Studies of two H-2Db mutants: B6. C-H-2bm13 and B6.C-H-2bm14

Two new C57BL/6 H-2 mutants, B6.C-H-2bm13 and B6.C-H-2bm14 are described. They arose independently in C57BL/6 as spontaneous mutations of the gain and loss type. Complementation studies map the mutations in both bm13 and bm14 to the H-2Db gene. However, these two mutant strains are not identical, but occurred as independent mutations at the same locus, as shown by reciprocal graft rejection and by the inability of the (bm13 X bm14)F1 hybrid to accept C57BL/6 grafts. Serological studies by direct testing (cytotoxicity and hemagglutination) and by quantitative absorption demonstrated a decrease in the H-2Db private specificity H-2.2 in both bm13 and bm14 when compared to C57BL/6. This was confirmed by SDS-PAGE analysis using antisera detecting the H-2.2 specificity. Attempts to produce antibodies to either the gained or lost specificities of the two mutant strains failed.     

Biochemical studies of H-2K antigens from a group of related mutants. II. Identification of a shared mutation in B6-H-2 bm6 , B6.C-H-2 bm7 , and B6.C-H-2 bm9

In an earlier paper, we presented evidence that two independent mutants of the bg series, B6-H-2 ^bm5 (bm5) and B6-H-2 ^bm16 (bm16) carry identical mutations such that tyrosine at residue number 116 of the H-2K^b molecule from the parent strain C57BL/6Kh is replaced by a phenylalanine in each of the two mutant molecules. In this paper, we demonstrate, using similar techniques, that the independent bg series mutants B6-H-2 ^bm6 (bm6), B6.C-H-2 ^bm7 (bm7), and B6.C-H-2 ^bm9 (bm9), which share biological properties with bm5 and bm16, can be grouped together because they share two identical mutations, one of which is common to bm5 and bm16, a Tyr to Phe interchange at residue number 116. In addition, a second mutation is at residue number 121, where a Cys in the H-2K molecule from 136 is substituted with an Arg in the mutant. Since all of the bg series mutants arose independently and share biological and biochemical characteristics, it is anticipated that study of these mutants could lead to some understanding of the high mutation rate in the K^b molecule.     

Biochemical studies on the H-2K mutant B6.C-H-2 bm10

The H-2K glycoprotein from the MHC mutant bm10 was analyzed biochemically to determine where primary structural differences distinguished it from the parental standard molecule, K^b. Comparative peptide maps showed differences in two peptides known to be part of the parental CNBr fragment spanning amino acids 139 to 228. Partial sequence analyses of CNBr fragments and tryptic peptides identified two tightly clustered amino acid substitutions at amino acids 165 (Val to Met) and 173 (Lys to unknown). The substitutions in bm10 represent the most carboxy-terminal substitutions characterized in the K^b molecules of the spontaneous, histogenically active H-2 mutants.     

The definition of a new IA antigenic specificity using the B6.C-H-2 bm12 I-region mutant strain

A new Ia specificity has been defined using theIA-subregion mutant B6.C-H-2 ^bm12. The immunization to produce the antiserum wasbm12 anti-A.BY, as all other immunizations, such asbm12 anti-C57BL/6, failed to produce antibody. By selecting strains of C57BL origin for testing, it was shown that, (a) the serum was only weakly cytotoxic but gave substantial reactions using a rosetting assay; (b) the antibody reacted with B cells and not T cells; (c) strains of theb, d, p andq H–2 haplotypes were positive, whereasf, k, r ands were negative; (d) absorption studies demonstrated only a single specificity to be present and by testing recombinant strains, the reaction mapped to theIA subregion; (e) SDS-PAGE demonstrated that the antiserum reacted with a molecule of MW 33 000. Preliminary studies indicate this new specificity, present on C57BL/6 and lost frombm12, is present on the same molecule as other I-A specificities.This work was supported by funds obtained from the N. H. and M. R. C. (Australia) and the National Institutes of Health, Grant No. CA-21224.     

T cell responses to select Ia determinants using the I-A mutant mouse strain B6.C-H-2bm12

The T cell repertoire of B6.C-H-2bm12 mice (an I-A mutant mouse strain) to wild-type Iab antigens was investigated using both secondary proliferative cultures and cloned T cell lines. Because bm12 mice have a gain-loss mutation of their gene encoding the Ia beta-chain polypeptide, bm12 anti-B6 T cell responses are specific for the select component of Iab specificities that was lost as a result of the mutation. Although stimulator cells bearing Iab antigens elicited the strongest responses, Iaq, d, and s antigens also resulted in reproducible stimulations of these bm12 anti-B6-primed T cells. Cloned T cell lines isolated from bm12 anti-b6 cultures revealed similar findings, with most clones recognizing determinants unique for Iab antigens; however, clones showing cross-reactions with Iad and/or q were also selected. Using F1 hybrid responder T cells (mutant x cross-reactive strain), we further dissected this cross-reactivity into several distinct cross-reactive determinants. Because bm12 mice lack the serologically defined Ia differentiation antigen W39, T cell recognition of this determinant was investigated by using bm12 anti-B6-primed cells. Stimulation by Ia.W39+ cells was appreciably better than by Ia.W39- (Xid-defective) cells, suggesting that bm12 T cells recognize an Xid-regulated, W39-like Ia differentiation antigen.     

Idiotypic and fluorometric analysis of the antibodies that distinguish the lesion of the I-A mutant B6.C-H-2bm12

The serologic lesion of the I-A mutant mouse strain, bm12, was investigated with the use of monoclonal anti-Iab antibodies and anti-idiotypic (Id) reagents produced against these antibodies. In a fluorometric analysis, three different monoclonal anti-Iab antibodies (25-9-17, 34-5-3, 28-16-8) failed to bind bm12 cells, whereas two anti-Iab antibodies (25-5-16 and 17/227), which bound bm12 cells, showed about one-half the fluorescence intensity that they showed in binding to Iab antigens. Of the three monoclonal antibodies that failed to react with bm12 cells, two antibodies (25-9-17 and 34-5-3) were found to bind the same steric site on Iab molecules (cluster I). In contrast, the antibodies (25-5-16 and 17/227) that reacted with both Iab and Iabm12 antigens were found to bind a second distinct site (cluster II). The binding of antibody 28-16-8 to Iab antigens inhibited reciprocally the binding of cluster I and II anti-Iab antibodies, suggesting a possible third site, sterically located intermediate between the other two sites. To assess the relatedness of the antibodies defining the serologic lesion of bm12 mice, xenogeneic and syngeneic anti-Id reagents were produced against antibodies 25-9-17 and 28-16-8. By using these anti-Ids in a binding site-related inhibition assay, a cross-reactive idiotype was detected that is shared by 25-9-17 and 34-5-3 antibodies; thus these two monoclonal antibodies share several features, including 1) idiotypic determinants, 2) failure to bind bm12 cells, 3) binding the same spatial Iab site, and 4) having indistinguishable serologic fine specificity that corresponds with a previously defined predominant alloantigenic determinant recognized in the bm12 anti-Iab humoral response. Therefore, several parameters of antibody recognition of Ia can now be correlated with structural changes in Ia molecules. These findings will potentiate future studies of the T cell recognition of these same Ia epitopes.     

Reactivity of the B6.C-H-2bm−1 mutant to the wild-type tumor EL4: Characterization of the serological response

Hyperimmunization of B6.C-H-2^bm?1 (H-2^bm?1), a congenic mutant of C57Bl/6J (B6), with the C57Bl lymphoma EL4 resulted in the induction of antibodies with apparent EL4 specificity. EL4 reactivity was demonstrable in H-2^bm?1 anti-EL4 sera by complement-mediated cytotoxicity, absorption, and enzyme-linked immunosorbent assay. By these same serological tests, H-2^bm?1 anti-EL4 serum was found to be nonreactive with B6 normal lymphoid cells, embryonic fibroblasts, and two fibrosarcomas previously induced in B6 mice by methylcholanthrene. These data suggest that the serological response of H-2^bm?1 to EL4 is directed against tumor-associated antigens on EL4. These findings indicate that congenic mutants which differ from the wild-type strain at MHC Class I subloci, but which do not evoke serological responses to MHC components, may provide convenient sources for preparing serological reagents directed against tumor-specific antigens.     

Biochemical studies of H-2K antigens from a group of related mutants. I. Identification of a shared mutation in B6-H-2 bm5 and B6-H-2 bm16

Structural studies of the H-2 gene products from a group of five closely related but independent C57BL/6 H-2 mutant mice were undertaken. Each of the mutants exhibits reciprocal graft rejection with the parent. The group is remarkable, however, because each member of this group can accept skin grafts from any other member. The results of biochemical analysis of the H-2 glycoproteins from two of these related mutants, bm5 and bm16, are presented in this report. Evidence is given that the H-2K molecules from these two mutants are identical to each other based on comparative tryptic peptide mapping profiles with the parent. From partial amino acid sequence analysis, K products of both mutants have at least one common difference from the parental type located at residue number 116. Definitive studies established that in both bm5 and bm16 a tryosine found in the parent molecule is substituted with a phenylalanine in the mutant. These results show that a biochemical difference between the K products of the two mutants and of the parent can be detected, that the mutants appear to be identical with one another even though they arose independently, and that they differ from the other H-2K ^b mutants analyzed.Abbreviations used in this paper B6 C57BL/6Kh - bm5 B6-H-2^bm5 - bm6 B6-H-2 ^bm6 - bm7 B6.C-H-2 ^bm7 - bm9 B6.C-H-2 ^bm9 - bm16 B6-H-2 ^bm16 - D H-2D - K H-2K - MHC major histocompatibility complex     

Haplotype-specific suppression of antibody responses in vitro. III. Haplotype-specific suppressor factor (TsF-H) binds to but fails to suppress responses by the I-A mutant strain B6.C-H-2bm12

Spleen cells from C57BL/6 and B6.C-H-2bm12 mice, both responder strains to GAT, differ in their ability to be suppressed by the monoclonal I-A-restricted, nonantigen-specific, but haplotype-specific suppressor factor, TsF-H, from the hybridoma 266A4.5. Whereas GAT-specific responses by C57BL/6 spleen cells are susceptible to TsF-H-mediated suppression, responses by bm12 spleen cells are nonsuppressible under the same conditions. Responses of both C57BL/6 and bm12 spleen cells are suppressed by monoclonal GAT-specific suppressor factors. The inability of TsF-H to suppress responses by the bm12 spleen cells presumably reflects the effects of the mutation in the beta-chain of the I-A antigen in this strain on the required I-A restriction between TsF-H and target cell for manifestation of suppressive activity. The data are discussed in terms of involvement of I-A or recognition of I-A in mediating suppression.     

Genetic analysis of anH-2 mutant,B6.C-H-2 ba,using cell-mediated lympholysis: T- and B-cell dictionaries for histocompatibility determinants are different

B6.C-H-2 ^ba [H (z1)] is a mutant derived from C57BL/6. The two strains mutually reject their skingrafts and are incompatible in the mixed leucocyte reaction (MLR) and in cell-mediated lympholysis (CML) assays. They are serologically indistinguishable. This report shows that H(z1) carries a new, privateK end CML specificity clearly distinguishable from that of B6 by a third party strain, HTG. Antisera directed against the private H-2K specificity of B6 present on H(z1) cells) can block CML between the two strains in either direction. The new CML specificities of H(z1) cross-react with (public) CML specificities controlled by bothK andD regions of other unrelated haplotypes. The results suggest that H(z1) carries a mutation in theH-2K locus itself or in a closely linked gene, the product of which is also physically associated with the H-2K molecule corresponding to the cis-configuration of the alleles in both loci. These findings indicate that T- and B-cell dictionaries for histocompatibility determinants are different.     

The congenic mutant B6.C-H-2bm-1 (H-2bm-1) serological response to the T-cell receptor on EL4

Antisera reactive with the C57Bl lymphoma EL4 were induced by hyperimmunization of B6.C-H-2bm-1 (H-2bm-1) mice, a congenic mutant strain of C57Bl/6 (B6). Molecular weight determinations of EL4 surface structures detected with the congenic mutant antibodies were accomplished by electrophoretic analyses (one and two dimensions) on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). Analysis of immunoprecipitates in the first dimension under reducing conditions revealed protein bands corresponding to gp 70 and its 33-kDa cleavage fragment, and two-three bands (40-45 kDa) that overlapped with those precipitated from EL4 with AS 8177, an antiserum which detects framework determinants on the T-cell heterodimeric receptor. Further analysis by diagonal electrophoresis confirmed identity of the 40- to 45-kDa proteins precipitated from EL4 with either AS 8177 or H-2bm-1 anti-EL4 sera. Additional diagonal electrophoretic studies showed that the congenic mutant antibodies do not precipitate T-cell receptor molecules from C6VL, an in vitro line derived from the C6XL T-cell lymphoma of C57Bl/ka origin, which was previously shown by some of us to express the heterodimeric T-cell receptor. Together these results demonstrate detection of the heterodimeric T-cell receptor on EL4 with congenic mutant antibodies directed against nonconstant region determinant(s), and indicate a possible linkage between MHC haplotype and the immune response to intraspecies variable regions of the T-cell receptor.     

Molecular analysis of antigen recognition by insulin-specific T-cell hybridomas from B6 wild-type and bm12 mutant mice.

Molecular analysis of the heterodimeric T-cell antigen receptor of insulin-specific class II-restricted T-cell hybridomas (THys) derived from C57BL/6 (B6) wild-type and B6.C-H-2bm12 (bm12) mutant mice revealed that such T cells use a diverse V gene repertoire. Analysis of three THys that use related V genes, however, showed a number of novel features. Two THys that share major histocompatibility complex restriction use V alpha genes that are 98.6% homologous. Two THys sharing the same antigen fine specificity use a particular germ line V beta D beta J beta combination. A 21-base-pair deletion in the 5' segment of the J beta gene occurs in one THy, suggesting a novel mechanism for generating diversity in T-cell antigen receptor beta genes. The first amino acid encoded by N sequences at the V-D junction is conserved in a pair of T cells which recognize identical antigenic epitopes. The implications of these findings for the structural mechanisms underlying major histocompatibility complex-restricted antigen-specific T-cell recognition are discussed.     

Studies on the cytochrome b6/f complex. II. Localization of the complex in the thylakoid membranes from spinach and Chlamydomonas reinhardtii by immunocytochemistry and freeze-fracture analysis of b6/f mutants

The distribution of the b₆/f complex among stacked and unstacked thylakoid membranes was studied by immunocytochemistry and freeze-fracture analysis of mutants of Chlamydomonas reinhardtii lacking the complex. Immunogold labeling demonstrates the presence of b₆/f complex in both regions of the thylakoid membrane in spinach and in C. reinhardtii. Numerous modifications were observed in the ultrastructure of the thylakoid membranes of mutants from C. reinhardtii lacking the complex. These modifications are consistent with the presence of b₆/f complexes in different states of association in the stacked and unstacked regions of the thylakoid membrane. In particular we present evidence for an association of some b₆/f complexes with the reaction centers of Photosystem I and II in large PFu and EFs particles, respectively.     

Penicillin-binding protein 2b of Streptococcus pneumoniae in piperacillin-resistant laboratory mutants.

In Streptococcus pneumoniae, alterations in penicillin-binding protein 2b (PBP 2b) that reduce the affinity for penicillin binding are observed during development of beta-lactam resistance. The development of resistance was now studied in three independently obtained piperacillin-resistant laboratory mutants isolated after several selection steps on increasing concentrations of the antibiotic. The mutants differed from the clinical isolates in major aspects: first-level resistance could not be correlated with alterations in the known PBP genes, and the first PBP altered was PBP 2b. The point mutations occurring in the PBP 2b genes were characterized. Each mutant contained one single point mutation in the PBP 2b gene. In one mutant, this resulted in a mutation of Gly-617 to Ala within one of the homology boxes common to all PBPs, and in the other two cases, the same Gly-to-Asp substitution at the end of the penicillin-binding domain had occurred. The sites affected were homologous to those determined previously in the S. pneumoniae PBP 2x of mutants resistant to cefotaxime, indicating that, in both PBPs, similar sites are important for interaction with the respective beta-lactams.     

Reproduction of bacteriophage T6 and T7 in auxotrophic mutants ofE. coli B

Summary Phage T₆ and T₇ did not reproduce in mutants ofE. coli auxotrophic for the amino acids methionine, leucine, serine, proline, histidine, tyrosine, phenylalanine and tryptophane unless these amino acids were present in the medium. Very small amounts of these auxotrophic factors (0.01 γ/ml) are able to support phage multiplication. Much care has to be taken to avoid impurities in the medium and to deplete the endogenous reserves of the host cells.