AU618143B2 - Cdna clones coding for polypeptides exhibiting multi- lineage cellular growth factor activity and/or mast cell growth factor activity - Google Patents

Description

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618143 COMMONWEALTH OF -AUSTRALIA PATENTS ACT1 im5 DIVISIONAL SPECIFICATION

(ORIGINAL)

FOR OFFICE USE Form :10 It Short ruio: hIt. Cl: Application Number:- Lodged: 4 4. 4 4 4 4 4 4 4 4 Complete Specification-Lodged: Accep ted: Lapsed: Published: Priority: Related Art: 4 4 4 4 40 44 TO BE COMPLETED BY APPLICANT 42 Name ofApplicant: Address of Applicant: Actual Inventor: Address for Service: SCI-ERING BIOTECH CORPORATION 1454 Page Mill Road, Palo Alto, California 94304, U.S.A.

TAKASHI YOKOTA, FRANK DON LEE, DONNA MAYE RENNICK and KEN-ICI

ARAI

GRIFF1TH1-lACK CO.

71 YORK STREET SYDNEY NSW 2000

AUSTRALIA

Complete Specification for the invention entitled: "eDNA CLONES CODING FOR POLYPEPTIDES EXHIBITING MULTI-LINEAGE CELLULAR GROWTH FACTOR ACTIVITY AND/ORt MAST CE~LL GROWTH4 FACTOR ACTIVITY" I'lia following statement is a full description of this invention, including the best method of performing it luiown to me/us:- 9258/NL i cDNA CLONES CODING FOR POLYPEPTIDES EXHIBITING MULTI-LINEAGE CELLULAR GROWTH FACTOR ACTIVITY AND/OR MAST CELL GROWTH FACTOR ACTIVITY This invention relates generally to the 5 application of recombinant DNA technology to elucidate S* the control mechanisms of the mammalian immune response and, more particularly, to the isolation of *r deoxyribonucleic acid (DNA) clones coding for polypeptides exhibiting multi-lineage cellular growth factor activity and/or mast cell growth factor activity.

Recombinant DNA technology refers generally to the technique of integrating genetic information from a donor source into vectors for subsequent processing, 1 such as through introduction into a host, whereby the 15 transferred genetic information is copied and/or expressed in the new environment. Commonly, the genetic S. information exists in the form of complementary DNA S (cDNA) derived from messenger RNA (mRNA) coding for a .desired protein product. The carrier is frequently a plasmid having the capacity to incorporate cDNA for later replication in a host and, in some cases, actually to control expression of the cDNA and thereby direct synthesis of the encoded product in the host.

This technology has progressed extremely rapidly in recent years, and a variety of exogenous proteins have been expressed in a variety of hosts. By way of example, some of the eukaryotic protola~ soproduced include: proinsulin (Naber, S. et al., Gene 21: 95-104 [1983])) interferons (Simon, L. et al., Proc. Nat. Acad. Seti 80: 2059-2062 [1983] and Derynck R, at ele, Nucl. Acids Ros. 1: 1819-1837 [1983)); and growth hormone (Gooddel, et al., Nature 281: 544-548 [1979)). (These publications and other referenced materilis have been included to provide additional details on the background of the pertinent are and, in particular instances, the practice of invention, and are all incorporated herein by reference.) For some time, it has been dogma that the .mammalian immune response was due primarily to a series S of complex cellular interactions, called the "immune network". While it remains clear that much of the response does in fact revolve around the network-like interactions of lymphocytes, macrophagen, granulocytes and other cells, immunologists now generally hold the 2. opinion that soluble proteins the so-called lymphokines) play a critical role in controlling these cellular interactions.

Lymphokines apparently mediate cellular activities in a variety of ways, They have been shown to have the ability to support the proliferation and growth of various lymphocytes and, indeed, are thought to play a crucial role in the basic differentiation of pluripotential hematopoietic stem cells into the vast number of progenitors of the diverse cellular linea.is responsible for the immune response. Cell lineages important in this response include two classes of lymphocytes: B cells, which can produce and secrete immunoglobulins (proteins with the capability of recognizing and binding to foreign matter to effect its -3nomral), and T cells (of various Gubsets) thatid'c or suppress B cells anl come of the, other cells (including other T cells) making up the immune network.

Another important,- cell lineage is the mast cell a ganule-containing connective tissue Coll located proximate to capillaries throughout the bcdy, with especially high concentrations in the lungs, skin, and gastrointestinal and genitourinary tracts. M asnt coils play a central role in allergy-related disordera, particularly anaphylaxig, ani this role can be briefly stated as follows: once certain antigens crosslink, special immunoglobulins bound to receptoro on the mast cell surface, the mast cell degranulates and releases the mediators histamine, serotonint heparin, kinins, etc.) which cause allergic reactions# eog.

Research to better Understand (and thus potentially tre at therapeutically) allergy, anaphylaxis and other immune disorders, through the study of mast cellst T ceils and the other cells involved in the immune response, has been hampered by the general inability to maintain these cells in viro However, several immunologists recently discovered that such cells could be isolated and cultured by growing them on S 25 secretions from other L.ells, conditioned media from splenic lymphocytes stimulated with Concanavalin A It has now become clear from this work that the *'generation of cell clones is dependent on specific factors, such as lymphokines.

Apparently alm~ost all blood cell types are Continuously generated in the adult vertebrate bone marrow through the growth and differentiation of the hierarchy of hematopoietic progenitor cells. At the apex of this hierarchy is the pluripotent stem cell, -4which can repopulate a lethally irradiated animal for most, if not all, immunological cell types red cells, platelets, lymphocytes, various granulocytes and monocytes/macrophages). The pluripotent cell not only S has the capacity to regenerate the pluripotent stem cell compartments (self-renewal), but also gives rise to progenitor cells committed to development along one particular lineage pathway. Progeny of a particular committed stem cell appear to share the same lineage commitment as the parent cell (Metcalf, "Hemopoietic Colonies", Springer Publishing Co., New York, N.Y.

[1977]).

1 if In vitro studies on hematopoiesis have shown I that a number of soluble colony stimulating factors i 15 (CSF) can regulate the growth of these various progenitor cells. Some of these factors have been partially purified and shown to affect specifically stem t cells belonging to a particular cell lineage. For example, erythropoietin stimulates more-differentiated members of the erythroid hierarchy (Miyake, et al., J. Biol. Chem 252: 5558 (1977]), and another factor I (colony stimulating factor-macrophage or CSF-1) preferentially stimulates macrophage growth in semisolid cultures of bone marrow cells (Stanley, and 25 Heard, J. Biol. Chem. 252: 4305 [1977]). Yet wr** another type of growth factor seems able to stimulate hematopoietic colonies consisting of single cell types L S* and mixtures of cells. The range of cells, e.g.

erythrocytes, megakaryocytes, granulocytes, mast cells and monocyte/macrophages, that are responsive to one factor of this second type have caused it to be named a multi-lineage cellular growth factor (multi-CSF) (Iscove, N. et al., J. Cell. Physiol. Suppl., 1: 65-78 (1982]), indicating its capability of affecting a number

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oZ committed progenitor cells, and perhaps pluripotential stem cells as well.

One of the better characterized factors is interleukin-1 a factor released from macrophages, which induces replication of thymocytes and peripheral T cells (Mizel, S. et al., J. Immunol. 120: 1497-1503 [1978]). Similarly, interleukin-2 (IL-2) and interleukin-3 (IL-3) are two well-studied lymphokines released by certain stimulated lymphocytes. A very significant characteristic of IL-2 is its ability to support the continuous growth of certain T cells in vitro (Farrar et al., Ann. N.Y. Acad. Sci. 332: 303-15 [1979]). Likewise, an important characteristic of IL-3 is its ability to support the growth of cell lines 15 having the phenotypic characteristics of mast cells '(Ihle, J. et al., Immunological Rev. 63: 5-32 [f9'82).

A number of other cellular growth properties have been ascribed to IL-3 as well (see Ihle, J. et al., a.

Immunol. 131: 282-287 and 129: 2431 (1981]), but its precise relationship with multi-CSF has been unclear.

lea Whereas both mouse IL-2 and IL-3 have been at o least partially characterized biochemically (Gillis, S.

et al., J. Immunol. 124: 1954-1962 [1980] and Ihle, J.

et al., J. Immunol. 129: 2431-2436 [1982], *r 25 respectively), IL-2 is presently the accepted primary factor responsible for T-cell growth, whereas the protein(s) responsible for mast cell growth factor (MCGF) and CSF activity have not been agreed upon to the same extent. It is now thought that mouse IL-2 has a molecular weight (probably as a dimer) of approximately 30-35,000 (Simon, P. et al., J. Immunol. 122: 127-132 [1979]), although some variations are recognized (Robb, R. and Smith, Molec. Immun. 18: 1087-1094 [1981]); and human IL-2 apparently has a molecular weight of -6- '.444 4* 44 4 4* 9* 44 4 4 *4*4 4 44*4 4444 444 9444 4*44 4,44 44 4* 4 *4 4 41 *44*44 4 4 4 *4*444 4 4 44 *4 1 4*44 about 15,000 (Gillis, S. et Immun. Rev. 63: 167-209 [1982]). Moreover, a cDNA clone coding for human IL-2 has recently been reported (Taniguchi, T. et al., Nature 302: 305-310 (1983]). On the other hand, mouse mast cell growth factors have been variously reported as having molecular weights of 45,000 (Nabel et al'., Nature, 291: 332-334 (1981],, of 35,000 (Yung, Y. et al., J. Immunol, 127: 794-799 11981]) and of 28,000 (Ihle, J. et al., J. Immunol. 129: 1377-1383 (1982)).

!0 Similar discrepancies surround the CSF's.

Although such molecular weight differences could perhaps be partially explained by varying amounts of glycosylation, clarification of the issue requires additional structural data, substantially full- 15 length sequence analysis of the molecules in question.

Protein sequencing offers, of course, a possible means to solve the problem, but it is very difficult work experimentally and often can provide neither completely accurate nor full-length amino acid sequences.

Moreover, having the capability of making bulk quantities of a polypeptide exhibiting mammalian MCGF or CSF activity will greatly facilitate the study of the biology of mast cells and other cells involved in the immune response; by minimizing the necessity of 25 relying on ConA-conditioned media for stimulating cell growth. Accurate and complete sequence data on an MCGF or CSF will also serve to simplify the search for other immunological factors. Finally, additional information on any lymphokine will help in evaluating the roles of the various growth factors and cells of the immune network and thus provide insight into the entire immune system with the concomitant therapeutic benefits.

Thus, there exists a significant need for extensive nucleotide sequence data on the DNAs coding 0 ii-7for, and amino acid sequences of, proteins exhibiting MCGF or CSF activity, as well as a sim le and economic method of making substantial and essentially pure quantities of such materials. The present invention fulfills these needs.

The present invention provides cDNA clones coding for polypeptides exhibiting mammalian mast cell growth factor (MCGF) activity and or multi-lineage cellular growth factor activity. A nucleotide sequence I0 for a cDNA and a putative amino acid sequence for an associated polypeptide are shown in Figure 1. The cDNA sequence can be integrated into various vectors, which :in turn can direct the synthesis of the corresponding polypeptides in a variety of hosts, including eukaryotic cells, such as mammalian cells in culture.

More specifically, the invention provides a 44:4 process for producing a polypeptide exhibiting mammalian MCGF activity and/or multi-lineage cellular growth factor activity, the process comprising the steps of: a) providing a vector comprising a nucleotide sequence coding for said polypeptide, wherein the nucleotide sequence is capable of being oexpressed by a host containing the vector; I o b) incorporating the vector into the host; and 25 c) maintaining the host containing the vector under conditions suitable for transcription of the nucleotide sequence into said polypeptide.

Preferably, the cDNA sequences are derived from an mRNA sequence coding for the polypeptides, and the host is an organism such as a eukaryotic, e.g.

mammalian, cell transfected or transformed with the vector. Further, the vector preferably comprises also a second nucleotide sequence capable of controlling f 8 expression of the nucleotide sequence coding for the polypeptide. This second sequence coding can include a promoter sequence, one or more intron sequences and a polyadenylation sequence, to permit, respectively, transcription, splicing and polyadenylation of the nucleotide sequence coding for the polypeptide.

Particularly, when the host is a mammalian cell, such as a COS-7 monkey (kidney) cell, the vector contains the promoter sequence of the simian virus 40 (SV40) early region promoter and the polyadenylation sequence of the SV40 late region polyadenylation sequence.

The mouse cDNA sequence of Figure 1 (see below) is capable of hybridizing with other DNA sequences, such as DNA coding for other mammalian growth factors from a cDNA or 0 genomic library. It is noted that the described cDNA S sequences seem to contain information for a leader sequence.

20 The polypeptides of the present invention are capable of enhancing mammalian mast cell and other cell growth, particularly in inJ vt.rQ cultures. Suitable pharmaceutical compositions can be prepared by adding the polypeptides (preparations of which are essentially free of other 25 mammalian growth factors) to therapeutically compatible carriers.

Other features and advantages of the invention will become apparent from the following detailed description, which describes, in conjunction with the accompanying drawings and by way of example, the present invention. The Examples are not intended to limit the scope of the invention in any way.

In the Drawings: Figi 1 illustrates the nucleotide sequence and putative sponding amino acid sequence of a cDNA 7275S/sy -ipV -9clone exhibiting multi-lineage cellular growth factor activity; Figure 2 depicts the amoun. of MCGF activity in fractions of a sucrose gradient sedimentation of mRNA isolated from ConA-stimulated Ci.Ly 1+2-/9 cells, The locations of 18S and 28S ribosomal peaks are indicated; Figure 3 illustrates pcD-MCGL, a plasmid carrying a cDNA clone exhibiting mast cell growth factor activity and multi-lineage cellular growth factor activity; and Figure 4 is a restriction endonuclease cleavage map of the cDNA insert of Figure 3.

TIn Figure 3, transcription of the 950 bp cDNA t: insert contained in the pcD expression vector from the 4 4 SV40 early promoter is indicated by the arrow. The locations of the splice donor ano acceptor sites are shown. The polyadenylation signal, also derived from o.ar SV40, is located at the 3'-end of the cDNA insert. The cDNA insert is heavily shaded. The remainder of the vector sequences are derived from pBR322, includin- the S-lactamase gene (AmpR) and the origin of replication.

44r4 In accordance with the present invention complementary DNA (cDNA) clones are provided for polypeptides exhibiting mammalian mast cell growth j 25 factor (MCGF) activity and/or multi-lineage cellular growth factor (multi-CSF) activity. After the cDNA sequences have been incorporated into replicable expression vectors, and the vectors tranfected into an appropriate host a mammalian cell culture), the expressed polypeptide or polypeptides will possess has the ability to allow the expansion of mast cells and hematopoietic cells to multiple lineages.

An exemplary, putative amino acid sequence based on the isolated nucloeotide sequence is shown in Figure 1. A portion of the predicted sequence (amino acids 33 to 41) is identical with the reported NH 2 terminal sequence of mouse Interleukin-3 which has been shown to exhibit mouse MCGF activity and multi- CSF activity (Ihle, J. et al., J. Immunol. 129, 2431- 2436 (1982); Ihle, J. et al., J. Immunol. 131, 282-287 (1983); and Garland, J. et al., Eds. Oppenheim, J. and Cohen, "Interleukinsi Lymphokines, and Cytosines", Proceedings of Third Int. Lymphokines Workshop, Academic Press, New York, pages 123-129 (1983)). The coding region located between the translation start codon (ASG) and the beginning of the sequence contained in IL-3 is rich in hydrophobic amino acids, as would be expected for a leader sequence of a secreted protein. Therefore, the polypeptide's mature form in vivo, as secreted, possibly begins with an Asp residue, as does IL-3, and the preceding 20 or so amino acids constituting the putative leader sequence are removed by proteolytic processing. Assuming this to be accurate, the mature polypeptide exhibiting MCGF and multi-CSF activities would consist of 134 amino acids, with a calculated molecular weight of about 15,000. Furthermore, the presence of four potential N-glycosylation sites, i.e., Asn-X-Ser (where X is an amino acid residue) at deduced amino acid positions 42-44, 70-72, 77-79, and 112-114 of o. the polypeptide (see Neuberger et al., Glycoproteins 450-490, Elsevier Publishing Co., U.S.A. [1972)), suggests that it would be glycosylated in vivo.

When transfected into COS-7 monkey cells, one of the cDNA clones of this invention directs the synthesis of biologically active MCGF and multi-CSF.

Addition of this expressed cloned gene product to 0 -11- .P 4* 1 *4 4 4 44 a 4 S*4 1*s

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4 rr 4 44 44l 4 44o 4r' cultures of mouse bone-marrow cells allows the expansion of hematopoietic cells committed to multiple lineages; it supports the formation of burst-forming erythroid colonies (BFU-E), granulocyte/macrophage colonies (CFUmast cell colonies (CU-mast), as well as colonies of multiple lineages (CFU-Mixed), and sustains multipotential stem cells (CFU-S) in liquid culture.

A variety of methods may be used to prepare the CDNAS of the present invention. By way of example, total mRNA is extracted as reported by Berger, S.

et al,, Biochemistry 18: 5143-5149 [1979]) from a cell line a hybrid cell line) producing polypeptides exhibiting mammalian mast cell growth factor activity.

The double-stranded cDNAs from this total mRNA can be 15 constructed by using primer-initiated reverse transcription (Verma, Biochim. Biophys. Acta, 473: 1-38 11977]) to make first the complement of each mRNA sequence, and then by priming for second strand synthesis (Land, H. et al., Nucleic Acids Res., 9: 2251- 20 2266 [1981]). Subsequently, the cDNAs can be cloned by joining them to suitable plasmid or bacteriophage vectors (Rougeon, F. et al., Nucleic Acids Res., 2, 2365-2378 [19751 or Scherer, G. et al., Dev. Biol. 86, 438-447 (1981]) through complementary homopolymeric 25 tails (Efstratiadis, A. et al., Cell, 10, 571-585 [1977]) or cohesive ends created with linker segments containing appropriate restriction sites (Seeburg, P. et al., Nature, 270, 486-494 [19771 or Shine, J. et al., Nature, 270, 494-499 [1977]), and then transforming a suitable host. (See generally Efstratiadis, and Villa-Kormaroff, "Cloning of double stranded cDNA" in Setlow, J. and Hollaender, A. (eds.) Genetic Engineering, Vol. 1, Plenum Publishing Corp., N.Y., U.S.A. (1982].) j1~ 1 4 -12- 4444 44 4 444 4444 4)IP 44 4444 4 4444 A preferred method of obtaining the fulllength cloned oDNAs of this invention is the procedure developed by I. Qkayama and P. Berg (Mol. and Coll.

sBiol., 2: 161-170 (19821). This method has the advantage of placing the vDNA inserts in a bacterial cloning vector at a position whereby the McDNA can also be directly translated and processed in mammalian calls. Briefly, the firsat cDNA strand is primed by polydeoxythymidylic acid covalently joined to one end of a linear plamid vector DNA. The plasmid vector is then cyclized with a linker DNA segment that bridges one end of the plasmid to the 5' and of the cDNA coding sequence. By employing a DNA fragment containing the Simian Virus 40 (SV40) early region promoter and a 15 linker containing a modified SV40 late region intron, the eDNA can be expressed in vitro in COS-7 mouse kidney cells without further modification. (See generally Okayama, H. and Berg, Mol. and Ceall., Biol.s, 3: 280- 289 [19831 and Jolly, D. et al., Proc. Nat. Acad. Sci.

SG: 477-481 (19831.) The desired cDNA clones can also be detected and isolated by hybridization screening with appropriate mRNA samples (Heindell, H. et al., Cell, 15: 43-54 [1978]). Altaernatively, the cDNA libraries can be 25 screened by hybrid selection (Harpold, M. atal., Nucleic Acid Res., 5: 2039-2053 [19781 or Farnes, J. t al., Proc. Nat. Acad. Sci. 78: 2253-2257 (1981]) or in Xenopus oocytes (Aurdon, Nature, 233: 177-181 [19711). (See generally Villa-Komaroff, L. at al., Proc. Nat. Acad. Sci. 75: 3727-3731 [1978].) Once the cDNA library in the Okayama/Berg plasmid vector has been completed the cDNA clones are collected, and random pools are checked for the presence of the desired cDNAs by hybrid selection, translation, i .i n:~ r -e .44* Ca 6 4* 4t 4 4 4r 44 4 rtra 1I -13- *Q .9 99 9 9 9i #9 9lP~ .99.

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*994* and assay by measuring MCGF or multi-CSF growth factor activity, the existence of antigenic determinants, or other biological activities). Pools positive by these criteria can then be probed with an appropriate subtracted probe, CDNA from a B cell line and/or uninduced T cell line. Thereafter, the positive, probed pools are divided into individual clones which are tested by transfection into a suitable host (such as a mammalian cell culture), and the host supernatant assayed for the desired activity (e.g.

multi-CSP or MCGF activity). Positive clones are then sequenced.

In further describing the procedures relating to preparing cDNA clones of the invention, the mast cell 15 and other lines will be considered first, followed by general descriptions of the procedures of the in vitro translation of mRNA coding for a protein exhibiting MCGF activity; the construction of a cDNA library containing the cDNA sequences; hybrid selection of the library; 20 isolation of full-length cDNA clones in a plasmid vector and subsequent expression in mammalian cells; multi-CSF assays; human multi-CSF and MCGF isolation, subcloning and expression in bacteria and yeast; and purification and formulation. A more detailed description of the 25 entire experimental process will follow thereafter.

Mast Cell and T-Cell Lines The preferred cells for use in connection with the present invention are those developed as described by Galli, J. et al. Cell Biol., 95: 435-444 11982]). One cloned mast cell line, designated MC/9 and deposited at the American Type Culture Collection (accession number ATCC CRL 8306) was grown in Dulbecco's modified Eagle's medium (DME) supplemented with i ii 999.9 9 #9 994 9 9 9 99 4 9 99 iS**«t -14supernatants from a ConA-activated T-cell line, designated Cl.Ly 1+2"/9 and deposited at the American Type Culture Collection (Accession Number ATCC CRL 8179) (Nabel, G. et al., Nature, 291: 332-334 (19811). This T-cell line was derived from C57B1/6 mice (Nabel, G. et al., Proc. Natl. Acad. Sci. 78: 1157-1161 11981]), and was maintained in modified supplemental DME (Nabel et al., Cell, 23: 19-28 [19811). It is a suitable mRNA source for the polypeptides of this invention, but other mammalian cell sources (including human peripheral blood) exhibiting the appropriate activity MCGF or multi-CSF) may also be used.

The MC/9 cells are used to assay for MCGF activity, preferably by a 3 H-thymidine incorporation 15 assay according to established methods Nabel et j Nature, 291: 332-334 (1981]). Briefly, MC/9 cells /well) are cultured in flat bottom Falcon microtiter trays in DME supplemented with 4% fetal calf serum, PM 2-mercaptoethanol 2mM glutamine, non- 20 essential amino acids, essential vitamins and varied 644* I concentrations of supernatant in a final volume of 0.1 *l ml. To each culture is added 0.5 Ci 3 H.thymidine for the last 4 hr of a 24 hr incubation period. The cells are then harvested onto glass filters and the 25 radioactivity measured by liquid scintillation spectrometer.

Isolation and size Fractionation of mRNA Total cellular mRNA can be isolated by a variety of well-known methods, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (Biochemistry, 18: 5294-5299 (1979]). If this method is used, approximately 100 Ug of polyA+ mRNA, selected on columns of oligo (dT) cellulose, is obtained from 1-2 x 108 activated helper T-cells, such as Cl.Ly 1+27/9. To fractionate the mRNA by size, 100 Ug of polyA+ mRNA is layered on a 10 ml 5-25% sucrose gradient (10mM Tris'HCl, pH 7.4, 100mM NaCI, 1 mM EDTA), and centrifuged for 19 hr at 26,000 rpm in a Beckman SW41 rotor. 450 Ul fractions are collected and the RNA is precipitated with 2 volumes of ethanol.

Hybrid Selection and Microinjection of Xenopus laevis Oocvtes Filter hybridizations are preferably performed essentially as described by Parnes et al. (Proc. Natl.

Acad. Sci. 78: 2253-2257 [1981]). Aliquots of 44,4 «eluted mRNA are injected into individual Xenopus laevis 15 oocytes by methods well known in the art. Supernatants from viable oocytes are collected after 48 hr, pooled and assayed for activities.

Construction of cDNA Library The cDNA library can best be constructed using the pcDV1 vector-primer and the pLi linker fragment (available from P-L Biochemicals Inc., Milwaukee, WI) according to procedures which result in greatly enriched S1) full-length copies of mRNA transcripts Okayama, H.

and Berg, Mol. Cell Biol., 2, 161-170 [1982] and S 25 Mol. Cell Biol., 3, 280-289 [1983]). The plasmid vector, which contains SV40 early promoter and SV40 RNA processing signals, is designed to promote expression of the cloned cDNA segment in mammalian cells.

Using the Okayama and Berg procedure, the cyclized vector-cDNA preparation is transformed into a competent bacterial cell, such as E. coli MC1061 cells (Casadaban, M. and Cohen, J. Mol. Biol., 138: 179- -16- 207 (19801) using calcium chloride (Cohen, S. ot .,i Proc. Nat. Acad. Sci. 69: 2110-2114 19721).

Starting with 5 Ug of polyA+ RNA from ConA-stimulated Cl.Ly 1+2-/9 cells, about 1.5 x 105 x 6 independent transformants are obtained. About 104 clones are picked up individually and inoculated into wells of microtiter plates (Flow Laboratories Inc., McLean, Virginia) containing 200 1l of L-broth, 50 ug/ml of ampicillin, and 7% DMSO. If desired, sublibraries based on the size of to cDNA insert are prepared from total cDNA library as described by Okayama, H. and Berg, P. (Mol. Cell Biol., with Sall, Clal, and HindliI separately, and electrophoresed in 1% agarose gel. After staining with 15 ethidium bromide, the gel is sliced into 7 sections correspor.aing to cDNA insert sizes of 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, and more than 6 kilobases DNA is extracted from each slice, recyclized with T4 DNA ligase, and used to transform MC1061. All nucleotide sequencing can be performed according to the J procedure of Maxam, A. and Gilbert, W. (Methods Enzymol., 65: 499-560 [19801).

I 4 Preparation of Subtracted cDNA Probe If desired, a 32 P-cDNA probe is enriched for ConA-induced sequence by two cycles of cDNA absorption in order to remove cDNA sequences common between Cl.Ly 1+2-/9 and closely related, but differentiated, cells of the immune system, such as B cell myelomas (see Davis, M. et al., "Isolation of B4 T-Cell Specific Genes", Vitteta, E. and Fox, C. eds., UCLA Symp., pg. 48 [1982)). About 2 vg of mRNA having MCGF or multi-CSF activity from a sucrose gradient fraction is preferably used as template for reverse transcriptase using oligo rl-l_ I -17- (dT) 12-18 primers (available from Collaborative Research, Waltham, Mass.). After hydrolysis of RNA by alkali, 32 P-cDNA is hybridized with 20 Ug of mRNA each from WEHI-231, a B-cell lymphoma (see e.g. Taussig et al., Immunology 39: 57-60 [1980]), and an NS-1-derived hybridoma (ATCC accession number HB-8113) at 680C for 14 hr (cot value=5,000). The unhybridized cDNA is separated from cDNA/RNA hybrids by column chromatography on hydroxyapatite. A second subtraction can then be performed with unhybridized 3 2 P-cDNA using an excess of mRNA (10 Ug) from uninduced Cl.Ly 1+2-/9 cells as above (cot=l,100). The single-stranded 32 P-cDNA enriched for ConA-induced sequences, constituting approximately 1-2% of the starting material, is then used for colony 15 hybridization (Maniatis, T. et al., "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, U.S.A. 11982)).

DNA Transfections into Monkey Cells C1L Approximately Ix10 6 COS-7 monkey kidney cells are seeded onto 60 mm plates the day prior to transfection. Transfections are best performed with Vg of plasmid DNA in 1.5 ml of DME containing 50 mM TrisHC1, pH 7.4, and 400 Vg/ml DEAE-Dextran (Pharmacia -4 Fine Chemicals, Uppsala, Sweden). This solution is then removed after 4 hr and replaced with 2.0 ml DME 4% fetal calf serum. The medium is collected after 72 hr and assayed for MCGF or multi-CSF activity as described above. DNA transfections may be carried out in a variety of other cell sources as well (see below).

-18- Multi-CSF Assays Multi-CSF activity comprises testing for the ability to act on multipotential progenitor cells, or a number of lineage restricted cells, or both. (See generally Iscove, N. et al., J. Cell Physiol. Suppl. 1: 65-78 [1982] and Ruppert, Exp. Hematol. 11: 154-161 (19831.) Basically, the assay conditions allow generation of burst-forming erythroid colonies (BFU-E), granulocyte/macrophage colonies (CFU-G/M) and colonies of mixed lineages (CFU-Mixed) and are performed generally according to the procedures of Metcalf, D. et eal. Cell Physiol., 98: 401-420 j1979]) and Johnson, G. Cell Physiol., 103: 371-383 t1980l).

CSF-c Assay (Colony Forming Unit culture) g Bone marrow cells can be harvested from the femurs of C57BI/6 mice. The cells are washed once and a single-cell suspension prepared in Iscove's modified Dulbecco's Medium, [IMDM] (GIBCO, Grand Island, New York) 3% Fetal Calf Serum [FCS] (GIBCO). The single- 20 cell suspension is plated in plastic tissue-culture dishes and incubated 1-2 hours in a 37°C incubator with 6% CO 2 to allow cells to adhere to the dish. The nonadherent cells are then removed and in some cases placed over a discontinuous Percoll (Sigma Chemical Co., St.

Louis, MO) gradient consisting of 2 ml layers of 40%, I 50%, 60%, 70% Percoll solution (as reported by Kakiuchi Set al., J. Immunol., 131: 109 [19831). The cells at the various interfaces are harvested separately and washed twice with IMDM 3% FCS. (Alternatively, cells not placed over Percoll can be washed once with IMDM 3% FCS.) The separate cell pellets are then resuspended at a concentration of 4.5-6 x 106 cells/ml in IMDM

FCS.

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44 4 .4 4 4 4 .4 I 444* 444* *144 4 4 *14 144 4 -19- CFU-c's can be assayed by using a modification of the methyl-cellulose procedure of Iscove etal (J.

Cell Physiol.t 83: 309 [1974]). FCS (final concentration 2-mercap' 'ethanol (5 x 10-5M), penicillin-stre, .omycin (1:100 of GIBCO stocks), methyl-.cellulose 4000 centipoise), cells (1.5-2 x, 10 5 /ml) and various experimental factors to be tested for CPU-c ability are mixed and 1 ml of the mixture dispensed per small petri dish. The plates are incubated 7 days in a 370C/6% C0 2 incubator. T.ey are then scored for colonies using a dissecting microscope A colony is defined as consisting of 50 or more cells. individual colonies can be extracted, placed on croscope slides, fixed and stained with Wright/Geims 15 (See Todd-Sanfordf Clinical Diagnosis By Laboratory Methods, 15th edition, Davidsohn and. Henry (eds.) 137 (1974]). Morphological analysis of cell types present per single colony is then determined.

BFU-E (Burst Forming Unit Erythroid or CFU-Z) 20 The above procedure is acceptable, with the following modifications. Either at the time of plating in methyl cellulose or 3 days later, sheep erythropoietin (Step III, Connaught Medical Research Laboratories, Philadelphia# PA) is added at a 25 concentration of 0.5-1 unit per plate. Erythroidcontaining colonies (BFU-E or CFU-E) are scored after 10-14 days (from time of plating) as colonies containing visibly read elements. individual colonies are extracted and stained as above for morphological analysis.

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4.5444 4 4 444444 4 4 44 44 S 44,4 CFU-s (Colony Forming Unit spleen) Bone marrow is extracted from femur bones of C57B1/6 mice. Cells are washed twice with Dulbecco's modified Eagle's medium IDME] (GIBCO) and either injected immediately into the tail vein of lethally irradiated (1000 rads) C57B1/6 recipients or treated further. Treatment consists of the following various procedures: 1) lysis of the cells with anti-e and complement, followed either by immediate injection into recipients, or by culture for various times under various conditions before injection; 2) alternatively, no antiserum lysis is performed, and cells are placed immediately into culture under various conditions. The culture conditions can be as follows: cells are 15 resuspended at 1 x 106 cells/ml in medium consisting of DME (GIBCO) 2-ME (5 x 10- 5 MEM-Vitamins (1:100) (GIBCO), non-essential amino acids (1:100) (GIBCO), Lglutamine (1:100) (GIBCO), penicillin/streptomycin (1:100j (GIBCO), a mix of arginine, asparagine, and 20 folic acid, 15% FCS (GIBCO., 2mM sodium pyruvate various factors to be tested for maintenance of CFU-s (final concentration This cell preparation is then plated in 24 well tissue culture plates (Falcon) at 1 ml/well and incubated in a 37*'/10% CO 2 incubator for various times (minimum of 7 days). Every 3-4 days, nonadherent cells are removed, spun down, resuspended in fresh media containing the appropriate factor, and replated. For assays in which incubation lasted more than 7 days, cells are "moved up" to larger plastic tissue culture vessels in order to maintain all nonadherent cells at a concentration not exceeding 5 x 10 per ml. At the end of incubation, cells are washed twice and resuspended in DME (no supplements) and injected into the tail vein of lethally-irradiated

A

-21- C57B1/6 mice. Cells are injected either at specific viable cell numbers or at specific volume fraction of the culture. Nine to twelve days following injection, spleens are excised and placed in Borin's fixative (Mallinkrodt, St. Louis, MO). Spleen colonies are scored as visible nodules on the spleen surface with the aid of a dissecting microscope (4x).

Human Multi-CSF and MCGF cDNA Isolation DNA clones of rodent genes have been used to identify and isolate DNA encoding the homologous human genes. Because of the relatively low degree of homology S* between human and rodent genes, the stringency of hybridization conditions must be adjusted to allow for cross-hybridization between sequences which are only 15 80% homologous. Several different experimental protocols have been used to achieve this purpose. For example, the human CK immunoglobulin light chain gene has been isolated using the corresponding mouse CK gene as a probe (Heiter, P. et al., Cell 22: 197-207 [1981]) S" 20 and mouse transplantation antigen genes have been i *O isolated by hybridization to DNA clones encoding their human counterparts (Steinnetz, T. et al., Cell 24: 125- '134 [1981]).

A preferred method entails plating Y phage 25 clones from a library of human genomic DNA (Maniatis, T.

et al., "Molecular Cloning, A Laboratory Manual", Cold 4 j Spring Harbor Laboratory, U.S.A. [1982]) at a density of 2 x 104 to 5 x 10 plaques per 150 mm plate on an appropriate host strain, such as E. coli LE392. Ten to twenty plates are generally sufficient.

After 10-12 hours' incubation at 37 0 C, the plates are refrigerated for two hours and then a 132 mm nitrocellulose filter is applied to the agar surface of -22each plate. The filter is allowed to remain in contact with the plate for at least five minutes, during which time the filters are keyed to the plates by puncturing with an ink-filled 22-gauge needle. The filters are then peeled from the plates and incubated successively for at least two minutes first in 250 ml of 0.1 N NaOH, M NaCI; then in 250 ml of 0.5 M Tris'HCl pH 7.5, M NaCI. The filters are dried on paper towels and then baked at 80"C for 4-8 hours.

For hybridization, the filters are wetted in Ix SET (0.15 M NaCI, 30 mM Tris'HCl pH 8.0, 1 mM Na 2 EDTA), then incubated in a solution of 3x SET, Denhardt's (Denhardt, B.B.R.C. 23: 641-646 (19661), 10% dextran sulfate, 0.1% sodium dodecyl 15 sulfate (SDS), and 50 Ug/ml each poly poly (rC), and poly at 65°C for 2 hrs (1.5-2 ml/filter) with constant agitation. This solution is then discarded, and the filters are hybridized with 0.5 Ug 106 cpm) of a nick-translated mouse DNA probe in the same 20 solution (fresh), 1.5-2 ml/filter at 65*C for 1 hour and then at 550C for 12-20 hours. The filters are then washed successively in 3x SET, 1x Denhardt's; 0.1% SDS; Sand 1x SET, 0.1% SDS (10-15 mi/filter) at 55°C for one hour with gentle agitation. The filters are dried on paper towels, then autoradiographed for 12-24 hours with •appropriate film and an intensifying screen.

Hybridizing plaques are pickrd from the agar plates with sterile pasteur pipets, and each is expelled into 1 ml T of 0.1 M NaCl, 0.01 M Tris'HC1 pH 7.5, 10 mm MoC1 2 100 Pg/ml gelatin, with 50 :1 of CHC13 added. After a least 4-8 hours in the cold, the phages from each plaque are rescreened at low density (2000-4000 plaques/150 mm plate) by a procedure identical to that described above.

.1 41 0 -23- In the same manner as described previously in the mouse system, positively-hybridizing phage clones verified by re-screening can then be used as a probe to screen random colonies from a human cDNA library. The human cDNA library should be prepared using RNA from an appropriate cellular source, such as human peripheral blood T lymphocytes (see Gray, P. et al., Nature 295: 503-508 (1972]). Full length cDNA clones can be identified by expression in Cos-7 cells, again as was done for the mouse cDNA clones. The isolated human multi-CSF cDNA clones will be able to express a factor capable of stimulating human bone marrow cells.

9 *r 9 Expression in E. coli, in Yeast and in Cell Culture Prokaryotes, such as E. coli, are very S 15 suitable for expression of the polypeptides of the present invention (rae, for example, U.S. patents number 4,338,397 and 4,411,994), provided that glycosylation is not desired. To obtain high expression levels, promoters should be utilized, such as the 20 (penicillinase) and lactose promoter systems (Chang et al., Nature, 275: 615 [1978]; Itakura et al., Science, 198: 1056 (1977]; Goeddel et al., Nature 281: 544 (1979] or a tryptophan (trp) promoter system (Goeddel et al., .Nucleic Acids Res., 8: 4057 [1980]). These are the most commonly used promoters, but other microbial promoters are available.

Those skilled in the art will realize that not only prokaryotes but also eukaryotic microbes, such as yeast, may also be used in protein production.

Saccharomyces cerevisiae is a preferred eukaryotic microorganism. Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255: 2073 _r -24a *ao0 00: a 00 4 0 0~ 0 0 O 0os 0000-~ 01 0e 4 0 0 0: 11980]) or other glycolytic enzymes (Hess et al., J.

Adv. Enzyme Reg., 7: 149 t19681; Holland et al., Biochemistry, 17: 4900 [1978]). Other promoters that have the additional advantage of transcription S controlled by growth conditions may be used. Basically any plasmid vector containing a yeast-compatible promoter, an origin of replication and termination sequences is suitable.

In addition to microorganisms, cell cultures derived from multicellular organisms (especially mammalian cells) may also be used as hosts. Examples of such useful host cell lines are HeLa cells, Chinese hamster ovary cell lines, and baby hamster kidney cell lines. Expression vectors for such cells ordinarily include, as necessary, an origin of replication, a promoter located in front of the gene to be expressed, along with any required ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. When used in mammalian cells, the expression vector often has control functions provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently SV-40. (See generally U.S.P. 4,399,216, WO 81/02425 and WO 83/03259.) To express cDNA clones of the present invention in E. coli, suitable promoters tr, lac, tac, XpL, etc.) and Shine-Dalgarno sequences will be fused with the entire coding sequence of those plasmids carrying an ATG codon preferably in front of the cleavage site of the signal peptide. More specifically, the cDNA clone of MCGF or multi-CSF, e.g., pcD-MCGF, is first digested with PstI and XhoI endonuclease, and about 1 kb segment containing the entire protein coding sequence is subcloned into the -4^

IL

I

appropriate E. coli expression vectors to express the protein and signal sequence. Alternatively, in order to express only the mature protein, the segment can be subcloned into the PstI and Sail endonuclease sites of 3mp8. Single stranded M13mp8 DNA containing the complementary atrand of protein coding sequence is annealed with a synthetic oligonucleotide GAT ACC CAC CGT TTA and double-stranded protein coding sequence is then synthesized by the Klenow fragment.

After digestion with Neol endonuclease and treatment with Si nuclease, a blunt-ended DNA segment containing the double stranded MCGF coding sequence or multi-CSF coding sequence is inserted into an appropriate 4e expression vector, such as pDR540, which has the tac 15 promoter (see Russel, D.R and Bennett, Gene 231-243 (1982]); and deBoer, H. et al., Proc. Natl.

Acad. Sci. U.S.A. 80, 21-25 (1983]). (See generally Messing, J. et al., Proc. Nat. Acad. Sci. U.S.A. 74, 3642-3646 [1977]); Gronenborn, B. and Messing, J., a.t, 20 Nature 272: 375 119781; Messing, J. et al., Nucl. Acid Res. 9, 309 11981); and Messing, J. and Vieira, Gene 19, 269-276 (19821.) 4 To express an MCGF or multi-CSF cDNA clone in yeast, the PstI-Xhol fragment carrying a CDNA insert is isolated from pcD-MCGF plasmid, and then cloned into the PstI-Sall sites of pUC8. The resultant plasmid B8/pUC8 is cut with PstI and digested with Bal31 to remove the oligo (dG:dC) block placed upstream of the cDNA. An Xhol linker is attached to Bal3l-digested DNA, and the plasmids are recovered in E. coli. The transformants are analysed to determine the size of the deletion. The XhoI-EcoRI fragment (carrying MCGF or multi-CSF cDNA) is then isolated from one of the deletion derivatives, which should have about a 20 base pair deletion, and

I

'1 41 -26- 9# r4 I Ir

IT

a cloned into the HindIII site of pAAH5 and the EcoRI site of pAAR6 by blunt-end ligation using the Klenow fragment. (pUC8 is an M13mp7-derived system useful for insertion mutagenesis and sequencing with synthetic universal primers: See Vieira, J. and Messing, Gene 19: 259-268 11982]); for pcD-X, see Okayama, H. and Berg, Mol. Cell. Biol. 3: 280-289 [19831)I pAAHS and pAAR6 are yeast expression vectors carrying the ADCI promoter and terminator: Ammer, "Expression of Genes in Yeast using the ADCI promoter", Methods in Enzymology, 101: 192-201 [1982].) Purification and Formulations The multi-CSF and MCGF polypeptides expressed in E. coli, in yeast or in other cells can be purified 15 according to standard procedures of the art, including ammonium sulfa'e precipitation, fractionation column chromatography ion exchange, gel filtration, electrophoresis, affinity chromatography, etc.) and ultimately crystallization (see generally "Enzyme 20 purification and Related Techniques", Methods in Enzymology, 22: 233-577 [1977]). Once purified, partially or to homogeneity, the polypeptides of the invention may be used in pharmaceutical compositions (see below), for treating parasitic infections of the gastrointestinal tract; or for research purposes, as a supplement to hematopoietic or mast cell media and as an antigenic substance for eliciting specific immunoglobulins useful in immunoassays, immunofluorescent stainings, etc. (see generally "Immunological Methods", Vols. I II, Eds. Lefkovits, I. and Pernis, Academic Press, New York, N.Y. (1979 1981]; and "Handbook of Experimental Immunology", Ed.

Weir, Blackwell Scientific Publications, St. Louis, MO [1978].) -q I Ia aO a al I Ii art -27- For preparing pharmaceutical compositions containing the polypeptides described by this invention, these polypeptides are compounded by admixture with preferably inert, pharmaceutically acceptable carriers. Suitable carriers and processes fox. their preparation are well known in the art (see e.g.

Remington's Pharmaceutical Sciences and US.

Pharmacopeja: National Formulary, Mack Publishing Company, Easton( PA [19801). The preferred course of administration is parenteral and can include use of mechanical delivery systems.

Preferably~f the pharmaceutical composition is in unit dosage form. in such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The quantity of active compound in a unit dose of preparation may be varied or adjusted from I uPg. to 100 mg., according to the particular application and the potency of the active ingredient. The composition can, if desired, also contain other therapeutic agents.

The dosages may be varied depending, upon the requirement of the patient, the severity of the condition being treated and the particular compound being employed. Determination of the proper dosage for U t 25 a particular situation is within the skill of the art.

Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound.

Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be *oft divided and administered in portions during the day if desired.

-28- The following experimental information and data are offered by way of example and not by way of limitation.

EXPERIMENTAL

A. Cloned Inducer T Cells 1) A clone ol. T Cells Cl.Ly 12"/9 (ATCC accession number CRL 8179) expressing the Thy 1+ Ly 1+2 phenotype is continuously maintained at 0.5 x 105 cells/ml in Dulbecco's Modified Eagle's medium (DME) with 10% heat-inactivated fetal calf serum, 5 x 10 M 2-ME, 2 mM glutamine, non-essential amino acids, and essential vitamins conditioned with S* jsupernatants from ConA-activated mouse Balb/c spleen cells.

S 15 2) ConA-activation of Cl.Ly 1+2-/9 cells: The i. calls are cultured at 5 x 10 5 /ml in DME with 4% heat-inactivated fetal calf serum, 5 x 10 5 M 2-ME, 2mM glutamine, non-essential amino acids, essential vitamins and 2 ug/ml ConA. After 12-14 hrs.' incubation at 37"C in 10% CO 2 the cell suspension is centrifuged at 1500 rpm for 10 minutes. The cell pellets are collected and frozen immediately at The supernatants are filtered (Nalgene-0.22 microns) and stored at -20'C as a source of growth factors. Aliquots of the supernatant are assayed for MCGF activity (see below) to verify the induction of the line by the ConA treatment.

B. Cloned Mast Cells 1) A mast cell line (MC/9) (ATCC accession number CRL 8306) was cloned by limiting dilution from the a C- 0 -29liver of a 13-day-old mouse fetus in DME with 4% heat-inactivated fetal calf serum (FCS), 5 x 10-5 M 2-ME and 2 mM glutamine conditioned by ConA activated Balb/c spleen cells (Nabel et al., Nature 291: 332-334 [19811). The cell clone expresses the Thy Ly Ly 5 phenotype for surface membrane glycoproteins.

2) The mast cell clone is continuously maintained with doubling times of 16-18 hours in DME with heat-inactivated FCS, 5 x 10 5 M 2-ME and 2 mM glutamine, non-essential amino acids and essential vitamins supplemented with 5% supernatant from ConAactivated inducer T cell clone (see above). The growth of the mast cell clone is dependent on the active growth factor(s) obtained from the supernatant of stimulated Cl.Ly 1+2-/9 cells.

C. Biological Assays for MCGF 1. Tritiated Thymidine Incorporation Assay.

a) 1 x 10 MC/9 cells were cultured in flat-bottom 20 microtiter trays in 0.1 ml of DME with 4% heatinactivated FCS, 5 x 10 5 M 2-ME, 2 mM glutamine, non-essential amino acids, essential o vitamins, and doubling dilutions of test supernatant.

25 b) The trays were incubated at 37*C in 10% CO 2 After twenty hours, 0.5 uCi 3 H-thymidine (New England Nuclear, Boston, Mass.) was added to each culture. Four hours later, the cells were harvested onto filter paper strips, using an 30 automated cell harvester unit. The dried samples were dispensed into liquid scintillation fluid and the cpm were counted in a standard counter.

-7 4 i :i i 4* 4r*4 444,r 4tr~ 4*44 4 4* *4 4 4 4r 4 1 2. Tetrazolium Salt (MTT) Colorimetric Assay.

a) 1 x 104 MC/9 cells were cultured in flat-bottom microtiter trays in 0.1 ml of DME supplemented with co-factors and test supernatant as described in 1) a).

b) The trays were incubated at 37*C in 10% CO 2 After twenty hours, 0.01 ml of 5 mg/ml MTT dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma Chemical Co., St. Louis, MO) in phosphate-buffered saline (PBS) was added to each culture. Four hours later 0.1 ml of 0.04 N HC1 in isopropanol was added to each culture and thoroughly mixed. After a few minutes, the plates were read on a Dynatech MR580 Microelisa Auto Reader (Dynatech Instruments, Inc., Torrance, CA), at a wavelength of 570 nm (reference wavelength of 630nm) and a calibration setting of 1.99.

D. Isolation of mRNA from Cl.Ly 1+279- cells.

1. Total cellular RNA was isolated from cells using the guanidine isothiocyanate procedure of Chirgwin et al. (Biochemistry, 18: 5294-5299 [1979]).

Frozen cell pellets from uninduced or ConAinduced C1.Ly I12-/9 were suspended in guanidine isothiocyanate lysis solution. Twenty ml of lysis solution was used for 1-2 x 108 cells. Pellets were resuspended by pipetting, then DNA was sheared by 4 passes through a syringe using a 16 gauge needle.

The lysate was layered on top of 20 ml of 5.7 M CsCI, 10 mM EDTA in 40 ml polyallomer centrifuge tube.

This solution was centrifuged at 25,000 rpm in a Beckman SW28 rotor (Beckman Instruments, Inc., Palo Alto, CA) for 40 hrs at 15*C. The guanidine isothiocyanate phase containing DNA was pipetted off r4 44 4 *444: -31from the top, down to the interface. The walls of the tube and interface were washed with 2-3 ml of guanidine isothiocyanate lysis solution. The tube was cut below the interface with scissors, and the CsCI solution was decanted. RNA pellets were washed twice with cold 70% ethanol. Pellets were then resuspended in 500 ul of 10 mM Tris'HCl pH 7.4, 1 mM EDTA, 0.05% SDS. 50 i1 of 3M sodium acetate was added and RNA was precipitated with 1 ml ethanol.

The RNA was collected by centrifuging and the pellets washed once with cold ethanol.

2) Poly A+ mRNA isolation: Washed and dried total RNA pellet was resuspended in 900 P1 of oligo (dT) elution buffer (10 mM Tris'HCl, pH 7.4, 1 mM EDTA, 0.5% SDS). RNA was heated for 3 min. at 68°C and then chilled on ice.

100 V1 of 5 M NaCl was added. The RNA sample was r loaded onto a 1.0 ml oligo (dT) cellulose column (Type 3, Collaborative Research, Waltham, MA) S 20 equilibrated with binding buffer (10 mM Tris'HCl pH 7.4, 1 mM EDTA, 0.5 M NaCl, 0.5% SDS.). Flow-through from the column was passed over the column twice more. The column was then washed with 20 ml binding buffer. Poly A+ mRNA was collected by washing with 25 elution buffer. RNA usually eluted in the first 2 ml of elution buffer. RNA was precipitated with 0.1 volume 3 M sodium acetate (pH 6) and two volumes of S ethanol. The RNA pellet was collected by S, *centrifugation, washed twice with cold ethanol, and 30 dried. The pellet was then resuspended in water.

Aliquots were diluted, and absorbance at 260 nm was determined.

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*4 *0 *0 0 0 *0*0

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*0*0 *000 0 6000 0 000 0*

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66 4 44 I I It E. Fractionation of poly A+ mRNA by Sucrose Gradient Centrifugation: 100 Vi containing 100 Vg of poly A+ mRNA from D.

2) was heated at 65°C for 1 min. and then layered onto a 10 ml 5-25% sucrose gradient (10 mM Tris'HCl pH 7.4, 100 mM NaCI, and 1 mM EDTA). The gradient was centrifuged in a Beckman SW41 rotor at 26,000 rpm for 19 hours at 5°C. 450 Vl fractions were collected, precipitated with 2 volumes of ethanol and resuspended for injection into oocytes (see below).

A parallel gradient was layered with a mixture of radio-labelled 3 H-uridine) ribosomal RNA (BRL, Bethesda, MA) centrifuged as described above, and 450 Pi fractions were counted in the scintillation counter.

The size-fractionated Poly A+ mRNA, following injection in Xenopus oocytes, gave a peak of MCGF activity by the colorimetric assay sedimenting slower than 18S, as shown in Figure 2. These fractions were enriched approximately 10-fold for MCGF mRNA and were utilized subsequently for the preparation of 32p_ labelled cDNA probe.

F. Oocyte Injection Oocytes were removed from female Xenopus laevis and incubated in Barth's solution (88 mM NaCl, 1 mM KC1, 0.33 mM Ca(N0 3 2 0.41 mM CaCI 2 0.82 mM MgSO 4 2.4 mM NaHCO 3 10 mM HEPES (pH 7.9) (Sigma Chemical Co., St. Louis, MO). Injection clusters of 2-3 oocytes were prepared. RNA samples were to be injected dissolved in injection buffer (40 mM Tris'HCl pH 7.4, 0.35 M NaCI). Total poly A+ mRNA was resuspended at a concentration of 500 Ug/ml in injection buffer, while RNA samples eluted from DNA 1060*4

I

I

4 0 .6* II 4 0144 -33filters from hybrid selections (see below) always contained 5 Ug of calf liver tRNA as carrier and were resuspended in 2 U1 of injection buffer. 40 nl aliquots were injected into each oocyte using micropipets pulled by hand with tips forged using a microforge. The pipettes were calibrated with known volumes of sterile water. Approximately 30-40 oocytes were injected for each mRNA sample. The injected oocytes were incubated in groups of two or three in individual wells of 96-well microtiter dishes containing 10 vl of Barth's solution 1% bovine serum albumin per oocyte. The oocytes were kept at 19*C for 48 hours and then the supernatants from wells containing viable oocytes were collected and pooled. These supernatants were sterilized by centrifuging for 10 minutes in a microcentrifuge and then assayed for MCGF activity as described above.

Supernatants from uninjected oocytes were always collected as a control.

20 The assay results from supernatants collected from untreated or ConA-stimulated Cl.Ly 1+2-/9 cells are shown in Table I. Titration of all samples, including the reference standard, was performed in triplicate. One unit of MCGF is the amount of factor that results in 25 of the maximal level of 3 H-thymidine incorporation obtained using Cl.Ly 1+2-/9 supernatant.

TABLE I Cl.Ly 1+2-/9-Produced MCGF Activity (units/ml) 4S *4* w* *444 9Ct *4* *44 *J .4 .4 9i 9 I$4 &i ,t

I.

4*14 Cell Supernatant Injected mRNA without ConA with ConA 26,383 1 ,403 -34- G. cDNA Library Construction: 1) Preparation of vector primer and oligo dG-tailed linker DNAs: The procedure of Okayama Berg (Mol. Cell.

Biol. 2: 161-170 (1982]) was used with only minor modifications and adapted to the pcDV1 and pL1 plasmids described by Okayama Berg (Mol. Cell.

Biol. 3: 380-389 (1983)].

An 80 Pg sample of pcDV1 DNA was digested at with 20 U of KpnI endonuclease in a reaction mixture of 450 P1 containing 6 mM Tris'HCl (pH 6 mM MgC1 2 6 mM NaCl, 6 mM 2-ME, and 0.1 mg of bovine serum albumin (BSA) per ml. After 16 hr the digestion was terminated with 40 ul of 0.25 M EDTA q 15 (pH 8.0) and 20 V1 of 10% sodium dodecyl sulfate (SDS); the DNA was recovered after extraction with water-saturated 1:1 phenol-CHCl 3 (hereafter referred to as phenol-CHCl 3 and ethanol precipitation.

Homopolymer tails averaging 60, but not more than deoxythymidylate (dT) residues per end were added to the KpnI endonuclease-generated termini with calf thymus terminal transferase as follows: The reaction mixture (38 V1) contained sodium cacodylate-30 mM Tris'HCl pH 6.8 as buffer, with 1 mM CoC1 2 0.1 mM dithiothreitol, 0.25 mM dTTP, the KpnI endonucleasedigested DNA, and 68 U of the terminal deoxynucleotidyl transferase (P-L Biochemicals, Inc., I tv* Milwaukee, WI). After 30 min. at 37 0 C the reaction was stopped with 20 V1 of 0.25 M EDTA (pH 8.0) and U1 of 10% SDS, and the DNA was recovered after several extractions with phenol-CHCl 3 by ethanol precipitation. The DNA was then digested with 15 U of EcoRI endonuclease in 50 VI containing 10 mM Tris'HCl pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol, and 0.1 mg of BSA per ml for 5 hr at 37°C. The large fragment, containing the SV40 polyadenylation site and the pBR322 origin of replication and ampicillinresistance gene, was purified by agarose gel electrophoresis and recovered from the gel by a modification of the glass powder method (Vogelstein, B. Gillespie, Proc. Nat. Acad. Sci. 76: 615-619 [1979]). The dT-tailed DNA was further purified by absorption and elution from an oligo (dA)-cellulose column as follows: The DNA was dissolved in 1 ml of mM Tris'HCl pH 7.3 buffer containing 1 mM EDTA and 1 M NaCl, cooled at 0 C, and applied to an oligo a (dA)-cellulose column (0.6 by 2.5 cm) equilibrated with the same buffer at 0°C. The column was washed with the same buffer at 0 C and eluted with water at room temperature. The eluted DNA was precipitate with ethanol and dissolved in 10 mM Tris'HCl pH 7.3 with 1 mM EDTA.

The oligo (dG) tailed linker DNA was prepared by digesting 75 Pg of pL1 DNA with 20 U of PstI :endonuclease in 450 UI containing 6 mM Tris'HCl pH 25 7.4, 6 mM MgCl 2 6 mM 2-ME, 50 mM NaCl, and 0.01 mg of BSA per ml. After 16 hr at 30 C the reaction .I mixture was extracted with phenol-CHC1 3 and the DNA Swas precipitated with alcohol. Tails of 10 to deoxyguanylate (dG) residues were then added per end 0 30 with 46 U of terminal deoxynucleotidyl transferase in 4 bu the same reaction mixture (38 111) as described above, except that 0.1 mM dGTP replaced dTTP. After 20 min.

at 37°C the mixture was extracted with phenol-CHCl 3 and after the DNA was precipitated with ethanol it was digested with 35 U of HindII endonuclease in -36-

I

4~ ot I i

P

4 44 i 4*4 4 1 4 II Ui containing 20 mM Tris'HCl pH 7.4, 7 mM MgC1 2 mM NaCI, and 0.1 mg of BSA at 37°C for 4 hr. The small oligo (dG)-tailed linker DNA was purified by agarose gel electrophoresis and recovered as described above.

2) cDNA Library Preparation: Step 1. cDNA synthesis.

The reaction mixture (10 P1) contained 50 mM Tris'HCl pH 8.3, 8 mM MgC1 2 30 mM KC1, 0.3 mM dithiothreitol, 2 mM each dATP, dTTP, dGTP, and dCTP, PCi 3 2 P-dCTP (3000 Ci/mmole), 2 Pg polyA RNA from Con-A induced Cl.Ly 60 units RNase (Biotec, Inc., Madison, WI), and 2 ug of the vector-primer DNA pmol of primer end), and 45 U of reverse transcriptase. The reaction was incubated 60 min at 42"C and then stopped by the addition of 1 P1 of 0.25 M ETDA (pH 8.0) and 0.5 P1 of 10% SDS; 40 P1 of phenol-CHCl 3 was added, and the solution was blended vigorously in a Vortex mixer and then centrifuged.

After adding 40 P1 of 4 M ammonium acetate and 160 1l of ethanol to the aqueous phase, the solution was chilled with dry ice for 15 min., warmed to room temperature with gentle shaking to dissolve unreacted deoxynucleoside triphosphates that had precipitated during chilling, and centrifuged for 10 min. in an Eppendorf microfuge. The pellet was dissolved in ul of 10mM Tris'HCl pH 7.3 and 1 mM EDTA, mixed with ul of 4 M ammonium acetate, and reprecipitated with 40 pl of ethanol, a procedure which removes more than 99% of unreacted deoxynucleoside triphosphates. The pellet was rinsed with ethanol.

1" I Z -37a.,r *r 0 *0*I QW44 Step 2: Oligodeoxycytidylate [oligo (dC)] addition.

The pellet containing the plasmid-cDNA:mRNA was dissolved in 20 11 of 140 mM sodium cacodylate-30 mM Tris'HCl pH 6.8 buffer containing 1 mM CoC1 2 0.1 mM dithiothreitol, 0.2 Og of poly(A), 70 PM dCTP, 5 PCi 3 2 P-dCTP, 3000 Ci/mmole, and 60 U of terminal deoxynucleotidyl transferase. The reaction was carried out at 37*C for 5 min. to permit the addition of 10 to 15 residues of dCMP per end and then terminated with 2 P1 of 0.25 M EDTA (pH 8.0) and 1 P2 of 10% SDS. After extraction with 20 11 of phenol- CHC1 3 the aqueous phase was mixed with 20 21 of 4 M ammonium acetate, the DNA was precipitated and reprecipitated with 80 11 of ethanol, and the final pellet was rinsed with ethanol.

Step 3: HindIII endonuclease digestion.

The pellet was dissolved in 30 U: of buffer containing 20 mM Tris'HCl pH 7.4, 7 mM MgC1 2 60 mM NaCI, and 0.1 mg of BSA per ml and then digested with 10 U of HindIII endonuclease for 2 hr at 37°C. The reaction was terminated with 3 P1 of 0.25 M EDTA (pH 8.0) and 1.5 PI of 10% SDS and, after extraction with phenol-CHCl 3 followed by the addition of 30 21 of 4 M ammonium acetate, the DNA was precipitated with 120 21 of ethanol. The pellet was rinsed with ethanol and then dissolved in 10 21 of 10 mM Tris'HCl (pH 7.3) and 1 mM EDTA, and 3 ~i of ethanol was added to prevent freezing during storage at Step 4: Cyclization mediated by the oligo (dG)-tailed linker DNA.

A 9 2i sample of the HindIII endonuclease-digested oligo (dC)-tailed cDNA:mRNA plasmid (90% of the aa 4r

U.

a ar

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ai -38- 4 4 4 I* 4 4444 **44 t o4 o 4.

44 o* 4 *4 4 44*44 S& 4 *f 4 44< o 4 4 44* sample) was incubated in a mixture (90 1l) containing 10 mM Tris'HCl pH 7.5, 1 mM EDTA, 0.1 M NaCI, and 1.8 pmol of the oligo (dG)-tailed linker DNA at for 5 min, shifted to 42"C for 60 min, and then cooled to 0°C. The mixture (90 Ul) was adjusted to a volume of 900 P1 containing 20 mM Tris'HCl pH 7.5, 4 mM MgC1 2 10 mM (NH 4 2

SO

4 0.1 M KC1, 50 Pg of BSA per ml, and 0.1 mM B-NAD; 6Ug of E. coli DNA ligase were added and the solution was then incubated overnight at 12'C.

Step 5: Replacement of RNA strand by DNA.

To replace the RNA strand of the insert, the ligation mixture was adjusted to contain 40 VM of each of the four deoxynucleoside triphosphates, 0.15 mM B-NAD, 4 Pg of additional E. coli DNA ligase, 16 U of E. coli DNA polymerase I (Poll,) and 9 U of E. coli RNase H. This mixture (960 Pi) was incubated successively at 12°C and room temperature for 1 hr each to promote optimal repair synthesis and nick translation by Poll.

Step 6: Transformation of E. coli.

Transformation was carried out using minor modifications of the procedure described by Cohen et al. (Proc. Nat. Acad. Sci. 69: 2110-2114 [1972]). E. coli K-12 strain MC1061 (Casadaban, M.

and Cohen, J. Mol. Biol. 138: 179-207 [1980]) was grown to 0.5 absorbancy unit at 600 nm at 37°C in ml of L-broth. The cells wete collected by centrifugation, suspended in 10 ml of 10 mM Tris'HCl pH 7.3 containing 50 mM CaC1 2 and centrifuged at 0 C for 5 min. The cells were resuspended in 2 ml of the above buffer and incubated again at 0"C for 5 min.; then, 0.2 ml of the cell suspensions was mixed with 0.1 ml of the DNA solution (step 5) and incubated at -39- 0OC for 15 min. Next the cells were kept at 37*C for 2 min. and thereafter at room temperature for min.; then 0.5 ml of L-broth was added, and the culture was incubated at 37'C for 30 min., mixed with 2.5 ml of L-broth soft agar at 42°C, and spread over L-broth agar containing 50 Vg of ampicillin per ml.

After incubation at 37°C for 12 to 24 hr, individual colonies were picked with sterile tooth-picks.

Approximately 1 x 106 independent cDNA clones were generated and, of these, 10,000 clones were picked up individually and inoculated into wells of microtiter plates containing 200 I~ of L-broth, 50 Vg/ml ampicillin, and 7% DMSO. Random pools of approximately 1000 clones each were generated, and 15 plasmid DNA prepared for hybrid selection experiments.

rrs4 H. Hybrid selections.

Hybrid selections were performed with eight cDNA plasmid preparations, taken from the random pools 20 described above.

1) Preparation of DNA filters All plasmid DNAs were linearized by digestion with Clal prior to binding to nitrocellulose S i filters. Digestions vere performed in 50 V1 25 containing: 10 mM Tris'HC1 pH 7.9, 10 mM MgC1 2 lg plasmid DNA, 50 mM NaC1, and 10 units Clal.

Following a 2 hr incubation at 37°C, samples were diluted to 200 1u with TE (10 mM Tris'HCl pH 8.0, 1 mM EDTA) and extracted with an equal volume (200 vl) of phenol saturated with TE. 20 V1 of 3M sodium acetate (pH 6) was added to the aqueous phase, and this was precipitated with 2 volumes of ethanol. The DNA pellets were recovered by centrifugation and then hi 0 ~q.

~q# I I *1*1*1

I

t I.

washed with 70% ethanol. The dried pellet was resuspended in 150 PI of sterile water for each 10 Ul of DNA. Duplicate filters were prepared for each DNA sample, 10 Ug DNA per filter. The DNA in 150 Ul of water was boiled for 10 min, then 150 Ul IN NaOH was added and the solution incubated 20 rmin at room temperature. The sample was chilled on ice, then 150 PUl 1M HC1, IM NaCI, 0.3M Na-citrate and Tris'HCl pH 8.0 was added.

0.9 cm Millipore HAWP filters wet with distilled water were fitted into Schleicher-and-Schuell microfiltration apparatus. The denatured and neutralized DNA solution from above was filtered through by centrifugation at 500 rpm for 5 min.

Filters were washed with 1 ml of 6xSSC (0.15 M NaCI, 0.015 M Na citrate) and then air-dried before baking 2 hrs. at 80 0

C.

2) Hybridizations Hybridizations were performed in 200 PI containing 65% redistilled formamide, 20 mM PIPES, pH 6.4, 0.4 M NaC1, 200 Vg/ml calf liver tRNA, and 100 ug/ml polyA+ mRNA from ConA-induced Cl.Ly Each hybridization solution was heated for 3 min at 70°C and then two DNA filters (10 Pg DNA/filter) were cut into quarters and added to the solution. Hybrids were incubated at 50°C for 4 hours followed by 4 hour incubations at 46" and 42*C.

After this period the supernatants were removed and the filters washed 3 times with 1 ml of: 10 mM Tris'HCl pH 7.4, 0.15M NaCI, 1 mM EDTA, 0.5% SDS.

This was followed by three 1 ml washes with the same buffer lacking SDS. Both buffers were kept at for the washes. To elute the hybridized mRNA, 400 vI of distilled water with 5 Vg calf liver tRNA was -jL, i

I

S

i I -41- 444 4 44i 4 4

**C

*9 4*44 9ra 4 *9 44 44 added to the vial with the filters. The tubes were boiled for 3 min and then quick chilled in dry ice/ethanol. Samples were thawed and the eluted RNA precipitated with 2 volumes of ethanol and 0.1 volume 3M Na acetate (pH RNA pellets were collected by centrifugation and washed twice with 70% ethanol.

The dried pellets were resuspended in 2 ui of oocyte injection buffer and the entire sample was injected into oocytes (see above).

Of 8 initial pools which were screened in this manner, several were positive, and one pool showing the highest level of MCGF activity was chosen for further analysis. This pool, which consisted of 672 individual clones, was subdivided further into 14 sub-pools of 48 clones each. Plasmid DNA from these sub-pools was used in a second series of hybrid selections. Only one of these sub-pools gave a positive signal. The 48 clones were then screened with the two subtracted cDNA probes as described below.

Preparation of Subtracted cDNA Probe 32 P-cDNA synthesis: 2 Vg of polyA mRNA from the MCGF peak fraction of the sucrose gradient from above was resuspended in 2 P1 of water. This was heated for 5 min at then added to a reaction containing 50 mM Tris'HCl pH 8.3, 8 mM MgC! 2 30 mM KC1, 0.7 mM DTT, 1 mM each of dATP, dGTP and dTTP, 34 PM dCTP, 10 Ug/ml oligo-(12- 18]-(dT) (Collaborative Research), 100 ug/ml Actinomycin D, 500 PCi a 3 2 p-dCTP (Amersham, 3000 Ci/mmole) and 150 units reverse transcriptase (Life Sciences, Inc., St. Petersburg, FL) in a total volume of 100 V1. Following a 2 hr incubation at 40 0 C, i 9 9 *49 jj i L, ir -42- ~l of the reaction was removed for precipitation in trichloroacetic acid to determine the amount of 32p incorporated. Then, 100 V~ of 0.2 N NaOH was added, and the sample was heated 20 min at 70 0 C to hydrolyze the RNA. After cooling, the reaction was neutralized with 20 Ui of 1 N HC1, and 4 i1 of 1 mg/ml tRNA was added as carrier. The sample was extracted twice with an equal volume of phenol-chloroform It was then precipitated with an equal volume of 4 M ammonium acetate and 2 volumes of ethanol. The pellet was resuspended in 100 111 water, the precipitation repeated, and the pellet washed twice I I with 80% ethanol.

2) First subtractive hybridization: 15 32 P-cDNA (synthesized as described above) was co-precipitated with 20 vg of polyA mRNA from WEHI-3 and 20 1I of poly A+ mRNA from a B-cell hybridoma. The pellet was resuspended in 7 P1 water, 1 Pi 4 M Na phosphate pH 7, 0.1 ,l 20% SDS, 20 and 0.1 V1 0.1M EDTA, and then the entire sample was sealed in a capillary tube. The sample was V" heated 30 min at 90'C, then shifted to 68'C for 14 hrs (Cot 5000). The hybridization mixture was S""then diluted to 1 ml with 0.12 M sodium phosphate 25 pH 7.0 and 0.1% SDS, and the temperature of the mixture raised to 60°C. The mixture was then loaded on a column of 0.4 gm hydroxyapatite equili- ;Ao brated in the same buffer and kept at 60°C. The flowthrough was collected and the column was then washed with 5 ml of the same buffer at 60 0 C. 1 ml fractions were collected and 1 Vi aliquots of each fraction were counted in a scintillation counter.

The peak of single stranded cDNA in fractions 2, 3, and 4 was pooled. This material, representing -43- 66.5% of the starting 32 P-cDNA, was concentrated to 0.4 ml by extraction with 2-butanol and then desalted by chromatography on a 2 ml Sephadex column.

3) Second subtractive hybridization: The desalted sample from above was concentrated by ethanol precipitation and then co-precipitated with 9.5 Ig of poly A mRNA from uninduced Cl.Ly 1+2 The washed and dried pellet was resuspended in 10 1l water, 1.5 1 4M sodium phosphate pH 7, 0.15 P1 20% SDS and 0.15 ul 0.1 M EDTA. The sample was incubated in a sealed capillary tube for 30 min at 90°C and then at 68°C for 20 hr. Chromatography on hydroxyapatite was 15 repeated as described above. The single stranded cDNA which eluted from the column at represented 17% of the starting material. This 32 P-cDNA was used for colony hybridizations of the 48 colonies in the sub-pool identified by hybrid 20 selections. Three colonies hybridized with the probe and were used for further hybrid selection.

T One of these, designated clone 5G, was reproducibly positive.

J. Size fractionated sub-library 30 Vg of plasmid DNA representing the entire cDNA library (pcD-X DNA) was digested separately with the restriction enxymes SalI, HindIII, and ClaI to linearize the plasmid. The restricted DNAs were size-fractionated on a 1% agarose gel to separate plasmids having different size cDNA inserts. Segments were excised from the gel representing plasmids with cDNA inserts of the following size ranges: -44- 0 1kb 1 2kb 3 4kb 4 5 6kb 6 kb and longer DNA was eluted from each gel slice using the glass powder method of Vogelstein and Gillespie (Proc. Nat. Acad. Sci. 76: 615-619 10 [1970]). The eluted DNAs from the 3 digests were pooled on the basis of size, and treated with T4 ligase to recyclize in a total volume of 15 il containing 50 mM Tris*HCl pH 7.4, 10 mM MgC12, mM DTT, 1 mM spermidine, 1 mM ATP and 100 Ug/ml BSA. The ligation reactions were incubated 16 hr at 12*C. 3 Pl of each combined size fraction was used to transform E. coli strain MC 1061 using the I method of Cohen et al. (Proc. Nat. Acad. Sci.

rU.S.A., 69: 2110-2114 [1972]). A library of 1.1 x 105 independent transformations was obtained for the fraction containing cDNA inserts 1-2 kb in S'length, which was used for subsequent screening for S full-length MCGF clones.

K. Screening of Size-fractionated Sub-library f 25 Preliminary restriction endonuclease analysis, as well as DNA sequence data, indicated that the cDNA insert for clone 5G (identified bN hybrid selection) was approximately 650 base pairs long.

An internal BamH1-NcoI restriction fragment was isolated from clone 5G. The fragment was dephosphorylated with calf intestinal alkaline phosphatase according to the method of Chacomas, G.

and Sande, J. (Methods Enzymol. 65: 75-79 119801). The fragment was then labelled using

Y

32 P-ATP and T4 polynucleotide kinase according to the method of Maxam, A. and Gilbert, W. (Methods Enzymol. 65: 499-507 [1980)). This labelled fragment was then used to probe an RNA blot of ConA-induced Cl.Ly 1+2-/9 mRNA. A single band, approximately 1 kb long, was detected, suggesting that clone 5G was not a full-length clone.

1 We therefore used the same probe from the S" cDNA insert to screen the sub-library enriched for 1-2 kb inserts (from above). We employed the 15 method of Hanahan and Moselson (Gene, 10: 63 [1980]) as described by Maniatis, T. et al.

(Molecular Chem., Cold Spring Harbor Laboratory [19821). Approximately 500-1000 bacteria were spread on 80 mm nitrocellulose filters and o 20 incubated on L-broth plates containing 50 Ui/ml ampicillin at 37 0 C, and the bacterial colonies transferred to a second nitrocellulose filter. The duplicate filters were incubated on L-broth «ampicillin for 8 hrs. at 37°C, transferred to L- S° 25 broth plates containing 10 Ig/ml chloramphenicol and incubated overnight to amplify the copy number of the plasmid. The DNA from the colonies was bound to the nitrocellulose following lysis of the colonies with SDS, denaturation with NaOH and neutralization as described by Maniatis, T. et al., supra.

Approximately 20,000 colonies were screened and 19 colonies were reproducibly positive following rescreening with the probe. Plasmid DNA from these -46colonies was prepared and used in transfection experiments.

L. DNA Transfections One day prior to transfection, approximately 106 COS-7 monkey cells were seeded onto individual mm plates in DME containing 10% fetal calf serum and 2 M glutamine. To perform the transfection, the medium was aspirated from each plate and replaced with 1.5 ml of DME containing 50 mM Tris'HCl pH 7.4, 400 Vg/ml DEAE-Dextran and 15 Pg of the plasmid DNAs to be tested. The plates were incubated for four hours at 37"C, then the DNAcontaining medium was removed, and the plates were washed twice with 2 ml of serum-free DME. 2.0 ml 15 of DME containing 4% fetal calf serum and 2 mM glutamine was added to the plates, which were then incubated 72 hours at 37"C. The growth medium was rPa collected and assayed for MCGF activity as Sdescribed above.

Five of the six initial positive clones were examined by transfection and the results are shown Sin Table II. Mock infected COS-7 cells were treated identically, but omitting DNA.

t I y 0

_I

It -47- TABLE II Transient Expression of MCGF in Monkey Cells Length cDNA of Oligo(dG) MCGF Activity Clone Start Point* Block*** in units/ml Mock B4 41 13 5,228 Br ND** ND 7,371 B6 1 13 3,307 B8 1 13 6,929 B9 1 13 3,362 Cl.Ly 1+2-/9 19,769 S* The 5' end of MCGF cDNA expressed as nucleotide t* residue in Fig. 1.

15 Not determined; cDNA start point is located at the o. side of position 41.

Oligo (dG) block at the 5' end of MCGF cDNA.

A plasmid (pcD-MCGF) carrying a full-length SMCGF and multi-CSF (see below) cDNA insert is shown in 20 Figure 3, and an E. coli bacterium carrying the plasmid t has been deposited with the ATCC (accession number S39467). The 950 bp insert is contained in the pcD i expression vector. Transcription from the SV40 early S( promoter is indicated by the arrow. The location of T 25 the splice donor and acceptor sites are shown. The polyadenylation signal, also derived from SV40, is located at the 3' end of the cDNA insert. The cDNA insert is heavily shaded. The remainder of the vector sequences are derived from pBR322 including the Blactamase gene (AmpR) and the origin of replication.

-q -48- Figure 4 shows the restriction endonuclease cleavage map of the cDNA insert of the present invention, and Figure 1 contains the nucleotide sequence and putative amino acid sequence.

Three cDNA inserts contain a single open reading frame consisting of 166 codons beginning with the methionine codon at position 28. In addition to this putative initiation codon, two other methionine codons occur, 12 and 18 codons downstream from the first. A fourth cDNA clone starts 40 base pairs downstream from the 5' ends of the other three inserts. This shorter cDNA clone lacks the first methionine codon but still makes active MCGF upon introduction into COS cells. Thus, one of the two ATG 15 codons downstream can apparently serve as the initiation codon.

"Clone B9 expressed in COS-7 cells (COS-MCGF) Swas used to evaluate, in the absence of other T cell products, its spectrum of activities. The expressed material is not mitogenic for T or B cells, fails to induce immunoglobulin production by B cells (Table III), and does not induce macrophage la expression.

:However, this gene product does stimulate the formation of hematopoietic colonies in bone marrow cells suspended in methylcellulose, demonstrating that a S"single gene product can exhibit both MCGF and colony stimulating activities.

II

W s 4 *4 Cty a 4 4~ 8 n* TABLE ITT. cXS-MCGF stimulates BR-W-E, CFU-C and CFU-mixed in mthylcellulose bone marrow cultures Supernatant Non- TCGF** BCGFt PFCtt added to erythroid CFU- Activity Activity culture Expt. CFU-C* BRU-E* Mixed* Units/il Units/il (106 B cells) COS-7 cells transfected 1 263 25.5 5.3 1.7 12.7 1.2 0 0 with CGF cNA 2 224 17.6 3.3 2 4.3A: 1.1 MOCK transfected 1 0 0 0 0 0 33 COS-7 cells 2 1.7 0.6 0 0 L cells 1 304 25.2 0 0 ND ND ND Cl.r y 1'2-/9 1 404 -1 23.0 5.0 -1 1.2 18.0 2.1 3,250 512 4,560 Medium 1 0 0 0 0 0 6 ND not determined. N, t, tt :For these footnotes, see next paqe.

I Im 0 Notes from Table III Number of colonies per 1.5 x 105 bone marrow cells.

Each value represents mean SEM of colonies determined from triplicate cultures. 1.5 x 10 5 nonadherent, light density (<1.077 g/ml) bone marrow cells from C57B1/6 mice were suspended in 1 ml aliquots in 35 mm dishes containing 0.9% methylcellulose, 20% FCS, Iscove's modified Dulbecco's medium, 50 IM 2-ME, and 30% medium conditioned by cell supernatants as indicated. Mouse L cells were used as a source of CSF, which induces the formation O~oof macrophage colonies. Following 5 days of ~incubation at 37°C in CO 2 0.5 1.0 unit of erythropoietin (Connaught step III) was added to each 15 plate. After incubating the plates for an additional 0 0 7 days, the colonies were counted using a dissecting microscope.

TCGF activity was assayed as previously described.

One unit of TCGF activity is defined as that amount S 20 which causes 25% of the maximum level of 3H-thymidine I aJ. incorporation in 5 x 103 HT-2 cells.

o ao° °t B cells were purified from spleen cells of C57B1/6 mice and assayed for proliferation as described. One unit of BCGF activity is defined as that amount which causes 50% of the level of 3 H-thymidine incorporation in 1 x 105 B cells stimulated by 2 Pg/ml

LPS.

tt Total immunoglobulin-secreting plaque-forming cells were enumerated by a modification of che hemolytic plaque assay.

A

-51- Specifically, the expressed B-9 clone was tested under conditions which allow generation of BFU- E, CFU-G/M and CFU-Mixed. Table III shows that three types of colonies could be identified and enumerated in cultures of bone marrow cells incubated with the expressed material. The most prevalent type consisted of colorless colonies lacking hemoglobinized elements Their morphology was typical of granulocyte/macrophage colonies, the existence of which was later confirmed by histochemical staining. Also present were some large macroscopic colonies containing a multicentric arrangement of uniformly red cell clusters, which were designated BFU-E. We further S. observed a few colonies containing hemoglobinized cells 15 mixed with large and small translucent cells, which were counted as mixed.

The composition of these various colonies was analyzed by applying selected colonies to glass slides o and staining with Wright-Giemsa or nonspecific esterase stains. Over 300 colonies were examined. The majority of these colonies were composed of granulocytes, macrophages or a granulocyte/macrophage mixture, while o four percent consisted of mast cells. The remainder 0 °were composed of mixed lineages other than neutrophil/macrophage. Differential counts of representative mixed colonies compiled from several Sexperiments are presented in Table IV. The presence of several cell types within single colonies suggests that these colonies derive from pluripotent progenitor cells.

0 W h m r -52- Table IV.

Cellular composition of mixed hematopoietic colonies picked from bone marrow cultures grown in OOS-MCGF conditioned medium Differential Counts Colony Number E n m e mast M B1 36 1 22 74 64 CIO 0 0+ 004 44 0 0.0 0'400 a 040 901 4000 Differential counts of greater than 200 nucleated cells/colony. Abbreviations used are: E, erythrocyte; n, neutrophil; m, macrophage/mnocyte; e, eosinophil; mast, mast cell; M, megakaryocyte; and Bl, blast cell.

4400 x 04 06 4 0 00 Ir 0, *4 I The effects of the expressed B-9 clone were assessed on early uncommitted stem cells according to a CFU-S assay of Till and McCullock (Radiat. Res., 14: 213-222 [1961]), as modified by Schrader, and Clark-Lewis, I. of Immunol., 129: 30-35 [1982]).

When non-adherent bone marrow cells, depleted of T cells, were incubated for one week in COS-MCGF medium and injected by vein into lethally irradiated mice, macroscopic colonies appeared in the spleen (Table V).

41 1 -53- In contrast, cells incubated in supernatants of mock transfected COS-7 cells formed no colonies.

TABLE V.

Detection of CFU-S in bone marrow cells cultured for one week in COS-MCGF conditioned medium.

Spleen Supernatant added Expt. nodules/ to culture No. nmouse* CFU-S** I 4

S*

444 4 4y4r 4ar COS-7 cells transfected with full-length MCGF cDNA COS-7 cells transfected with incomplete MCGF cDNAt Mock transfected COS-7 cells 8.8 6.2 t 2.8 0.8 i 0.7 L cells Medium 0.8 E 0.7 0.7 1 0.6 0.3 k Not done 0.3 i 0.5 1 4* 4.

4 4 4 4 41*1 4 i

A.

.4,4 The mean SEM of spleen colonies detected in 5 individual 20 mice.

The mean of CFU-S calculated to reflect the frequency of CFU-S in the total culture of 3 x 106 bone marrow cells.

t Incomplete MCGF cDNA clone lacks the coding region for the

NH

2 -terminal 55 amino acids.

Light density 1.077) C57B1/6 bone marrow c.lls treated with anti-Thyl antibody and complement were plated at 1 x 106 cells/ml 0-4.

-54in Iscove's modified Dulbecco's mdium su21emented with 20% fetal calf serum, 50 W- 2-ME and 30% conditioned medium, as indicated above. Non-adherent cells were removed three times during the one-%,ek incubation period and replated in fresh medium. The cells were harvested after one week, washed twice and diluted to the original culture volume in phosphate-buffered saline. Each lethally-irradiated (1,000 R) C57B1/6 recipient was injected i.v.

with 0.1 ml of the cell suspension. After 9 days, the spleens were removed and the spleen nodules were counted using a dissecting microscope.

To summarize, in addition to the mast cell growth factor activity characterized initially, COS- MCGF has erythroid burst-promoting activity and allows Sexpansion of stem cells and early committed progenitor 15 cells of several lineages, including monocytic/granulocytic, erythroid and megakaryocytic cells. This range of activities indicates that the cDNA clones of the present invention encode proteins having the characteristics of growth factors for hematopoietic cells for multiple lineages.

From the foregoing, it will be appreciated that the cDNA clones of the present invention provide accurate and complete sequence data on mammalian multi- S. CSF and mast cell growth factors. The invention also o 25 provides to those skilled in the art means for producing significant quantities of such factors (essentially free from other hematopoietic factors) for the improved in vitro maintenance of mast cells and other hematopoietic cells. Further, the information gleaned from the cDNA clones increases understanding of the mammalian immune response, enhancing therapeutic potentialities.

Claims (23)

1. A process for producing a polypeptide exhibiting mammalian multi-lineage cellular growth factor activity and/or mammalian mast cell growth factor activity, said process comprising the steps of: a) providing a vector comprising a nucleotide sequence coding for said polypeptide, wherein the nucleotide sequence is capable of being expressed by a host containing the vector; b) incorporating the vector into the host; and c) maintaining the host containing the vector under conditions suitable for transcription of the nucleotide sequence into said polypeptide.

2. A process as claimed in claim 1 wherein the nucleotide sequence is a cDNA sequence derived from an mRNA sequence coding for said polypeptide.

3. A process as claimed in claim 1 or cla.m 2 wherein the host is a mammalian cell transformed or transfected with the vector.

4. A process as claimed in claim 3 wherein said polypeptide is glycosylated. A process as claimed in any of claims 1 to 4 wherein the vector comprises the nucleotide sequence coding for said polypeptide linked to a second nucleotide sequence, and this second nucleotide sequence comprises a promoter "A 4P 4 oil .I r I_ S-56- sequence which promotes expression of the nucleotide sequence coding for said polypeptide.

6. A process as claimed in claim 5 wherein the second nucleotide sequence comprises an SV40 virus early region promoter and an SV40 virus late region polyadenylation sequence.

7. A process as claimed in any of claims 1 to 6 wherein the nucleotide sequence codes for a polypeptide having hematopoietic cell growth activity. S 8. A process as claimed in any of claims 1 to 7 wherein the nucleotide sequence coding for said polypeptide has substantially the sequence shown in Figure 1.

9. A process as claimed in any of claims 1 to 8 wherein the nucleotide sequence coding for said polypeptide is different from but is capable of hybridizing with the 4, nucleotide sequence shown in Figure 1.

10. A process as claimed in any of claims 1 to 9 wherein the nucleotide sequence also encodes a leader sequence of the polypeptide.

11. A recombinant polypeptide consisting of at least a substantial portion of the amino acid sequence shown in Figure 1 and exhibiting mammalian multi-lineage growth factor activity and/or mammalian mast cell growth factor activity.

12. A recombinant polypeptide consisting of at least a substantial portion of the amino acid sequence shown in Figure 1 and exhibiting multi-lineage growth factor activity and/or mast cell growth factor activity on mouse cells. -2 4r 4 4 .4sai .444r 4 .444 4 l~ 4-44 4I)* 4444 4 4 -57-

13. A polypeptide as claimed in claim 11 or claim 12 in substantially pure form and essentially free from other mammalian hematopoietic cell proteins.

14. A nucleic acid sequence that codes for a polypeptide exhibiting mouse multi-lineage cellular growth factor activity and/or mast cell growth factor activity and is capable of hybridizing to a second nucleic acid sequence coding for another mammalian cellular growth factor.

15. A nucleic acid sequence as claimed in claim 14 that is a DNA sequence coding for at least a portion of the polypeptide of Figure i.

16. A nucleic acid sequence that codes for a polypeptide exhibiting mammalian multi-lineage cellular growth factor activity and/or mast cell growth factor activity and is capable of hybridizing to a second nucleic acid sequence coding for a mouse cellular growth factor.

17. A vector comprising the nucleic acid sequence of claim

18. A replicable vector capable of expressing a DNA sequence of any of claims 14 to 16, when said vector is incorporated into a microorganism or cell.

19. A microorganism or cell transformed or transfected with the replicable expression vector of claim 17 or claim 18. A pharmaceutical composition consisting of a recombinant polypeptide having mammalian multi-lineage growth factor activity and/or mammalian mast cell growth factor activity and a therapeutically compatible carrier. I -58-

21. A process for enhancing cell growth comprising contacting said cell with a recombinant polypeptide having a substantial portion of the amino acid sequence of Figure 1.

22. A process as claimed in claim 21 wherein the cell growth is enhanced in vitro.

23. A process for preparing a Folypeptide exhibiting mammalian multi-lineage cellular growth factor activity, which comprises cultivating, in an aqueous nutrient medium, a prokaryotic microorganism or eukaryotic cell I which has been transfected or transformed with a vector comprising a substantial portion of the DNA sequence shown in Figure 1. u*"t 24. A transformed microorganism or cell which contains at least a portion of a gene or other DNA sequence coding for one or more polypeptides having mammalian multi- lineage cellular growth factor activity and/or mammalian '*mast cell growth factor activity. A protein exhibiting mammalian multi-lineage cellular growth factor activity produced by cultivating the organism or cell of claim 24. S26. A recombinant DNA molecule consisting of segments of DNA from different genomes which have been joined end to end outside of living cells and have the capacity to infect some host and to be maintained therein, and the progeny thereof, comprising a DNA sequence selected from the group consisting of: a) the DNA sequence of Figure 1; b) DNA sequences which hybridize to the DNA *b\d1 >1 S- 59 sequence of Figure 1 and which code for a polypeptide exhibiting mammalian multilineage cellular growth factor activity and/or mammalian mast cell growth factor activity; and c) DNA sequences which on expression code for a protein exhibiting mammalian multilineage cellular growth fator activity and/or mammalian mast cell growth factor activity.

27. A recombinant polypeptide exhibiting human 15 multi-cellular growth factor activity and/or human mast cell growth factor activity, whose SDNA coding sequence is capable of hybridizing with DNA coding for murine IL-3. 4*49

28. A polypeptide as claimed in any of claims 11 to 13 and 27 that is capable of acting on a hematopoietic cell line. a

29. Recombinant murine IL-3 having substantially the 25 amino acid sequence shown in Figure 1 of the accompanying drawings. 9 Dated this 30th day of September 1991 SCHERING BIOTECH CORPORATION By their Patent Attorneys GRIFFITH HACK CO S:18580F/438/30.9.91 h 1 20 GGGGGGGGGG GGGAACCCCT TGGACGACCA GAACGAGACA ATG G'fl C'rr GCC MET Val Leu Ala 40 AGC TCT ACC Set Set Thr C; C ACC A'IX CTC CTC His Tht MET Leu Leu ACC AGC ATC Thr Set le so CTG CTC CTG Leu Leu Leu 100 CTC CAA CCT Leu Gin Ala 120 CCC CGC GAT ACC CAC CGT W~A Cly Arg Asp Thr His Mrg ILeu 140 ACC AGA ACG TTG Thr Arg Thr Leu ATG CTC 'rrC CAC CTG CGA MET Leu Phe His Leu Gly TCA ATC ACT Set Ile Set 160 TCT AT]T GTC Set Ile Val 180 ATA CCC AAG CTC CCA GAA CCT Ile Gly Lys Leu Pro Ciu Pro 200 GAM CTC AAA ACT CAT CAT CAA Clu Leu Lys Thr Asp Asp Giu AAT TGC AC Asn Cys Set AAG GAG ATT ELys Glu Ile 240 AAC ACC ~TT CCC AG3A GTA AMC Lys Set Phe Arg Arg Val Asn 260 CTG TCC AAA TITC CTG CAA AC Leu Set Lys Phe Val Ciu Set CTG ACC AAT7 Leu Atg Asn GGA CAA GTG CAT CCT Cly Clu Val Asp Pro 320 -TC AAT7 Crr CAC AAA CTT AAC Set Asn Leu Gin Lys Leu Asn 360 CC AATr Ala Asn 220 Gly Pro Set 300 GAG GACL AulA Ciu Asp Arg 380 CCA CCC GTC Pro Gly Val 460 ACA GTG UTa Thr Val Leu FIGURE 1 TAC CIT ATC AAG Tyr Val Ile Lys 'rGC CTG CCT ACA TCT Cys Leu Pro Thr Set CAC TCT CC cTG Asp Set Ala Leu 420 CILG ACA Leu Arg 440 CAC CTT AAC His Leu Asn 'TTC ATT CCA CAT CTC Phe Ile Arg Asp Leu CAC TTT CCC AAC vA Asp Phe Arg Lys Lys ITC 'TAC ATC GTC Phe Tyr Met Val CATr C GAG Asp Leu Glu 480 CAG CCC Gin Pro 500 Val Set Pro 520 ACC GIG, GAA TId TAA ht Val Cli: Cys. CCC TCT AGA CCA OCI Ala Ser Arg Pro Pro CCA ICT CCC TCC Ala Set Cly Set AAC CGT CCA Asn Arg Gly r "fin' 0 800 600 400 200 FIGURE 2 Fraction Number 0 0 0 0 S -C S p "-i HindHTJ SV4Oori AmpR splice junction Pst I pBR322 ori *G-C tail -Hind III1 clDNA insert poly A FIGURE 3 0 0 0.70 0 0 S MCGF coding region Hpall G-C Tail II Hind IJ BP *ii NcI A-T Tail 100 bp FIGURE 4I