The H-2 dm1 mutation and Qa antigens

The effect of the H-2dm1 mutation on Qa-m2 expression was examined. The mutant strain B10.D2-H-2dm1 showed a fourfold increase in Qa-m2 expression when compared with the parental strain B10.D2. Qa-m2 molecules immunoprecipitated from B10.D2-H-2dm1, C57BL/10, and B10.D2 spleen cells were identical by two-dimensional (2-D) gel electrophoresis [isoelectric focusing (IEF) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE]). It is likely therefore that the increased Qa-m2 expression is not due to gross structural alterations of the Qa-m2 molecule; in the present study, alternative explanations are discussed.     

The Qa-1 alloantigens. I. Identification and molecular weight characterization of glycoproteins controlled by the Qa-1a and Qa-1b alleles

Splenocytes from the Qa-Tla congenic strain pairs, A and A-Tlab or B6 and B6-Tlaa, were biosynthetically labeled with 3H-amino acids or cell surface labeled with 125I. Membrane proteins were solubilized with detergent and chromatographed on lentil lectin-Sepharose, and the resulting adherent pools were immunoprecipitated with antisera specific for determinants controlled by the Qa-1a and Qa-1b alleles, Qa-1.1 and Qa-1.2, respectively. Polyacrylamide gel electrophoresis analysis of immunoprecipitates from biosynthetically labeled preparations indicated that both the Qa-1.1 and Qa-1.2 antigens were glycoproteins with a m.w. of approximately 46,000. Qa-1.2 isolated from radioiodinated spleen cells similarly had a m.w. of 46,000. Analysis of anti-Qa-1.1 precipitates from 125I-labeled Qa-1a lysates demonstrated in addition to the 46,000 m.w. component, an electrophoretically heterogeneous protein or series of proteins in the m.w. range of 55,000 to 75,000. The specificity of these reactivities was shown by both antiserum and genetic control immunoprecipitations. These findings indicate that the Qa-1.1 and Qa-1.2 antigens are cell surface glycoproteins that are distinct from the TL antigens, and suggest a further complexity at the Qa-1--Tla locus.     

Molecular basis of Qa-11 antigen and paradoxical Qa-gene expression in an H-2 recombinant

The Qa-11 Ag expressed in certain strains with the B2-microglobulin-b allele, apparently maps into the Tla region as well as into the Qa-2 region. Moreover Qa-11 has been shown to be biochemically indistinguishable from Qa-2. Genetic complementation studies combining the right Qa and Tla regions failed to lead to Qa-11 expression. To elucidate the molecular basis of this apparent paradox, we examined the expression of Qa-11 on products of transfected Q-region class I genes. Immunochemical analysis has shown that the Qa-11 Ag is expressed on class I molecules encoded by the Q7 gene from both C57BL/10 (Q7b) and BALB/c (Q7d), but not on the protein product of the Q9 gene isolated from the C57BL/10 strain (Q9b). Inasmuch as the predicted protein products of the Q7b and Q9b genes would differ at a single amino acid, a residue critical for Qa-11 expression has been identified. Based on these results it is proposed that among the beta-2-mb strains, the Qa-11+/Qa-2+ mice are likely to express at least the Q7 gene, whereas Qa-11-/Qa-2+ mice express only Q9. In support of this model, the Qa-2+/Q-11- recombinant B6.K2, essential for the apparent mapping of Qa-11 into the Tla region, expresses only Q9 but not Q7 encoded molecules on the cell surface, and only Q9 and no processed Q7 mRNA is detected in the cytoplasm. This expression pattern in B6.K2 cannot be explained on the basis of a single crossing-over event.     

Antigenic Heterogeneity of Qa-2 Antigens in C57BL/6

The Qa-2 antigens are class I-like molecules encoded by genes mapped telomeric to the H-2D region on chromosome 17 in the mouse. A panel of 8 new monoclonal anti-Qa-2 antibodies derived from a C3H.KBR anti-C3H. SW immunization was studied. Immunoprecipitation of¹²⁵I-labeled C57BL/6 splenocyte antigens showed that all of these antibodies precipitated 40 kDa molecules which could be completely precleared by the monoclonal antibody 20-8-4, which had previously been shown to crossreact with Qa-2. One of the monoclonal antibodies (1-12-1), however, was found not to completely preclear Qa-2 antigens precipitable by the other 7 antibodies or by 20-8-4, suggesting the existence of at least two different species of Qa-2 molecules. Cell lines transfected with Q7 or Q9 genes were reactive with all 9 antibodies and the Qa-2 antigens expressed on surface membranes of these cells were completely precleared by both 20-8-4 and 1-12-1. Therefore, the observed heterogeneity of these molecules cannot be explained by an antigenic difference between the Q7 and Q9 gene products. 2D gel analyses showed identical pI spectra between Qa-2 molecules precipitated with 20-8-4 and 1-12-1. In addition, all of the monoclonal antibodies reacted with labeled antigen preparations following treatment with Endo F or neuraminidase, indicating that carbohydrate moieties are probably not responsible for the antigenic difference between the two species of Qa-2 antigen.     

Further biochemical data on Qa-2

The Qa-2 differentiation alloantigen is coded by a gene situated between the D and Tla loci of the murine major histocompatibility complex (H-2). Qa-2-bearing protein was isolated by immunoprecipitation and found to be composed of subunits of 40 000 and 12 000 daltons by SDS polyacrylamide gel electrophoresis (PAGE). The 12 000 dalton material was identified as 2-microglobulin (2M) by its molecular weight (SDS PAGE), charge (isoelectric focusing), antigenicity (reactivity with xenogenic anti- 2M), and genetics. The 40 000 dalton mol. wt. of Qa-2 heavy chain is 5 000 daltons less than that of D and K molecules (45 000 daltons). The quantity of Qa-2 isolated by immunoprecipitation was found to vary in a strain-specific fashion and as much as a 15-fold difference was observed.Abbreviations used in this paper B6 C57BL/6 strain mice - B10 C57BL/10 mice - 2M beta 2-microglobulin - IEF isoelectric focusing - K 1000 daltons - MHC major histocompatibility complex - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TL thymusleukemia antigen     

A new Tla region antigen Qa-11, similar to Qa-2 and associated with B-type beta 2-microglobulin

A new antigen, Qa-11, is detected as a 40,000 dalton band in the SDS-PAGE of immunoprecipitates of radiolabeled lymphocyte membrane preparations. In C57BL H-2 congenic strains, its presence is controlled by a gene in the Tla region. In strains with genetic background other than C57BL it is not expressed. Tests with recombinant inbred strains and with H-3 congenic strains show that, in addition to the Tla region, a gene linked to or identical with the beta 2-microglobulin-b-allele is required for the expression of Qa-11 as well. The mobility of the Qa-11 antigen in SDS-PAGE and in isoelectrofocusing is the same as that of Qa-2 antigen. The Cleveland peptide maps of Qa-2 and Qa-11 are identical as well. This finding, that the Tla region controlled Qa-11 antigen is structurally very similar to the Qa-2 antigen, contrasts with the fact that Tla region products do not react with anti-Qa-2 sera. This paradox could be explained by a separate Qa-11 region between Qa-2 and Tla. Alternatively, it is possible that the Qa-11 antigen is the result of the action of a modifying gene in the Tla region upon a Qa-2 gene product, or that the structural gene for Qa-11 is located in the Qa-2 region and a Tla region gene controls its expression.     

Regulation of the cytotoxic T lymphocyte response against Qa-1 alloantigens

Spleen cells from B6.Tlaa (Qa-1a) mice primed against C57BL/6 (Qa-1b) splenocytes in vivo generate Qa-1-specific CTL when rechallenged with Qa-1b Ag in vitro. The addition of unirradiated Qa-1b splenocytes to these cultures inhibits the generation of Qa-1-specific CTL. By using highly purified cell populations, we demonstrate that the only cell population in resting spleen capable of causing this inhibition is NK1.1+. Although resting CD8 cells lack inhibitory activity, purified CD8 cells precultured with Con A and IL-2 inhibit anti-Qa-1 CTL. This inhibition is specific for the Qa-1b Ag expressed on the inhibitor cells, is not due to cold target competition, and is thus similar to that ascribed to veto cells. Although NK cells from resting spleen inhibit the generation of Qa-1-specific CTL, NK cells precultured in the presence of Con A and IL-2 show an approximate 30-fold increase in veto activity. Thus, NK cells represent the most likely cell population for down-regulating anti-self class I-reactive CTL.     

Tissue distribution, structural characterization, and biosynthesis of Mac-3, a macrophage surface glycoprotein exhibiting molecular weight heterogeneity

Mac-3 is a mouse macrophage differentiation antigen defined by a rat anti-mouse monoclonal antibody (MAb),M3/84. The structure, biosynthesis, quantitative surface expression, and distribution of Mac-3 have been studied by radiolabeling and isolation with MAb-Sepharose, saturation binding, absorption, and immunofluorescence flow cytometry. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Mac-3 migrates as a diffuse band with average Mr = 110,000. Labeling of intact cells with 125I and accessibility to MAb show it is present at least in part on the cell surface. Saturation labeling with 125I-MAb shows 4.2 X 10(4) cell surface sites on thioglycollate medium-elicited peritoneal macrophages. [35S]Methionine and [3H]glucosamine incorporation into Mac-3 by purified macrophages show it is a glycoprotein synthesized by these cells. Absorption shows Mac-3 is strongest in macrophages, present in lower quantities in lung, liver, bone marrow, and spleen, and undetectable in thymus, lymph node, brain, and heart. Immunofluorescent flow cytometry shows surface expression on thioglycollate-elicited macrophages but not bone marrow, spleen, lymph node, or thymus cell suspensions. Similar amounts of Mac-3 are immunoprecipitated from resident macrophages or macrophages elicited by sterile inflammatory agents, intracellular parasites, or immunomodulators, but the average Mr of Mac-3 varies from 92,000 to 110,000. Mac-3 is synthesized from precursor(s) of Mr = 74,000 and 79,000, identical in the different macrophages. Processing into the mature molecule, which migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a more diffuse band and varies in Mr among macrophage elicited by different agents and to a lesser degree between different preparations of the same type of macrophage, occurs in 15 to 30 min.     

In vivo and in vitro synthesis of adenovirus type 2 early proteins.

The synthesis of adenovirus type 2 (Ad2)-induced early polypeptides was examined in vivo and in vitro by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis alone and specific immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of total [35S]methionine-labeled polypeptides synthesized in vivo at 3 h postinfection allowed us to detect in infected cells at lease 13 distinct polypeptides that are either absent or less conspicuous in extracts from mock-infected cells. These Ad2-induced early polypeptides have molecular weights ranging from 72 x 10(3) to 10.5 x 10(3) and have accordingly been designated as E72K to E10.5K. Nine of the in vivo synthesized early polypeptides can be precipitated specifically from infected cell extracts by antisera with specificity against early adenovirus proteins. In vitro translation of mRNA extracted from mock-infected cells and from Ad2-infected cells was carried out in preincubated Ehrlich ascites cell extracts. All the early Ad2-induced polypeptides identified in the extracts from infected cells labeled in vivo were also detected among the polypeptides immunoprecipitated specifically from the in vitro reaction mixtures programmed by RNA extracted at 4 h postinfection from Ad2-infected cells.     

Biochemical analysis of an MHC-linked hematopoietic cell surface antigen,Qa-2

The region of the murine 17th chromosome telomeric to H-2D encodes a group of serologically defined cell surface antigens termed Qa-1-5. These antigens are of interest because their expression is restricted to hematopoietic cells. In addition, the molecular weight and subunit structure (ie, association with β-2 microglobulin) of Qa-2 molecules are similar to H-2 and TL antigens. In the present studies, we have prepared isotopically labeled Qa-2 and H-2 molecules from mitogen-stimulated C57BL/6 spleen cells. Comparative peptide mapping of tryptic peptides from Qa-2 and H-2 molecules (K^b, D^bK^k, D^d) reveal that Qa-2 has a unique primary structure. However, considerable homology is indicated since 30–40% of the Qa-2 peptides cochromatograph with peptides derived from H-2K^b, H-2D^b, H-2K^k, and H-2D^d. Studies by other investigators have demonstrated that similar levels of structural homology are observed when H-2K, H-2D, and H-2L tryptic peptides are analyzed. We conclude from these studies that the Qa-2 alloantigen is structurally related to a class of cell surface molecules (ie, H-2) that play critical roles in immune recognition processes. These data further suggest that the genes encoding Qa-2 and H-2 molecules have arisen from a common primordial gene.     

Ontogeny of murine T lymphocytes: II. Postnatal appearance and organ distribution of lymphocytes bearing Qa-4 and Qa-5 surface antigens

Cells from the lymphoid organs of C57BL/6 mice (from birth to 20 weeks) were monitored by the cytotoxicity assay for the presence of Qa-4 and Qa-5 surface antigens. Qa-4- and Qa-5-bearing cells are detectable in spleen, lymph nodes, and Peyer's patches, but not in thymus, liver, or bone marrow. Both antigens are present on small fractions of cells in each of these organs during the first week after birth. At 4–6 weeks of age, the fractions of Qa-4- and Qa-5-bearing cells rise to maximal levels which are then maintained throughout the ages studied (4–20 weeks). The relative proportion of these cell populations is greatest in the lymph nodes and smallest in the Peyer's patches, and in all three organs, more Qa-4- than Qa-5-positive cells are detected. The majority of Qa-4- and Qa-5-positive cells are Thy-1 positive, however, not all Thy-1- positive cells are Qa-4, Qa-5 positive. During postnatal development the ratio of Qa-4 or Qa-5-positive cells to Thy-1-positive cells increases in spleen, lymph nodes, and Peyer's patches indicating that cells bearing these antigens become a larger fraction of the T-cell population with age.     

Properties of the PC-1 molecule

The technique of cell-surface iodination, followed by immuno-precipitation withPC-1.1 antiserum, was applied to normal spleen cells and to MOPC-70A BALB myeloma cells. The results indicate that the cell-surface component bearing PC-1 alloantigen has a molecular weight of from 105,000 to 110,000 daltons and does not resemble any constituent of plasma membranes or MuLV-type virus so far categorized by similar methods. The PC-1 specificity of the molecule was confirmed by comparison of the spleen cells of two mouse strains with spleen cells of their respective PC-congenic partner strains. MOPC-70A myeloma cells, but not spleen cells, yield a fainter band in the PC-1 position in controls in which antiserum is omitted, but peptide maps show that this similarly placed band has no relation to the PC-1 molecule.Abbreviations used in this paper are as follows PC plasma cell - B6 C57BL/6 - SDS/PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - a anti - NMS normal mouse serum - MuLV murine leukemia virus - Ig immunoglobulin     

Studies of a high molecular weight human melanoma-associated antigen

Hybridomas were generated by fusing SP2/0 mouse myeloma cells with spleen cells from mice that had been immunized with cultured human melanoma cells. One of the hybridomas secreted a monoclonal IgG1 antibody, 48.7, which binds to a cell surface antigen of cells from human melanomas and compound nevi. The presence of the target antigen in vivo was demonstrated immunohistologically by staining frozen sections of primary and metastatic melanoma by the peroxidase anti-peroxidase technique. Weak staining of some blood vessel cells was also seen, but other normal cells, including skin melanocytes, were unstained, as were cells from other tumor types. Antibody 48.7 immunoprecipitated polypeptides with apparent m.w. on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 250,000 and greater than 400,000.     

Recognition by cytotoxic T lymphocytes of Qa-2 antigens. Sensitivity of Qa-2 molecules to phosphatidylinositol-specific phospholipase C

Con A splenic lymphoblasts were incubated with phosphatidyl-inositol specific phospholipase C (PIPLC) derived from Bacillus thuringiensis and subsequently analyzed for Qa-2 Ag with the Qa-2 reactive mAb Qa-m2. This treatment completely removed Qa-2 detectable Ag on lymphoblasts from H-2d animals, indicating that these molecules are likely anchored to the cell membrane through phosphatidyl inositol (PI). Although exposure of lymphoblasts from H-2b mice to PIPLC greatly reduced Qa-2 expression, a subpopulation of cells retained a limited quantity of the Ag. Bulk cultured anti-Qa-2 CTL generated against the Qa-2 region from H-2b haplotype mice lysed Qa-2+ targets from B6.K2 (H-2b) and BALB/cJ (H-2d) animals. Pretreatment of these lymphoblast targets with PIPLC completely abolished lysis of the BALB/cJ target cells, whereas lysis of B6 targets was reduced only slightly. Anti-Qa-2 CTL clones tested against PIPLC-treated B6 target cells revealed two patterns of reactivity. One group of clones was unaffected in its ability to lyse PIPLC-pretreated targets and cross-reacted on Q6d/Ld molecules expressed on transfected L cells. A second group was unable to lyse PIPLC-pretreated lymphoblasts and cross-reacted on Q7d/Ld targets. These data suggest that H-2b-derived lymphoblasts express two different types of Qa-2 molecules with respect to PIPLC sensitivity; one type is sensitive to PIPLC and cross-reactive with Q7d, the other type is resistant to PIPLC and cross-reactive with Q6d. In contrast, H-2d lymphoblasts express only the PIPLC-sensitive type of molecules. It was also noted that bulk cultured anti-Qa-2 CTL more readily lysed H-2b target cells expressing a smaller quantity of PIPLC-resistant Ag than H-2d targets expressing a larger amount of PIPLC-sensitive Ag. Further, anti-Qa-2 CTL clones readily lysed PIPLC-treated target cells expressing very low levels of serologically detectable Qa-2. This suggests that recognition of class I molecules anchored to the membrane via a PIPLC-resistant linkage may more readily activate CTL for expression of lytic activity than molecules anchored through PI.     

The differentiation of cytotoxic T cells in vitro. IV. Interleukin-2 production in primary mixed lymphocyte cultures involves cooperation between Qa-1+ and Qa-1- "helper" T cells

Studies with C57BL/6-TIaa mice have established that both Qa-1+ and Qa-1- helper T cells are required for the optimal production of Interleukin 2(IL-2) activity in primary MLC. This was established both by depletion of Qa-1 bearing cells by treatment of responder cells with anti-Qa-1 serum in the presence of complement and by positive selection (using FACS II analysis) of those cells displaying Qa-1. Furthermore, microfluoremetry revealed that the great majority of C57BL/6-TIaa and of A/J splenic T cells bore Qa-1 alloantigen but that only those with the highest antigen density were susceptible to complement-mediated lysis.     

Correlation of Qa-1 determinants defined by antisera and by cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) activated in H-2 identical, Qa-1 disparate mixed leukocyte cultures recognize H-2-nonrestricted target antigens indistinguishable by strain or tissue distribution from serologically defined Qa-1 antigens. Cloned Qa-l-specific CTL define determinants encoded by four Qa-1 genotypes; we used anti-Qa-1 sera in antibody blocking experiments to determine if these determinants reside on molecules recognized by Qa-1-specific antibodies. Antisera containing Qa-1.1-specific and TL-specific antibodies blocked recognition of two CTL-defined determinants associated with Qa-1 ^a . Although both Qa-1 and TL molecules are expressed on activated T cells from appropriate strains, our studies indicated that the CTL recognized Qa-1, not TL. In addition, anti-Qa-1.2 serum inhibited CTL recognition of Qa-1^b- and Qa-1^c-encoded determinants. Qa-1 ^d target cells are unique in that they express determinants recognized by anti-Qa-1^a CTL and by anti-Qa-1^b CTL. Killing of Qa-1 ^d targets by anti-Qa-1^a CTL was not inhibited by anti-Qa-1.1 serum, but was partially inhibited by anti-Qa-1.2 serum. Cytotoxicity of Qa-1 ^d cells by one anti-Qa-1^b CTL clone was inhibited by both anti-Qa-1.2 and anti-Qa-1.1 sera, indicating close association of both serological determinants with the determinants recognized by the CTL. Thus, all of the CTL-defined Qa-1 determinants resided on molecules recognized by Qa-1-specific antibodies, but anti-Qa-1^a CTL and Qa-1.1-specific antibodies did not have identical specificities.Abbreviations used in this paper B6 C57BL/6J - CAB concanavalin A stimulated lymphoblasts - CML cell-mediated lympholysis - CTL cytotoxic T lymphocyte - NMS normal mouse serum - MHC major histocompatibility complex - MLC mixed leukocyte culture - MR maximum release - SMDM supplemented Mishell-Dutton medium - SR spontaneous release     

Human transforming growth factor beta.alpha 2-macroglobulin complex is a latent form of transforming growth factor beta

125I-Labeled human platelet-derived transforming growth factor beta (125I-TGF-beta) and human alpha 2-macroglobulin (alpha 2M) formed a complex as demonstrated by 5% native polyacrylamide gel electrophoresis. The 125I-TGF-beta.alpha 2M complex migrated at a position identical to that of the fast migrating form of alpha 2M. Most of the 125I-TGF-beta.alpha 2M complex could be dissociated by acid or urea treatment. When 125I-TGF-beta was incubated with serum, the high molecular weight form of 125I-TGF-beta could be immunoprecipitated by anti-human alpha 2M anti-sera as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 2M purified from platelet-rich plasma also showed the latent transforming growth factor activity and immunoreactivity of TGF-beta. These results suggest that TGF-beta.alpha 2M complex is a latent form of TGF-beta.     

Biochemical characterization of the molecules reactive with Qa-6 antiserum and the monoclonal antibody 20-8-4: evidence for structural similarity with the Qa-2 molecule

The Qa-6 alloantigen and the molecule that crossreacts with the monoclonal antibody (mAb) 20-8-4 have been shown to be serologically distinct from the Qa-2 alloantigen by strain distribution and tissue distribution, respectively. In this report, we address the biochemical relationships among Qa-2, Qa-6, and the 20-8-4 cross-reactive molecule by using immunoprecipitation and polyacrylamide gel electrophoresis. Each of these molecules had an apparent m.w. of approximately 41K and was associated on the cell surface with beta 2-microglobulin. Removal of N-linked oligosaccharides with endoglycosidase F reduced their apparent m.w. to approximately 33K to 34K. The determinants recognized by anti-Qa-6 and mAb 20-8-4 were shown to reside on the same molecule(s) precipitated by anti-Qa-2 sera by immunodepletion experiments. The mAb 20-8-4 was also shown to preclear the molecules detected by the Qa-6 and Qa-2 antisera. Two-dimensional gel electrophoresis analysis demonstrated complete co-migration of the approximately 41K molecules detected by the three antibodies. By peptide map analysis with V8 protease, all three molecules appeared identical. Also, the determinant recognized by Qa-6 antiserum co-modulated with that recognized by the anti-Qa-2 mAb D3.262. Taken together, these results demonstrate that the molecules recognized by these three antisera and/or mAb are biochemically indistinguishable. These data, in conjunction with the serologic and genetic findings suggest that mAb 20-8-4 recognizes a molecule that is biochemically similar and possibly identical to the Qa-2 antigen. Moreover, although the genetic, serologic, and biochemical data demonstrate that Qa-6 is not controlled by the Qa-2 locus, but rather by a gene telomeric to Qa-2, the molecule bearing the Qa-6 determinant is very similar, if not identical, to the Qa-2 molecule. Several possible explanations for these discrepancies are discussed.     

The Qa-1 alloantigens. III. Biochemical analysis of the structure and extent of polymorphism of the Qa-1 allelic products

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to examine and compare the products of the Qa-1 locus. Analysis of Qa-1 isolated from detergent lysates of surface labeled cells indicated this molecule was a slightly acidic 48,000 to 50,000 dalton glycoprotein that displayed little charge heterogeneity on resting lymphocytes. The level of expression and degree of charge heterogeneity were both increased on activated lymphocytes. Direct comparison of the Qa-1b, Qa-1c, and Qa-1d allelic products by 2-D PAGE revealed that these three molecules could be distinguished from one another on the basis of isoelectric point, indicating that they were distinct at the molecular level. Comparison of Qa-1 isolated from several Qa-1b strains did not detect additional polymorphism. Removal of asparagine-linked oligosaccharides by treatment with endoglycosidase F indicated that carbohydrate contributed 10,000 to 12,000 to the m.w. of these allelic products. Comparative 2-D PAGE analysis could not distinguish between the deglycosylated Qa-1b, Qa-1c, and Qa-1d allelic products, implying that these molecules have similar primary structures. Peptide mapping supported this conclusion. Proteolytic digestion of the deglycosylated Qa-1b and Qa-1c allelic products resulted in identical peptide map patterns; such treatment of the deglycosylated Qa-1d allelic product produced a slightly different pattern. Peptide mapping analysis also demonstrated that the Tlaa and Qa-1a allelic products were distinct from one another, as well as being very different from the other three Qa-1 allelic products.     

A Monoclonal Antibody Raised to Corpus Callosum Extract Reacts with 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase

Monoclonal antibody against 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with delipidated white matter from rat corpus callosum. The antibody was characterized by solid-phase radioimmunoassay, immunoblot of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoprecipitation from C6 glioma cells, and indirect immunofluorescence staining of monolayer cultures containing oligodendrocytes. The monoclonal antibody bound specifically to an intracellular antigen of oligodendrocytes, but not to Schwann cells, astrocytes, neurons, or fibroblast cytoplasm. The immunoblot of SDS-PAGE of CNS myelin showed that the antibody identified two protein bands at 48,000 and 50,000 molecular weight. These proteins were not identified in peripheral nervous system myelin. The monoclonal antibody immunoprecipitated CNP enzyme activity from extracts of C6 glioma cells. This monoclonal antibody should prove useful in further study of this myelin-specific enzyme in CNS myelin and in cells responsible for myelin production.