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cDNA clones coding for the complete murine B chain of complement Clq: nucleotide and derived amino acid sequences
Immunology Letters, 17 (1988) 115-120 Elsevier IML 00997
cDNA clones coding for the complete murine B chain of complement Clq: nucleotide and derived amino acid sequences Linda Wood 1, Steve Pulaski 2 and Gabriel Vogeli 1 tMolecular Biology Research 7242, 267-5, and 2Biopolymer Research, 7240, 209-7, The Upjohn Company, Kalamazoo, Michigan, U.S.A. (Received 18 May 1987; revision received 11 September 1987; accepted 29 September 1987)
1. Summary We have isolated cDNA clones covering the complete B chain of the complement subunit Clq from mouse; this subunit initiates the classical complement pathway. Deoxynucleotide sequence analysis shows that these clones contain 156 nucleotides of the 5' untranslated region, followed by sequences coding for the 25 amino acids of the signal peptide, all of the 228 amino acids of the mature protein and 140 nucleotides of the 3' untranslated region, including a poly A addition signal. The coding region for the mature protein contains 261 nucleotides for the G I y - X - Y repeat and 408 nucleotides for the globular portion of Clq. By comparing the nucleotide sequence of the mouse B chain of Clq with the B chain from human (Reid, K. B. M., 1985, Biochem. J. 231, 729), we find a high homology (80070) within the mature protein, a lower homology within the signal peptide (59070) and the 3' untranslated region (47070) and no homology (2607o) in the 5' untranslated region.
2. Introduction Cell lysis by the classical complement cascade is activated not only during the immunological defense against foreign cells, but also to the detriment Key words: Mouse; Complement; B-chain of Clq; Signal peptide; EHS tumor; DNA sequencing Correspondence to: L. Wood, Molecular Biology Research 7242, 267-5, The Upjohn Company, Kalamazoo, MI 49001, U.S.A.
of the organism in many diseases including various autoimmune disorders, rheumatoid arthritis and allograft rejection [1]. Clq, the first molecule in the classical complement cascade, is composed of a total of 18 subunits (6 chains each of subunit A, B and C). The N-terminal collagen domain from three subunits (one A, one B and one C) combine to form a collagen triple helix. The collagen triple helices of 6 molecules assemble to form the central collagen fibril of Clq. The classical complement reaction is initiated when the globular domain at the Cterminus of the Clq subunit binds to the Fc portion of immunoglobulins that have previously aggregated on the cell surface. The binding of the Clr2- Cls2 complex to the collagen domain of Clq follows, forming the complete C1 molecule, which then leads to the rest of the classical complement cascade and to cell lysis. Our goal is to study small peptides derived from Clq that act as immunosuppressors [2], in the hope of finding peptides that interfere with the binding of Clq to cell bound antibodies in order to suppress only that part of the immune system that is involved in the classical complement reaction (e.g. graftversus-host reaction). However, in order to initiate such studies using an animal model, the detailed structure of the molecules involved has to be known. While the nucleotide and the amino acid sequence for most of the subunits of Clq from human have been reported [3], no data is available from rodents. Here we report the isolation and characterization of several cDNA clones specific for the 5' and 3' untranslated regions, the signal peptide and the mature B subunit of Clq from mouse.
0165-2478 / 88 / $ 3.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
1 15
3. Materials and Methods
Results and Discussion
Standard methods were used for the cloning and sequencing of the cDNA clones [4]. In short, cDNA clones were isolated from a mouse Englebreth-Holm- Swarm (EHS) tumor [5] cDNA library [6, 7] by screening with three synthetic oligonucleotides, LW-1 (CTCGCCCTTGGTGCCATTGCAGCCAGGGATGCCAGGGGGGCC) and LW-8 (GGGGCCAGGGGTGCCAGGGATGCCAGGGAAGCCCCGCTCGCC), which are specific for amino acids nos. 88-101 and 146-159, respectively, of the G I y - X - Y repeat of the 7S region from the alpha 1 (IV) collagen chain [8], and LW-26 (TGGGCATAGTCACAGAAGGTGACTACTTTCTGCATGCTGTCC), which is spefific for the 3' end of the cDNA clone pCIq-B-C301 (See Figs. 1 and 2). The filters were hybridized in 10°70 dextran sulfate, 0.9 M NaCI, 0.2 M Tris HC1 pH 7.2, 5 mM EDTA, Denhardt's solution and 1% SDS at 55 °C for 16 h and then washed in 0.1 x SSC (15 mM NaC1, 1.5 mM Na-Citrate, pH 7.2), 1°70 SDS at 37°C. Tetracycline-resistant colonies that hybridized with both probes were picked and purified. Plasmid DNA was isolated [9] and the cDNA inserted into the PstI site of pBR322 was subcloned into M13mpl8 for sequencing by the dideoxy method [10]. To determine the cellular origin of the mRNA that gave rise to the cDNA clones from the mouse Englebreth- H o l m - Swarm (EHS) tumor tissue, a lambda gtll library made from RNA isolated from macrophages was purchased (ML1005, Clonetech Laboratories, Inc., Palo Alto, CA) and screened under the above conditions following the manufacturer's recommendations.
4.1. Isolation of cDNA clonesfor the B-chain of CIq We have isolated and characterized 5 cDNA clones covering the complete B chain of Clq (See Fig. 1: pClq-B-C65, pCIq-B-C61, pClq-B-C301, pCIq-B-C56, pClq-B-C78) by screening a total of 390,000 independent cDNA colonies from an EHS cDNA library. The cDNA library [6] had been made by oligo-dT priming of mRNA isolated from a murine basement membrane tumor (Englebreth- H o l m - Swarm tumor, see Section 3), that is not expected to synthesize mRNA for Clq. At low hybridization stringency, the oligonucleotides designed to recognize alpha 1 (IV) collagen specific DNA sequences also recognized the typical collagenous G l y - X - Y tripeptide repeats found in the B-chain of Clq. Since we did not find mRNA for the B-chain of Clq in ceils derived from the EHS tumor tissue (unpubl. results), we assume that the cDNA clones for the B-chain of Clq stem from contaminating cells, possibly macrophages (see below).
5' UNTRANSLATED SIGNAL PEPTIDE I
COLLAGENDOMAIN
4.2. Deoxynucleotide sequence analysis of the Bchain of Clq The DNA sequencing strategy is shown in Fig. 1. The deoxynucleotide sequence and the derived amino acid sequence is shown in Fig. 2 and is compared with the published human sequence [3]. 4.3. 5' and3' untranslated region and signal peptide The 5' untranslated region contained within our cDNA clones is 156 nucleotides long; the native 5' untranslated region is probably longer. The DNA sequence shows 5 stop codons, two in frame, preceding
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Fig. 1. cDNA clones for the murine B-chain of Clq and sequencing strategy.
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Fig. 2. D N A and derived a m i n o acid sequence of the murine B-chain of Clq compared with the sequence from h u m a n [3]. The amino acids are numbered following Reid [3]. For the h u m a n , only the nucleotides and the a m i n o acids that differ are indicated. The signal sequence is boxed in thick line, the collagen d o m a i n in thin line. The additional Gly in the murine sequence are boxed (thick line).
the single initiation codon. In addition, two other stop codons that are not in frame (TGA TGA) follow each other and include the nucleotides that form the initiation codon ATG. The adjacent signal peptide contains the expected elements [ll]: a charged residue (Lys) within the first 5 amino acids, a helix breaking residue (Gly) eight amino acids before the cleavage site and an Ala at the end of cleavage site. There seems to be no homology (26°70) between the human and the mouse sequence in the 5' untranslated region, whereas the homology in the signal peptide (59°7o) and in the 3' untranslated region (47 070)is substantially higher. That these differences are not due to a cDNA cloning artifact is demonstrated by the fact that 3 different cDNA clones (See Fig. 1) confirmed the mouse sequence. These differences might reflect species differences; however, one would expect the homology in the 5' untranslated region to be similar to the homology in the 3' untranslated region. There are other explanations for the differences between the routine and the human sequence. The sequencing data for the human B-chain [3] was derived in part from genomic clones, thus part of the discrepancies could be a problem of detecting the correct splicing sites within the exon/intron structure of the 5' untranslated region of the gene. The possibility also exists that tissue-specific RNA splicing mechanisms produce different 5' untranslated regions, as has been shown for other pro'teins [12]. 4.4. Mature protein The cDNA clones cover 684 nucleotides coding for all 228 amino acids of the mature protein, two amino acids more than found in the human [3]. The homology between the murine and the human sequence of the mature protein is 80°7o(see Fig. 2). The major difference in the globular domain between the murine and the human is that there are two amino acids missing (in positions B162 and B163, boxed in Fig. 2) in the human sequence. Within the collagenous domain that is shaded in Fig. 2, two amino acid changes (boxes within the shaded area in Fig. 2) affect the rigid G I y - X - Y backbone of the collagen triple helix. The Gly in position B9 is replaced by an Ala in the human and the Gly in position B90 by a Lys. Therefore, the mouse sequence contains two additional G I y - X - Y repeats making the murine collagenous domain more stable [13]. 118
It has been proposed that two different genes code for two different forms of Clq (a serum form and a fibroblast form) [14], thus it could be possible that the murine B-chain isolated from the EHS tumor is the fibroblast form. Against this notion are several facts: first, the calculated molecular weight of the murine B-chain of Clq is 23 740 and it is 23 720 for the serum B-chain of Clq for the human. The molecular weight for the mouse is in agreement with published data concerning the molecular weight of mouse serum Clq [15]. Second, to analyze the origin of the murine clones further, we compared them with a cDNA clone isolated from a cDNA library made from murine macrophage RNA. Preliminary sequendng data (data not shown) established that the macrophage B-chain of Clq is identical to the Bchain of Clq isolated from the EHS cDNA library. It has been previously shown that serum Clq is similar to macrophage Clq [16]. Therefore, we conclude that our Clq was derived from normal serum Clq and not the other Clq-like molecule synthesized in fibroblasts, which has a higher molecular weight [17].
Acknowledgements We would like to thank Lee Giordano for her help with bacterial supplies and Paul Kaytes and Kathy Hiestand for help with the manuscript.
References [1] Reid, K. B. M. (1983) Biochem. Soc. Trans. 11, 1-12. [2] Ninnemann, J. L. and Ozkan, A. N. (1987) J. Trauma 27, 119-122. [3] Reid, K. B. M. (1985) Biochem. J. 231, 729-735. [4] Maniatis, T., Fritsch, E. E and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, NY. [5] Orkin, R. W., Gehron, P., McGoodwin, E. B., Martin, G. R., Valentine, T. and Swarm, R. (1977) J. Exp. Med. 145, 204 - 220. [61 Nath, P., Laurent, M., Horn, E., Sobel, M. E., Zon, G. and Vogeli, G. (1986) Gene 43, 301-304. [7] Vogeli, G., Horn, E., Laurent, M., Nath, E (1985) Anal. Biochem. 151, 442-444. [8] Glanville, R. W., Qian, R., Siebold, B., Risteli, J. and Kuhn, K. 0985) Eur. J. Biochem. 152, 213-219. [9] Birnboim, H. C. and Doly, J. (1979) Nucleic Acids Res. 7, 1513-1523.
[10] Sanger E, Nieklen S. and Coulson A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463- 5467. [11] Watson, M. E. E. (1984) Nucleic Acids Res. 13, 5145-5164. [12] Shaw, P., Sordat, B. and Schibler, U. (1985) Cell 40, 907- 912. [13] Piez, K. A. and Reddi, A. H. (1984) Extracellular Matrix Biochemistry, Elsevier, Amsterdam, New York. [14] Skok, J., Solomon, E., Reid, K. B. M. and Thompson, R.
A. (1981) Nature 292, 549-551. [15] Yonemasu K. and Sasaki, T. (1981) Biochem. J. 193, 621 - 629. [16] Rabs, U., Martin, H., Hitschold, T., Golan, M. D., Heinz, H - P . and Loos, M. (1986) Eur. J. Immunol. 16, 1183-1186. [17] Solomon, E. and Reid, K. B. M. (1977) Biochem J. 167, 647 - 660.
119
cDNA clones coding for the complete murine B chain of complement Clq: nucleotide and derived amino acid sequences Linda Wood 1, Steve Pulaski 2 and Gabriel Vogeli 1 tMolecular Biology Research 7242, 267-5, and 2Biopolymer Research, 7240, 209-7, The Upjohn Company, Kalamazoo, Michigan, U.S.A. (Received 18 May 1987; revision received 11 September 1987; accepted 29 September 1987)
1. Summary We have isolated cDNA clones covering the complete B chain of the complement subunit Clq from mouse; this subunit initiates the classical complement pathway. Deoxynucleotide sequence analysis shows that these clones contain 156 nucleotides of the 5' untranslated region, followed by sequences coding for the 25 amino acids of the signal peptide, all of the 228 amino acids of the mature protein and 140 nucleotides of the 3' untranslated region, including a poly A addition signal. The coding region for the mature protein contains 261 nucleotides for the G I y - X - Y repeat and 408 nucleotides for the globular portion of Clq. By comparing the nucleotide sequence of the mouse B chain of Clq with the B chain from human (Reid, K. B. M., 1985, Biochem. J. 231, 729), we find a high homology (80070) within the mature protein, a lower homology within the signal peptide (59070) and the 3' untranslated region (47070) and no homology (2607o) in the 5' untranslated region.
2. Introduction Cell lysis by the classical complement cascade is activated not only during the immunological defense against foreign cells, but also to the detriment Key words: Mouse; Complement; B-chain of Clq; Signal peptide; EHS tumor; DNA sequencing Correspondence to: L. Wood, Molecular Biology Research 7242, 267-5, The Upjohn Company, Kalamazoo, MI 49001, U.S.A.
of the organism in many diseases including various autoimmune disorders, rheumatoid arthritis and allograft rejection [1]. Clq, the first molecule in the classical complement cascade, is composed of a total of 18 subunits (6 chains each of subunit A, B and C). The N-terminal collagen domain from three subunits (one A, one B and one C) combine to form a collagen triple helix. The collagen triple helices of 6 molecules assemble to form the central collagen fibril of Clq. The classical complement reaction is initiated when the globular domain at the Cterminus of the Clq subunit binds to the Fc portion of immunoglobulins that have previously aggregated on the cell surface. The binding of the Clr2- Cls2 complex to the collagen domain of Clq follows, forming the complete C1 molecule, which then leads to the rest of the classical complement cascade and to cell lysis. Our goal is to study small peptides derived from Clq that act as immunosuppressors [2], in the hope of finding peptides that interfere with the binding of Clq to cell bound antibodies in order to suppress only that part of the immune system that is involved in the classical complement reaction (e.g. graftversus-host reaction). However, in order to initiate such studies using an animal model, the detailed structure of the molecules involved has to be known. While the nucleotide and the amino acid sequence for most of the subunits of Clq from human have been reported [3], no data is available from rodents. Here we report the isolation and characterization of several cDNA clones specific for the 5' and 3' untranslated regions, the signal peptide and the mature B subunit of Clq from mouse.
0165-2478 / 88 / $ 3.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
1 15
3. Materials and Methods
Results and Discussion
Standard methods were used for the cloning and sequencing of the cDNA clones [4]. In short, cDNA clones were isolated from a mouse Englebreth-Holm- Swarm (EHS) tumor [5] cDNA library [6, 7] by screening with three synthetic oligonucleotides, LW-1 (CTCGCCCTTGGTGCCATTGCAGCCAGGGATGCCAGGGGGGCC) and LW-8 (GGGGCCAGGGGTGCCAGGGATGCCAGGGAAGCCCCGCTCGCC), which are specific for amino acids nos. 88-101 and 146-159, respectively, of the G I y - X - Y repeat of the 7S region from the alpha 1 (IV) collagen chain [8], and LW-26 (TGGGCATAGTCACAGAAGGTGACTACTTTCTGCATGCTGTCC), which is spefific for the 3' end of the cDNA clone pCIq-B-C301 (See Figs. 1 and 2). The filters were hybridized in 10°70 dextran sulfate, 0.9 M NaCI, 0.2 M Tris HC1 pH 7.2, 5 mM EDTA, Denhardt's solution and 1% SDS at 55 °C for 16 h and then washed in 0.1 x SSC (15 mM NaC1, 1.5 mM Na-Citrate, pH 7.2), 1°70 SDS at 37°C. Tetracycline-resistant colonies that hybridized with both probes were picked and purified. Plasmid DNA was isolated [9] and the cDNA inserted into the PstI site of pBR322 was subcloned into M13mpl8 for sequencing by the dideoxy method [10]. To determine the cellular origin of the mRNA that gave rise to the cDNA clones from the mouse Englebreth- H o l m - Swarm (EHS) tumor tissue, a lambda gtll library made from RNA isolated from macrophages was purchased (ML1005, Clonetech Laboratories, Inc., Palo Alto, CA) and screened under the above conditions following the manufacturer's recommendations.
4.1. Isolation of cDNA clonesfor the B-chain of CIq We have isolated and characterized 5 cDNA clones covering the complete B chain of Clq (See Fig. 1: pClq-B-C65, pCIq-B-C61, pClq-B-C301, pCIq-B-C56, pClq-B-C78) by screening a total of 390,000 independent cDNA colonies from an EHS cDNA library. The cDNA library [6] had been made by oligo-dT priming of mRNA isolated from a murine basement membrane tumor (Englebreth- H o l m - Swarm tumor, see Section 3), that is not expected to synthesize mRNA for Clq. At low hybridization stringency, the oligonucleotides designed to recognize alpha 1 (IV) collagen specific DNA sequences also recognized the typical collagenous G l y - X - Y tripeptide repeats found in the B-chain of Clq. Since we did not find mRNA for the B-chain of Clq in ceils derived from the EHS tumor tissue (unpubl. results), we assume that the cDNA clones for the B-chain of Clq stem from contaminating cells, possibly macrophages (see below).
5' UNTRANSLATED SIGNAL PEPTIDE I
COLLAGENDOMAIN
4.2. Deoxynucleotide sequence analysis of the Bchain of Clq The DNA sequencing strategy is shown in Fig. 1. The deoxynucleotide sequence and the derived amino acid sequence is shown in Fig. 2 and is compared with the published human sequence [3]. 4.3. 5' and3' untranslated region and signal peptide The 5' untranslated region contained within our cDNA clones is 156 nucleotides long; the native 5' untranslated region is probably longer. The DNA sequence shows 5 stop codons, two in frame, preceding
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Fig. 1. cDNA clones for the murine B-chain of Clq and sequencing strategy.
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human A AA A GT C AC C C C NOUSE GTC 6CC TTC TCT GCC CTG AGGACC ATC AN; AGC CCC TTG CGA CCI; AAC rAG GTC ATT CGCTT(: GAA AAG b'TGATC ACC HOUSEi l l J ( A l l l~r(gln Lys 1il All Phe Ser Ala Leu Arg Thr I l e Asn Ser Pro Leu Arg Pro Asn 61n Val 11e Ar8 Phe Glu L~s re1 l l e Thr human~ Ile Thr Val Ar9 Asp Thr Asp Hts BlO0 8110 8120 890 600 * * 630 * * 660 * * human AT AC T C GT C T C C T A G flOUSE AAC 6C6 AAC 6A6 AAC TAT ~ CCA C6C AAC 66C M& TIC ACC T6C ~ 6T6 CCT ~ : CTC TAC TAC TTC ACC TAT CAT 6CC A6C TCC C6G G6(; flOUSE Ash Ala Asn Glu Asn T~r glu Pro Arg Ass Ely LTs Phi lhr C¥s Lys Val Pro ally Leu T~" T~" Phe Thr TJn" His A l l Ser Set Ar8 61y human Met Asn Ser 8130 B140 5150 720 human C C A8 C T GCA G G TC C NOUSE AN; CTG TGT 61"6AAT CTC GTT CGT J~C AIG CAGAAA GTA GTC ACC TTC TGT GAC TAT GCC CA6 AN; ACC TIC CA6 6T6 NOUSE ASh Leu Cys Yal kin Leu Val Arg 61y /Irg~Asp Arg~Asp Ser Net liln Lys Val Val l~r Phe Cys Asp Tyr Ala Gln ASh Thr Phe Gln Val human Met I . . . . . IGlu Ar 9 Ala T~ 8160 I I 8170 8180
780 * * 810 * * 840 * human C CAG CC G 6 G AAC C TT C *A A NOUSE ACC ACA 6GT G66 6TA GTC TTG AAG CTA GAG CAA Ik~G6A6 GTT liTi" CAC CT6 CAG GCCJ ~ ~ ~ ~ ~C ~ ~ ~ HOUSE Thr Thr 61y 61y Val Val Leu Lys Leu &lu 61a 61u lilu Val Val His Leu 61n Ala Thr Asp LJrS Ash S4r Leu Leu Ely human Met Gly Asn Phe 8190 8200
*
870 G A~ ~ ~T ~C ale 61u Ely Ala Met 6210
* * 900 * * 930 * * 960 * human TTC G C C T A T S C G C TGT CTGCTC T . . . . . . . CCGG TCC C TGC CS A HOUSE AAC AGC Arc TTC ACT GGCTIT CTG CTT TIT CCT IUkCAT6 GAT ~ T/~ TCACGGOGTCAMTTACACCTATCCAACACCATCTTCCTGCTCCTGCAGCAATCCTCCC ROUSE ASh Saw l l e Phe Thr Gly Phe Leu Leu Phe Pro ASp Net Asp Ale TER human Ser 61u 8220 8228
* 990 * * 1020 * * 1050 1059 human CT CA C T GCC A AATGCAACAGTAGGCTTGGTGC G T ~T A A CTC... (112 nts) . . . poly A NOUSE TGGACCCCTCACATCACCCCCTTGACTGC CTSNtACCCMACCACAGCCCTGTA~ATGTT~TIIGGTCAATAAA--Poly A Mdltton s|gnal
Fig. 2. D N A and derived a m i n o acid sequence of the murine B-chain of Clq compared with the sequence from h u m a n [3]. The amino acids are numbered following Reid [3]. For the h u m a n , only the nucleotides and the a m i n o acids that differ are indicated. The signal sequence is boxed in thick line, the collagen d o m a i n in thin line. The additional Gly in the murine sequence are boxed (thick line).
the single initiation codon. In addition, two other stop codons that are not in frame (TGA TGA) follow each other and include the nucleotides that form the initiation codon ATG. The adjacent signal peptide contains the expected elements [ll]: a charged residue (Lys) within the first 5 amino acids, a helix breaking residue (Gly) eight amino acids before the cleavage site and an Ala at the end of cleavage site. There seems to be no homology (26°70) between the human and the mouse sequence in the 5' untranslated region, whereas the homology in the signal peptide (59°7o) and in the 3' untranslated region (47 070)is substantially higher. That these differences are not due to a cDNA cloning artifact is demonstrated by the fact that 3 different cDNA clones (See Fig. 1) confirmed the mouse sequence. These differences might reflect species differences; however, one would expect the homology in the 5' untranslated region to be similar to the homology in the 3' untranslated region. There are other explanations for the differences between the routine and the human sequence. The sequencing data for the human B-chain [3] was derived in part from genomic clones, thus part of the discrepancies could be a problem of detecting the correct splicing sites within the exon/intron structure of the 5' untranslated region of the gene. The possibility also exists that tissue-specific RNA splicing mechanisms produce different 5' untranslated regions, as has been shown for other pro'teins [12]. 4.4. Mature protein The cDNA clones cover 684 nucleotides coding for all 228 amino acids of the mature protein, two amino acids more than found in the human [3]. The homology between the murine and the human sequence of the mature protein is 80°7o(see Fig. 2). The major difference in the globular domain between the murine and the human is that there are two amino acids missing (in positions B162 and B163, boxed in Fig. 2) in the human sequence. Within the collagenous domain that is shaded in Fig. 2, two amino acid changes (boxes within the shaded area in Fig. 2) affect the rigid G I y - X - Y backbone of the collagen triple helix. The Gly in position B9 is replaced by an Ala in the human and the Gly in position B90 by a Lys. Therefore, the mouse sequence contains two additional G I y - X - Y repeats making the murine collagenous domain more stable [13]. 118
It has been proposed that two different genes code for two different forms of Clq (a serum form and a fibroblast form) [14], thus it could be possible that the murine B-chain isolated from the EHS tumor is the fibroblast form. Against this notion are several facts: first, the calculated molecular weight of the murine B-chain of Clq is 23 740 and it is 23 720 for the serum B-chain of Clq for the human. The molecular weight for the mouse is in agreement with published data concerning the molecular weight of mouse serum Clq [15]. Second, to analyze the origin of the murine clones further, we compared them with a cDNA clone isolated from a cDNA library made from murine macrophage RNA. Preliminary sequendng data (data not shown) established that the macrophage B-chain of Clq is identical to the Bchain of Clq isolated from the EHS cDNA library. It has been previously shown that serum Clq is similar to macrophage Clq [16]. Therefore, we conclude that our Clq was derived from normal serum Clq and not the other Clq-like molecule synthesized in fibroblasts, which has a higher molecular weight [17].
Acknowledgements We would like to thank Lee Giordano for her help with bacterial supplies and Paul Kaytes and Kathy Hiestand for help with the manuscript.
References [1] Reid, K. B. M. (1983) Biochem. Soc. Trans. 11, 1-12. [2] Ninnemann, J. L. and Ozkan, A. N. (1987) J. Trauma 27, 119-122. [3] Reid, K. B. M. (1985) Biochem. J. 231, 729-735. [4] Maniatis, T., Fritsch, E. E and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, NY. [5] Orkin, R. W., Gehron, P., McGoodwin, E. B., Martin, G. R., Valentine, T. and Swarm, R. (1977) J. Exp. Med. 145, 204 - 220. [61 Nath, P., Laurent, M., Horn, E., Sobel, M. E., Zon, G. and Vogeli, G. (1986) Gene 43, 301-304. [7] Vogeli, G., Horn, E., Laurent, M., Nath, E (1985) Anal. Biochem. 151, 442-444. [8] Glanville, R. W., Qian, R., Siebold, B., Risteli, J. and Kuhn, K. 0985) Eur. J. Biochem. 152, 213-219. [9] Birnboim, H. C. and Doly, J. (1979) Nucleic Acids Res. 7, 1513-1523.
[10] Sanger E, Nieklen S. and Coulson A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463- 5467. [11] Watson, M. E. E. (1984) Nucleic Acids Res. 13, 5145-5164. [12] Shaw, P., Sordat, B. and Schibler, U. (1985) Cell 40, 907- 912. [13] Piez, K. A. and Reddi, A. H. (1984) Extracellular Matrix Biochemistry, Elsevier, Amsterdam, New York. [14] Skok, J., Solomon, E., Reid, K. B. M. and Thompson, R.
A. (1981) Nature 292, 549-551. [15] Yonemasu K. and Sasaki, T. (1981) Biochem. J. 193, 621 - 629. [16] Rabs, U., Martin, H., Hitschold, T., Golan, M. D., Heinz, H - P . and Loos, M. (1986) Eur. J. Immunol. 16, 1183-1186. [17] Solomon, E. and Reid, K. B. M. (1977) Biochem J. 167, 647 - 660.
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