[51] Purification and molecular cloning of Rat Ia antigens

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the isolation of these antigens by specific affinity chromatography may prove useful. A similar approach has proven invaluable in the structural and functional analysis of the H-2K k antigens.19,2° Acknowledgments The work described in this chapter was supported by research grants from the National Institutes of Health (AI-13448) and from the Welsh Foundation. In addition, the authors wish to acknowledge Drs. Richard Cook, J. Donald Capra, Katherine Mclntyre, and Jonathan Uhr for their contributions, advice, and criticism of this work. The expert technical assistance of Ms. Linda Trahan, Ms. Sandy Graham, Ms. Shirley Shanahan, Ms. Kay Snave, and Ms. Martha Miles is gratefully acknowledged. We also thank Ms. G. A. Cheek for her patient and expert typing skills.

19 K. C. Stallcup, T. A. Springer, and M. F. Mescher, J. Immunol. 127, 921 (1981). 2o j. E. Mole, F. Hunter, J. W. Paslay, A. S. Bhown, and J. C. Bennett, Mol. Immunol. 19, l (1982).

[51] P u r i f i c a t i o n a n d M o l e c u l a r C l o n i n g o f R a t Ia A n t i g e n s

By W. ROBERT MCMASTER Introduction The Ia antigens are a set of highly polymorphic cell surface molecules involved in many cell-cell interactions and coded for by the major histocompatibility complex (MHC). The polymorphism of the Ia antigens is directly related to their function, as these molecules are thought to be the products of the immune response genes which genetically determine the outcome of many types of immune responses.l Ia antigens have been identified in several different mammalian species and are composed of two noncovalently bonded glycosylated polypeptides referred to as the a and/3 chains. The Ia glycoproteins show a restricted tissue distribution being present on B lymphocytes, dendritic cells, some macrophages and thymocytes, and on epithelial and reticular cells of thymus and spleen. These molecules are not generally found on the majority of T lymphocytes or other cells. The rat MHC, called the RT1 complex, codes for two sets of Ia antigens referred to as Ia-A and Ia-E which correlate to the mouse H-2 I-A and I-E Ia antigens, respectively. 2 There are at least two I j. Klein, Science 203, 516 (1979). 2 T. Fukumoto, W. R. McMaster, and A. F. Williams, Eur. J. Immunol. 12, 237 (1982).

METHODS IN ENZYMOLOGY,VOL. 108

Copyright © 1984by AcademicPress. Inc. All rightsof reproductionin any form reserved. ISBN 0-12-182008-4

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sets of Ia antigens encoded by the human H L A complex. The HLA-DR antigens are homologous to rodent Ia-E antigens 3 while the HLA-DC1 (HLA-DS) antigens have recently been shown to be homologous to Ia-A antigens.4,5 This chapter describes the purification of Ia glycoproteins from detergent solubilized rat spleen membranes using monoclonal antibody affinity chromatography and the characterization of the purified Ia molecules. Methods are also described for the purification of mRNA from rat spleen and the molecular cloning of cDNA coding for rat Ia-A antigens. Production of Monoclonal Antibodies to Rat la-A Antigens (This Volume [57]) A series of monoclonal antibodies to rat Ia antigens were derived during a study of the characterization of glycoproteins from rat thymus.~ Rat thymocyte glycoproteins were prepared by lentil lectin affinity chromatography of membrane proteins solubilized by sodium deoxycholate followed by chromatography on Sephadex G-200. A fraction of Ia-like glycoproteins was then used to immunize mice and monoclonal antibodies prepared essentially as described by Kohler and Milstein. 7 The techniques for producing monoclonal antibodies have recently been reviewed in a previous volume of this series. 8 Monoclonal antibodies which bound to rat lymphocytes were detected using indirect radioimmune cellular binding assays and a fluorescence-activated cell sorter (FACS). The advantage of indirect binding assays for the detection and quantitation of cell surface molecules has been reviewed. 9 Using this approach, a series of monoclonal antibodies of the IgG class, designated MRC 0X 3, 4, 5, and 6 were derived. These antibodies detect Ia antigenic determinants present on all rat B lymphocytes and on a subpopulation of rat thymocytes. MRC 0X3 antibody detect an Ia antigenic determinant polymorphic in the rat, while MRC 0X 4, 5, and 6 detect a determinant common to all rat strains tested. When assayed on spleen cells from recombinant mouse strains, all four antibodies detect polymorphic Ia determinants encoded by the H-2 I-A subregion. MRC 0X3 antibody reacts with cells of the H-2 haplotype b and s while MRC 0X 4, 5, and 6 antibodies react with cells of the H-2 haplo3 j. Silver, W. A. Russell, B. L. Reis, and J. A. Frelinger, Proc. Natl. Acad. Sci. U.S.A. 74, 5131 (1977). 4 S. M. Goyert, J. E. Shively, and J. Silver, J. Exp. Med. 156, 550 (1982). 5 M. R. Bono and J. L. Strominger, Nature (London) 299, 836 (1982). 6 W. R. McMaster and A. F. Williams, Immunol. Rev. 47, 117 (1979). 7 G. Kohler and C. Milstein, Eur. J. Immunol. 6, 511 (1976). G. Galfre and C. Milstein, this series, Vol. 73 [I]. 9 A. F. Williams, Conternp. Top. Mol. hnrnunol. 6, 83 (1977).

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type k and s. Therefore, MRC 0X 3, 4, 5, and 6 antibodies react with the rat homolog of mouse I-A antigens and define Ia-A antigens in the rat.

Purification of Rat Ia Antigens Using Monoclonal Antibody Affinity Chromatography The methods described are based on techniques first used to purify rat Thy 1 antigen from brain tissuC ° and modified for the use of monoclonal antibodies. Purification of lgG from Ascities Fluid. CLARIFICATION OF ASCITIES FLVlO. Ascities fluid is collected from the peritoneal cavity of mice, injected with Pristane 2 weeks prior to the injection of the appropriate monoclonal antibody cell line, and is stored at - 2 0 °. Upon thawing, the ascities fluid is filtered through cotton wool to remove aggregated material and centrifuged at 10,000 g for 20 rain at 4 °. This step should immediately precede ammonium sulfate precipitation as further material aggregates upon storage at 4° or after freeze thawing. As each monoclonal antibody may have slightly different characteristics, it is important to follow the antibody activity during each step of the purification by an appropriate serological assay. As a safety precaution, all supernatants should be saved until all the antibody activity can be accounted for. A M M O N I U M SULFATE PRECIPITATION. Immunoglobulin preparation from ascities fluid is described starting from an initial volume of 50 ml usually containing between 50 to 250 mg IgG. To 50 ml of clarified ascities fluid 50 ml of phosphate-buffered saline pH 7.3 (PBS: 0.2 g KCI, 0.2 g KH2PO4, 1.15 g Na2HPO4, 8.0 g NaCI per 1000 ml distilled HzO) is added. IgG is precipitated by adding dropwise I00 ml of 100% saturated ammonium sulfate pH 7.4 while stirring at 20 ° (to prepare 100% saturated ammonium sulfate: 800 g of ammonium sulfate is added to 1000 ml of distilled H20. The mixture is heated to 100° for 30 min, then cooled, and the pH is adjusted to 7.4). After the saturated ammonium sulfate is added, the mixture is stirred for 30 min at 20 °, and then left at 4° for 1 hr without stirring. The precipitate is recovered by centrifugation at 10,000 g for 20 min at 4 °, dissolved in 50 ml PBS, and reprecipitated by adding 50 ml 100% saturated ammonium sulfate as before. The final precipitate is dissolved in DEAE buffer (0.032 M Tris base, 0.025 M HCI, 10 mM NAN3, pH 7.3) and dialyzed extensively against the same buffer at 4°. Proteins are determined from the absorbance at 280 nm (1 mg/ml IgG = 1.35 OD units). DEAE ION EXCHANGE CHROMATOGRAPHY.DEAE-cellulose (DE-52 10 A. N. Barclay, M. Letarte-Muirhead, and A. F. Williams, Biochem. J. 151, 699 (1975).

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Whatman) and DEAE-Sephacel (Pharmacia) exhibit similar characteristics. A column of DEAE is prepared using 1 ml of gel for 5 mg protein and equilibrated with DEAE buffer. The column is washed until the pH and/ or conductivity of the effluent is equal to that of the DEAE buffer. The protein sample is applied and the column is washed with DEAE buffer until absorbance at 280 nm is less than 0.05. Four to 5 ml fractions are collected. IgG is eluted with a linear gradient of 0 to 0.1 M sodium chloride in DEAE buffer (300 ml total for a 25 ml column). The column is then eluted with 0.5 M sodium chloride in DEAE buffer. The absorbance at 280 nm is determined for each fraction. Protein-containing fractions are assayed for antibody activity and analyzed for purity by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). IgG-containing fractions are pooled, concentrated by ultrafiltration to 5 mg/ml, and stored at - 2 0 °. A protein which is occasionally present following this procedure is serum transferrin of molecular weight 85,000. Transferrin can be separated from IgG by ammonium sulfate precipitation at 50% saturation, as described, or by gel chromatography on Sephacryl S-200 or equivalent gel. Methods to prepare F(ab')2 and Fab' fragments by pepsin digestion have been recently reviewed, n Preparation of Monoclonal Antibody Affinity Columns. Sepharose 4B (Pharmacia) activated by reaction with cyanogen bromide has been used for the preparation of monoclonal antibody affinity columns. The following steps must be carried out in a fume hood. To activate Sepharose 4B, 25 ml of wet beads is washed with 250 ml cold distilled H20 on a sintered glass funnel, dried until just moist, and then transferred to a glass beaker containing 25 ml distilled H20. The beaker is placed in an ice bucket and the slurry gently stirred. A pH electrode is placed in the slurry to monitor the reaction. Seven hundred and fifty milligrams of cyanogen bromide is weighed in a fume hood and dissolved in 5 ml dimethylformamide. The dissolved cyanogen bromide is added to the Sepharose 4B dropwise over I min. The pH of the reaction is kept between 10 and 11 with 5 M sodium hydroxide for 10 to 15 rain. The activated Sepharose 4B is transferred as rapidly as possible to a sintered glass funnel and washed with 250 ml of cold distilled H20 followed by 250 ml cold sodium borate buffer (0.05 M Na2B407 • 10H20 pH 8.0, 10 mM NAN3). The activated Sepharose 4B is dried on a sintered glass funnel and then transferred to a tube containing 25 ml of monoclonal antibody (5 mg/ml) in sodium borate buffer. The gel and monoclonal antibody should form a thick slurry which is gently rotated for 18 to 24 hr at 4 °. The gel mixture is returned to a sintered glass n p. Parham, M. J. Androlewicz, F. M. Brodsky, N. J. Holmes, and J. P. W a y s , J. /mmunol. Tech. 53, RI01 (1982).

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funnel and filtered. To determine coupling efficiency the absorbance at 280 nm of the filtered supernatant is measured and compared to that of the original solution. The gel is washed with 250 mi of sodium borate buffer and any free reactive groups are blocked by resuspending in 50 ml of 0.15 M ethanolamine in sodium borate buffer pH 8.5. The gel is incubated for 2 hr at 4° and then washed with 10 mM Tris-HCl, 10 mM NaN3 pH 8.0. Prior to use the free IgG that may be present should be washed off the gel with elution buffer. Preparation of Spleen Membrane. Crude rat spleen membranes are prepared using the Tween 40 method. 12 All steps are carried out at 4 °. Whole spleens, usually 150 g or about 150 spleens, are disrupted in 150 ml PBS in a Waring blender at full speed. To minimize proteolytic degradation, 10 mM iodoacetamide and 5 mM diisopropylfluorophosphate (DFP) or 5 mM phenylmethylsulfonyl fluoride (PMSF) are added prior to disruption. An equal volume of 5% (v/v) Tween 40 in PBS is added to the disrupted spleens and the mixture homogenized by four strokes of a Potter-Elvehjem homogenizer with a power driven Teflon pestle. The mixture is stirred for 60 min on ice and nuclei sedimented by centrifugation at 3000 g for 30 min. The supernatant is filtered through two layers of cheesecloth and centrifuged at 60,000 g for 90 min to yield a pelleted crude membrane. The membrane fraction is resuspended in 10 mM TrisHC1 pH 8.0 and centrifuged at 60,000 g for a further 90 min. The resulting membrane fraction is resuspended in a total of 100 ml 10 mM Tris-HCl pH 8.0, 10 m M NAN3, and solubilized as described below or stored at - 7 0 °"

Solubilization of Spleen Membrane and MonoclonalAntibody Affinity Chromatography. Spleen membranes (150 ml) are solubilized by the addition of an equal volume (150 ml) of 10% (w/v) sodium deoxycholate in 10 m M Tris-HCl pH 8.0, 10 m M NaN3 and homogenized as described above. Iodoacetamide (10 mM) and 5 mM DFP or 5 mM PMSF are added prior to homogenization. The mixture is stirred on ice for 60 min and centrifuged at 60,000 g for 120 min. The supernatant is passed first through a column of rabbit IgG-Sepharose 4B, then through a monoclonal antibody-Sepharose 4B column (25 ml conjugated Sepharose 4B containing 125 mg IgG) at a flow rate of 10 to 15 ml/hr. It is important to precede the specific affinity column with a nonspecific column as the latter removes any material binding to cyanogen bromide-activated Sepharose 4B or nonspecifically to IgG. Columns are washed with deoxycholate buffer (0.5% w/v sodium deoxycholate in 10 m M Tris-HC1 pH 8.0, 10 mM NAN3) until absorbance at 280 nm is less than 0.05. The monoclonal antibody column is then washed with deoxycholate buffer containing 0.2 12 R. Standring a n d A. F. Williams, Biochim. Biophys. Acta

508, 85

(1978).

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M NaCI and eluted with deoxycholate elution buffer (0.5% w/v sodium deoxycholate in 0.05 M diethylamine-HC1 pH 11.5, 10 mM NAN3). Absorbance at 280 nm and the pH are determined. Each fraction (4 ml) is then immediately neutralized by the addition of solid glycine. A high pH elution buffer is used because sodium deoxycholate is insoluble below pH 7. Furthermore, this elution procedure has given at least 50% recovery of antigenic activity in the purification of several different cell surface glycoproteins. The eluted material is pooled and concentrated by ultrafiltration and chromatographed on a Sephacyl S-200 column (1.6 x 100 cm) eluted with deoxycholate buffer. Each fraction is analyzed by SDS-PAGE and for antigenic activity and those fractions containing activity are pooled and concentrated by ultrafiltration. In order to remove sodium deoxycholate, the protein is precipitated by adding 95% ethanol to a final concentration of 75% and by incubating at - 2 0 ° for 48 hr. The precipitate is washed in 75% ethanol by centrifugation, dried under vacuum, dissolved in 10 mM Tris-HC1 pH 8.0, 10 mM NAN3, and stored at - 2 0 °. Yields of antigenic activity are calculated by carrying out quantitative inhibition assays using cellular radioimmune binding assays. These assays can be used in the presence of detergents by treating target cells with glutaraldehyde prior to u s e . 9 However, use of some monoclonal antibodies in inhibition assays in the presence of detergents results in an underestimate of actual yields of antigenic activity. 6 This is partly due to differences in the binding properties of some monoclonal antibodies in the presence and absence of detergents. The binding properties of several monocional antibodies to cell surfaces have been discussed recently. J3 In the purification of rat Ia antigens, rabbit antibodies prepared by immunization with purified Ia antigen were more reliable in determining the yields and overall purification than monoclonal antibodies. 6 Most of the rat Ia antigenic activity is lost in preparation of membranes where the recovery varies between 30 and 50%. Monoclonal antibody columns retain 90% of the rat la antigenic activity present in the sodium deoxycholate solubilized spleen membrane preparation and 70% of the applied activity is eluted with high pH buffer. The purification of rat Ia antigen in the affinity column step is 200- to 400-fold and values of several thousandfold have been obtained in the purification of the rat thymocyte glycoprotein W3/13 antigen. 14 In the purification of rat Ia antigen, the spleen membranes are solubilized in sodium deoxycholate since this detergent will release DNA from 13 D. W. Mason and A. F. Williams, Biochem. J. 18% 1 (1980). 14 W. R. A. Brown, A. N. Barclay, C. A. Sunderland, and A. F. Williams, Nature (London) 289, 456 (1981).

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FIG. 1. Purification of la-A antigens from spleen membrane of Wistar rats. A summary of the purification steps is shown and analysis by SDS-PAGE of track A, an aliquot (100/zg) of sodium deoxycholate solubilized rat spleen membrane electrophoresed in the presence of 100 mM dithiothreitol; track B, an aliquot (8 /xg) of eluted Ia-A antigen electrophoresed without reduction.

the nuclei of whole cells. To minimize the loss of antigen occurring during the membrane preparation, whole rat spleens are solubilized directly with the nonionic detergent Triton X-100. The detergent extract is then applied to a monoclonal antibody column washed with Triton X-100, followed by deoxycholate buffer. The bound Ia antigen is then eluted with deoxycholate buffer pHI 1.5. Following this procedure, however, the Ia antigens are contaminated with other proteins which cannot be removed. A procedure has been developed recently for the purification of W3/13 antigen by which whole thymocytes are solubilized first in the nonionic detergent Brij 96, the nuclei removed, and then sodium deoxycholate added to solubilize further any aggregated material. 14This procedure eliminates the need for preparing membrane and may be suitable for the purification of other membrane proteins. A summary of the procedures for the purification of Ia-A antigens from rat spleen using MRC 0X4 monoclonal antibody is shown in Fig. 1. SDS-PAGE of an aliquot of the starting sodium deoxycholate extract (track A) and of an aliquot of the eluted material from the antibody

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column (track B) is shown in Fig. 1. The purified rat Ia-A antigen was composed of two polypeptides, the a and/3 chains, of molecular weight 30,000 and 25,000 (unreduced), respectively. As shown in Fig. 1, the eluted antigen was free from contaminating proteins and the use of the monoclonal antibody column resulted in a single step purification. Strategy to Produce Monoclonal Antibodies to a S e c o n d Rat la Antigen

The first series of monoclonal antibodies (MRC 0X3, 4, 5, and 6) were clearly shown to react with the rat homolog of mouse I-A encoded Ia antigens. Quantitative inhibition studies using rat alloantibodies against Ia antigens and rat Ia antigen purified using MRC 0X4 antibody showed that approximately 50% of these rat alloantibodies reacted with rat la-A antigens, while the remainder appeared to react with other rat Ia molecules. 6 To investigate this possibility further, the following approach was used to prepare monoclonal antibodies to these additional rat Ia antigens, which, subsequently, were shown to be homologous to mouse l-E encoded antigens. Rat spleen membrane glycoproteins are prepared by lentil lectin affinity chromatography and separated according to size by gel filtration in deoxycholate buffer. Fractions containing Ia antigens, as detected by SDS-PAGE analysis, are pooled and Ia-A antigens removed by affinity chromatography on a MRC 0X6 antibody column. The remaining glycoproteins are used to immunize mice and a monoclonal antibody, MRC 0XI7, reacting with a second rat Ia antigen is produced. 2 The MRC 0X17 antibody reacts only with B lymphocytes, detects an antigenic determinant common to all rat strains tested, and does not cross-react with mouse spleen ceils. A monoclonal affinity column is prepared using MRC 0X17 antibody following the methods described above and is used to purify the corresponding Ia antigen from sodium deoxycholate solubilized rat spleen membranes. Rabbit antibodies are prepared to the purified Ia antigen which cross-react with mouse spleen cells. The binding pattern of these rabbit antibodies to spleen cells from various mouse strains maps to the mouse H-2 I-E subregion thus establishing that the MRC 0X17 Ia antigen is the rat homolog of mouse I-E Ia antigen. The rat Ia-A antigen, purified using MRC 0X4 or 6 antibodies, and the rat Ia-E antigen, purified using MRC 0X17 antibody, can be shown to be encoded by the rat MHC (RT1 complex) by their ability to inhibit the binding of rat alloantibodies. Each Ia antigen used alone inhibits approximately 50% of the binding of the rat alloantibodies; when added together, greater than 90% of the specific

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binding is inhibited. Quantitative absorption studies using monoclonal and rabbit antibodies show that there is no cross-reactions between the rat Ia-A and Ia-E antigens. 2

Separation of Rat Ia a and fl Chains and Location of Antigenic Determinants The o~ and/3 chain of Ia antigens are strongly associated by noncovalent bonding. The two chains can be dissociated by heating to 100° in the presence of SDS. The a and/3 chains can then be separated by preparative S D S - P A G E under nonreducing conditions. The following procedure 15 has been used to purify the a and/3 chains of rat Ia-A and Ia-E antigens previously purified by monoclonal antibody affinity chromatography. Separation ofla ~ and/3 Chains. One milligram of purified Ia antigen is precipitated with 75% ethanol. The precipitate is dissolved in 0.5 ml 5% SDS prepared in S D S - P A G E stacking gel buffer of Laemmli 16 (0.125 M Tris-HCl pH 8.3, 15% glycerol, 0.1% bromophenol blue) and heated to I00 ° for 5 rain. After cooling, the sample is loaded onto a cylindrical gel composed of a 3% polyacrylamide stacking gel (1 × 1.5 cm) and a 10% polyacrylamide separating gel (1.5 × 10 cm) and electrophoresed for 6 hr. The gel is cut into 1.5 mm slices and each slice individually eluted with 3 × 1 ml of 0.1% SDS in distilled H20 at 20° for a total of 24 hr. An aliquot of each sample is analyzed by slab SDS-PAGE. The fractions found to contain either a or/3 chain are pooled, concentrated by ultrafiltration, and dialyzed against 0.1% SDS or distilled HzO. Yields of between 40 and 70% for each chain are obtained routinely. In order to determine which chain carries the antigenic determinant, quantitative inhibition assays using monoclonal antibodies are carried out using whole Ia antigen and each separated chain. After absorption at 4° for 16 hr, each sample is centrifuged and the remaining antibodies assayed using indirect binding assays. With some monoclonal antibodies clear cut results are obtained. MRC 0X6 antibody, which detects a common Ia-A antigenic determinant in rats and a polymorphic determinant in mice, reacts specifically with purified rat Ia-A/3 chain. ~5Assuming that rat and mouse Ia-A antigens are homologous, these results imply that the mouse polymorphism is located in the Ia-A/3 chain. With MRC 0X3 antibody, which detects an Ia-A polymorphic determinant in both rats and mice, purified rat Ia-A antigen inhibits the binding to rat lymph node cells whereas no inhibition is found with separated ct or 13 chains when added separately or together. The determinant with which MRC 0X3 reacts, 15 W. R. McMaster, lmmunogenetics 13, 347 (1981). 16 U. K. Laemmli, Nature (London) 277, 680 (1970).

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therefore, depends either on the a and/3 chains being associated together in their native conformation or the determinant being denatured during the separation procedures. Using the same approach, MRC 0XI7 antibody, which reacts with a common Ia-E determinant in rats and does not cross-react with mouse spleen cells, reacts specifically with separated rat Ia-E a chain. 2 Molecular Cloning of Rat Ia Antigens

Cloning of cDNA Coding for Rat la Antigens A possible approach to the determination of the primary structure of rat Ia antigens is to clone and sequence the complementary DNA (cDNA) coding for these molecules. The protein sequence can then be predicted from the nucleotide sequence and these data may be used to predict the domain structure of these molecules. Furthermore, the cloned cDNAs may be used as probes to study the structure and expression of the corresponding genomic DNA. Studies at the DNA level may also lead to a better understanding of the molecular basis of the polymorphisms exhibited by the Ia antigens. The approach used to clone cDNA coding for rat Ia-A antigens is to purify mRNA from rat spleen and identify specific mRNA coding for Ia-A a and /3 chains by in vitro translations in the presence of radioactive amino acids. Rat Ia polypeptides are immunoprecipitated with rabbit antibodies prepared against separated Ia-A chains and the radioactive proteins are analyzed by SDS-PAGE. Total spleen mRNA is fractionated by sucrose density gradient centrifugation and fractions containing mRNA coding for Ia-A antigens are pooled and used to prepare double-stranded cDNA. cDNA is inserted into the bacterial plasmid, pBR322, and the resulting recombinant plasmids used to transform E. coli. Specific cDNA clones are detected using a positive mRNA selection assay. This strategy results in the identification of a cDNA clone coding for a rat Ia-A a chain. Purification of Spleen mRNA. Since spleen tissue contains high levels of ribonuclease, methods 17to inhibit this enzyme must be used in order to obtain intact mRNA. Animals are sacrificed by placing them in a desiccator containing COz (dry ice and water) and spleens immediately removed and placed in sterile ice cold PBS. Whole spleens are disrupted using a Polytron homogenizer (Brinkman Instruments) in 10 mi/g of tissue of guanidine-HC1 buffer (7.5 M guanidine-HCl, 0.025 M sodium citrate pH 7.0, 10 mM dithiothreitol). After homogenization the volume is measured. Ten percent sodium lauryl sarcosine in distilled H20 is added to a final ~7j. M. Chirgwin, A. E. Przybyla, R. J. McDonald, and W. J. Rutter, Biochemistry 18, 5294 (1979).

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concentration of 0.5%, and the solution centrifuged at 3000 g for 30 min at 0°. The supernatant is transferred to sterile polypropylene 50-ml plastic test tubes and the R N A precipitated by the addition of 95% ethanol to a final concentration of 33%. The ethanol is mixed well and the preparation is left at - 2 0 ° for 6 to 18 hr. The precipitate is collected by centrifugation (3000 g for 30 min at 0°) and the supernatant is removed. The test tubes are drained thoroughly and any remaining liquid is wiped away with tissue paper. The pellets are dissolved in one-half of the starting volume with guanidine-HCl buffer and centrifuged as above. The RNA is precipitated by adding 95% ethanol to 33% and left at - 2 0 ° for 4 to 18 hr. The precipitated RNA is then collected as before, dissolved in one-quarter the starting volume with guanidine-HC1 buffer, and reprecipitated by adding 95% ethanol to 33%. From this point onward, all buffers must be treated with 0.2% diethyl pyrocarbonate for 20 min at 20° and autoclaved to inactivate ribonuclease. All glassware must be baked overnight or sterile plastic test tubes and pipets must be used. The final pellet of RNA is dissolved in sterile distilled H20 and centrifuged at 3000 g for 30 min to remove insoluble material. The absorbance at 260 and 280 nm is measured and the RNA concentration is calculated (1 mg/ml RNA = 20 OD units at 260 nm). The ratio of absorbance at 260 nm:280 nm should be between 1.75 and 2.0. One gram (wet weight) of whole spleen yields approximately 2 mg of total cellular RNA. Purification ofPoly(A) RNA. Polyadenylated RNA is separated from ribosomal R N A by two rounds of affinity chromatography using oligo(dT)-cellulose. ~8All buffers must be treated with 0.2% diethyl pyrocarbonate for 20 min at 20° and autoclaved. RNA is diluted to 1 to 2 mg/ml with sterile distilled HzO and EDTA pH 7.5 is added from a 0.2 M stock solution to give a final concentration of 10 mM. RNA is heated to 70° for 5 min and cooled rapidly in an ice bath. Sodium acetate pH 7.5 is added from a 2 M stock solution to give a final concentration of 0.4 M and the RNA is applied to an oligo(dT)-cellulose column (Type T-3, Collaborative Research). The R N A solution is passed through the column three times. The column is then washed with 0.4 M sodium acetate pH 7.5, 10 mM EDTA until the absorbance at 260 nm is less than 0.05. Bound poly(A) R N A is eluted with 1 m M EDTA pH 7.5. The eluate is made 0.4 M in sodium acetate pH 7.5 and 10 m M in EDTA and applied again to the oligo(dT)-cellulose column. The column is washed and bound poly(A) R N A is eluted as detailed above. Eluted poly(A) RNA is precipitated by addition of sodium acetate pH 5.0 to 0.2 M and two volumes of 95% ethanol. The precipitate is allowed to form at - 2 0 ° for 18 hr and is colmsH. Aviv and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 69, 1408 (1972).

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lected by centrifugation at 3000 g for 30 min, washed in 70% ethanol, and dried under vacuum. The final pellet of poly(A) RNA is dissolved in sterile distilled HzO, aliquoted, and stored at - 7 0 °. Approximately 3% of total spleen R N A is obtained as poly(A) RNA after two rounds of affinity chromatography on oligo(dT) cellulose. Cell-Free Translation of Rat Spleen mRNA and Immunoprecipitation of Rat Ia-A Polypeptides. The purified mRNA is tested for biological activity by translation in a cell-free rabbit reticulocyte lysate assay supplemented with [35S]methionine or [3H]leucine essentially as described. ~9 Commercial kits from New England Nuclear or Amersham have proven to be reliable and convenient to use. Incorporation of radioactive amino acids into proteins is determined by trichloroacetic acid (TCA) precipitation followed by analysis on SDS-PAGE and autoradiography. To increase sensitivity, the polyacrylamide gels are treated with Enhance (New England Nuclear) and exposed to preflashed X-ray film (Kodak XOmat XAR-2) at - 7 0 ° as described. 2° Immunoprecipitations are carried out using monoclonal or rabbit antibodies and formalin-fixed protein A containing Staphylococcus aureus (Bethesda Research Laboratories, Bethesda, MD). Prior to use, S. aureus is washed two times in 0.5% Triton X-100 in PBS and resuspended in the original volume of the same buffer. Spleen mRNA, I to 2/xg, is translated in a 25/xl lysate reaction in a sterile 1.5 ml microfuge test tube at 37° for 60 min. A 5-tzl aliquot is taken for analysis by S D S - P A G E and two 1-/xl aliquots are precipitated with TCA to determine total incorporation of radioactivity. The remaining mixture is placed on ice and is precleared by adding 5 ~1 normal rabbit serum followed by 100/zl of S. aureus in 0.5% Triton X-100. The mixture is incubated at 4° for 30 min and then centrifuged in a microfuge for 5 min. The supernatant is transferred to a new microfuge test tube and 5 /xl of specific antibody added: rabbit antiserum or 25 tzg monoclonal antibody. The antibody is allowed to react for 4 to 6 hr at 4° and I00/zl of S. aureus is added. The mixture is incubated for 30 min with occasional mixing followed by the addition of 0.75 ml 0.5% Triton X-100. The mixture is centrifuged in a microfuge for 2 min and the supernatant removed by aspiration. The pellet is vortexed and then 0.75 ml of 0.5% deoxycholate buffer added. The mixture is centrifuged again and washed one additional time with deoxycholate buffer. The final pellet is vortexed, 50/xl of SDSPAGE sample buffer containing 100 mM dithiothreitol is added, and the sample boiled for 5 min. The material is centrifuged for 5 min and the supernatant analyzed by S D S - P A G E or stored at - 2 0 ° until used. ~ H. R. B. Pelham and R. J. Jackson, Eur. J. Biochem. 67, 247 (1976). 2o R. A. L a s k e y a n d A. D. Mills, F E B S Lett. 82, 314 (1977).

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FIG. 2. Cell-free translations of rat spleen mRNA and immunoprecipitation of rat Ia-A a chains. Wistar rat spleen poly(A) RNA was translated in 25/zl rabbit reticulocyte lysate assays containing 20/xCi [3H]leucine (110 Ci/mmol). Translated products were immunoprecipitated using rabbit antibodies against separated rat Ia-A c~chains and protein A containing Staphylococcus aureus, analyzed by SDS-PAGE, and visualized by fluorography. Track 1,5 /xl total translated products; track 2, immunoprecipitated translated products; track 3, as in track 2 except that immunoprecipitation was carried out in the presence of 1/zg nonradioactive rat la-A a chain.

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An example of the cell-free translation of total rat spleen poly(A) mRNA and the identification of in vitro synthesized rat Ia-A a chains is shown in Fig. 2. In these experiments rat spleen mRNA is translated in a rabbit reticulocyte lysate assay containing [3H]leucine. Rat Ia polypeptides are immunoprecipitated using rabbit or MRC 0X6 antibodies to the isolated o~ or /3 chains. Rabbit antibodies are prepared by immunizing rabbits twice at three week intervals with 75 ~g of separated Ia-A a or/3 chains (see above) emulsified in complete Freund's adjuvant. Serum is collected 2 weeks after the last immunization. Samples of the total translation products and immunoprecipitates are analyzed by SDS-PAGE and fluorography. Figure 2 (track 1) shows the total polypeptides synthesized using rat spleen mRNA. A control with no mRNA added shows no bands (data not shown). Track 2 shows the results after immunoprecipitation with rabbit anti Ia-A a chain antibodies. A single band of apparent molecular weight 26,000 is clearly visible. Track 3 shows the results of blocking the immunoprecipitation of radioactive o~ chain with the addition of 1 ~g of purified rat ~ chain to the reaction mixture prior to the addition of the rabbit antibodies. In this instance there is no radioactive c~ chain detectable and these results clearly demonstrate that the band in track 2 is the cell-free translation product of mRNA coding for rat Ia-A u chain. Attempts were also made to identify the cell-free translation product of mRNA coding for rat Ia-A/3 chain by immunoprecipitation with three different antibody preparations: MRC 0X6 antibody, which reacts with separated Ia-A/3 chains (see above), rabbit antibodies prepared against separated Ia-A 13 chain, and rabbit antibodies prepared against reduced and carboxymethylated Ia-A/3 chain. In no instance is a/3 chain detectable. Cell-free translations can also be carried out in the presence of dog pancreas microsomes in order for the translation products to be "processed" (leader sequence removed and carbohydrate side chains attached) and membrane proteins inserted into microsomal membranes. 2~ After translation the radioactive products are immunoprecipitated with each of the three preparations of antibodies to Ia-A/3 chains and analyzed by SDS-PAGE. Again no bands are visible using any of these three anti-/3 chain antibodies. These results could be due to the absence of Ia-A /3 chain mRNA in the poly(A) preparation of total rat spleen mRNA, the failure of/3 chain mRNA to be translated in the reticulocyte lysate assay, or to the fact that the in vitro synthesized Ia-A/3 chain products have a different conformation from that of the separated 13 chain and, therefore, do not react with any of the three different antibody preparations. At this point it is not clear which of these possibilities is occurring. These results 2~ R. J. Jackson and G. Blobel, Proc. Natl. Acad. Sci. U.S.A. 74, 5598 (1977).

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point out the difficulties encountered in identifying mRNA to a given protein by cell-free translation and immunoprecipitation using specific antibodies.

Enrichment of Specific mRNA by Sucrose Gradient Centrifugation. Messenger R N A can be fractionated by preparative sucrose gradient centrifugation or by preparative polyacrylamide agarose gel electrophoresis. To carry out sucrose gradient centrifugation either isokinetic (exponential) or linear gradients are used. Isokinetic 5-20% sucrose gradients in 10 mM sodium acetate pH 5.0, 100 mM NaC1, 1 mM EDTA are prepared as described. 22,23 Linear gradients (5-20%) are formed using an appropriate gradient mixer. R N A (250/zg in 50/zl 1 mM EDTA pH 8) is heated to 70° for 5 min and quickly cooled in an ice bath prior to layering over the gradient. Centrifugation is carried out in a Beckman SW41, SW50.1, or equivalent rotor at 75,000 g and 4° for 18 hr. The effluent is collected in 0.4 ml fractions and mRNA in each fraction precipitated by the addition of two volumes of 95% ethanol and incubation at - 2 0 ° for 18 hr. The precipitate is recovered by centrifugation, washed in 70% ethanol, dried under vacuum, and dissolved in 50/zl sterile distilled H20. Aliquots, 2 /xl, of each fraction are then translated in a cell-free assay and the radioactive products identified by immunoprecipitation and analysis by SDS-PAGE. Fractions containing specific mRNA are pooled and centrifuged on sucrose gradients as before. Fractions containing specific mRNA are pooled, precipitated as described, and used as templates for cDNA synthesis. Sucrose gradient fractionation usually results in a 5- to 10-fold enrichment for specific mRNA species. A much higher degree of enrichment can be achieved, in some instances, by immunoprecipitation of polysomes using antibodies reacting with the nascent polypeptide c h a i n . 24-26 These methods have been demonstrated to be very efficient and have resulted in a several hundredfold enrichment of low abundance mRNA species from various tissues or cell lines from which intact polysomes can be prepared. With spleen tissues it has not been possible to prepare intact polysomes (unpublished results) presumably due to high levels of ribonuclease. Therefore, this approach has not been useful in order to enrich mRNA coding for rat Ia antigens. 22 H. Noll, Nature (London) 215, 360 (1967). 23 K. S. McCarty, Jr., R. T. Vollmer, and K. S. McCarty, Anal. Biochem. 61, 165 (1974). 24 R. T. A. MacGillivray, S. J. Friezner Degen, T. Chandra, S. L. C. Woo, and E. W. Davie, Proc. Natl. Acad. Sci. U.S.A. 77, 5153 (1980). 25 N. M. Gough and J. M. Adams, Biochemistry 17, 5560 (1978). 26 A. J. Korman, P, J. Knudsen, J. F. Kaufman, and J. L. Strominger, Proc. Natl. Acad. Sci. U.S.A. 79, 1844 (1982).

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cDNA Synthesis. Double-stranded cDNA is synthesized essentially as described, z7 Poly(A) RNA (1 mg/ml in distilled H20) is heated to 100° for 2 rain and quickly cooled on ice. A 200 pA (final volume) reaction mixture is prepared on ice in a siliconized 1.5-ml microfuge tube containing 50/zg mRNA in 50 mM Tris-HCl pH 8.3 at 43 ° 8 mM MgC12, 5 mM dithiothreitol, 8 mM sodium pyrophosphate, 500 /.~M of each deoxyribonucleotide triphosphate, 20 /xCi deoxyribocytidine [32P]triphosphate (dCTP), 3000 Ci/mmol (New England Nuclear), 100 p.g/ml oligo(dT) 12-18 (Collaborative Research), 100/xg/ml nuclease-free bovine serum albumin (Enzo Biochemicals), 20 units human placental ribonuclease inhibitor (Enzo Biochemicals), 200 units avian myoblastosis virus (AMV) reverse transcriptase (Life Sciences Inc.), and the mixture is incubated for 30 rain at 43 °. The dCTP specific activity is calculated by determining the total 32p radioactivity in two 1-/zl aliquots of the reaction mixture and 32p incorporation into cDNA is calculated on the TCA precipitate of two 1-txl aliquots. The reaction is stopped by the addition of EDTA pH 8.0 to a final concentration of 10 mM, cDNA is heated to 100° for 1 min, quickly cooled on ice, and mRNA hydrolized by adding 4 M NaOH to a final concentration of 0.4 M and incubation at 20 ° for 16 to 18 hr. The mixture is neutralized by adding 5 M HCI, 50/zg of carrier tRNA (Sigma type X) is added, and the cDNA extracted with one-half volume phenol and one-half volume chloroform: isoamyl alcohol (24:1 v/v). The aqueous layer is chromatographed on a 10 ml column of Sephadex G-50 Fine (Pharmacia) in TEN 8 (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 100 mM NaCI). The cDNA eluting in the excluded volume is precipitated by the addition of NaCI to 0.2 M and two volumes 95% ethanol and incubation at - 2 0 ° for 18 hr. Single-stranded cDNA is recovered by centrifugation in a microfuge, dissolved in 50 tzl distilled H20, and the cDNA concentration calculated by determining the total 32p radioactivity in two 1-IA aliquots. The amount (in terms of micrograms) of cDNA is calculated and the average length estimated by analysis of an aliquot by denaturing gel electrophoresis followed by autoradiography. From these two values the number of picomoles of 3' ends is determined. A tract of approximately 20 dC nucleotides is then added to each 3' end. Single-stranded cDNA is heated to 100° for 2 min and quickly cooled on ice. A reaction mixture is prepared on ice (40 ~zl total volume per 4 pmol 3' ends) containing the cDNA in 140 mM potassium cacodylate, 30 mM Tris-HCI pH 7.0, 0.1 mM dithiothreitol,

27 H. Land, M. Grez, H. Hauser, W. Lindenmaier, and G. Schutz, Nucleic" AcidRes. 9, 2251 (1981).

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1 mM cobalt chloride, 25/xM dCTP, 20/zCi [32p]dCTP (3000 Ci/mmol), and 40 units terminal transferase (Boehringer Mannheim) per 40/zl reaction. The initial cDNA concentration is calculated by determining the 32p radioactivity of two l-/zl aliquots of TCA precipitate. The reaction mixture is incubated at 37° for 20 min. The dCTP specific activity is calculated by determining the total 32p radioactivity in two 1-/zl aliquots and the dCTP incorporated is calculated by determining the 32p radioactivity in two 1-/xl aliquots of TCA precipitate. The number of dC nucleotides added per 3' end is then calculated. C-tailed cDNA is phenol extracted, chromatographed on Sephadex G-50 Fine, ethanol precipitated, and dissolved in distilled H20 as before. Second-strand synthesis is primed using oligo(dG) and carried out using AMV reverse transcriptase. C-tailed single-stranded cDNA is heated to 100° for I rain and quickly cooled on ice. A reaction mixture is prepared on ice containing cDNA (1/zg cDNA/100/.d) in 50 mM Tris-HCl pH 8.3 at 43 °, 8 mM MgCI2, 5 mM dithiothreitol, 500/xM of each deoxyribonucleotide triphosphate, 30 /zCi [3H]dCTP (20 Ci/mmol), I00 /xg/ml bovine serum albumin, 50 /zg/ml oligo(dG) 12-18 (Collaborative Research), and AMV reverse transcriptase 100 units/100/xl reaction. The mixture is incubated at 43 ° for 120 min. Incorporation of [3H]dCTP into second-strand cDNA is calculated by determining the 3H radioactivity in two 1-/zl aliquots of TCA precipitate. Double-stranded cDNA is phenol extracted, chromatographed on Sephadex G-50 Fine, and ethanol precipitated as before. Additional poly(dC) tails of approximately 10 nucleotides in length are added as described for C-tailing first-strand cDNA except that the dCTP is added to a final concentration of 10 /~M. C-tailed double-stranded cDNA is then separated by preparative nondenaturing agarose gel electrophoresis and cDNA molecules greater than 600 base pairs in length are eluted as described. 28 The eluted cDNA is ethanol precipitated, dissolved in distilled HzO, and stored at - 2 0 °. Annealing and Transformation. The plasmid vector, pBR322, 29 is digested to completion with PstI restriction endonuclease enzyme and dG tails of approximately 5 to 10 nucleotides in length are added using the conditions detailed above for tailing double-stranded cDNA with deoxyriboguanosine triphosphate at a final concentration of I0/.tM. Equimolar amounts of PstI digested G-tailed pBR322 (50 ng) and C-tailed doublestranded cDNA (5 to 20 ng) are added to give a final volume of 50/zl in 10 mM Tris-HC1 pH 7.5, 1 mM EDTA, 100 mM NaC1. DNA is annealed by heating to 65 ° for 5 min followed by incubation at 43 ° for 120 min and 28 S. C. Girvitz, S. Bacchetti, A. J. Rainbow, and F. L. Graham, Anal. Biochem. 106, 492 (1980). 29 F. Bolivar and K. Backman, this series, Vol. 68, p. 245.

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cooling to room temperature for 120 min. Annealed DNA is placed on ice for 15 min and transformation is carried out as described. 3° Calcium chloride treated E. coli strain RR129 is prepared as described 3° and 100/zl of E. coli added to each 50/~1 annealing mixture. Annealed DNA and E. coli are kept on ice for 45 rain, heated to 37° for 5 min, and kept at room temperature for 5 min. E. coli are transferred to a culture tube containing 2 ml LB medium (10 g Difco tryptone, 5 g Difco yeast extract, and 10 g NaCl/liter distilled H 2 0 ) 29 and incubated at 37° for 60 min with occasional shaking. Transformed E. coli are then plated onto fresh LB agarplates containing I0/~g/ml tetracycline (0.1 ml cells per plate) and incubated overnight at 37°. Annealed cDNA should give between 50 and 200 × 103 transformants//xg cDNA. Colonies containing recombinant plasmids are resistant to tetracycline and sensitive to ampicillin.

Identification of Specific cDNA Clones by mRNA Selection To identify specific cDNA clones an mRNA selection assay essentially as described is used. 31Recombinant plasmid DNA is immobilized on nitrocellulose filters and hybridized with an excess of mRNA. Filters are washed and bound mRNA eluted and translated in a cell-free system. Specific polypeptides are identified by immunoprecipitation and analysis by SDS-PAGE. Several cDNA clones coding for MHC antigens have been identified by this approach. 32-36 The same strategy can be used to identify cDNA clones which specifically hybridize to mRNA coding for proteins which have a detectable biological activity. In this instance, eluted mRNA is either translated in a cell-free system or injected into frog oocytes and the products assayed using a biological assay. This approach has enabled the identification of cDNA clones coding for interferon3v and more recently for the T lymphocyte growth factor, interleukin-2. 38 30 M. Dagert and S. D. Ehrlick, Gene 6, 23 (1979). 31 j. Parries, B. Velan, A. Felsenfeld, L. Rarnanathan, U. Ferrini, E. Appella, and J. G. Seidman, Proc. Natl. Acad. Sci. U.S.A. 78, 2253 (1981). 32 H. L. Ploegh, H. T. Orr, and J. L. Strominger, Proc. Natl. Acad. Sci. U.S.A. 77, 6081 (1981). 33 S. Kvist, F. Bregegere, L. Rask, B. Cami, H. Garoff, F. Daniel, K. Wiman, D. Larhammar, J. P. Abastado, G. Gachelin, P. A. Peterson, B. Dobberstein, and P. Kourilsky, Proc. Natl. Acad. Sci. U.S.A. 78, 2772 (1981). 34 j. S. Lee, J. Trowsdale, and W. F. Bodmer, Proc. Natl. Acad. Sci. U.S.A. 79, 545 (1982). 35 K. Winman, D. Larhammar, L. Claesson, K. Gustafsson, L. Schenning, P. Bill, J. Bohme, M. Denaro, B. Dobberstein, U. Hammerling, S. Kist, B. Servenius, J. Sundelin, P. Peterson, and L. Rask, Proc. Natl. Acad. Sci. U.S.A. 79, 1703 (1982). 36 C. Wake, E. O. Long, M. Strubin, R. Accolla, S. Carrel, and B. Mach, Proc. Natl. Acad. Sci. U.S.A. 79, 6979 (1982). 37 S. Nagata, H. Taira, A. Hall, L. Johnsrud, M. Streuli, J. Ecsodi, W. Boll, K. Cantell, and C. Weissman, Nature (London) 284, 316 (1980). 38 T. Taniguchi, H. Matsui, T. Fujita, C. Takaoka, N. Kashirna, R. Yoshimoto. and J. Hamuro, Nature (London) 302, 305 (1983).

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Plasmid DNA Preparation. Individual colonies resulting from transformations, as described above, are transferred to 5 ml cultures of LB medium containing tetracycline (10/.~g/ml), grown overnight, and stored at 4 °. Six individual cultures are used to innoculate (0.1 ml of each culture) 40 ml of LB medium containing tetracycline (10/zg/ml) in 125-ml flasks. The cells are grown with shaking for 3 to 6 hr until the absorbance at 600 nm is between 0.7 and 1.0. Chloramphenicol (160 mg/ml in 95% ethanol) is added to give a final concentration of 200/zg/ml. The cells are grown overnight at 37 ° with shaking to ensure adequate oxygenation. Plasmid DNA is prepared essentially as described. 39 Forty milliliters of culture is transferred to sterile 50-ml polycarbonate tubes and the cells collected by centrifugation at 5000 g for 5 min at 4°. The resulting cell pellets are resuspended in 0.9 ml 25 mM Tris-HCl pH 8.0, 10 mM EDTA 50 mM glucose, and 0.1 ml freshly dissolved lysozyme (20 mg/ml) in 50 mM Tris-HC1 pH 8.0. Cells are left on ice for 30 min, 1.0 ml of freshly prepared 1% SDS in 0.2 M sodium hydroxide is added to each tube, and the contents mixed by swirling gently. The mixtures are kept on ice for 5 min and 1.5 ml 3 M sodium acetate pH 4.8 added to each test tube, the contents mixed by swirling gently and left on ice for a further 60 min. Lysates are cleared by centrifugation at 22,000 g for 30 min at 4 ° and the supernatants transferred to 15 ml polypropylene test tubes. DNA is precipitated by adding an equal volume of 100% isopropanol and cooling to - 2 0 ° for 1 hr. DNA is collected by centrifugation, dissolved in 2 ml TEN 8, and extracted with 1 mi phenol and 1 ml chloroform-isoamyl alcohol (24 : 1 v/v). The aqueous layers are transferred to new test tubes; sodium chloride added to 0.2 M, and plasmid DNA precipitated by addition of two volumes of 95% ethanol and incubation at - 2 0 ° for 18 hr. Precipitated DNA is collected by centrifugation, washed with 70% ethanol, dried under vacuum, and dissolved in 1 ml 20 mM Tris-HCl, 1 mM EDTA pH 8.0. Filter Binding and Hybridization. Plasmid DNA dissolved in 1 ml of 20 mM Tris-HCl, 1 mM EDTA pH 8.0 is heated to 100° for 10 min. One milliliter of 1 M sodium hydroxide is added and the mixture is kept at room temperature for 20 min. The DNA is neutralized by adding 6 ml of 1.5 M sodium chloride, 0.15 M sodium citrate, 0.25 M Tris-HCl pH 8.0, 0.25 M HCI, and is then immediately filtered through 13-mm-diameter prewashed nitrocellulose filters (Sartorius 0.2 /~m or Schleicher and Schuell BA 85 0.45/zm, numbered with a soft pencil) at a flow rate of 1-2 ml/min by applying a slight vacuum. A sampling manifold which holds 10 individual 13-mm-diameter filters (Amicon) may be used when a large number of preparations are being screened. Each filter with bound DNA 39 H. C. Birnboim and J. Doly, Nucleic Acids Res. 7, 1513 (1979).

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is washed with 30 ml of 6xSSC (0.9 M sodium chloride, 0.09 M sodium citrate), air dried, and baked at 70° for 18 hr. A small disk, 5 mm diameter, is cut from each filter using a paper hole punch. Up to 20 disks are placed in a siliconized glass vial and prehybridized at 42 ° for 2 hr in 1 ml of 50% deionized formamide (pH 7), 20 mM Pipes-HCI pH 6.4, 0.4 M sodium chloride, 100/xg/ml tRNA (Sigma type X), 0.2% SDS. The prehybridization solution is then removed and replaced with 1 ml of an identical hybridization solution containing poly(A) RNA at 400 ~g/ml. Disks are hybridized at 42 ° for 6 hr with occasional shaking and then transferred to a sterile 50 ml polypropylene test tube. Disks are washed (50 ml per wash) at 42° nine times with 10 mM Tris-HC! pH 8.0, 0.15 M sodium chloride, 1 mM EDTA, 0.5% SDS, and two times with 50 ml of the same buffer without SDS. Two disks are placed in each siliconized and autoclaved 1.5 ml polypropylene microfuge tube. Bound mRNA is eluted with 0.3 ml water containing 30 tzg carrier RNA. The tubes are heated at 100° for 2 min, quickly frozen in dry ice ethanol, and thawed at room temperature. The filters are removed, and the mRNA precipitated by adding sodium acetate pH 5.0 to 0.3 M and two volumes of 95% ethanol. Precipitated RNA is kept at - 2 0 ° for 18 hr, recovered by centrifugation in a microfuge for I0 min, washed with 70% ethanol, dried under vacuum, and dissolved in 4 ~1 distilled H20. Each disk contains plasmid DNA from 6 individual colonies, therefore, each elution represents 12 individual colonies. Eluted mRNA is translated in a cell-free translation assay and specific polypeptides are identified by immunoprecipitation and analysis by SDS-PAGE, as described above. Once positive pools are identified, the 12 cultures which constituted the original innoculums are individually screened using the same procedure. A large number of individual cDNA transformants can be screened in a relatively short period of time. It is helpful to include a positive control to ensure that all procedures are working and, therefore, all potential specific cDNA clones will be identified.

Molecular Cloning of Rat Ia-A~ Chain cDNA Cell free translation and immunoprecipitation were used to identify mRNA coding for rat Ia-A a chains from Wistar strain rats (RTI") as shown in Fig. 3. Total rat spleen mRNA (prepared as described above) is fractionated on isokinetic 5-20% sucrose density gradients and fractions containing Ia-A o~ chain mRNA pooled and recentrifuged. The resulting pool of enriched mRNA is used as a template for double-stranded cDNA synthesis as described above, size separated by agarose gel electrophoresis, and cDNA molecules greater than 400 base pairs eluted. An aliquot of this cDNA (20 ng) is annealed to PstI digested, G-tailed pBR322 (65 ng)

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FIG. 3. Identification of cDNA clones coding for rat Ia-A a chain by mRNA selection. Recombinant DNA was prepared from 40 ml cultures, immobilized on nitrocellulose filter, and hybridized with total rat spleen mRNA. After washing, bound mRNA was eluted and translated in 50/zl rabbit reticulocyte lysate assay containing 100 p.Ci [3~S]methionine (500 Ci/mmol). Translated products were immunoprecipitated with rabbit antibodies to separated rat Ia-A a chains, analyzed by SDS-PAGE, and visualized by fluorography. Tracks 1 to 12: immunoprecipitated translation products of selected mRNA representing 12 individual cDNA transformants (12 cultures had previously been used to prepare a pool of plasmid DNA which had positively selected Ia-A a chain mRNA); track T, immunoprecipitated translated products of total spleen mRNA.

and the resulting recombinant plasmids are used to transform E. coli strain RR1 as described. F r o m this transformation 1500 independent colonies are usually obtained. Individual colonies are transferred to 5 ml LB medium and used as stock cultures to prepare pools of plasmid DNA. Plasmid D N A is bound to nitrocellulose filters and m R N A selection assays carried out as described. F r o m an initial screen of 192 colonies (16 pools representing 12 colonies) one pool is identified which specifically selects m R N A coding rat Ia-A ot chain. The 12 individual cultures are then screened and the results are shown in Fig. 3. From this screen plasmid D N A from two cultures specifically selects m R N A coded for rat Ia-A a chains (Fig. 3, tracks I and 7). Analysis of the plasmid D N A from

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these two positive cultures shows that culture number 7 contains a single recombinant plasmid designated pRIa. 1 while culture number 1 contains two plasmids, one of which is pRIa. 1 and the other which is negative on rescreening. Restriction endonuclease enzyme analysis shows that the cDNA insert of pRIa. 1 is approximately 600 base pairs in length and that one PstI site is not regenerated. A 150 base pair EcoRI-PstI fragment from pRIa. 1 is prepared and used to screen another cDNA library prepared from total rat spleen mRNA. From a screen of 2000 independent transformants, two colonies which hybridized to this fragment are obtained. The relative abundance of Ia-A ct chain mRNA in this library, prepared from total spleen mRNA, is approximately 0.1%. Of these two positive colonies the recombinant plasmid containing the largest cDNA insert (800 base pairs in length) is designated pRIa.2. The complete nucleotide sequence of the cDNA insert of pRIa.2, determined by the methods of Maxam and Gilbert, 4° is shown in Fig. 4. The DNA sequence is 779 nucleotides in length and contains a single open reading frame of 387 nucleotides which, when translated into protein sequence, codes for the carboxy-terminal 129 amino acids of a rat Ia-A chain (Fig. 4). 41 This represents approximately 55% of the mature a chain based on an apparent molecular weight of 30,000 and on the predicted structures of human HLA-DC142 and mouse H-2 I-A 43 ct chains. Following the open reading frame there is an untranslated region of 290 nucleotides terminating in a tract of poly(A) of 63 nucleotides. Fifteen nucleotides prior to the poly(A) region there is a putative polyadenylation site AATAAA. This is, therefore, the 3' end of the corresponding mRNA. The protein structure of the rat Ia-A a chain can be divided into different regions or domains by analogy with the domain structure of the H L A DC1 a chain. 42 Based on this sequence the cDNA insert of pRIa-2 codes for amino acids 102 to 232. This represents the majority of the second extraceUular domain ~ 2 (amino acids 104 to 181) which contains a putative disulfide loop of 55 amino acids from Cys-ll0 to Cys-164 and a connecting peptide of 13 amino acids (182 to 194). Following these two extracellular regions is a hydrophobic transmembrane region of 23 amino acids (195 to 217) and a cytoplasmic region of 15 amino acids (218 to 232). The rat Ia-A ~ chain is highly homologous, in terms of both DNA and amino acid sequence identity, to human HLA-DC 1 and mouse H-2 I-A chains. Comparison of the DNA protein coding region shows there is 85% ~0 A. Maxam and W. Gilbert, this series, Vol. 65, p. 499. 4~ A. E. Wallis and W. R. McMaster, lmmunogenetics 19, 53 (1984). 4z C. Auffray, A. J. Korman, M. Roux-Dosseto, R. Bono, and J. L. Strominger, Proc. Natl. Acad. Sci. U.S.A. 79, 6337 (1982). 49 C. O. Benoist, D. J. Mathis, M. R. Kanter, V. E. Williams, II, and H. O. McDevitt, Proc. Natl. Acad. Sci. U.S.A. 80, 534 (1983).

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LYMPHOID CELL SURFACE ANTIGENS

(G)

[511

1 40 T CAG CCC AAC ACC CTC ATC TGC TTT GTA GAC AAC ATC TTT 18 GLN PRO ASN THR LEU ILE CYS PHE VAL ASP ASN ILE PHE 104 116

85 CCT CCT GTG ATC AAT ATC ACA TGG TTG AGA AAC AGC AAG CCA GTC PRO PRO VAL ILE ASN ILE THR TRP LEU ARG ASN SER LYS PRO VAL 131 130 ACA GAA GGC GTT TAT GAG ACC AGC TTC CTT TCC AAC CCT GAC CAT THR GLU GLY VAL TYR GLU THR SER PHE LEU SER ASN PRO ASP HIS 146 175 TCC TTC CAC AAG ATG GCT TAC CTC ACC TTC ATC CCT TCC AAC GAC SER PHE HIS LYS MET ALA TYR LEU THR PHE ILE PRO SER ASN ASP t61 220 GAC ATT TAT GAC TGC AAG GTG GAG CAC TGG GGC CTG GAC GAG CCG ASP ILE TYR ASP CYS LYS VAL GLU HIS TRP GLY LEU ASP GLU PRO 176 265 GTT CTA AAA CAC TGG GAA CCT GAG GTT CCA GCC CCC ATG TCA GAG VAL LEU LYS H I S TRP GLU PRO GLU VAL PRO ALA PRO MET SER GLU 181 182 191 310 CTG ACA GAG ACT GTG GTC TGT GCC CTG GGG TTG TCT GTG GGC CTC LEU THR GLU THR VAL VAL CYS ALA LEU GLY LEU SER VAL GLY LEU 194 195 206 355 GTG GGC ATC GTG GTG GGC ACC ATC TTC ATC ATT CAA GGC CTG CGA VAL GLY ILE VAL VAL GLY THR ILE PHE ILE ILE GLN GLY LEU ARG 217 218 221 400 TCA GAT GGC CCC TCC AGA CAC CCA GGG CCC CTT TGA GTC ACA CCC SER ASP GLY PRO SER ARG HIS PRO GLY PRO LEU * ' " 232 445 TGG GAA AGA AGG TGC GTG GCC CTC TAC AGG CAA GAT GTA GTG TGA 490 GGG GIG ACC TGG CAC AGT GTG TTT TCT GCC CCA ATT CAT CGT GTT 535 CTT TCT CTT CTC CTG GTG TCT CCC ATC TTG CTC TTC CCT TGG CCC 590 CCA GGC TGT CCA CCT CAT GGC TCT CAC GCC CTT GGA ATT CTC CCC 625 IGA CCT GAG TTT CAT TTT TGG CAT CTT CCA AGT CGA ATC TAC TAT 670 AGA TTC CGA GAC CCT GAT TGA TGC TCC ACC AAA CCA ATA AAC CTC 681 (C) I C A TAA GTT GG ( A ) 63 17

FIG. 4. The nucleotide sequence of the cDNA insert of pRIa.2 coding for a rat Ia-A a chain and the predicted amino acid sequence. The DNA sequence is presented 5' to 3' and

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D N A s e q u e n c e identity b e t w e e n rat and h u m a n a chains and 91% D N A sequence identity b e t w e e n rat and m o u s e a chains. At the protein level, without the need to introduce any gaps, b e t w e e n rat and h u m a n a chains there is 81% protein sequence identity and b e t w e e n rat and m o u s e a chains there is 91% protein sequence identity. The Ia-A a chains, therefore, h a v e b e e n highly c o n s e r v e d during evolution and are more homologous to each other b e t w e e n species than are the Ia-A and Ia-E a chains within the s a m e species. 41 The major histocompatibility c o m p l e x Class 144 and Ia 45 antigens and rodent T h y - 1 4 6 antigens h a v e b e e n shown to be homologous in terms of sequence identity to immunoglobulin domains. Analysis of the predicted amino acid sequence of the rat I a - A a chain also shows that this protein shares several c o n s e r v e d amino acids which are characteristic of immunoglobulin constant region domains. 41 T h e s e c o n s e r v e d amino acids are thought to be important in maintaining a g-pleated sheet structure in immunoglobulin domains. Therefore, this h o m o l o g y implies that the a 2 domain o f Ia-A o~ chain also r e s e m b l e an immunoglobulin-like fold. Further analysis of the structure of Ia antigens m a y determine which domains are involved in cell-cell interactions within the immune s y s t e m m a y lead to a better understanding of the molecular basis of the immune response genes. Acknowledgments I am grateful to Drs. R. T. A. MacGillivray and A. F. Williams for many discussions and helpful suggestions during the course of these studies. This work was supported in part by grants from the MRC (Canada) and the B.C. Health Care Research Foundation. W. R. McMaster is a Scholar of the MRC.

44 H. T. Orr, J. A. Lopez de Castro, D. Lancet, and J. L. Strominger, Biochemistry 18, 5711 (1979). 4~ D. Larhammar, K. Gustafsson, L. Claesson, P. Bill, K. Winman, L. Schenning, J. Sundelin, E. Widmark, P. A. Peterson, and L. Rask, Cell 30, 153 (1982). 46 A. F. Williams and J. Gagnon, Science 216, 696 (1982).

only the coding strand is shown. Numbers above each line correspond to the nucleotide position starting after the poly(G) tail and ending prior to the tract of poly(A). The putative polyadenylation site, AATAAA, is underlined. The predicted amino acid sequence of the carboxy-terminal 129 positions of the rat Ia-A a chain is shown below the nucleotide sequence. Numbers below each line corresponds to the amino acid positions of an HLA-DC1 a chain. (From Auffray et al. 42)