8). The mature G-CSF sequence is preceded by a 30-residue putative signal sequence (-1 to -30).

occur at several points (for example, amino acid positions 7, NFS-60 cells. Rapidly growing NFS-60 cells were extensive- 12, and 102) of the murine G-CSF sequence. ly washed free ofendogenous factors, and the dependency of Expression of Murine G-CSF cDNA in Monkey COS Cells. [3H]thymidine incorporation on growth factors was evaluat- To prove that the cDNA insert of pMG2 can code for murine ed. NFS-60 cells were factor-dependent for proliferation, and G-CSF, we expressed the cDNA in the mammalian cell this requirement could be met by the human G-CSF purified system. The 745-bp Apa IlAha III fragment containing the from the conditioned medium of CHU-2 cells (19), human coding sequence for G-CSF (see Fig. 2) was recloned into the COS G-CSF, or murine COS G-CSF, but not by the super- expression vector pdKCR (28, 29), and the resultant plasmid natant of COS cells transfected with pdKCR vector plasmid (pMGD2) was used to transfect monkey COS cells. After 72 (Fig. 4). The half-maximal incorporation was obtained with hr, the protein secreted into the medium was assayed for G-CSF activity by using mouse bone marrow cells. The supernatant of COS cells transfected with either the murine Table 1. Colony-stimulating activity in the supernatant of or the human G-CSF expression plasmid (pMGD2 or COS cells pHGV21 in ref. 8) could stimulate colony formation, while Colonies formed, no. little CSF activity was detected in the supernatant of COS cells transfected with pdKCR vector plasmid DNA (Table 1). COS-cell Transfection vector Control The colonies stimulated by the COS cell supernatant were supernatant,,l pdKCR pMGD2 pHGV21 G-CSF Saline mainly neutrophilic granulocyte colonies, and little colonies 20 15, 15 74, 77 66, 90 85, 75 18, 10 consisting of either macrophages or eosinophilic granulo- 4 4, 20 72, 81 75, 76 cytes were observed. 0.8 12,17 54, 48 48, 49 Recently, Holmes et al. have established several IL-3- dependent myeloid leukemia cell lines from mouse spleen The supernatants of COS cells transfected with the indicated cells transfected with Cas-Br-M murine leukemia plasmid DNA were collected at 72 hr after transfection and aliquots virus (20). (20, 4, and 0.8 ,ul) were assayed for CSF activity as described in 1 ml Although most ofthese cell lines require murine IL-3 for their of the bone marrow culture in duplicate. As controls, the purified proliferation, NFS-60 cells are able to grow in response to the human G-CSF (10 ng) or saline was added to the assay mixture. purified murine G-CSF (30). We tested the ability of murine Numbers of colonies consisting of more than 50 cells were counted and human COS G-CSF to support the proliferation of on day 5. Downloaded by guest on September 29, 2021 7636 Biochemistry: Tsuchiya et al. Proc. Natl. Acad Sci. USA 83 (1986) cells. By screening the library with human G-CSF cDNA as 100 a hybridization probe, a full-length murine G-CSF cDNA clone was isolated (Fig. 3). The nucleotide sequence of C murine G-CSF cDNA exhibits a strong homology (69.3%) in 80)F A :8 both coding and noncoding regions with that of human 0 C. A G-CSF cDNA (7-9). The high degree of homology is strong ou 60 evidence that the cloned cDNA is the murine equivalent of )t human G-CSF cDNA. The murine cDNA could direct the ._c synthesis of a protein in COS cells, which has an authentic . 40 G-CSF activity (Table 1). A comparison of the deduced amino acid sequences of 'E human G-CSFa (7) and murine G-CSF is presented in Fig. 5. 20 The overall homology of the mature human and murine m, G-CSF polypeptides is =72.8%, which is much higher than the homology (50%) observed between human and murine 1 2 4 8 16 32 64 GM-CSF (31, 32). This result agrees with the observation that Dilution G-CSF has no apparent species specificity between human and murine systems (10), while GM-CSF shows species FIG. 4. Response of NFS-60 cells to G-CSF. NFS-60 cells were specificity (31, 33). Murine and human G-CSF proteins incubated for 24 hr with various doses of the purified human G-CSF contain five cysteine residues of which four residues are (o), the supernatants of COS cells transfected with human G-CSF found in the homologous positions (Fig. 5). There is evidence expression vector (pHGV21) (e), murine G-CSF expression vector that the murine G-CSF molecule contains internal disulfide (pMGD2) (A), or pdKCR vector (x). Cells were then pulsed with 0.25 bonds (6), suggesting this may also be the case with human GCi of [3H]thymidine for 6 hr, and the incorporated radioactivity was G-CSF. determined. [3H]Thymidine incorporation is expressed as a percent- The alignment of the amino acid sequence of murine age of the maximal incorporation (110,000 cpm). The incorporation without growth factors was 11,500 cpm. The purified human G-CSF G-CSF with that of human G-CSF has indicated that the was diluted starting from 2.5 ng/ml, while the supernatants of COS murine cDNA isolated in this report is the counterpart of cells were diluted starting from a 1:300 diluted sample (for human human G-CSFb cDNA, which has a nine-nucleotide (three COS-G-CSF) or a 1:200 diluted sample (for mouse COS-G-CSF). amino acids) deletion at the amino acid residue 35 of the mature G-CSFa polypeptide (Fig. 5; refs. 7 and 8). In the human system (8), a single chromosomal gene for G-CSF is 0.2 ng of the purified human G-CSF per ml (10 pM) which is interrupted by four introns. At the 5' end of the intron 2, two almost the same concentration obtained with the purified splice donor sequences are arranged in tandem, 9 bp apart, murine IL-3 (20). and two G-CSF mRNAs are generated by alternative use of the two splice donor sequences. Southern hybridization DISCUSSION analysis of DNA from mouse spleen cells with mouse G-CSF cDNA as probe has suggested the existence of a single gene Previously, the human CHU-2 carcinoma cell line from an for G-CSF in mouse haploid genome (M.T. and S.N., oral cavity tumor and the 5637 bladder carcinoma cell line unpublished results). Whether two different mRNAs exist were successfully used to isolate human G-CSF cDNA (7-9). also for murine G-CSF as in the human system remains to be In this report, NFSA fibrosarcoma cells were found to seen. synthesize murine G-CSF mRNA constitutively (Fig. 1), and Among many IL-3-dependent cell lines established from a cDNA library was constructed with mRNA from the NFSA mouse spleen cells transformed with Cas-Br-M murine leu-

-30 -20 -10 Mouse: Met Ala Gin Leu Ser Ala Gin Arg Arg Met Lys Leu Met Ala Leu Gin Leu Leu Leu Trp Gin Ser Aia Leu Trp Ser Gly Human: Met Ala Gly Pro Ala Thr Gin Ser Pro Met Lys Leu Met Ala Leu Gin Leu Leu Leu Trp His Ser Ala Leu Trp lhr Val -30 -20 -10 -1 1 10 20 Mouse: Arg Glu Ala Val Pro Leu Val Thr Val Ser Ala Leu Pro Pro Ser Leu Pro Leu Pro Arg Ser Phe Leu Leu Lys Ser Leu Human: Gin Glu Ala Thr Pro Leu ------Gly Pro Ala Ser --- Ser Leu Pro Gin Ser Phe Leu Leu Lys Cys Leu -1 I 10 30 40 Mouse: Glu Gin Val Arg Lys lie Gin Ala Ser Gly Ser Val Leu Leu Glu Gin Leu Cys Ala Thr Tyr Lys Leu tys Human: Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu Gin Glu Lys Leu|Val Ser GlulCys Ala Thr Tyr Lys Leu Cys 20 30 40 50 60 70 Mouse: His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Lys Ala Ser Leu Ser Gly Cys Ser Ser Gin Ala Leu Human: His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gin Ala Leu 50 60 70 80 90 100 Mouse: Gin Gin Thr Gin Cys Leu Ser Gin Leu His Ser Gly Leu Cys Leu Tyr Gin Gly Leu Leu Gin Ala Leu Ser Gly lie Ser Human: Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly lie Ser 80 90 110 120 Mouse: Pro Ala Leu Ala Pro Thr Leu Asp Leu Leu Gin Leu Asp Val Ala Asn Phe Ala Thr Thr Ile Trp Gin Gin Met Glu Asn Human: Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gin Gin Met Glu Glu 100 110 120 130 140 150 Mouse: Leu Gly Val Ala Pro Thr Val Gin Pro Thr Gin Ser Ala Met Pro Ala Phe Thr Ser Ala Phe Gln Ars Arg Ala Gly Glb Human: Leu Gly Met Ala Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin Arg Arg Ala Glo GCl 130 140 150 160 170 Mouse: Val Leu Ala lie Ser Tyr Leu Gin GlY Phe Leu Glu Thr Ala Arg Leu Ala Leu His His Leu Ala Human: Val Leu Val Ala Ser His Leu Gin Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro 160 170

FIG. 5. Comparison of murine and human G-CSF amino acid sequences. The numbers above and below each row refer to amino acid positions ofmurine G-CSF and human G-CSF, respectively. The two sequences are aligned to get the maximum homology between them, and the identical amino acids are underlined. Human G-CSF amino acid sequence is the G-CSFa amino acid sequence deduced from pBRG-4 cDNA (7). The boxed three amino acids, Val-Ser-Glu at the residue numbers 36-38 cannot be found on the human G-CSFb sequence (8). Downloaded by guest on September 29, 2021 Biochemistry: Tsuchiya et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7637 kemia virus (20), only NFS-60 cells could respond to G-CSF W. J. (1979) Biochemistry 18, 5294-5299. for its proliferation (30), and the dependency of NFS-60 cells 12. Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, on G-CSF was confirmed by the recombinant human and 1408-1412. 13. Hall, A. & Brown, R. (1985) Nucleic Acids Res. 13, 5255-5268. murine G-CSF (Fig. 4). On the other hand, G-CSF can induce 14. Huynh, T., Young, R. A. & Davis, R. W. (1985) in DNA the terminal differentiation of the murine myelomonocytic Cloning Techniques, A Practical Approach, ed. Glover, D. M. leukemia cell line WEHI-3B D+ (6) but not of NFS-60 cells (IRL, Oxford). (ref. 30; S.N., unpublished data). By using these two cell lines 15. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular as a model system, it will be possible to study the prolifer- Cloning: A Laboratory Manual (Cold Spring Harbor Labora- ation and differentiation of the progenitor cells of granulo- tory, Cold Spring Harbor, NY). cytes. In particular, it will be interesting to examine the 16. Benton, W. D. & Davis, R. W. (1977) Science 196, 180-182. receptor for G-CSF on both cell lines and the intracellular 17. Wahl, G. M., Stem, M. & Stark, G. R. (1979) Proc. Natl. mechanisms for proliferation and differentiation of cells Acad. 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