Howard C. Passmore

Genetic control of acquired resistance to visceral Leishmaniasis in mice

A series ofH-2 and non-H-2 congenic resistant (CR) strains on a C57BL/10Sn background were infected with 107 amastigotes ofLeishmania donovani. Non-H-2 congenic strains B10.LP-H-3b and B10.CE(30NX) and (B10.LP-H-3b × B10)F1 hybrids showed a very rapid decrease in liver-parasite burdens beyond day 21. Parasite counts for these strains at day 35 were significantly lower than for all other strains tested. The rapid decrease in parasite numbers, massive lymphocellular infiltration into the liver and strong delayed hypersensitivity reactions to parasite antigens in strains congenic for a portion of chromosome 2 indicated that acquired immunity toL. donovani was controlled by a dominant gene at or near theIr-2 locus. In addition, B10.129(10M) mice, which differ from C57BL/10Sn at theH-11 locus, showed highly significant increases in parasite numbers at day 35. Other observations supporting the absence of acquired immunity in B10.129(10M) included negative delayed hypersensitivity tests to parasite antigens and the absence of lymphocellular infiltrate into the liver. Although the differences were not as pronounced,H-2 CR strains withH-2b,H-2a, andH-2k haplotypes also showed significantly greater decreases in parasite numbers by day 35 as compared to otherH-2 CR strains.

A testis-specific geneTpx-1 maps betweenPgk-2 andMep-1 on mouse chromosome 17

Tpx-1 is a novel testis-specific gene, which was identified in one of the cosmid clones isolated at random from mouse chromosome 17 (Kasahara et al. 1987). We show here that Tpx-1 maps between Pgk-2 (a gene encoding a testisspecific isozyme of phosphoglycerate kinase; EC 2.7.2.3) and Mep-1 (a locus that regulates kidney meprin activity) on mouse chromosome 17. The polymorphism of Tpx-1 in t mutant mice, as defined by restriction fragment length polymorphism (RFLP) patterns, suggests that Tpx-1 is probably located outside the distal inversion of the t complex. To map Tpx-1 to a specific region of mouse chromosome 17, DNA was isolated from a panel of mice listed in Table 1 as described by Blin and Stafford (1976). The DNA was then digested with Pst I and transferred to nitrocellulose according to the method of Southern (1975). A 0.95 kb Hind III fragment (1-1-1H4) isolated from the cosmid clone containing Tpx-1 (Kasahara et al. 1987) was radiolabeted by the hexamer priming method (Feinberg and Vogelstein 1984) and used as a hybridization probe (Fig. 1). The size of hybridizing bands obtained is summarized in Table 1. The fact that C57BL/6 and B6.K1 (or B6.K2) share regions proximal to and including H-2D, but differ in Pst I fragments detected with 1-1-1H4, places Tpx-1 distal to H-2D. Similarly, the comparison of B6.K2 with B6-T/a a places Tpx-1 distal to Qa-2. Further mapping of Tpx-1 was achieved by four recombinant mice with crossing-over points between T/a and Mep-1; MA.R1 and B10.MR2 place Tpx-1 distal to Tla, while MA.R5 and B10.DR1 localize Tpx-1 distal to Pgk-2. The information on the telomeric border concerning the location of Tpx-1 was provided by two strains of mice, B10.TFR1 and MA.R2. The observation that C57BL/10 and B10.TFR1, which share regions distal to and including Upg-1 (a locus encoding urinary pepsinogen-1), differ in 1-1-1H4 pst I fragments, localizes Tpx-1 proximal to Upg-1. On the other hand, comparison of MA.R2 with B10.D2 and MA/MyJ localizes Tpx-1 proximal to Mep-1. Taken together, these results demonstrate that Tpx-1 is located between Pgk-2 and Mep-1. Mutant t mice carry at least two nonoverlapping inversions in the proximal third of mouse chromosome 17 (Artzt et al. 1982, Pla and Condamine 1984, Herrmann et al. 1986). Since the exact extent of the distal inversion, which involves tf (tufted locus) and H-2, remains unknown, Tpx-1 may be located within it. To test this possibility, we examined RFLP of Tpx-1 in t mutant mice. The results obtained are summarized in Table 2. Two observations suggest that Tpx-1 is probably located outside the distal inversion. First, some t mice with the same H-2 haplotypes have distinct Tpx-1 RFLP patterns (for example, t o vs t~; t Tuw24 VS t Tuw27 and tTuw2), indicating that there is no suppression of crossing-over between H-2 and Tpx-1. Second, comparison of Tables 1 and 2 shows that Tpx-1 is essentially equally polymorphic between t and non-t mice (we assume that the -16and > 30 kb Pst ! fragments found only in t mice probably exist in non-t wild mice populations not included in this study). This finding is inconsistent with the location of Tpx-1 within the inversion because t-complex genes show low polymorphism in t mice, reflecting their origin from either one or very few ancestors (Klein et al. 1986). Since the distal inversion is believed to mark the distal border of the t complex, we assume that Tpx-1 is located outside the complex. This view is consistent with the results of Nadeau (1983), who found no suppression of crossing-over between H-2 and Pgk-2 and therefore suggested that Pgk-2 lies outside the t complex. The intrachromosomal location of Tpx-1 and the fact that major alleles of Tpx-1, as defined by RFLP

Multiple sites of crossing over within the Eb recombinational hotspot in the mouse

The Eb gene of the mouse major histocompatibility complex (MHC) contains a well-documented hotspot of recombination. Twelve cases of intra-Eb recombination derived from the b, d, k and s alleles of the Eb gene were sequenced to more precisely position the sites of meiotic recombination. This analysis was based on positioning recombination breakpoints between nucleotide polymorphisms found in the sequences of parental haplotypes. All twelve cases of recombination mapped within the second intron of the Eb gene. Six of these recombinants, involving the k and s haplotypes, mapped to two adjoining DNA segments of 394 and 955 base pairs (bp) in the 3′ half of the intron. In an additional two cases derived by crossing over between the d and s alleles, breakpoints were positioned to adjoining segments of 28 and 433 bp, also in the 3′ half of the intron. Finally, four b versus k recombinants were mapped to non-contiguous segments of DNA covering 2.9 kb and 1005 bp of the intron. An analysis of the map positions of crossover breakpoints defined in this study suggests that the second intron of the Eb gene contains a recombinational hotspot of approximately 800–1000 bp which contains at least two closely linked recombinationally active sites or segments. Further examination of the sequence data also suggests that the postulated location for the recombinational hotspot corresponds almost precisely to an 812 bp sequence that shows nucleotide sequence similarity to the MT family of middle repetitive DNA.

Evidence that nucleotide sequence identity is a requirement for meiotic crossing over within the mouse Eb recombinational hot spot

Meiotic recombination in the mouse is sometimes restricted to specific chromosomal sites. For example, when recombinants within the I region of the mouse major histocompatibility complex (MHC) are examined, the breakpoints between standard alleles can usually be mapped to the Eb gene. DNA sequence analysis of five cases of meiotic crossing over associated with this gene suggests that the recombinational hot spot may be confined to large regions of nucleotide identity located within the second intron of the Eb gene.

Mep-1, the gene regulating meprin activity, maps between Pgk-2 and Ce-2 on mouse chromosome 17

Meprin is a metallo-endopeptidase expressed in the kidney brush border, a specialized plasma membrane of the proximal tubule of the kidney (Beynon et al. 1981, Bond and Beynon 1986, Craig et al. 1987). Meprin proteolytic activity is deficient in certain mouse strains (Beynon and Bond 1983). This deficiency is inherited as an autosomal recessive trait and controlled by the Mep-1 gene (Bond et al. 1984). Inbred mice that have high levels of meprin activity are of the Mep-l~/Mep-1 a genotype; mice that have low meprin activity (i. e., manifest < 10% of normal specific activity) have the Mep-lb/Mep-1 b genotype; heterozygous animals of the Mep-U/Mep-1 b genotype have the high meprin activity phenotype. Recently, we reported the results of a backcross study in which Mep-1 was estimated to be 2.1 crossover units telomeric to H-2D on Chromosome 17 (Reckelhoff et al. 1985). The Mep-I gene was linked most closely to Pgk-2, the gene controlling electrophoretically separable isozymes of testicular phosphoglycerate kinase; i .e. , mice having Mep-la/Mep-1 ~ genotype were usually Pgk-2 a or Pgk-2 C, and mice having Mep-lb/Mep-1 b genotype were Pgk-2 b (Bond et al. 1984). Only a few exceptions to this generalization were found (Reckelhoff et al. 1985). The results of the backcross experiment were inconclusive, however, as to the location of Mep-1 with respect to Pgk-2. In the present work, we address the question of the relative order of Mep-1, Pgk-2, and Ce-2, a gene controlling kidney isozyme patterns of catalase, by examining newly developed congenic strains which carry crossovers in the area of the Pgk-2 and Ce-2 loci. The nine recombinant strains used in this study were originally produced to examine the genetic organization

Preparation of antisera for the detection of the Ss protein and the Slp alloantigen

The Ss and Slp traits are controlled by a gene or genes located within the H-2 complex o f the mouse. The Ss trait is expressed as inherited variation in the quantitative level of an immunologically detected serum protein (Shreffler and Owen 1963), while the Slp trait is expressed as the presence or absence of an alloantigenic determinant on a subpopulation of Ss molecules (Passmore and Shreffler 1970). Over the past ten years these genetic markers, which define the S region of the H-2 complex, have served as invaluable landmarks for the genetic fine structure analysis of the major histocompatibility complex of the mouse (Shreffler 1970, Shreffler and Passmore 1971, Shreffler and David 1975). Additional interest in these serum traits has been stimulated by the demonstration that the Ss serum protein is associated with serum complement levels (D6mant et al. 1973, Goldman and Goldman 1975, Hansen et al. 1975) and is immunologically and biochemically homologous to the fourth component of human complement (Meo et al. 1975, Lachmann et al. 1975, Curman et al. 1975). Since the functional and genetic importance of the Ss and Slp traits has increased the demand for antisera used in their detection, we have described in this communication detailed protocols for the production of anti-Ss and anti-Sip.

Genetic Organization of the Qa and Tla Regions: Gene Mapping Based on the Analysis of Recombinant Strains

Studies on the genetic organization of the mouse major histocompatibility complex (MHC) have been highly dependent on the development of intra-MHC recombinant inbred strains of mice (Klein 1979). For example, well over 200 recombinant strains have been developed for mapping the loci of the classically defined H—2 complex (summarized in Klein et al., 1983, and in Mouse Newsletter volumes 68, 70 and 74). Unfortunately however, only a handful of recombinant strains exist which carry crossover points associated with the Qa or Tla regions (Flaherty, 1981; O’Neill, 1986). In the past five years, the genetic analysis of the mouse MHC, has been highlighted by substantial advances in the molecular characterization and cloning of MHC genes including those of the Qa and Tla regions (Steinmetz et al., 1982; Weiss et al., 1985; Fisher et al., 1985). This development, rather than detracting from the value of traditional analysis with intra-MHC recombinant strains, provides an opportunity to combine new and traditional approaches into a more powerful battery of tools for the examination of the organization, function and evolution of the class I genes of the Qa and Tla regions. Moreover, the advent of molecular genetics has even created new uses for intra-MHC recombinant strains including the study of the molecular character and distribution of meiotic recombination sites (Steinmetz et al., 1986; Kobori et al., 1986).

SPECTRAL MODEL OF LEISHMANIASIS IN CONGENIC STRAINS OF MICE

Nineteen congenic, resistant strains of mice on C57BL/10ScSn genetic background were infected with Leishmania donovani and the course of infection quantitated. Early in the infection, parasite burdens in the liver were similar for all strains, indicating that the parasite was able to establish, grow, and reproduce in the liver macrophages of each strain with equal facility. Differences in acquired resistance, indicated by decreases in parasite burden, among the strains were first noted at day 21 and became distinct by day 35 postinfection. The extremes were represented by B10.129(10M) mice in which the parasite burden continued to increase at day 35, and B10.LP-H-3b in which only 10% of the peak parasite population remained at this time. The other strains formed a complete continuum between the two extremes. Differences in hepatic pathology were noted among strains, but the severity was not related directly to the strength of the immune response as indicated by reduction in parasite burden; instead, it was more correlated with spleen-to-body weight ratios. Because of the range of responses observed, congenic strains of mice may be of use not only for immunization and chemotherapy studies of leishmaniasis, but also may yield fundamental information on spectral diseases in general.

Cardiac allografts in mice congenic at non-H-2 histocompatibility loci

Neonatal mouse heart fragments were grafted under the ear skin of adult recipients. Cardiac allograft survival was evaluated by visual observation of pulsation, electrocardiography, and histology. Employing a series of congenic resistant strains differing from C57BL/10Sn at theH-1, H-3,H-4, H-7, H-8, H-9, H-10, H-11, andH-12 loci, the median survival times of the heart grafts to and from C57BL/10Sn were obtained. The various interallelic combinations resulted in a wide variation of graft survival. Reciprocal transplants frequently showed different survival times.H-1c grafts were rejected by B10.129(5M)/nSn female mice with a median survival time of 90 days.H-1b grafts were not rejected by C57BL/10Sn mice for the experiments duration of 200 days. The weaker the histocompatibility barrier, the more variable the survival times and the smaller the ratio of rejected to total grafted heart fragments. Female recipients were observed to reject their grafts more rapidly and to reject a higher proportion than males of the same strain. Although the strength of the different non-H-2 barriers generally paralleled that determined by skin transplants, the rankings of the strongest minor barriers were not the same for both tissues.

Immune response to the slp alloantigen in the mouse. H-2-linked qualitative and quantitative control.

Immunization of inbred mouse strains lacking the Slp allotype results in the production of Slp antibodies in some strains but elicits no detectable response in other strains. Analysis of standard inbred and congenic resistant strains reveals that both the qualitative and quantitative ability to respond to the Slp allotype is associated with theH-2 haplotype of the recipient. Three different response phenotypes can be identified utilizing complement fixation and quantitative immunodiffusion tests. Strains which carry theH-2q haplotype are high responders,H-2k strains are intermediate in response, whileH-2b andH-2v strains produce no detectable antibody. The characteristic response patterns of high and intermediate responders were manifest by day 35 of immunization and continued as discrete response types after a second booster. Quantitative data in the immune response of the intra-H-2 recombinant B10.A(4R) suggest that the recombination event which established theH-2h4 chromosome disturbs the proper function of the genetic determinant controlling response to Slp.