©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Major Histocompatibility-encoded Human Proteasome LMP2
GENOMIC ORGANIZATION AND A NEW FORM OF mRNA (*)

(Received for publication, June 6, 1994; and in revised form, October 20, 1994)

Dharam P. Singal (§) Ming Ye Shafat A. Quadri

From the Department of Pathology, McMaster University, Hamilton, Ontario L8N 3Z5, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

LMP2 is one of the two proteasome subunits encoded by genes in the major histocompatibility complex class II region. Here we report the genomic organization of human LMP2 gene. Sequence analysis of polymerase chain reaction-amplified cDNA from a number of lymphoblastoid cell lines demonstrated two forms of LMP2 mRNA, one (LMP2.l) complete and homologous to the published LMP2 genomic sequence from cosmid clones, and the other (LMP2.s) a smaller transcript resulting from splicing of a 30-base pair fragment from the first exon. Antibodies to recombinant LMP2.s protein (22.3 kDa) were raised in rabbits. This anti-LMP2.s serum recognized both recombinant proteins (LMP2.l = 23.3 kDa and LMP2.s = 22.3 kDa) and a single protein of 21.5 kDa molecular mass in lysates from human lymphoblastoid cell lines. Pulse-chase experiments demonstrated that LMP2 polypeptide also undergoes processing from 22.3- to 21.5-kDa protein when incorporated into proteasomes. These data suggest that the processing of human LMP2 subunit takes place both at the transcription and post-translational levels. Northern blot analysis showed that the LMP2 mRNA is expressed in lymphoblastoid cell lines and in fibroblasts following -interferon induction, but not in brain, smooth muscle, fibroblasts (uninduced), and colon epithelial cells.


INTRODUCTION

Cytotoxic T lymphocytes recognize peptides derived from intracellular antigens presented at the cell surface in association with MHC^1 class I molecules. Before peptide presentation by class I molecules, cytoplasmic antigenic proteins must be degraded in cytosol and the peptides derived from them transported into endoplasmic reticulum (Townsend and Bodmer, 1989; Monaco, 1992). Furthermore, it has been demonstrated that bound peptide is important for the stability, conformation, and cell-surface expression of class I molecules (Townsend et al., 1989; Spies and DeMars, 1991; Powis et al., 1991; Attaya et al., 1992). Four tightly linked genes involved in generation of peptides and their transport to the assembly site have been mapped within the MHC class II region. Of these, two genes (TAP1 and TAP2) related to the superfamily of the ATP-binding cassette transporters have been identified in the class II region of human, rat, and mouse (Deverson et al., 1990; Monaco et al., 1990; Spies et al., 1990; Trowsdale et al., 1990). Transfection of mutant class I-deficient cell lines with TAP1 and TAP2 cDNA restored the cell surface expression of class I molecules (Spies and DeMars, 1991; Powis et al., 1991; Attaya et al., 1992).

Two genes, LMP2 and LMP7, are tightly linked to TAP genes and encode two of the subunits of a large cytoplasmic structure called the LMP complex (Monaco and McDevitt, 1986; Martinez and Monaco, 1991; Glynne et al., 1991; Kelly et al., 1991). The predicted amino acid sequences of gene products of these genes, mapping of these genes to the region of the MHC that encodes genes for antigen processing, and the regulation of the LMP complex by -IFN suggest that the MHC-encoded proteasome chains play some distinctive role in protein degradation for antigen processing (Monaco and McDevitt, 1986). Two reports, however, showed that mutant cell lines with defective class I expression and lacking genes for both TAP and LMP could be restored to normal functional expression of class I molecules by transfection of TAP genes in the absence of LMP (Arnold et al., 1992; Momburg et al., 1992). Some recent studies bring the LMP2 and LMP7 chains back into consideration by demonstrating that the lack or presence of LMPs was associated with changes in cleavage specificity of the proteasomes (Brown et al., 1993; Driscoll et al., 1993; Gaczynska et al., 1993). Indeed, it has been shown that the substrate specificity of LMP27 proteasomes differs from that of LMP27 proteasomes (Driscoll et al., 1993). In addition, it has been demonstrated that the LMP2-deficient mice express normal or near normal levels of class I molecules, but generate 5-6-fold fewer cytotoxic T lymphocytes in response to influenza infection (quoted by Brown et al., 1993).

The genomic structures of human LMP2 and LMP7 genes have recently been reported (Martinez and Monaco, 1991; Kelly et al., 1991; Früh et al., 1992). Western analysis and pulse-chase experiments have suggested that the polypeptide encoded by LMP2 gene undergoes proteolytic processing when incorporated into proteasome. In the present study, we report a second form of human LMP2 mRNA, which results from splicing of a 30-bp fragment from the first exon during transcription. In addition, the data suggest proteolytic processing of the LMP2 polypeptide at the post-translational level when incorporated into proteasome.


MATERIALS AND METHODS

Lymphoblastoid and Fibroblast Cell Lines

Five EBV-transformed human B lymphoblastoid typing cell lines (JESTHOM, DEU, BM14, VAVY, and SLE005) obtained from the Tenth International Histocompatibility Workshop were utilized in these studies. T1 and T2 cell lines were provided by Dr. P. Cresswell. EBV-transformed B cell lines from six unrelated subjects and fibroblast cell lines from another four unrelated individuals, generated in our laboratory, were also studied. Lymphoblastoid and fibroblast cell lines were propagated and maintained in medium RPMI 1640 or minimum essential medium, respectively, supplemented with 10% fetal bovine serum.

Preparation of RNA and Synthesis of cDNA

Total RNA from lymphoblastoid and fibroblast cell lines was prepared by the acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomeczynski and Sacchi, 1987). In addition, RNA was extracted from human brain, smooth muscle, and colon epithelial cells. The first strand cDNA was prepared from RNA using the Superscript choice system (Life Technologies, Inc.). Briefly, 5 µg of RNA was primed with 1 µg/µl oligo(dT), and the mixture was incubated at 37 °C with 10 mM deoxyribonucleoside triphosphates (dNTPs) in the presence of 200 units of Superscript RT.

PCR Amplification of cDNA and Sequence Analysis of LMP2 Gene

Samples of cDNA were separately amplified for the LMP2 gene by PCR utilizing primers deduced from published sequences (Kelly et al., 1991). These primers, PR1 (bp -9 to 8, 5`-CGCGCGGATCCTTGCAGGGATGCTGCG-3` and PR2 (bp 651-668, 5`-CGCGCGAATTCGGGAAGGTTCACTCATCA-3`), were designed with restriction sites for BamHI and EcoRI, respectively.

PCR-amplified DNA was ligated into Bluescript KSII vector. DHalpha competent cells (Life Technologies, Inc.) were transformed. DNA extracted from at least two colonies in each case was sequenced by the dideoxy chain termination method (Sanger and Coulson, 1975).

Analysis of Nucleotide Variants of LMP2 cDNA and Genomic DNA with Sequence-specific Oligonucleotide (SSO) Probes

Samples of cDNA and genomic DNA from a number of lymphoblastoid and fibroblast cell lines were separately amplified for the first exon of the LMP2 gene using primers PR1 and PR3 (beginning of intron 1, 5`-TCAAGCCCAGACCCATTAAC-3`). PCR-amplified DNA was denatured, slotted onto GeneScreen Plus membrane, and hybridized overnight with [-P]ATP (ICN)-labeled SSO probes (SSO-1 = 5`-CCAACCGGGGACTTACCC-3`; SSO-2 = 5`-CTGCGGGCGGGAGAAGTC-3`). After hybridization, the blot was washed under stringent conditions and autoradiographed for 30-45 min using Quanta Blue (Eastman Kodak) intensifying screens (Singal et al., 1992, 1993).

Cloning, Expression, and Purification of LMP2 Proteins in Escherichia coli

Full-length cDNAs corresponding to the two forms of LMP2 (LMP2.long = LMP2.l, and LMP2.short = LMP2.s) were isolated from PCR-amplified cDNA from a lymphoblastoid cell line (JESTHOM). A fragment starting at the initiator ATG, with 5` blunt end and 3` sticky end, was cloned at the XmnI and XbaI sites into the pMAL-C2 vector (New England Biolabs, Beverly, MA). E. coli TB1 cells were transformed, and the expression of protein was induced by isopropyl beta-D-thiogalactopyranoside. LMP2 proteins were purified from the maltose-binding protein on cross-linked amylose resin (Guan et al., 1988). For both LMP2 proteins, single protein bands corresponding to the correct molecular weights were observed.

Production of Anti-LMP2 Antiserum

Antiserum to lower molecular mass LMP2 protein (LMP2.s) was raised in rabbits by injecting 100 µg of purified recombinant protein in Freund's complete adjuvants, followed by several bi-weekly injections of the same amount of antigen without adjuvants. Animals were bled 1 week after each immunization, and the specificity of the rabbit antisera was tested by immunoblotting.

Western Blot Analysis

Cells (3-5 times 10^6) were suspended in SDS sample buffer, boiled for 5 min, and the proteins separated by 12% SDS-PAGE. After separation, proteins were electrophoretically transferred onto a nitrocellulose membrane (Schleicher and Schuell) in a trans blot cell (Bio-Rad). The membranes were incubated overnight in a 5% non-fat milk blocking solution and probed with 1:500 dilution of anti-LMP2.s serum. Bound antibodies were detected by alkaline phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad).

Northern Blot Analysis of LMP2 mRNA

Northern blots were carried out by separating total RNA from human B lymphoblastoid and fibroblast (with or without prior induction with -IFN) cell lines, brain, smooth muscle, and mucosal epithelial cells on a denaturing agarose gel and transfer onto GeneScreen Plus membrane (Sambrook et al., 1989). The membranes were prehybridized overnight at 42 °C to salmon sperm DNA and then hybridized with radiolabeled (nick translation) LMP2 cDNA. Blots were washed under stringent conditions and autoradiographed on Kodak XAR-5 film (Eastman Kodak) with an intensifying screen. The blots were dehybridized and probed with beta-actin cDNA in control experiments.

Metabolic Radiolabeling and Immunoprecipitation

Cells (6 times 10^6) were metabolically radiolabeled in medium 199 (minus methionine) with 500 µCi of [S]methionine (Dupont Canada Inc., Mississauga, Ontario) for 50 min, washed, and chased with unlabeled methionine for up to 120 min. Aliquots of cells were removed at 0, 60, and 120 min following label. Cells were lysed in 100 µl of lysis buffer (137 mM NaCl, 3 mM KCl, 8 mM Na(2)HPO(4), 1.5 mM KH(2)PO(4), pH 7.4) containing 1% SDS and 1 mM phenylmethylsulfonyl fluoride. Lysates were diluted in lysis buffer containing 0.01% SDS, 1% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride. Cellular proteins were immunoprecipitated with anti-LMP2.s antiserum and analyzed by SDS-PAGE.


RESULTS

Sequence Analysis of LMP2 cDNA

Electrophoretic analysis of PCR-amplified cDNA from lymphoblastoid cell lines, JESTHOM, DEU, and an unrelated subject JW, showed two fragments, one of 670 bp and the other of 640 bp (Fig. 1). Similar results were obtained with remaining lymphoblastoid cell lines.


Figure 1: Electrophoretic analysis of PCR-amplified LMP2 cDNA from human lymphoblastoid cell lines. Lane 1 = JESTHOM, lane 2 = DEU, and lane 3 = unrelated subject JW.



Sequence analyses of these two LMP2 cDNAs from two different clones of a cell line JESTHOM are shown in Fig. 2. For comparison, it also shows nucleotide sequences (row 1) of LMP2 gene as described previously (Kelly et al., 1991; Früh et al., 1992). It is evident that two sets of LMP2 sequences were observed from two different clones from this cell line. Of these, one sequence (LMP2.l) showed identity with the published sequence (row 2), whereas the other one (LMP2.s) had a deletion of 30 bp (from 16 to 45 bp) in the first exon (row 3). Since nucleotide sequence CGGGCGGGA (position 7-15) is repeated again (position 37-45), it is likely that splicing takes place from bp 7 to 36 bp. Similarly, two sets of sequences for LMP2 cDNA (LMP2.l and LMP2.s) were observed in cell line DEU and each of the six lymphoblastoid cell lines from unrelated subjects.


Figure 2: Nucleotide sequences of LMP2. Row 1 = LMP2 gene sequence obtained from Früh et al.(1992); rows 2 and 3 = LMP2 sequences from two different clones obtained following PCR amplification of cDNA from a lymphoblastoid cell line JESTHOM. Dashes indicate identity to respective nucleotide in row 1, and the shaded area indicates deletion.



Oligonucleotide Typing of PCR-amplified Genomic DNA and cDNA for Two Forms of LMP2

Samples of genomic DNA and cDNA from lymphoblastoid cell lines were separately amplified utilizing PR1 and PR3 primers and hybridized with SSO-1 (specific for LMP2.l) and SSO-2 (specific for LMP2.s) probes. The results of dot-blot analysis of PCR-amplified genomic DNA (rows A and B) and cDNA (rows C and D) from four lymphoblastoid (1-4) cell lines show that all genomic DNA samples had only one (LMP2.l) sequence (Fig. 3). In contrast, cDNA samples from lymphoblastoid cell lines had both (LMP2.l and LMP2.s) sequences. Similar results were obtained with remaining lymphoblastoid cell lines.


Figure 3: Dot-blot analysis of PCR-amplified genomic DNA and cDNA with sequence-specific oligonucleotide probes. Samples of DNA were separately amplified with primers PR1 and PR3 and hybridized with SSO probes; rows A and C = SSO-1, rows B and D = SSO-2. DNA and cDNA samples were obtained from lymphoblastoid cell lines (1-4), fibroblasts (5 and 6), smooth muscle (7), colon epithelial cells (8), brain (9), and -IFN-treated fibroblasts (10).



Dot-blot analysis of PCR-amplified genomic DNA from fibroblasts (5 and 6), smooth muscle (7), colon epithelial cells (8), and brain (9) also showed only one, i.e. LMP2.l, sequence (Fig. 3). No amplification, either for exon 1 or for the entire LMP2 sequence, was observed with cDNA from these tissues.

Tissue Distribution of LMP2-specific mRNA

Northern blot analysis of RNA showed that LMP2 mRNA was present in lymphoblastoid cell lines (lanes 1-4) but was not detectable in uninduced fibroblasts (lane 6), smooth muscle (lane 7), colon epithelial cells (lane 8), and brain (lane 9) (Fig. 4). In fibroblasts, LMP2 mRNA was observed following induction with -IFN (lane 5). Dot-blot analysis of PCR-amplified cDNA showed that IFN-induced both LMP2.l and LMP2.s transcripts in fibroblasts (Fig. 3, lane 10).


Figure 4: Northern blot analysis of total RNA from lymphoblastoid cell lines (1-4), -IFN-induced fibroblasts (5), uninduced fibroblasts (6), smooth muscle (7), colon epithelial cells (8), and brain (9) with human LMP2 cDNA probe (lower panel) or with beta-actin cDNA probe (upper panel).



Identification of LMP2-encoded Proteins

Both LMP2 recombinant proteins corresponded to the expected molecular masses (LMP2.l = 23.3 kDa; LMP2.s = 22.3 kDa). The anti-LMP2.s antiserum recognized both species of recombinant LMP2 (LMP2.l and LMP2.s) proteins and a single protein of 21.5 kDa in cell lysates from lymphoblastoid cell lines and from -IFN-treated fibroblasts (Fig. 5). For comparison, we carried out Western analysis with anti-LMP2 antibody obtained from Dr. McDevitt (Patel et al., 1994). It is evident that this antibody gives an identical profile with both recombinant proteins and with cell lysate as observed with anti-LMP2.s antibody raised in the present study (Fig. 5). In addition, a single protein of 21.5 kDa was present in T1 cell lysate but absent in mutant T2 cell line (data not shown). These data suggest a high degree of specificity of anti-LMP2.s serum raised in the present study.


Figure 5: Western analysis of LMP2 subunit with rabbit anti-LMP2.s serum raised in the present study (lanes 1-4, and 8), and with anti-LMP serum obtained from Patel et al. (lanes 5, 6, and 7). Lanes 1 and 5 = recombinant LMP2.l protein generated from LMP2.l mRNA, lanes 2 and 6 = recombinant LMP2.s protein generated from spliced LMP2.s mRNA, lane 3 = lysate from a lymphoblastoid cell line, VAVY, lanes 4 and 7 = lysate from a lymphoblastoid cell line, SLE005, and lane 8 = lysate from -IFN-treated fibroblasts.



To identify the possible precursor of LMP2, we performed the pulse-chase experiments prior to immunoprecipitation with anti-LMP2.s antiserum. Cells from three homozygous typing cell lines (BM14, SLE005, and JESTHOM) were labeled for 50 min followed by 0-, 60-, and 120-min chase. Fig. 6shows the results from one such experiment. It can be seen that the anti-LMP2.s serum precipitated a 22.3-kDa protein (lane 1), whose intensity decreased during the chase period, and a protein of molecular mass of 21.5 kDa appeared (lanes 2 and 3) after 60- and 120-min chase. This shows that the low molecular mass form of LMP2 is derived from the precursor of 22.3 kDa, the product of the smaller transcript, i.e. LMP2.s mRNA.


Figure 6: Pulse-chase analysis of LMP2. Lymphoblastoid (SLE005) cells (lanes 1-3) and -IFN-treated fibroblasts (lanes 4 and 5) were metabolically labeled with [S]methionine for 50 min followed by a 0-min (lanes 1 and 4), 60-min (lane 2), and 120-min (lanes 3 and 5) chase with unlabeled methionine. After cell lysis, proteins were immunoprecipitated with anti-LMP2.s serum and analyzed by SDS-PAGE.



To elucidate the effect of -IFN on processing of LMP2 protein, pulse-chase experiments were carried out with -IFN-treated fibroblasts. The results show that the anti-LMP2.s serum precipitated a 22.3-kDa protein (Fig. 6, lane 4). The intensity of this band decreased during the chase period and a protein of 21.5 kDa appeared after 120-min chase (lane 5). These data suggest that the processing of protein in -IFN-treated fibroblasts was similar to that in untreated lymphoblastoid cell lines.


DISCUSSION

In the present article, we have analyzed the genomic organization of human proteasome LMP2 gene by sequencing the PCR-amplified cDNA from a number of human EBV-transformed lymphoblastoid cell lines. Nucleotide sequence analysis showed the presence of two transcripts; one homologous (LMP2.l) to the LMP2 sequence published earlier from the cosmid clone, U15 (Martinez and Monaco, 1991; Kelly et al., 1991; Früh et al., 1992), and the other representing a smaller transcript (LMP2.s). It is likely that this new form of LMP2 mRNA arises following deletion of a 30-bp fragment from the first exon.

Northern blot analysis of lymphoblastoid cell lines and various human tissues revealed differences in LMP2 mRNA levels in different tissues. The results in the present study therefore confirm and extend the earlier data in that LMP2 mRNA is abundant in lymphoblastoid cell lines, but no detectable amounts are present in brain, smooth muscle, fibroblasts, and colon epithelial cells (Früh et al., 1992; Frentzel et al., 1993). Treatment of fibroblasts with -IFN led to an increase in both LMP2.l and LMP2.s mRNA levels. Pulse-chase experiment showed that the precursor protein in -IFN-treated fibroblasts, as in lymphoblastoid cells, is the product of LMP2.s transcript. In addition, processing of the precursor protein in -IFN-treated fibroblasts is similar to that in lymphoblastoid cell lines, suggesting that -IFN up-regulates levels of LMP2 mRNA, but does not affect post-translational regulation of LMP2 protein.

Cloning and expression of recombinant proteins to the two forms of LMP2 mRNA resulted in proteins of expected sizes (23.3 and 22.3 kDa). Anti-LMP2.s sera raised against the 22.3-kDa recombinant protein recognized a single protein of 21.5 kDa in cell lysates from a number of human lymphoblastoid cell lines and from -IFN-treated fibroblasts by the Western analysis. Reactivity of this antiserum with T1 and mutant T2 cell lines, and comparison of results with anti-LMP2 serum from Dr. McDevitt (Patel et al., 1994), suggest a high degree of specificity of the anti-LMP2.s serum raised in the present study.

Investigations of the LMP2 subunits in cell lysates have been carried out utilizing anti-LMP2 and anti-proteasome antibodies (Früh et al., 1992; Frentzel et al., 1993, Patel et al., 1994). These studies demonstrated that rabbit anti-LMP2 antibodies recognized a single protein of approximately 21 kDa in mouse RMA cells (Früh et al., 1992; Frentzel et al., 1993). The rabbit anti-human LMP2.s serum in the present study also recognizes a single protein of approximately the same molecular mass in cell lysates from human lymphoblastoid cell lines.

Pulse-chase experiments have been performed to identify the possible precursors of LMP2 proteins (Früh et al., 1992; Frentzel et al., 1993). The results from these experiments suggest that the polypeptide encoded by LMP2 undergoes proteolytic processing from a protein of molecular mass of 23.3 kDa to a 21.5-kDa protein when incorporated into proteasomes. These experiments were, however, carried out with mouse RMA cells utilizing rabbit antiserum directed against mouse LMP2 (Frentzel et al., 1993) or human LMP2 protein (Früh et al., 1992). In the present study, we utilized rabbit anti-human LMP2.s serum to recognize LMP2 subunits in human lymphoblastoid cell lines. This antibody recognizes in cell lysates a single protein of a lower (21.5 kDa) molecular mass. Pulse-chase experiments demonstrated that this lower molecular mass protein (21.5 kDa) is derived from precursor protein of 22.3 kDa, the product of smaller transcript of LMP2, i.e. LMP2.s. The results in the present study, therefore, demonstrate that processing of LMP2 takes place both at the transcription and post-translational levels. These data suggest different mechanisms for processing of LMP2 subunits in mice and humans; LMP2 undergoes proteolytic processing when incorporated in proteasomes in mice, whereas in humans the processing of LMP2 takes place both at the transcription and post-translational levels.

Patel et al.(1994) utilized rabbit anti-LMP2 (alphaLMP2) antibody and a monoclonal anti-proteasome (mAb2-17) antibody and demonstrated the existence of two forms of LMP2, LMP2a, and LMP2b. Rabbit anti-LMP2 antibody recognized only LMP2a subunit, whereas mAb2-17 immunoprecipitated both subunits. They suggested that LMP2a is probably the precursor of mature LMP2 subunit, since anti-LMP2 antibody recognized protein of the expected (23.3 kDa) molecular mass. However in the pulse-chase, the complex was still present after a 6-h chase (Patel et al., 1994). In our laboratory, this antibody recognized LMP2 polypeptide of 21.5 kDa, suggesting that this protein is the processed LMP2 subunit. The reasons for differences in these two investigations are not clear, however.

LMP gene products play an important role in processing antigens for class I-restricted presentation. It is likely that a distortion in the novel processing mechanisms of LMP2 described in the present report, i.e. splicing at the transcriptional level followed by processing at the post-translational level, for example by pathogens, will result in an aberrant immune response and may contribute to the development of autoimmune diseases. Furthermore, these data will facilitate to investigate if the MHC-associated diseases are caused by defective structural (sequence) and/or regulatory (processing) components of LMP2 gene (Früh et al., 1992).


FOOTNOTES

*
This work was supported by research grants from the Medical Research Council and the Arthritis Society, Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) UO1025.

§
To whom correspondence should be addressed: Dept. of Pathology, McMaster University, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada. Tel.: 905-525-9140 (Ext. 22473); Fax: 905-522-6750.

(^1)
The abbreviations used are: MHC, major histocompatibility complex; TAP, transporter associated with antigen processing; LMP, low molecular mass polypeptide; EBV, Epstein-Barr Virus; FBS, fetal bovine serum; -IFN, -interferon; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); SSO, sequence-specific oligonucleotide.


REFERENCES

  1. Arnold, D., Driscoll, J., Androlewicz, M., Hughes, E., Cresswell, P., and Spies, T. (1992) Nature 360, 171-174 [CrossRef][Medline] [Order article via Infotrieve]
  2. Attaya, M., Jameson, S., Martinez, C. K., Hermel, E., Aldrich, C., Forman, J., Lindhal, K. F., Bevan, M. J., and Monaco, J. J. (1992) Nature 355, 647-649 [CrossRef][Medline] [Order article via Infotrieve]
  3. Brown, M. G., Driscoll, J., and Monaco, J. J. (1993) J. Immunol. 151, 1193-1204 [Abstract/Free Full Text]
  4. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  5. Deverson, E. V., Gow, I. R., Coadwell, W. J., Monaco, J. J., Butcher, G. W., and Howard, J. C. (1990) Nature 348, 738-741 [CrossRef][Medline] [Order article via Infotrieve]
  6. Driscoll, J., Brown, M. G., Finley, D., and Monaco, J. J. (1993) Nature 365, 262-264 [CrossRef][Medline] [Order article via Infotrieve]
  7. Frentzel, S., Kuhn-Hartmann, I., Gernold, M., Gott, P., Seelig, A., and Kloetzel, P.-M. (1993) Eur. J. Biochem. 216, 119-126 [Abstract]
  8. Früh, K., Yang, Y., Arnold, D., Chambers, J., Wu, L., Waters, J. B., Spies, T., and Peterson, P. A. (1992) J. Biol. Chem. 267, 22131-22140 [Abstract/Free Full Text]
  9. Gaczynska, M., Rock, K. L., and Goldberg, A. L. (1993) Nature 365, 264-267 [CrossRef][Medline] [Order article via Infotrieve]
  10. Glynne, R., Powis, S. H., Beck, S., Kelly, A., Kerr, L., and Trowsdale, J. (1991) Nature 353, 357-360 [CrossRef][Medline] [Order article via Infotrieve]
  11. Guan, C.-D., Li, P., Riggs, P. D., and Inouye, H. (1988) Gene (Amst.) 67, 21-30 [CrossRef][Medline] [Order article via Infotrieve]
  12. Kelly, A., Powis, S. H., Glynne, R., Radley, E., Beck, S., and Trowsdale, J. (1991) Nature 353, 667-668 [CrossRef][Medline] [Order article via Infotrieve]
  13. Martinez, C. K., and Monaco, J. J. (1991) Nature 353, 664-667 [CrossRef][Medline] [Order article via Infotrieve]
  14. Momburg, F., Ortiz-Navarrete, V., Neefjes, J., Goulmy, E., van de Wal, Y., Spits, H., Powis, S. J., Butcher, G. W., Howard, J. C., Walden, P., and Hämmerling, G. J. (1992) Nature 360, 174-177 [CrossRef][Medline] [Order article via Infotrieve]
  15. Monaco, J. J. (1992) Immunol. Today 13, 173-179 [CrossRef][Medline] [Order article via Infotrieve]
  16. Monaco, J. J., and McDevitt, H. O. (1986) Hum. Immunol. 15, 416-426 [Medline] [Order article via Infotrieve]
  17. Monaco, J. J., Cho, S., and Attaya, M. (1990) Science 250, 1723-1726 [Medline] [Order article via Infotrieve]
  18. Patel, S. D., Monaco, J. J., and McDevitt, H. O. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 296-300 [Abstract]
  19. Powis, S. J., Townsend, A. R. M., Deverson, E. V., Bastin, J., Butcher, G. W., and Howard, J. C. (1991) Nature 354, 528-531 [CrossRef][Medline] [Order article via Infotrieve]
  20. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  21. Sanger, F., and Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448 [Medline] [Order article via Infotrieve]
  22. Singal, D. P., Green, D., Reid, B., Gladman, D. D., and Buchanan, W. W. (1992) Ann. Rheum. Dis. 51, 23-28 [Abstract]
  23. Singal, D. P., Qiu, X., D'Souza, M., and Sood, S. K. (1993) Immunogenetics 37, 143-147 [Medline] [Order article via Infotrieve]
  24. Spies, T., and DeMars, R. (1991) Nature 351, 323-324 [CrossRef][Medline] [Order article via Infotrieve]
  25. Spies, T., Bresnahan, M., Bahram, S., Arnold, D., Blanck, G., Mellins, E., Pious, D., and DeMars, R. (1990) Nature 348, 744-747 [CrossRef][Medline] [Order article via Infotrieve]
  26. Townsend, A., and Bodmer, H. (1989) Annu. Rev. Immunol. 7, 601-624 [CrossRef][Medline] [Order article via Infotrieve]
  27. Townsend, A., Ohlen, C., Bastin, J., Ljunggren, H.-G., Foster, L., and Kärre, K. (1989) Nature 340, 443-448 [CrossRef][Medline] [Order article via Infotrieve]
  28. Trowsdale, J., Hanson, I., Mockridge, I., Beck, S., Townsend, A., and Kelly, A. (1990) Nature 348, 741-744 [CrossRef][Medline] [Order article via Infotrieve]

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