Spontaneous Classical Pathway Activation and Deficiency of Membrane Regulators Render Human Neurons Susceptible to Complement Lysis
2000; Elsevier BV; Volume: 157; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64604-4
ISSN1525-2191
AutoresSim K. Singhrao, James Neal, Neil K. Rushmere, B. Paul Morgan, Philippe Gasque,
Tópico(s)Cellular transport and secretion
ResumoThis study investigated the capacity of neurons and astrocytes to spontaneously activate the complement system and control activation by expressing complement regulators. Human fetal neurons spontaneously activated complement through the classical pathway in normal and immunoglobulin-deficient serum and C1q binding was noted on neurons but not on astrocytes. A strong staining for C4, C3b, iC3b neoepitope and C9 neoepitope was also found on neurons. More than 40% of human fetal neurons were lysed when exposed to normal human serum in the presence of a CD59-blocking antibody, whereas astrocytes were unaffected. Significant reduction in neuronal cell lysis was observed after the addition of soluble complement receptor 1 at 10 μg/ml. Fetal neurons were stained for CD59 and CD46 and were negative for CD55 and CD35. In contrast, fetal astrocytes were strongly stained for CD59, CD46, CD55, and were negative for CD35. This study demonstrates that human fetal neurons activate spontaneously the classical pathway of complement in an antibody-independent manner to assemble the cytolytic membrane attack complex on their membranes, whereas astrocytes are unaffected. One reason for the susceptibility of neurons to complement-mediated damage in vivo may reside in their poor capacity to control complement activation. This study investigated the capacity of neurons and astrocytes to spontaneously activate the complement system and control activation by expressing complement regulators. Human fetal neurons spontaneously activated complement through the classical pathway in normal and immunoglobulin-deficient serum and C1q binding was noted on neurons but not on astrocytes. A strong staining for C4, C3b, iC3b neoepitope and C9 neoepitope was also found on neurons. More than 40% of human fetal neurons were lysed when exposed to normal human serum in the presence of a CD59-blocking antibody, whereas astrocytes were unaffected. Significant reduction in neuronal cell lysis was observed after the addition of soluble complement receptor 1 at 10 μg/ml. Fetal neurons were stained for CD59 and CD46 and were negative for CD55 and CD35. In contrast, fetal astrocytes were strongly stained for CD59, CD46, CD55, and were negative for CD35. This study demonstrates that human fetal neurons activate spontaneously the classical pathway of complement in an antibody-independent manner to assemble the cytolytic membrane attack complex on their membranes, whereas astrocytes are unaffected. One reason for the susceptibility of neurons to complement-mediated damage in vivo may reside in their poor capacity to control complement activation. Complement is part of the body's natural immune defense system and comprises >30 secreted and cell-bound proteins. Some of these proteins belong to the complement pathways whereas others function as regulators. Complement activation occurs in a sequential manner via the classical pathway involving C1 (C1q, C1r, C1s), C4, C2, and C3, or the alternative pathway involving C3, factor B, factor D, and properdin.1Pillemer L Blum L Lepow IH Ross OA Todd EW Wardlaw AC The properdin system and immunity. I. Demonstration of a new serum protein, properdin and its role in immune phenomena.Science. 1954; 120: 279-285Crossref PubMed Scopus (419) Google Scholar, 2Lachmann PJ Hughes-Jones NC Initiation of complement activation.Springer Semin Immunopathol. 1984; 7: 143-162Crossref PubMed Scopus (121) Google Scholar One of the most remarkable properties of complement is its capacity to lyse targeted organisms, such as bacteria, viruses, and parasites.3Nuttall G Experimente ueber die Bakterienfeind-lichen. 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These regulators can be fluid-phase proteins such as C1 inhibitor, C4b binding protein, factor H, factor I, S-protein, and clusterin, or membrane-bound proteins such as membrane cofactor protein (MCP, CD46), decay accelerating factor (DAF, CD55), complement receptor 1 (CR1, CD35), and CD59.6Meri S Jarya H Complement regulation.Vox Sang. 1998; 74: S291-S302Crossref PubMed Scopus (56) Google Scholar, 7Morgan BP Harris C Regulation in the activation pathways—membrane RCA proteins.in: Complement Regulatory Proteins. Academic Press, San Diego1999: 82-120Google Scholar, 8Morgan BP Harris C Regulation in the terminal pathway—membrane regulators of the terminal pathway.in: Complement Regulatory Proteins. Academic Press, San Diego1999: 153-165Google Scholar Transudation of complement proteins through a damaged blood-brain barrier can contribute to the deposition of potentially cytolytic components of the complement pathway on the surface of neurons and glial cells. In support of this, a number of reports exist implicating complement-mediated damage in the etiology of neurodegenerative disease, demyelinating disease, and ischemic stroke.9Eikelenboom P Stam FC Immunoglobulins and complement factors in senile plaques. 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Others have addressed the expression of complement regulatory proteins on primary cultures of human brain cells in vitro but have concentrated on astrocytes25Gordon DL Sadlon TA Wesselinghe SL Russel SM Johnstone RW Purcell DFJ Human astrocytes express membrane cofactor protein (CD46), a regulator of complement activation.J Neuroimmunol. 1992; 36: 199-208Abstract Full Text PDF PubMed Scopus (35) Google Scholar, 26Vedeler C Ulvestad E Bjorge L Conti G Williams K Mork S Matre R The expression of CD59 in normal human nervous tissue.Immunol. 1994; 82: 542-547PubMed Google Scholar and oligodendrocytes.27Zajicek J Wing M Skepper J Compston A Human oligodendrocytes are not sensitive to complement.Lab Invest. 1995; 73: 128-138PubMed Google Scholar, 28Scolding NJ Morgan BP Compston DAS The expression of complement regulatory proteins by adult human oligodendrocytes.J Neuroimmunol. 1998; 84: 69-75Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar It is reported that astrocytes express MCP, CD59, and low levels of DAF whereas human oligodendrocytes express only DAF.28Scolding NJ Morgan BP Compston DAS The expression of complement regulatory proteins by adult human oligodendrocytes.J Neuroimmunol. 1998; 84: 69-75Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar The capacity of rat oligodendrocytes to directly activate the complement system in vitro has long been reported29Scolding NJ Morgan BP Houston WAJ Linington C Campbell AK Compston DAS Vesicular removal by oligodendrocytes of membrane attack complexes formed by activated complement.Nature. 1989; 339: 620-622Crossref PubMed Scopus (204) Google Scholar, 30Scolding NJ Morgan BP Houston A Campbell AK Linington C Compston DAS Normal rat serum cytotoxicity, against syngeneic oligodendrocytes. 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Human neuroblastoma cell lines (IMR32, SKN-SH, differentiated NT2, and SH-SY5Y) have been reported to spontaneously activate the classical pathway of complement.33Gasque P Thomas A Fontaine M Morgan BP Complement activation on human neuroblastoma cell lines in vitro: route of activation and expression of functional complement regulatory proteins.J Neuroimmunol. 1996; 66: 29-40Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 34Agoropoulou C Wing MG Wood A CD59 expression and complement susceptibility of human neuronal cell line (Nterra2).Neuroreport. 1996; 7: S997-S1004Crossref PubMed Scopus (12) Google Scholar, 35Shen Y Halperin JA Lee C-M Complement-mediated neurotoxicity is regulated be homologous restriction.Brain Res. 1995; 671: 282-292Crossref PubMed Scopus (43) Google Scholar The majority of these cell lines expressed low levels of CD59, lacked DAF, and were consequently lysed when cultured in the presence of human serum. The SH-SY5Y line expressed CD59 and was lysed by serum only after enzymatic removal of CD59.35Shen Y Halperin JA Lee C-M Complement-mediated neurotoxicity is regulated be homologous restriction.Brain Res. 1995; 671: 282-292Crossref PubMed Scopus (43) Google Scholar Primary cultures of human neurons have not been investigated for their ability to spontaneously activate the complement system and to control complement activation by expressing complement regulators. We have performed a systematic investigation to test whether human fetal neurons spontaneously activate the complement system. We also assessed their capacity to inhibit the complement cascade at the C3/C5 convertase stage and at the stage of MAC formation by expressing membrane-bound complement regulators. We extended our study to characterize the expression of complement regulatory proteins at the mRNA level using both reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) on human fetal brain cells. Fresh human fetal brain tissue from 10 samples (10- to 12-week-old fetus) was received in Hanks' buffer from the Medical Research Council Tissue Bank (Dr. Wong, Hammersmith Hospital, London, UK) and used for cell cultures following local ethical guidance (BRO TAF Health Authority, Ref 98/2773). The brain tissue was chopped into 1-cm3 pieces, washed twice in fresh Hanks' buffer (Sigma, Poole, Dorset, UK), and pelleted by centrifugation at 1,000 rpm. The tissue was digested in 0.05% trypsin (Sigma) in Hanks' buffer at 37°C for 15 minutes with constant agitation and the mixture was treated with DNase I (0.2 μg/ml final; Sigma) for 1 minute. Cells were thoroughly dissociated using a 3-ml plastic Pasteur pipette and finally resuspended in complete medium that consisted of Dulbecco's modified Eagle's medium (Gibco Life Technologies, Paisley, UK) supplemented with heat inactivated 10% fetal calf serum, 4 mmol/L glutamine, 2.5 μg/ml fungizone, 2 mmol/L sodium pyruvate, 100 IU/ml penicillin, and 100 IU/ml streptomycin. The cells were filtered through a 70-μm nylon cell strainer (Becton-Dickinson, Cowley, Oxford, UK) and pelleted by centrifugation at 1,500 rpm for 20 minutes. The cell pellet was resuspended in fresh complete medium further supplemented with 2.5 mmol/L KCl to encourage neuronal cell growth.36Bolanos JP Heales SJR Land JM Clark JB Effect of peroxynitrite on the mitochondrial respiratory chain: differential susceptibility of neurons and astrocytes in primary culture.J Neurochem. 1995; 64: 1965-1972Crossref PubMed Scopus (479) Google Scholar Cells were seeded at 107cells/10 ml in 25-cm2 culture flasks coated with 10 μg/ml poly-l-lysine (Sigma). Primary mixed cell cultures were also seeded on coverslips. Coverslips were first coated with a 2% solution of 3-aminopropyltriethoxysilane (Sigma) in acetone for 5 minutes followed by poly-l-lysine as for flasks. Coverslips were individually placed into 12-well plates. Cells in complete medium (200 μl, 106cells/ml) were transferred onto each coverslip and incubated in 95% air/5% CO2in a humidified incubator at 37°C. The sources of polyclonal antibodies were: rabbit anti-C1q (OTNT05) from Behring Diagnostics (Hamburg, Germany); rabbit anti-C4 from Sigma; rabbit anti-C3c obtained as a gift from Dr. M. Fontaine (INSERM U519, Rouen, France); and rabbit anti-factor B from Serotec (Oxford, UK). The mouse monoclonal antibodies (mAb) against complement were: anti-iC3b neoepitope from Quidel (San Diego, CA); clone C3/30 anti-C3b, a gift from Dr. P. W. Taylor (Ciba-Geigy Ltd., Horsham, UK); and mAb clone B7, anti-C9 neoepitope (raised in-house). The mouse anti-C1q (clone 12A5B7) hybridoma was purchased from the American Type Culture Collection (Rockville, MD). Rabbit anti-glial fibrillary acidic protein (GFAP, code B5) and mouse mAb anti-GFAP (clone MCAB5.2E4) were from Dr. J. Newcombe (Mutiple Sclerosis Society Laboratory, London, UK). Rabbit antisera against CR1, DAF, and MCP were all raised in-house using highly purified or recombinant proteins as immunogens. The specificity of all antibodies against complement components and complement regulators was further tested by Western blot analysis using either human serum or cell lines such as HeLa, THP1, K562, CB193, and IMR32.37Singhrao SK: Expression and role of complement in neurodegeneration. Thesis for Doctor of Philosophy (Ph.D.), 1999, University of WalesGoogle Scholar Mouse mAb anti-MCP (clone GB24) was from Professor J. P. Atkinson (Washington University School of Medicine, St. Louis, MO). Mouse mAb OX23 anti-human complement factor H was from Dr. R. B. Sim (Medical Research Council Unit, Oxford, UK). Mouse mAb anti-neuron-specific enolase (NSE) clone BBS/NC/VI-H14 was from DAKO Ltd., (Milton Keynes, UK). Mouse mAb anti-NCAM (CD56) clone MY31 was from Becton-Dickinson. Mouse mAbs anti-CD44 (clone BRIC 222), anti-DAF (CD55) (BRIC 216), and anti-CD59 (BRIC 229) were all purchased from the International Blood Group Reference Laboratory (Oxford, UK). Rabbit anti-kappa (κ) and rabbit anti-lambda (λ) antisera were purchased from ICN Pharmaceuticals Ltd. (Oxford, UK). Mixed human fetal brain cells were cultured on glass coverslips in a 12-well plate. Neurons were easily identified as small rounded cells co-culturing in the presence of astrocytes (large flat cells) and their identity was confirmed by immunocytochemistry using specific cell markers (anti-NSE and anti-GFAP) as described previously.38Gasque P Singhrao SK Neal JW Gotze O Morgan BP Expression of the receptor for complement C5a (CD88) is upregulated on reactive astrocytes, microglia, and endothelial cells in inflamed human central nervous system.Am J Pathol. 1997; 150: 31-41PubMed Google Scholar, 39Gasque P Jones J Singhrao SK Morgan BP Identification of an astrocyte cell population from human brain that expresses perforin, a cytotoxic protein implicated in immune defense.J Exp Med. 1998; 187: 451-460Crossref PubMed Scopus (34) Google Scholar Cells were loaded for 1 hour at 37°C with the green fluorescent dye calcein.AM (Molecular Probes, Eugene, OR) diluted in complete medium to 2 μg/ml final concentration. Inside the cell, calcein.AM is de-esterified to a polar fluorescent product which is retained within intact cells and released only after membrane damage (cell killing). Cells were washed three times in 0.9% sterile saline (tissue culture grade) and incubated in veronal buffer (VBS; Oxoid Ltd., Basingstoke, UK) containing 1% bovine serum albumin (BSA) and PI at 10 μg/ml final concentration for 30 minutes or 1 hour at 37°C containing the following: 1) normal human serum (NHS) diluted (1/4 or 1/8); 2) heat-inactivated NHS (inactivation at 56°C for 30 minutes) at the same dilutions; 3) NHS diluted 1/8 in VBS/BSA to which was added the noncomplement-fixing but neutralizing antibody against CD59 (mouse IgG2b isotype, clone BRIC229) at 13 μg/ml; 4) VBS/BSA containing mouse anti-CD59 antibody (clone BRIC229) at 13 μg/ml; and 5) NHS diluted 1/8 in VBS/BSA containing mouse anti-CD59 antibody (clone BRIC229) at 13 μg/ml and soluble complement receptor 1 (sCR1; T cell Sciences, Needham, UK) at 10 μg/ml final concentration. The coverslips were inverted onto prelabeled glass microscope slides and the cells examined under a fluorescent microscope (Leica UK Ltd., Milton Keynes, UK) using the fluorescein isothiocyanate filter for the calcein signal and the rhodamine filter for the PI signal. Lysed cells were PI-positive (depicted as white spherical cells) and calcein-negative (because of leakage of the green fluorochrome). Viable cells were green fluorescent (visualized as gray cells) because of calcein retention and PI-negative and are illustrated in Figure 2A. Mixed human fetal brain cells cultured on coverslips were incubated for 30 minutes at 37°C with NHS diluted 1/16 in VBS/1%BSA or with NHS deficient in IgA, IgM, and IgG (Sigma) diluted 1/8. Controls included heat-inactivated NHS and NHS containing 10 mmol/L ethylenediaminetetraacetic acid (EDTA). The cells were thoroughly washed five times in phosphate-buffered saline (PBS), pH 7.3, and fixed in acetone for 5 minutes at room temperature followed by further washes (5 times) in PBS. All coverslips were immersed for 30 minutes in PBS/BSA to block the nonspecific-antibody binding. The coverslips were incubated in 100 μl of the appropriate dilution of primary antibody (anti-κ and anti-λ light chains, anti-complement, or anti-complement regulatory protein) at 4°C overnight in a humidity chamber. The cells on coverslips were thoroughly washed (10 times) in PBS and incubated for 1 hour at room temperature in the specific secondary peroxidase conjugate (Bio-Rad, Hemel Hempstead, UK) diluted 1/100 in PBS/BSA. Cells were washed 10 times in PBS after which they were developed for 5 minutes in a freshly-made solution of 0.05% diaminobenzidine (DAB) and 0.005% (v/v) hydrogen peroxide diluted in PBS. After a brief wash in PBS, the cells were washed thoroughly in water before and after counterstaining in hematoxylin. After either a full dehydration in ethanol or air-drying (37°C oven), the cells were cleared in xylene and the coverslips were mounted on glass slides. The level of immunostaining on fetal brain cells using anti-complement antibodies was assessed by semiquantitative image analysis (Openlab/Improvision, Coventry, UK). The salient instrumentation of the system included a color digital camera mounted on a light microscope and connected to a computer based image analysis system. Briefly, the method involved recording a series of images of the areas of interest (neurons and astrocytes) in the sample and measuring the amount of DAB (brown) staining of gated cells from 10 random but representative fields in each sample. The data were sorted and expressed as a mean ±SEM of the measurements (staining index). For double-immunocytochemistry, antibodies to GFAP and anti-complement regulatory protein derived from different species were simultaneously applied to cells on coverslips. The secondary conjugates specific for each of the primary antibodies were peroxidase-conjugated goat anti-mouse/rabbit immunoglobulins diluted 1/100 (Bio-Rad) and alkaline phosphatase conjugated goat anti-mouse/rabbit immunoglobulins diluted 1/500 (Sigma). The substrates were: 0.05% DAB/0.005% hydrogen peroxide diluted in PBS and nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP). NBT stock solution was at 75 mg/ml dissolved in 70% N-N-dimethyl formamide and BCIP stock solution was at 50 mg/ml dissolved in N-N-dimethyl formamide. A fresh solution of alkaline phosphatase substrate was prepared by adding 4.5 μl of stock NBT and 3.5 μl BCIP per 1 ml of detection buffer (0.1 mol/L Tris-HCl, 0.1 mol/L NaCl, 50 mmol/L MgCl2, pH 9.5) to which 0.3% levamisole (Sigma) was added. The co-localization of the brown (DAB) and blue (NBT/BCIP) product was identified on the cells. Representative bright-field images were photographed using a Leica DMLB microscope (Leica UK Ltd.). The detection of membrane-bound complement regulators (CR1, DAF, MCP, CD59) and cell markers (CD44, CD56) on mixed human fetal brain cells was assessed by flow cytometry. All steps were performed on ice. Cultured adherent fetal brain cells were washed three times in sterile NaCl (0.9%) and harvested using 10 mmol/L EDTA in PBS/BSA. The cells (105 cells/tube) were incubated in primary antibody at the appropriate dilution in PBS/BSA for 1 hour, washed three times in PBS/BSA by centrifugation (1,000 rpm for 3 minutes) before incubation for 1 hour with the secondary red phycoerythrin-conjugated antibody (DAKO, High Wycombe, Bucks, UK) diluted in PBS/BSA. The cells were washed three times in PBS/BSA as before and analyzed on a flow cytometer (Becton-Dickinson). Two distinct populations present in the mixed human fetal brain were identified from the cell scatter (FSC/SSC plot). The small cells (38 to 40% of the total cell population) were CD56high, CD44dim, and GFAPnegative and were identified as human fetal neurons. The large cells were CD56high, CD44high, and GFAPhigh and represented the fetal astrocyte population as already described.40Haegel H Tolg C Hofmann M Ceredig R Activated mouse astrocytes and T cells express similar CD44 variants. Role of CD44 in astrocyte/T cell binding.J Cell Biol. 1993; 122: 1067-1077Crossref PubMed Scopus (66) Google Scholar, 41Moretto G Xu RY Kim SU CD44 expression in human astrocytes and oligodendrocytes in culture.J Neuropathol Exp Neurol. 1993; 52: S419-S423Crossref PubMed Scopus (58) Google Scholar Total RNA was isolated from four different samples of human fetal brain primary cell cultures using the Ultraspec RNA isolating reagent according to the manufacturer's instructions (Biotecx Labs., Houston, TX). Reverse transcription (RT) was performed at 37°C for 2 hours using 3 μg of total RNA in the presence of 50 mmol/L Tris-HCl, 75 mmol/L KCl, 3 mmol/L MgCl2, 5 μmol/L dithiothreitol, 60 U rRNasin, and 2 mmol/L dNTPs and MMLV in a total volume of 30 μl. A 3-μl aliquot of RT reaction was used for polymerase chain reaction (PCR) using specific oligonucleotide pairs (see below) for each of the complement regulators. Their gene target, primer sequence, and the predicted size of each cDNA product are: MCP (GCTACCTGTCTCAGATGACG) (ACCACTTTACACTCTGGAGC) 419 bp; CR1 (TGGCATGGTGCATGTGATCA) (TCAGGGCCTGGCACTTCACA) 514 bp; DAF (GCAACACGGAGTACACCTGT) (GCTAAGAATGTGATTCCAGG) 360 bp; clusterin (GTCTCAGACAATGAGCTCCA) (TGCGGTCACCATTCATCCAG) 419 bp; CD59 (ATTTCAACGACGTCACAACC) (GACTGGTCTTCAAAGTCTCC) 369 bp; glyceraldehyde phosphate dehydrogenase (GAPDH) (GAACGGGAAGCTTGTCATCA) (TGACCTTGCCCACAGCCTTG) 473 bp. PCR amplifications were performed in an Omnigene thermocycler (Hybaid, Teddington, UK) using the following conditions: denaturation at 94°C for 4 minutes, five cycles (94°C for 30 seconds, annealing 60°C for 1 minute, 72°C extension for 2 minutes), 20 cycles (94°C for 30 seconds, annealing 60°C for 30 seconds, 72°C extension for 45 seconds), and a final extension at 72°C for 15 minutes. All samples were subjected to RT-PCR for housekeeping gene GAPDH as a positive control and as an internal standard. RT-PCR products were resolved on 1.2% agarose gels in 1× Tris-borate-EDTA (TBE) buffer. Comparative DNA ladder markers (123 bp and 1 kb from Life Technologies Ltd.) were loaded to identify the correct size of the different cDNA fragments. Gels were visualized by ethidium bromide and photographed (using a gel 1,000 UV documentation system, Bio-Rad). The identity of each of the cDNA fragments was confirmed by sequencing using the Big DYE sequencing kit (Perkin Elmer, Buckinghamshire, UK) and analysis on the ABI 377 automated sequencer (Perkin Elmer). Plasmid containing the full-length human MCP cDNA clone was used with appropriate primer pairs (see above) to generate a specific riboprobe of 419 bp for use in ISH on mixed fetal brain cultures. The PCR product corresponded to bases +301 to +720 in the MCP coding region39Gasque P Jones J Singhrao SK Morgan BP Identification of an astrocyte cell population from human brain that expresses perforin, a cytotoxic protein implicated in immune defense.J Exp Med. 1998; 187: 451-460Crossref PubMed Scopus (34) Google Scholar, 42Lublin DM Liszewski MK Post TW Arce MA LeBeau MM Rebentisch MB Lemons RS Seya T Atkinson JP Molecular cloning and chromosomal localization of human membrane cofactor protein (MCP).J Exp Med. 1988; 168: 181-194Crossref PubMed Scopus (199) Google Scholar (accession number Y00651) and was cloned into pGEM-T (Promega, Southampton, UK) with flanking SP6/T7 RNA polymerase sites. The human CD59 riboprobe was a 518-bp PCR product containing the region −37 to +481 (accession number M95708) cloned into pGEM-3Z (Promega).43Davies A Simmons DL Hale G Harrison RA Tighe H Lachmann PJ Waldmann H CD59, an Ly-6 like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells.J Exp Med. 1989; 170: 637-654Crossref PubMed Scopus (584) Google Scholar A 473-bp PCR fragment of GAPDH cDNA coding region +249 to +702 (accession number M33197) was also cloned into pGEM-T. In all cases, the identity and orientation of the cloned fragments we
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