Separation of T and B lymphocytes from various mouse strains by density gradient electrophoresis.

T and B mouse spleen lymphocytes were separated by density gradient electrophoresis on the basis of their surface charge. In all strains examined, the T lymphocytes were found in the high mobility fractions and the B in the low. The T and B cells were separated completely in most fractions, with some overlapping in the middle. Significant differences were found in the electrophoretic distribution profiles between the strains: C57BL/6j, C57BL/10j, (BALB/cXC57BL/6j)F1, and all the following: B6.C-H-2d/cBy (congenic to C57BL/6j), BALB/c, CBA/H/T6j, C57BL/10Sn, and C3H. The C57BL/6j and the (BALB/cXC57BL/6j)F1 cells appear more heterogeneous as far as electrophoretic mobility is concerned. Almost all the other strains give two major peaks. Moreover, the high mobility areas are less populated in the C57BL/6j and the (BALB/cXC57BL/6j)F1 animals than in all the others. The above differences were found consistently when cells prepared by different methods were electrophoresed. It is concluded that the surface charge of lymphocytes may be genetically determined. Possible dependency on the H-2 complex or non-H-2 areas is discussed.     

Genetic studies of murine catalase: Regulation of multiple molecular forms of kidney catalase

Starch gel electrophoresis of kidney catalase in inbred strains C3H and C57BL/6, their F₁ hybrid, and first and second backcross generations demonstrated that single-component (type A) v. multiple-component (type B) electrophoretic patterns are controlled by a single locus. The type A electrophoretic pattern is dominant. Twenty-five inbred strains of mice were classified according to their kidney catalase electrophoretic pattern. The data indicate that the segregating genetic factor determines a specific substance in the type A kidney which affects the electrophoretic mobility of catalase. A comparison of the F₁ hybrid enzyme with a 1:1 mixture of C3H and C57BL/6 enzyme showed that the alteration of electrophoretic mobility is the result of posttranslational modification of the catalase molecule. An association of kidney catalase electrophoretic pattern and the H-2 ^k haplotype indicates that the locus controlling the electrophoretic pattern is most likely located on chromosome 17 in close proximity to the H-2 complex.     

Sex-limited genetic variation in a mouse salivary protein

This report describes a gene which influences the electrophoretic mobility of a protein in the salivas of adult mice. Three categories of phenotype have been observed: the two single-banded types, F (Fast) and S (Slow), and the two-banded type, SF (Slow-Fast), with the two bands represented in varying proportions. All females, regardless of age or strain, and all males before puberty show only the F phenotype. Males of the BALB/c and C57BL/6J strains show the F phenotype throughout puberty and adult life, whereas males of the C3H/St and C57BL/KsJ strains show the SF phenotype in puberty and the S phenotype in adult life. We have designated this variation the sex-limited saliva pattern (Ssp). The results from genetic crosses indicate that the variation among the strains is determined by an autosomal locus, Ssp, with two alleles, Ssp ^S andSsp ^F ,where Ssp ^S is dominant to Ssp ^F .Testosterone treatment can accelerate the acquisition of the S type in males of the strains C3H/St and C57BL/KsJ and also induces that phenotype in C3H/St females and C57BL/6J males. Thus it appears that the observed strain-specific differences reflect a genetic variation in androgen levels and/or androgen sensitivity rather than variation in a structural gene.This study was supported in part by PHS Research Grant 5 RO1 AM21177 and by the Indiana University Human Genetics Center (PHS PO1 GM21054). The preliminary work was done during a 1-month visit by RCK to the Institute of Ecology and Genetics which was supported by the University of Aarhus. This is publication No. 80-18 from the Department of Medical Genetics. RCK was supported by PHS Career Development Award 1 KO4 AM00284. SRD was supported by PHS General Medical Training Grant T32 GM07468.     

Comparison of patterns of hybrid resistance to the BALB/c plasmacytomas LPC-1 and MPC-11

Summary Patterns of genetic control of hybrid resistance to the BALB/c plasmacytoma LPC-1 were studied for comparison with those to MPC-11, a plasmacytoma investigated previously. The overall patterns of hybrid resistance to the two tumors were similar, i.e., hybrids between BALB/c and BALB congenic resistant (CR) strains, A and A CR strains, SJL and DBA/2 were as susceptible to LPC-1 as BALB/c mice themselves, whereas hybrids between BALB/c and AKR, C57BL/Ks, DBA/1, C57BL/6 (B6), C57BL/10 (B10) and B10 CR strains were resistant to LPC-1 as previously shown with MPC-11. Heterozygosity within the H-2 complex alone was insufficient for resistance to either tumor. Among hybrids between BALB/c and the B10 CR strains, however, the presence of certain H-2 haplotypes influenced the degree of resistance seen and this H-2 effect was different for the two tumors. A sex effect on resistance to LPC-1, but not to MPC-11, was seen among F₁ hybrids between BALB/c and DBA/1 although not in any other F₁ hybrids. Among ((B10×BALB/c)F₁×BALB/c) and (BALB/c×(B10×BALB/c)F₁) and ((BALB/c×B10)F₁×BALB/c) and ((BALB/c×B10)F₁×BALB/c) backcross mice, however, significantly more males than females were resistant to LPC-1 and the results of this study are compatible with the idea that in F₁ hybrids between BALB/c and B10, resistance to LPC-1 is controlled by two dominant autosomal genes, one of which is sex-limited and neither of which is linked to H-2. In contrast, hybrid resistance to MPC-11 in this cross is controlled by a single gene. Cross-protection experiments indicated that the two tumors share at least one tumor-associated transplantation antigen.     

Tumor-associated antigenic differences between the primary and the descendant metastatic tumor cell populations

The existence of antigenic differences between cell populations in the local growth of the 3LL tumor (L-3LL) and its lung metastases (M-3LL) was studied. Normal C57BL/6 spleen cells sensitized in vitro for 5 days against L-3LL monolayers lysed preferentially L-3LL targets but not M-3LL tumor cell targets. Conversely, anti-M-3LL-sensitized lymphocytes killed M-3LL targets more efficiently than they killed L-3LL targets. Furthermore, spleen cells from mice bearing subcutaneous L-3LL tumors were significantly more cytotoxic to L-3LL targets than to M-3LL targets and vice versa. M-3LL cells were found also to be more resistant in vitro and in vivo to natural killer cells than were L-3LL tumor cells. M-3LL cells were more resistant than L-3LL cells to hybrid resistant mechanisms when they were inoculated into F₁ (C3Heb X C57BL/6) or F₁ (BALB/c X C57BL/6) mice. Anti-M-3LL lymphocytes generated both in vitro and in vivo, but not anti-L-3LL lymphocytes, admixed with L-3LL or M-3LL tumor cells and inoculated into footpads of syngeneic recipients suppressed the development of lung metastases. These results suggest that metastatic cells are indeed phenotypic variants of the local growing tumor cell populations. Presumably, these variants are selected for their capacity to home to and grow in the lungs, and for their resistance to specific immune effects initially evoked against the local tumor and to nonspecific natural killer cells. These data may prove to be of importance with respect to any rational approach to the problem of immunotherapy.     

Xenogeneic antisera against T-lymphocyte receptors recognizing allogeneic transplantation antigens: Preparation and properties

Antireceptor sera ARS-1 (anti-CBA-anti-C57BL/6) and ARS-2 (anti-C57BL/6-anti-BALB/c) were prepared in a xenogeneic system by immunizing rabbits with alloimmune CBA anti-C57BL/6 and C57BL/6 anti-BALB/c lymphocytes and subsequent exhaustive absorption of rabbit immune sera with red blood cells, lymphocytes, liver cells, and serum from intact mice. In the cytotoxicity test, both ARS sera lysed only activated T lymphocytes of corresponding, but no other specificities, and did not affect intact lymphocytes. They did not inactivate in vitro the PFC-producing antibodies to SRBC or inhibit the immune response to SRBC and Viantigen of Salmonella typhi in adoptive transfer experiments. Pretreatment of intact CBA lymphocytes in vitro with ARS-1 plus complement suppressed their ability to induce lethal GvH disease in irradiated (CBA × C57BL/6)F₁, but not (CBA × BALB/c)F₁ recipients; similar pretreatment of C57BL/6 lymphocytes did not lower lethality among irradiated (CBA × C57BL/6)F₁ recipients. This method of preparing ARS may help to obtain highly specific antireceptor sera for regulation of the immune response to different antigens in outbred animals and in humans.     

Murine responses to (Tyr-Glu-Ala-Gly)n: II. H-2 and non-H-2 gene effects

Mice of the H-2^b haplotype responded to the sequential polymer poly(Tyr-Glu-Ala-Gly) in the in vitro T-cell proliferative assay, irrespective of whether they were homozygous or heterozygous at the H-2^b locus. The antibody responses of the H-2^b congenic mice to this polymer were variable, with A.BY and BALB.B showing responses better than those of C57BL/6 and C57BL/10 strains. The antibody responses of the F₁ progeny of (responder × nonresponder) strains of mice to this polymer are generally lower than the responder parents. F₁ mice with C57BL/10 background were the poorest responders. Studies with F₂ mice and backcross progenies of selective breeding of high and low antibody responder (C57BL/6 × BALB/c) F₁ to high responder C57BL/6 mice indicated that both non-H-2 genes and H-2 gene dosage effects influenced the magnitude of the humoral antibody responses. Animals having low responder non-H-2 background and only half the dosage of the responder immune response genes has greatly diminished antibody responses.     

Purification and characterization of the Danish (skive) variant of mouse liver alcohol dehydrogenase

The partially inbred Danish (Skive) strain of mice exhibits a form of liver alcohol dehydrogenase (ADH) which differs in electrophoretic mobility from that of all other inbred mouse strains thus far examined, e.g., C57BL/10, DBA/2J, and BALB/c. In order to compare the catalytic and molecular properties of the variant and normal enzyme forms, they were purified to homogeneity by ion-exchange and affinity chromatography. Tryptic peptides of reduced and carboxymethylated subunits of the normal and variant ADH forms were mapped by thin-layer two-dimensional electrophoresis and chromatography and by reversed-phase high-performance liquid chromatography. A unique nonapeptide in the Danish mouse liver ADH which did not appear in enzymes from C57BL/10, DBA/2J, or BALB/c mice was identified by both methods. Amino acid sequencing of this peptide revealed that the Arg residue at position 124, as predicted from the cDNA sequence of ADH in DBA/2J mice, has been replaced by Leu in the Danish variant. The Leu for Arg substitution in the variant form appears to account for its decreased cathodic mobility with electrophoresis in starch gels at pH 7.2. The K _m and V _max of ADH from the Danish strain for three primary alcohols and three aldehydes were similar in value to those of ADH from the C57BL/10, DBA/2J, and BALB/c strains. Based on the X-ray structure of horse liver ADH, position 124 is on the solvent-exposed surface of the catalytic domain. The finding that the kinetic constants are similar for the normal and variant forms is consistent with the observation that this residue is not in the active site and that there is no known role for it in the ADH catalytic mechanism.This work was supported by NIAAA Grant AA-04307.     

Trypanosoma brucei: influence of host strain and parasite antigenic type on infections in mice

Inbred mice were infected with cloned populations of Trypanosoma brucei brucei Lister S42 under carefully controlled conditions. The course of infection was found to depend both on host strain and the antigenic type of the infecting organisms. The two antigenic types used, “NIM2” and “NIM6” had differing virulence for (CBA/H × C57BL/6)F₁ mice, and when mice were infected with parasites of one clone, trypanosomes of the other type frequently appeared after the initial population had been eliminated. Different mouse strains had varying susceptibility to clone NIM6. In most cases there was an inverse relation between the survival time and the parasite load during the first peak of parasitemia. The differences in resistance to T. brucei were unrelated to H-2 haplotype: C57BL/6 and (CBA/H × C57BL/6)F₁ were most resistant, CBA/H, BALB/c and DBA/2 less so, and C3H/He most susceptible.     

Single gene control of resistance to cutaneous leishmaniasis in mice

A series of inbred, congenic resistant, and hybrid strains of mice were intradermally inoculated with 10⁶ promastigotes of Leishmania tropica. These mice were divided into susceptible and resistant groups using the criteria of lesion size, development of metastatic foci and skin-test reactivity. At 16 weeks of infection, resistant strains A/J, DBA/1J, AKR/J, CBA/J, C3H/HeJ, NZB/BINJ, C57BL/6J, C57BL/10Sn, B10.D2, B10.129(10M), and B10.CE(30NX) had completely resolved their lesions, while susceptible SWR/J and BALB/cJ mice demonstrated large, nonhealing cutaneous lesions. In addition, BALB/cJ developed metastatic lesions on the extremities which progressively increased in size. All BALB/cJ and SWR/J mice died by 7 1/2 months of infection. The BALB/cJ x C57BL/6JF₁ hybrid behaved in an intermediate fashion showing a slower expansion of cutaneous ulcers and a delayed development of metastatic foci, however, the infection ultimately proved fatal. The F₂ generation could be separated into three distinct groups: resistant, intermediate, and susceptible mice with a lesion size distribution pattern in conformity with a 1:2:1 ratio. Male/female susceptibility differences were not noted. These data indicated that development of acquired resistance may be under the control of a single, autosomal gene. The gene did not appear to be H-2-, Ir-2-, or H-11-linked as is seen with Leishmania donovani infections.     

Studies on theH-X locus of mice

The results of challenging F₁ hybrid male mice derived from strains BALB/c, A/J, C57BL/6J, C57BL/10ScSn and DBA/2J with small male ear skin grafts of paternal strain origin indicate thatH-X is highly polymorphic. Thus, with the exception of the B6 and B10 strains which may share oneH-X allele, each of these strains appears to have a different H-X genotype.     

Murine survival of infection with Francisella novicida and protection against secondary challenge is critically dependent on B lymphocytes

Respiratory infection of mice with Francisella novicida has recently been used as a model for the highly virulent human pathogen Francisella tularensis. Similar to F. tularensis, even small doses of F. novicida administered by respiratory routes are lethal for inbred laboratory mice. This feature obviously limits study of infection-induced immunity. Parenteral sublethal infections of mice with F. novicida are feasible, but the resulting immune responses are incompletely characterized. Here we use parenteral intradermal (i.d.) and intraperitoneal (i.p.) F. novicida infections of C57BL/6J mice to determine the role of B cells in controlling primary and secondary F. novicida infections. Despite developing comparable levels of F. novicida-primed T cells, B cell knockout mice were much more susceptible to both primary i.d. infection and secondary i.p. challenge than wild type (normal) C57BL/6J mice. Transfer of F. novicida-immune sera to either wild type C57BL/6J mice or to B cell knockout mice did not appreciably impact survival of subsequent lethal F. novicida challenge. However, F. novicida-immune mice that were depleted of T cells after priming but just before challenge survived and cleared secondary i.p. F. novicida challenge. Collectively these results indicate that B cells, if not serum antibodies, play a major role in controlling F. novicida infections in mice.     

Purification and characterization of two allelic forms of l-glycerol 3-phosphate dehydrogenase from inbred strains of mice

l-Glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) was purified from the muscle of BALB/cJ and C57BL/6J mice. The half-lives of the enzyme at 50 C were 6 and 33 min, respectively, for the BALB/cJ and C57BL/6J strains. Enzyme preparations from the two strains of mice were compared with respect to the following properties and found to be essentially indistinguishable: K _m values for dihydroxyacetone phosphate, NADH, l--glycerophosphate, and NAD⁺; maximum velocity; competitive inhibition by inorganic phosphate; pH optimum; energy of activation; electrophoretic mobility; molecular weight and subunit molecular weight. From these data, it is concluded that the kinetic properties of the purified enzyme are not the factors responsible for the differences in activity found in crude homogenates of mouse tissues.This work was supported by NIH Research Grant HD 06712 from the National Institute of Child Health and Human Development and by an allocation from NIH General Research Support Grant RR-05545 from the Division of Research Resources to The Jackson Laboratory. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Teratocarcinoma transplantation rejection loci: AnH-2-linked tumor rejection locus

Embryoid bodies (ascites tumor) from a 129/Sv transplantable teratocarcinoma produce tumors (100%) in syngenic 129/Sv mice but fail to form tumors (3–6%) in BALB/c mice, C3H/He mice and C57BL/6 mice, in spite of the fact that the malignant stem cells of this tumor do not express detectable H-2 antigens. The available evidence indicates that this allogeneic tumor restriction has an immunological basis; 100% of the F₁ hybrid mice between 129/Sv and the three other inbred mouse strains accept the 129/Sv teratocarcinoma. The backcross and F₂ mice segregate the BALB/c, C3H/He and C57BL/6 tumor transplantation rejection loci in a manner that indicates that each of these inbred strains of mice contain one to two major transplantation rejection loci. A linkage analysis in the BALB/c and C3H/He backcross and F₂ generations indicates that these mice have a teratocarcinoma transplantation rejection locus on chromosome 17, about eight to nine recombination units from theH- 2 complex. An F₁ complementation analysis between allogeneic mice that each reject teratocarcinomas tumors (BALB/c × C57BL/6 and C3H/He × C57BL/6), indicates that the C57BL/6 mice have the 129/Sv tumor-accepting (sensitive) allele at theH-2-linked locus but reject teratocarcinomas because of antigenic differences at a second locus.While these major teratocarcinoma transplantation rejection loci determine the acceptance or rejection of a tumor by a mouse injected with high doses of tumor tissue (750 g of tumor protein), evidence is presented for a number of minor genetic factors that can (1) affect the efficiency of tumor rejection and (2) cause complete tumor rejection at lower tumor doses (7.5–75 g of tumor protein).     

A quantitative genetic analysis of tissue-specific catalase activity inMus musculus

Tissue-specific catalase activity in 3-week-old animals from inbred mouse strains 129/ReJ, BALB/c, C3H/HeAnl/Cas-1^b, C3H/HeSnJ, C3H/S, C57BL/6J, and Swiss-Webster was found to be highly variable by analysis of variance (P=0.01). Appropriate crosses were made among strains which were classified as normal (BALB/c, C3H/HeSnJ, C3H/S), hypocatalasemic (129/ReJ, C57BL/6J), and acatalasemic (C3H/HeAnl/Cas-1^b) with respect to blood catalase activity to study the inheritance of the blood, kidney, liver, and lung catalase activity levels in a number of generations (reciprocal F₁'s, F₂, two backcrosses —BC₁ and BC₂— and some RI lines). Segregation analysis and statistical methods which tested different models of inheritance as well as calculations of heritability were used in an effort to assess and evaluate genetic parameters that affect catalase activity. Results indicate that the inheritance of blood catalase activity in the cross involving acatalasemic and normal (BALB/c, C3H/HeSnJ) strains is compatible with the single-locus difference between the parental strains; however, the difference between the acatalasemic and the hypocatalasemic strain (C57BL/6J) would require additional genetic interaction for a satisfactory explanation. A similar pattern of generalization also applies to the inheritance of kidney catalase activity. The segregation pattern for the liver and lung catalase activity in most crosses is significantly different from the expectations of the single locus model. These results are compatible with the concept that a number of genes must affect tissue-specific catalase activity in mice. These may include previously described (e.g., Ce-1 and Ce-2) or novel genetic regulators/modifiers which interact with a single structural gene (Cas-1) or its product to produce the catalase phenotype characteristic of specific tissues in each strain.This investigation was supported by a Natural Sciences and Engineering Research Council of Canada operating grant to S.M.S.     

Mice carrying a CD20 gene disruption

 CD20 is a hallmark antigen of B lymphocytes. Its expression is restricted to precursor and mature B cells but it is not expressed on plasma cells. The protein is a membrane-embedded phosphoprotein that appears likely to transverse the membrane four times. Its function is unknown although CD20 has been variously proposed to play a role in B-cell activation, proliferation, and calcium transport. A unique homologue of human CD20 has been described in mouse, which also shows a B-cell-specific pattern of expression. Here we describe the generating of mice carrying a CD20 gene disruption. So far, we have failed to detect any major effect of the gene disruption on the differentiation and function of B lymphocytes as judged by the expression of surface markers, antigen receptor signaling, proliferative responses, or calcium uptake. We did note, however, that the mice homozygous for the gene disruption [generated by intercrossing (129 × C57BL/6)F₁ CD20 ^+/- heterozygotes] showed a substantial depletion of the sub-population of peritoneal B cells that lack expression of the B220 (RA3–6B2) isoform of CD45. The loss of the IgM⁺ 6B2^- peritoneal B cells is not, however, attributable to the CD20 gene disruption itself. Rather, it segregates with a polymorphic difference between the 129 and C57BL/6 strains that is linked to the CD20 locus which, intriguingly, is itself close to the CD5 gene. This demonstrates that caution must be exercised when comparing the phenotypes of F₂ litter-mates generated from crosses between 129 embryonic stem-cell-derived chimeras and mice of other strains. Received: 23 December 1997 / Revised: 27 January 1998     

Polymorphism of esterase-10 in Mus musculus

An esterase, esterase-10, in the house mouse, Mus musculus, is specific for esters of 4-methylumbelliferone and exhibits a polymorphism detectable by electrophoresis. Fifteen inbred strains and two outbred strains have been examined for this polymorphism, and two phenotypes, ES-10A and ES-10B, have been observed. Each phenotype manifests itself as a single band of enzyme activity, but under the electrophoretic conditions used the ES-10A phenotype has less anodal electrophoretic mobility than the ES-10B phenotype. In F₁ hybrids (C3H/He/Lac×C57BL/Gr) a third phenotype was observed, ES-10AB, consisting of three bands of enzyme activity, two of which correspond to the parental forms and the third with intermediate mobility. The triple-band pattern in the F₁ hybrids indicates that esterase-10 is a dimeric enzyme protein.This work was supported by the Medical Research Council.     

Characterization of single nucleotide polymorphisms for a forward genetics approach using genetic crosses in C57BL/6 and BALB/c substrains of mice

Forward genetics is a powerful approach based on chromosomal mapping of phenotypes and has successfully led to the discovery of many mouse mutations in genes responsible for various phenotypes. Although crossing between genetically remote strains can produce F₂ and backcross mice for chromosomal mapping, the phenotypes are often affected by background effects from the partner strains in genetic crosses. Genetic crosses between substrains might be useful in genetic mapping to avoid genetic background effects. In this study, we investigated single nucleotide polymorphisms (SNPs) available for genetic mapping using substrains of C57BL/6 and BALB/c mice. In C57BL/6 mice, 114 SNP markers were developed and assigned to locations on all chromosomes for full utilization for genetic mapping using genetic crosses between the C57BL/6J and C57BL/6N substrains. Moreover, genetic differences were identified in the 114 SNP markers among the seven C57BL/6 substrains from five production breeders. In addition, 106 SNPs were detected on all chromosomes of BALB/cAJcl and BALB/cByJJcl substrains. These SNPs could be used for genotyping in BALB/cJ, BALB/cAJcl, BALB/cAnNCrlCrlj, and BALB/cCrSlc mice, and they are particularly useful for genetic mapping using crosses between BALB/cByJJcl and other BALB/c substrains. The SNPs characterized in this study can be utilized for genetic mapping to identify the causative mutations of the phenotypes induced by N-ethyl-N-nitrosourea mutagenesis and the SNPs responsible for phenotypic differences between the substrains of C57BL/6 and BALB/c mice.     

Purification and characterization of mouse alcohol dehydrogenase from two inbred strains that differ in total liver enzyme activity

Alcohol dehydrogenase activity in mouse liver homogenate-supernatants is 1.7 times greater in the C57BL/10 strain than in the BALB/c strain, regardless of whether activity is expressed in units per gram liver, total liver, or milligram DNA. The K _m values for ethanol and NAD⁺, approximately 0.4 and 0.03mm, respectively, of enzyme purified from both strains are similar. Moreover, the K _i for NADH, 1 µm, the pH optimum for ethanol oxidation, 10.5, and the V _max for ethanol oxidation, 160 min^–1, for ADH from the C57BL/10 and BALB/c strains are similar. Therefore, the difference in ADH activity in the two strains cannot be due to differences in the catalytic properties of the enzyme. The electrophoretic and isoelectric focusing patterns and two-dimensional tryptic peptide maps of the purified enzyme from both strains are identical. Thus the amino acid sequences of enzyme from C57BL/10 and BALB/c mice must also be identical or very similar. The difference in ADH activity in the two strains is most likely the result of genetic differences in the content of ADH protein in liver.Supported by NIAAA Grant AA 04307.     

H-Y antigen: No evidence for alleles in wild strains of mice

H-Y antigen(s) coded or controlled by the Y chromosome in a variety of wild mouse strains have been compared with those of the inbred laboratory strains C57BL/6 (B6) and C57BL/10 (B10). H-Y antigen(s) were detected by H-2-restricted cytotoxic T cells from B6 and B10 female mice primed in vivo and boosted in vitro with syngeneic male spleen cells: There was no difference in the degree of H-Y specific lysis of male cells from the C57BL strains and of F₁ hybrids or B6 congenic mice carrying the Y chromosome from the wild mouse strains examined. This result indicated that at the level of target cell specificity the H-Y antigen(s) from wild and laboratory strains were indistinguishable. H-Y antigen(s) were also found to be indistinguishable at the level of the in vitro induction of the anti H-Y cytotoxic response: F₁ female mice, primed in vivo and boosted in vitro with homologous F₁ male cells, all made H-Y-specific responses and where it could be examined, the target cell specificity of the anti-H-Y cytotoxic cells showed that B10 male cells as well as the homologous F₁ male cells (where the Y chromosome was derived from the wild strain) were good targets. Finally, possible differences in H-Y transplantation antigens between the wild strains and the B10 laboratory strain were examined by grafting F₁ male mice, the progeny of B10 females, and wild strain males with B10 male skin. These grafts were not rejected during an observation period of more than 9 months. Taken together, neither the cytotoxic data nor the skin graft data provide any evidence for allelism of H-Y even though the mouse strains examined were collected from widely disparate geographical locations.