Strong teratocarcinoma transplantation loci,Gt-1 and Gt-2, Flank H-2

Evidence is presented for the existence of two strong murine teratocarcinoma transplantation antigens (Gt) on the cell line PCC3. It is shown that the loci governing expression of these antigens are linked to the H-2 complex. These loci have been further mapped with respect to the brachyury marker (T) and H-2: Gt-1 lies 5±2 crossover units proximal to H-2 and 12±2 crossover units distal to T, Gt-2 lies 21±4 crossover units distal to H-2. It is possible that these strong transplantation antigens provide an embryonic analogue to the adult major histocompatibility system.     

Organization and Evolution of the Mammalian Genome: I. Polymorphism of H-2 Linked Loci

To test the hypothesis that the H-2 polymorphism is adaptive, the degree of polymorphism of loci linked to the H-2 complex on chromosome 17 of the house mouse was compared to the degree of polymorphism of loci located on other chromosomes. Published theoretical analyses show that polymorphisms subject to natural selection usually reduce the polymorphism of linked neutral loci. The first test of the hypothesis was based on data obtained from a survey of the polymorphism of 12 isozyme-encoding loci in wild house mice from Europe, North Africa and South America. Results of this test showed that, on the average, H-2-linked loci were as polymorphic as loci located on other chromosomes. In fact, the data suggested that H-2 linked loci might be more polymorphic than other loci. To test this hypothesis more rigorously, data for the 12 isozyme-encoding loci were augmented with data from published surveys of the polymorphisms of 59 loci in house mice from Europe and North America. Results of these tests showed that polymorphic loci linked to the H-2 complex tended to be more, rather than less, polymorphic than loci located on other chromosomes. The cluster of highly polymorphic loci seems to be related to linkage of these loci to the highly polymorphic H-2 complex, but the way in which the influence is exerted could not be readily explained.     

Genetic studies on theH-3 region of mice and implications for polymorphism of histocompatibility loci

Evidence has been obtained for recombination betweenH-3 and the closely linkedIr-2 locus, which controls the antibody response toEa-1 antigens. The data suggest thatIr-2 maps close toH-3, betweenH-3 andH-13. The YBR strain has been found to possess anH-3 allele not previously reported. A comparison has been made between the degree of polymorphism of histocompatibility loci and that which involves electrophoretically detectable protein variants.     

Viral sequences are associated with many histocompatibility genes

A C57BL/6By 5.5 kb Pvu II polymorphic restriction fragment which hybridizes with a spleen focus-forming env probe and maps in the H-30 region has been cloned, and a 358 by subfragment subcloned. Hybridization and sequencing studies show that the 358 by fragment is encoded by the region of the pol gene of murine retrovirus which codes for an endonuclease critical for viral integration. Hybridizations of digested murine genomic DNAs with the 358 by probe generate 31 restriction fragment length polymorphisms (RFLPs); 16 of these can be placed near the following 15 minor histocompatibility (H) loci: H-3, H-4, H-7, H-13, H-15, H-16, H-17, H-19, H-22, H-24, H-27, H-30, H-34, H-36, and H-38. We suggest that the proximity of viral sequences to H loci is probably evolutionarily and functionally significant and that the closeness of viral sequences and minor H loci can probably be utilized to facilitate the cloning of minor H genes. During the course of these studies, it has become possible to tentatively assign H-17, H-34, and H-38 to chromosome 12. In addition, it was observed that several H-2 congenic strains retain portions of chromosome 12 from the parental donor strains used in their derivation.     

The genetic control of liver cAMP levels in mice

Steady-state level of liver 3,5-cyclic monophosphate, cAMP, has been shown to be under genetic control linked to the mouseH-2 complex. Liver cAMP levels are associated withH-2 haplotype in fully segregating crosses of strains C3H and C57BL/10. In crosses involving strain A, other loci have an effect that swamps that ofH-2. Results withH-2 recombinants indicate that liver cAMP levels are affected by more than oneH-2-linked locus.     

Genetic analysis of the non-H-2-linked Ir genes controlling the cytotoxic T-cell response to H-Y in H-2 d mice

The T-cell mediated immune responses to the male specific minor histocompatibility antigen H-Y in mice have been studied extensively as a model for immune responses to other weak antigens like tumor antigens or autoantigens. In a recent analysis of the strain distribution of the cytotoxic T-cell (Tc-cell) responsiveness to H-Y, it has been found that genes both within and outside the H-2 complex exert an interactive control. Whereas the H-2 ^b strains all are high responders, independent of their non-H-2 background, other H-2 haplotypes (d, k, and s) only allow for a response if they are combined with certain non-H-2 genes. The H-2-linked immune response genes (Ir-genes) have been previously mapped to the I and K or D region of the H-2 complex, but the mapping of the non-H-2 genes has not yet been established. In this study evidence is presented, using recombinant inbred strains and immunoglobulin heavy chain (Igh) congenic strains of mice, to show that there is more than one non-H-2 Ir-gene involved, that the main controlling genes are not linked to the Igh complex, and that at least one non-H-2 Ir-gene is linked to the H-3 region on chromosome 2. This region includes genes for beta-2-microglobulin (2m), the Ly-mllalloantigen a polymorphic cell surface glycoprotein (Pgp-1), a B-cell specific antigen Ly-4, a transplantation antigen H-3, and genes (Ir-2) controlling the immune response to Ea-1 and H-13.     

Genetic organization of the pigSLA complex. studies on nine recombinants and biochemical and lysostrip analysis

Nine recombinants were found amongst 2233 piglets all belonging to 268 informative families and typed for the major histocompatibility complex,SLA. These recombinants have allowed the identification of three loci, two controlling SLA allelic series homologous toH-2D andH-2K, the third the mixed lymphocyte culture reaction. The latter is situated 0.4 cM from the other two loci.Lysostrip and biochemical experiments have confirmed the presence of two allelic SLA series, and indicate that a third series controlling SLA antigens probably exists.     

Sexual preference of meiotic recombination within the H-2 complex

The recombination frequency between the H-2K and H-2D marker loci in male mice was measured using heterozygotes that carry the H-2 ^wm7 haplotype derived from the Japanese wild mouse and common H-2 haplotypes derived from inbred mice. Previous mating experiments in which backcross progeny of heterozygous females were screened demonstrated that the H-2 ^2m7 displays marked enhancement of recombination within the H-2 complex. In contrast to recombination in female mice, no enhancement of recombination was observed during male meiosis in the present study. Thus, it appeared that enhancement of recombination is specific to female mice. A genealogical study of recombination indicated that the postmeiotic stage is not involved in the generation of sexual preference of enhancement of recombination, suggesting that the preference is meiotic-drive and that a female-specific mechanism is involved in meiotic recombination mediated by the H-2 ^wm7 haplotype.     

Regulation of the production of murine IgG2a by an H-2-linked gene and other unlinked genes

Alleles of at least two loci (rig-1 and Rig-2) regulate the levels of serum immunoglobulin of the Igh-1b class and allotype in BALB/c Ig^b (BAB/14) and (BALB/c × BAB/14)F₁ mice. The combined effect of the BALB/c alleles at these two loci is to lower Igh-1b levels significantly below those observed in other strains and below their own levels of Igh-1a in allotype heterozygous mice. The rig-1 locus is closely linked to or within the H-2 complex. Two alleles have been defined: rig-1 ^d and rig-1 ^b in H-2 ^d and H-2 ^b haplotypes, respectively. Homozygous rig-1 ^d d animals heterozygous for the BALB/c Rig-2 allele(s) have very low levels of Igh-1b. The designation of Rig-2 is provisional since it has not been mapped or defined as a single locus.     

The dynamic of the t-haplotype in wild populations of the house mouse Mus musculus domesticus in Israel

The t-haplotype, a variant of the proximal part of the mouse chromosome 17, is composed of at least four inversions and is inherited as a single genetic unit. The haplotype causes embryonic mortality or male sterility when homozygous. Genes within the complex are responsible for distortion of Mendelian transmission ratio in males. Thus, the t-haplotype in heterozygous males is transferred to over 95% of the progeny. We examined the dynamic and behavior of the t-haplotype in wild populations of the house mouse in Israel. The Israeli populations show high frequency (15%–20%) of both partial and complete t-carrying mice, supporting the suggestion that the t-complex evolved in the M. domesticus line in the Israeli region. In one population that had the highest frequency of t-carrying individuals, we compared the level of gene diversity between t-carrying and normal mice in the marker’s loci: H-2 locus of the major histocompatibility complex (MHC) on the t-haplotype of chromosome 17, three microsatellites on other chromosomes, and the mitochondrial D-loop. Genetic variability was high in all tested loci in both t and (+) mice. All t mice carried the same chromosome and showed the same H-2 haplotype. While t-carrying mice showed significant H-2 heterozygotes access, (+) mice expressed significant H-2 heterozygote deficiency. There were no differences in the level of gene diversity between t and (+) mice in the other loci. Heterozygosity level at the MHC may be an additional factor in the selective forces balancing the t-haplotype polymorphism.     

The minimal length of the differential segment in H-2 congenic lines

One hundred and four H-2 congenic lines were typed for alleles at seven loci, Qa-1, Qa-2, Tla, C3, Ce-2, Pgk-2, and Upg-1, residing distal to the H-2 complex. The results of the typing were used to estimate the length of the segment of chromosome 17 derived from the donor strain of each line—that is, the minimal length of the differential segment. The results indicate that only lines derived by intra-H-2 crossing-over in such a way that they inherited the right-hand portion of H-2 from the inbred partner have the telomeric half of chromosome 17 identical with that of the inbred-partner strain. In other lines the differential segment is at least 3 to 10 cM long. It is argued that in some lines the entire telomeric half of chromosome 17 might be of donor-strain origin.     

Definitive chromosomal location of the H-2 complex by in situ hybridization to pachytene chromosomes

Chromosome 17 of the mouse carries the H-2 complex and the T/t complex. An understanding of the organization of this region and an accurate genetic map of chromosome 17 would be of great value for both immunologists and developmental biologists. Until now the only maps available have been derived solely from recombinational studies using several translocations, an inherently inaccurate method. We have found the definitive location of the H-2 complex by the use of in situ hybridization. Our results show that both the T/t complex and the H-2 complex map to positions far more distal than the generally accepted map positions. This proves that recombination in Robertsonian chromosomes underestimates physical map distances on chromosome 17.     

A semiquantitative measure of immune responses against erythropoietic stem cell antigens

A semiquantitative assay was developed and used to measure the effects of immune responses against 16 independent non-H-2 antigenic loci on erythropoietic stem cells. The assay compares repopulation in genetically anemic WBB6F₁-W/W recipients that have normal immune responses, and in lethally irradiated WBB6F₁ +/+ mice whose immune responses are suppressed by the irradiation. The differences in repopulating ability between these two types of recipients measure how immune responses affect erythropoietic stem cells. Stem cell repopulating abilities for the cells with antigens specified by the Thy-1, H-1, H-24, Ly-1, H-37, and H-17 loci were affected slightly, if at all. Repopulating abilities were moderately reduced by responses against antigens specified by H-15, 16, Ea-2, and Ly-2, 3 loci, and against the differences between the B6 and B10 genotypes, although marrow of these types cured W/W recipients. A surprising result occurred for the antigen specified by the H-8 locus, in which immune responses strongly reduced repopulating abilities, although this type of marrow cell cured W/W recipients. A comparison of these results with skin graft survival times suggests that the antigens specified by the H-17 and H-24 loci are strongly immunogenic on skin but not on marrow stem cells, while those specified by the H-12 and H-8 loci are strongly immunogenic on marrow stem cells but not on skin.     

Subdivision of the S region of the mouse major histocompatibility complex by identification of genomic polymorphisms of the class III genes

The S region of the mouse major histocompatibility complex (MHC) encodes the class III proteins, the second (C2) and fourth (C4) components of complement, and factor B. Previously, the assignment of S-region haplotypes was based on analysis of protein polymorphisms. The recent availability of C2, C4, and factor B cDNA probes prompted a search for restriction fragment length polymorphisms which would serve as additional genetic markers for these loci. DNA was isolated from livers of mice of all standard inbred H-2 haplotypes and of haplotypes pz and bs. These DNA samples were digested with restriction endonucleases and analyzed by Southern blot. By the pattern of restriction fragment length polymorphism observed, specific markers have been identified in factor B of haplotypes f, u, z, bs, r, and v, and in C4 of haplotypes b, q,f,j,p,s, pz, r, and v. These genetic markers were used in the analysis of S-region composition in strains B10.TFR5 (H-2 ^ap5) and C3H.LG (H-2 ^dx), and a possible intra-S-region recombinant was revealed in the H-2 ^dxhaplotype. The genetic markers identified here subdivide the S region and will be of value in defining further the composition of the complement gene complex of the mouse MHC.     

Meiotic recombination within the H-2K-H-2D interval: characterization of a panel of congenic mice,including 12 new strains,using DNA markers

Intra-H-2 recombinant congenic strains are widely used to localize traits to specific subregions of the major histocompatibility complex and have provided evidence for the existence of meiotic recombinational hotspots in mammals. Forty-seven intra-H-2 recombinant strains, including 12 not previously reported, have been identified by serological typing in our laboratory. We have extended the analysis of the cross-over sites in these mice using DNA markers for Ab, Aa, Eb, Ea, Cyp21-ps, D17Tu3, Bat7, and Bat5. The recombinant chromosomes of these congenic strains include loci derived from the a, b, f, k, p, q, r, s, u, and v haplotypes of H-2, providing a diverse panel of strains. Although some alleles of Bat7 could not be distinguished from one another, results from the majority of strains indicated a probable gene order of C4Slp/D17Tu3-Bat7-Bat5-H-2D. No recombinants between Cyp21-ps, C4Slp, and D17Tu3 were observed. The crossover sites in 31 of the 47 intra-H-2 recombinants were within the C4Slp/D17Tu3—H-2D interval; of these 31 crossovers, three were bracketed by D17Tu3 and Bat7, ten by Bat7 and Bat5, seven by Bat5 and H-2D, and 11 by D17Tu3 and Bat5. The results from all 47 strains suggest recombinational hotspots within the C4Slp/D17Tu3—H-2D interval and emphasize the influence that specific haplotypes can have on preferred crossover sites. Correspondence to: G. A. Carlson.     

Murine antigen H-2.7: Its genetics,tissue expression,and strain distribution

Reinvestigation of alloantisera containing antibodies to murine antigen H-2.7 revealed that the crucial recombinant, A.TFR1 (H-2 ^an1 ), which was reported to separate theH-2G locus from theSs-Slp loci, has ak-like instead off-like H-2.7 antigen. Therefore, the crossover position inH-2 ^an1 and the position of the G locus in theH-2 map are now uncertain. By using the hemagglutination-serum inhibition test, anti-H-2.7 reactive substance was found to be present in normal mouse serum in a strain-specific manner. Tissue distribution study by absorption analysis indicated that H-2.7 antigen is present, in addition to RBCs, on spleen and lymph node cells, but is absent on thymus cells. Thirty B10.W congenic lines were analysed for the presence of the H-2.7 antigen. Two lines (B 10.CHA2 and B 10.KPA44) were found to be H-2.7 positive by both direct hemagglutination and absorption tests.Abbreviations used in this paper ACT Ammonium chloride Tris-buffer - BSA Bovine serum albumin - HA Hemagglutination - HASI Hemagglutination serum inhibition - HBSS Hanks balanced salt solution - PBS Phosphate buffered saline - PVP Polyvinylpyrrolidone - RBCs Red blood cells     

Anti-MHC immunity detected prior to intentional alloimmunization

Cell fusion was performed between spleen cells from young BALB/cBy (H-2 ^d) mice which have never been immunized and SP2/0 mouse plasmacytoma cells. A monoclonal H-2-specific cytotoxic IgM antibody was obtained (By-1) which detected a new public biregional H-2 specificity, H-2.m210. The mcAb By-1 reacted strongly with H-2K^d, D^d, and H-2^s antigens, gave weak cross-reactions with H-2K^k, D^q, H-2^r, and H-2^v antigens and was negative with H-2^b, H-2^f, H-2^p, and H-2L^d antigens. A polymorphic reaction pattern was also observed on a panel of lymphocytes from B 10.W strains. The intriguing finding on this reaction pattern was the reactivity on H-2^d cells, including the syngeneic BALB/cBy and truly autologous cells. As shown by capping and immunoprecipitation experiments on H-2^d cells and by studies on H-2^d-transfected mouse L cells, the target molecules for McAb By-1 were H-2K^d and H-2D^d molecules. The BALB/cBy mouse, from whose spleen cells the McAb By-1 was obtained, survived after the fusion experiment, and serum was examined for the presence of cytotoxic H-2-specific antibodies during the rest of its life. At the time of the fusion, no autoreactive serum antibodies were found, but about 4 months later, we found in the serum of this mouse autoreactive H-2-specific cytotoxic IgM antibodies. The serum antibodies followed the same reaction pattern as that of the McAb By-1. As far as we know, this is the first report of autoreactive H-2-specific antibodies in serum of a mouse which has never been immunized and of the first natural autoreactive H-2-specific monoclonal antibody.Abbreviations McAb monoclonal antibody - MHC major histocompatibility complex - H-2 major histocompatibility complex of mice - CTLs cytotoxic T cells - FMF flow microfluorometry - FITC fluorescein isothiocyanate - LPS lipopolysaccharide W.E. coli 0111:134 - PBS phosphate-buffered saline - Iodogen 1,3,4,6,-tetrachloro-3,6-diphenylglycoluril - GAMIg goat-antimouse immunoglobulin - Staph-A Staphylococcus aureus Cowan I     

Low H-2 polymorphism in some Israeli wild mouse populations

Two populations of the wild house mouse, Mus domesticus, found living close to each other (one inhabited a chicken coop and the other an open field at the Educational Farm of the Hebrew University of Jerusalem, East Talpiot, Jerusalem) were studied for their H-2 polymorphism. These two populations were selected because they are well characterized in terms of their ecological parameters; they have been under continuous surveillance for several years. Twenty-seven H-2 homozygous lines were produced by mating wild mice from these two populations with laboratory strains. The H-2 ^w homozygotes were then characterized by serological typing with monoclonal and polyclonal antibodies specific for the known allomorphs controlled by the class I H-2K and H-2D loci or the class II H-2A and H-2E loci. They were also used as donors for immunizations and for the selection of antisera defining the H-2 haplotypes carried by these lines. Four new H-2 haplotypes could be identified: H-2 ^w82 (K ^wl6 D^ws2) H-2 ^w83 (K ^w83 D^w16) H-2 ^w84 (K ^w84 D^w84) and H-2 ^w85 (K ^w83D^w84) the last haplotype being a recombinant derived from H-2 ^w83 and H-2 ^w84. Antinsera defining the new haplotypes were then used for a study of the wild populations. This study revealed that the populations contain only the four identified H-2 haplotypes, having three alleles at the H-2K locus (K ^w16 K^w83, K^w84) and three alleles at the H-2D locus (D ^w16, D^w82 and D ^w84). The alleles occur in the populations with a frequency of 0.12–0.54. There were no significant differences in gene frequencies between the two populations, and the allele frequencies remained more or less stable. There was a significant excess of heterozygotes for at least some of the genes, compared with the frequency expected from Hardy-Weinberg equilibrium. The same antisera were also used to type other populations in the vicinity of Jerusalem. In one population, located 30 km west of Jerusalem, the mice failed to react with any of the reagents. In the other two populations, located 15 km west and 40 km northeast of Jerusalem, three of the four H-2 haplotypes found in East Talpiot were present at high frequencies. It appears, therefore, that only three main H-2 haplotypes and two or three minor ones are present in the area around Jerusalem. This study thus provides the first example of a large mainland population in which the H-2 polymorphism is comparable to that of many other non-H-2 loci.     

Immunogenetic basis for immunologic privilege in the anterior chamber of the eye

DBA/2 mastocytoma (P815) cells are able to grow when inoculated into the anterior chamber of eyes of histoincompatible mice. Tumor growth is unrestrained in recipient mice differing from the DBA/2 strain at multiple minor H loci, but sharing the H-2 ^d haplotype; progressive tumor growth in these animals involves the entire eye, invades the orbit and kills the hosts by extension into the cranial vault. Alternatively, P815 cells grow initially but are unable to sustain continued growth in the anterior chambers of recipient mice differing from DBA/2 at the H-2 complex. Recipients differing from DBA/2 at either the K or D regions of H-2 develop anti-DBA/2 immunity that destroys the intraocular tumor within 20 days of inoculation. Severe inflammatory reactions in these eyes produce innocent bystander destruction of ocular tissue and produce blindness. Recipients differing from DBA/2 at both K and D regions of H-2 also mount vigorous anti-DBA/2 immunity that destroys the intraocular tumor. In these instances, the nonspecific component of the rejection reaction is minimal: when the tumor cells are destroyed, the eyes are restored to anatomic and functional integrity. These results indicate that immunologic privilege in the anterior chamber of the eye is afforded to allogeneic tissues to varing degrees depending upon allodisparity at H-2 loci encoding class I MHC products. The results further imply that the precision of the alloimmune response may be under the control of these same MHC products.