Histocompatibility gene mutation rates in the mouse: a 25-year review.

Mutation rates of H2 and non-H2 histocompatibility genes in the mouse are examined over a 25-year period. Detected by skin graft rejections, the mutations were screened in inbred and hybrid mice from a continuously maintained and monitored colony and from a regularly supplied set of mice provided from the National Cancer Institute for monitoring of genetic integrity. Twenty-five H2 mutations were recovered, involving the K, D, L, and Ab loci, as well as over 80 mutations of non-H2 histocompatibility genes. Aside from a single allele at a single locus (H2-Kb), the spontaneous mutation rate of H2 class I genes appears to be equivalent to that found estimated for non-H2 histocompatibility genes, and comparable to rates reported for a variety of mouse genes. This is in contrast with previous suggestions that H2 genes mutate at orders of magnitude greater than do "average" mammalian genes. The discrepancy is attributed to the H2-Kb gene which accounts for over half of all reported H2 mutations and which mutates spontaneously at a rate of 1-2 x 10(-4) per gene per generation. Furthermore, over half of the spontaneous H2-Kb mutations result in a single mutant phenotype (the "bg" group) which involve similar changes at amino acid residues 116 and 121. Thus, the high spontaneous mutation rate for H2-Kb appears to be the exception among major histocompatibility genes, rather than the rule.

Failla Memorial Lectures. Radiation genetics: the mouse's view.

This report describes the lecturer's visit to Murinia where he consulted with the leading geneticists, including Dr. Maxie Mouse CXIV. The mice are greatly interested in the field of radiation genetics, but they no longer wish the honor of the major responsibility for setting our genetic radiation standards.

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

Structural studies of the H-2 gene products from a group of five closely related but independent C57BL/6H-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 tyrosine 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-2Kb mutants analyzed.

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

In an earlier paper, we presented evidence that two independent mutants of the bg series, B6-H-2bm5 (bm5) and B6-H-2bm16 (bm16) carry identical mutations such that tyrosine at residue number 116 of the H-2Kb 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-2bm6 (bm6), B6.C-H-2bm7 (bm7), and B6.C-H-2bm9 (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 B6 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 Kb molecule.

An independent analysis of the National Cancer Institute study on non-nutritive sweeteners and bladder cancer.

We evaluated the possible relation between use of non-nutritive sweeteners and bladder cancer using data obtained from the National Cancer Institute Bladder Cancer Study under the Freedom of Information Act. In the general study group, there was no evidence for an association between non-nutritive sweeteners and bladder cancer. Control for a variety of factors through multivariate techniques diminished the plausibility of earlier interpretations of these data, which had raised the possibility that certain subgroups of users or non-nutritive sweeteners might be at an increased risk for bladder cancer. We found that the putative effects of non-nutritive sweeteners were not consistent among subgroups with similar baseline risk, did not display consistent dose-response trends, and were subject to considerable sampling error. We concluded that the data provided little evidence that non-nutritive sweeteners increase risk for bladder cancer among subgroups of users, and that definitive evidence on this question is beyond the reach of conventional research.

History and genealogy of the H-2Kb mutants from the C57BL/6Kh colony.

The genealogy of 14 H-2Kb mutations arising spontaneously in the C57BL/6Kh colony is presented together with data from skin-graft monitoring and husbandry procedures. Eight of the 14 mutations have phenotypic and structural similarities, but the pedigree analysis and evaluation of the histocompatibility genetics of their sibs and ancestors strongly indicate that they represent recurring mutational events rather than the segregation of a single mutation throughout the colony.

Late effects of radiation therapy for cancer of the uterine cervix.

This report presents follow-up information on 497 women diagnosed with cancer of the uterine cervix in Connecticut and California between 1932 and 1951 who received only radiation as their initial course of therapy. Patients entered into the study were all treated before age 55 and all were five-year-survivors following treatment in order to eliminate early deaths due to the cervical cancer. Three radiologic dosage groups (high, medium, and low) were formed with 93, 244, and 160 patients, respectively. For all dosage groups combined 108 subsequent cancers were observed more than 5 yr after cancer treatment compared with 64 expected (P less than 0.01). Sites for which subsequent cancers were significantly (P less than 0.05) in excess of expectation were rectum, ovary, lung, vulva and vagina, small intestine, oropharynx, and central nervous system excluding brain. The ratio of observed to expected cases of subsequent cancers rose only slightly with increasing radiologic dose. No significant differences in overall survival patterns for the three dosage groups were found. For all dosage groups survival was poorer than in the corresponding segment of the general population.

Some comparisons between induced and spontaneous mutation rates in mouse sperm and spermatogonia.

The induction of histocompatibility gene mutations in BALB/cKh mouse sperm was determined following doses of 0, 350, or 350 + 300 rad (24 h apart) of 250 kV X-rays. The mutation rate is significantly lower than predicted on the basis of the seven-locus test and is not significantly different from the very low rate of H-loci in BALB/cKh spermatogonia. Furthermore, selection and repair are important in determining mutation rates per viable progeny of H-loci during both spermatogenesis and gestation.

Fine specificity of alloimmune cytotoxic T lymphocytes directed against H-2K. A study with Kb mutants.

The fine specificity of alloimmune cytotoxic T lymphocytes (CTL) was investigated in CTL responses across the smallest known H-2 differences, those based on mutation at a single H-2 locus. CTL were generated in all possible mixed lymphocyte culture (MLC) combinations among seven H-2Kb mutants and the mouse strain of origin, C57BL/6 (B6-H-2b). CTL were also generated in all F1 hybrid responder/homozygous stimulator-cell combinations among four Kb mutants and B6-H-2b. CTL activity was measured in cell-mediated lympholysis (CML) against target cells from all Kb mutants and B6-H-2b. Cross-reactivity against targets other than the MLC stimulator was extensive and led to the distinction of 64 CML target determinants. Two Kb mutants (B6-H-2bm6 and B6.C-H-2bm9) showed identical typing for all 64 CML determinants. CML reactions after MLC between these two haplotypes were mutually negative. The mutants B6-H-2bm3 and B6.C-H-2bm11 showed identical typing for 47 of the 64 determinants. Their close relationship is in agreement with the finding that H-2bm3 anti-H-2bm11 CTL were the only ones that exclusively lysed target cells of stimulator-cell genotype. On the basis of CML typing, the sequence of relatedness of the mutants with H-2b is as follows: bm6/bm9-bm10-bm3-bm1-bm11, bm6/bm9 being the closest to, and bm11 the most distant from H-2b. The extensive cross-reactivity of alloimmune CTL appears to reflect immunogenetic complexity rather than lack of specificity. Comparison with other reports supports the notion that the system of Kb CML determinants, recognized by alloimmune CTL, is at least partially overlapping with the H-2Kb specificity repertoire involved in the associative T cell recognition of virus-infected cells.

Interaction of H-2Db with mutant histocompatibility gene H (KH-11) in the mouse.

The detectable presence of H (KH-11)b, a mutant non-H-2 histocompatibility gene, was previously shown to depend upon the simultaneous presence, in the skin-graft donor, of both the mutant gene and the H-2b haplotype. The experiments reported here demonstrate that H-2Db is the essential element of H-2b for this interaction. Of two H-2Db histocompatibility mutations, H-2bm13 can replace H-2Db in this interaction, but H-2bm14 cannot.

Biochemical studies on the H-2K antigens of the MHC mutants bm3 and bm11.

Biochemical analyses of the H-2K-gene products of the C57BL/6 mutant strains, bm3 and bm11, have been carried out in order to characterize the structural relationships among these antigens. From comparative tryptic peptide mapping of cyanogen bromide fragments from the mutant and parent Kb glycoproteins and from preliminary amino-acid sequence analyses, a number of discrete differences have been discerned. Two sites of difference at amino residues 77 and 89 in the Kbm3 glycoprotein are noted relative to Kb. An alteration at residue 77 similar or identical to that seen in Kbm3 is present in the K antigen of the bm11 mutant. Because our techniques sample only 75 to 80 percent of the extracellular portion of H-2Kb, other undetected changes are possible. However, our present findings are most consistent with the conclusion that only very limited differences exist between mutant and parent molecules. Further, taken together with CML (cell-mediated-lymphocytotoxicity) reaction patterns (Melief et al. 1980), the biochemical data support the hypothesis that the proposed structural alterations in the Kb mutant glycoproteins are directly related to their observed immunological specificity.

B6.C-H-2bm12. A new H-2 mutation in the I region in the mouse.

The B6.C-H-2bm12 mutant is described and evidence is presented for the mutational site occurring in the IA subregion. The mutant is of the gain and loss type as bm12 in equilibrium or formed from C57BL/6 grafts are rejected in 14-16 d. Mapping studies by the gene-complementation method using H-2 recombinant strains place the mutation in the K or IA regions of the H-2 complex and furthermore, the use of this test and the use of other H-2 mutants indicate that H-2Kb is not the site of the mutation, making the IA region the most likely site. Serological analysis with a battery of H-2b, Iab, and other Ia sera, both by cytotoxicity, rosetting, and also by absorption analysis, indicated no alteration in H-2 specificities, particularly in H-2.K33. By contrast, all of the Iab specificities coded for by the IA subregion (Ia.3, 8, 9, 15, and possibly 20) are extensively altered and are either absent or greatly reduced in amount indicating an extensive alteration in the Ia-bearing molecule. The bm12 mutant strongly stimulates the parental C57BL/6 strain in an mixed lymphocyte reaction (MLR), and the reciprocal also occurs, the degree of stimulation being similar to that obtained with K + IA differences originating in another H-2 haplotype and points to the mutation effecting the Lad-1 locus. The presence of an extensive histocompatibility change, a marked alteration in the serologically detected Ia specificities, and a strong MLR, all produced by the one mutation, provides strong evidence for the identity of the Ia-1, Lad-1, and H-2(IA) loci in the IA subregion. The bm12 mutant should be of value in determining the relationship of Ia specificities, Ir genes, and other phenomena effected by the I region.

The firstH- 2 mutant workshop.

CONCLUSION: The major accomplishment of the Workshop was probably the realization of many of its participants that most of the 21 availableH- 2 variants aretrue mutations very likely derived from single nucleotide substitutions. Any theory of the pleiotropic effect of theH- 2 genes must now take this fact into account; such theories must also consider the observation that a wide variety of immunological phenomena are affected byH- 2 mutations and thus, apparently, are controlled by a single gene.

Cytotoxic T-cell response to Ectromelia virus-infected cells. Different H-2 requirements for triggering precursor T-cell induction or lysis by effector T cells defined by the BALB/c-H-2db mutation.

The T(c)-cell response to ectromelia virus infection was studied in BALB/c-H-2(db) mice which carry a loss mutation in the H-2D region that results in the absence from cell surfaces of a molecule (D') bearing certain public H-2 specificities. When infected, these mice showed a poor response of T(c) cells that recognize H-2D(d) plus virus-specific determinants on infected macrophage targets, but gave a normal response to H-2K d plus virus-specific antigens. However, their own infected macrophages do display wild-type antigenic patterns involving virus and H-2D(d) since they were killed as efficiently as wild-type (BALB/c,H- 2(d))-infected cells by T(c) cells specific only for H-2D(d) plus viral antigens. When tested in vitro, infected BALB/c-H-2(db) cells stimulated a poor T(c)-cell response to H-2D plus virus-specific antigens, but stimulated a normal response (in comparison with infected BALB/c macrophages) to H-2K(d) plus viral antigens. Uninfected BALB/c-H-2(db) cells stimulated a normal T(c)-cell response to minor H antigens or trinitrophenyl in association with H-2D(d), thus suggesting that the defective response to infection may reside in a failure of the relevant H-2D(d) antigens of mutant cells to physically associate with viral antigens. Close association of viral and H-2D-coded molecules was also suggested by ability of specific anti-H-2K or -H-2D to partially block T(c)-cell-mediated lysis of infected targets. These results were interpreted to mean that H-2Dd-dependent, virus- immune T(c) cells recognized an antigenic pattern consisting of virus- specific and H-2D(d) determinants with the latter borne on an H-2D molecule carrying serologically-defined H-2D(d) private specificities. A second H-2D(d)-coded molecule (D') was not required for recognition and lysis by activated T(c) cells, but was apparently necessary for efficient stimulation of precursor T(c) cells, perhaps by promoting appropriate physical association of viral and H-2D(d) molecules.

Failure of X-rays to mutate class II histocompatibility loci in Balb/c mouse spermatogonia.

Adult Balb/c Kh male mice were irradiated (pelvic region, 250 kVcp X-rays, 60 rad per min) and three months later were mated to untreated C57BL/6 Kh females. Their B6C F1 progeny were screened for mutations at the Class II histocompatibility loci, i.e. those that carry similar alleles in the parental lines and are therefore homozygous in the F1 progeny. The treatment groups were: single doses of 0, 350, 500, 650 and 800 rad; split doses 1 day apart, totalling 500, 650 and 800 rad; split doses averaging 52 days apart, totalling 650 and 800 rad. Thirty-six mutants were identified in 13,614 progeny. Twelve of them occurred in five clusters of two or three, presumably owing to five gonadal mosaics among 940 parents. Irradiation did not increase the spermatogonial mutation rate. The only effect of exposure appeared to be a decrease in the mutation rate of the 1-day split dose-groups compared to those with the same total doses in a single exposure or in two fractions, 52 days apart.