Molecular characterization of four intra-t mouse recombinants

Recombination in the proximal region of mouse chromosome 17 is greatly reduced in heterozygotes carrying the wild-type and thet complex-type chromosomes. The reason for this is the presence of two non-overlapping inversions in thet complex. Rare crossing-over does, however, occur within thet complex of thet/+ heterozygotes. Here we characterize four such exceptional intra-t recombinants,t ^Tu1 throught ^Tu4 . To map the positions of the genetic exchange in these four recombinants, we analyzed them with DNA probes specific for 16 loci distributed over thet complex. The analysis revealed that in three of the four recombinants, an equal crossing-over occurred in the short region between the two inversions, producing chromosomes carrying either the proximal inversion only (t ^Tu1 andt ^Tu4 ) or the distal inversion only (t ^Tu2 ). In the fourth recombinant (t ^Tu3 ), unequal crossing-over occurred within the proximal inversion between lociD17Leh119 andD17Leh66, producing a chromosome in which the region containing lociTcp-1, T, andD17Tu5 has been duplicated. The duplication of theBrachyury locus leads to the suppression of the tail-shortening effect normally produced by the interaction of the dominant (T) and recessive (tct) alleles at this locus so that theT/t ^Tu3 mice have normal tails.     

H-2 typing of mutants of thet 6 haplotype in the mouse

Mutantt haplotypes derived from thet ⁶ haplotype were typed forH-2. The mutantt ^h2 that arose fromt ⁶ due to crossing over in the region betweenT andtf had, as expected, lost theH-2 haplotype characteristic oft ⁶. The haplotypest ^h17,t ^h18, andt ^p1, which also arose by recombination, but which represent the complementary crossover products, including the distal part of thet ⁶ haplotype, carried the sameH-2 type ast ⁶. This suggests that crossing over betweentf andH-2 is suppressed int ^h17 andt ¹⁸. This in turn suggests that mutantt haplotypes suppress crossing over for that part of thet chromatin that they still retain.The origin oft ^h7, which apparently did not include any crossover distal toT, and which retains the crossover-suppressing property oft ⁶, retains thet ⁶ H-2 type. Unexpectedly, J ^h20 , which expressestf and was at first thought to have arisen due to crossing over, also retains theH-2 type oft ⁶. This provides part of the evidence thatt ^h20 arises fromt ⁶ not by crossing over, but by a small deletion, and hence that duplication and deletion are possible modes of origin of mutantt haplotypes.Abbreviations used in this paper are t haplotype mutant haplotype of the chromosome 17, often designated J allele - T Brachyury mutant - T/+ short-tailed mouse - T/T lethal during embryogenesis - T-int T interaction (characteristic oft haplotypes that interact in heterozygotes withT to produce a tailless mouse) - tf locus homozygotes showing waves of hair loss - Kb knobby, which produces a knobbly tailed heterozygote, homozygous lethal - titer reciprocal of serum dilution giving 50% kill     

Intra-H-2 recombination in t haplotypes shows a hot spot and close linkage of l tw5 to H-2K

The embryonic lethal mutation in the t ^w5 haplotype is known to map near the H-2K region of the mouse major histocompatibility complex. Additional data obtained by classical genetic methods demonstrate that the t ^w5 lethal gene is effectively inseparable from H-2K. No recombinants were found between H-2K and t ^w5 in a sample representing over 1200 mice. On a statistical basis t ^w5 must be less than 250 kb from the H-2K gene. In the course of these mapping studies we obtained a set of 11 intra-H-2 recombinants. We have analyzed these and three others derived from another experiment to define their breakpoints as precisely as possible. Southern blot analysis with molecular probes to the D, S, I, and K regions of the H-2 complex defines seven recombinations between the D and S regions, two between S and I, none within the I region, and five events between I and K. The last category was studied in finer detail by developing unique copy probes to the I-K boundary region. Two of the five events occurred within probably less than 6 kb of each other: these two recombinants define the centromeric limit of the location of the t ^w5 gene within the H-2K region. The other three I-K recombinants occurred in at least two other nearby locations. Altogether at least three, and probably all five I-K recombinants fall within a 45 kb recombinational hot spot recently identified in Mus musculus castaneus.     

Isolation of virus-like (VL30) elements from theQ10 andD regions of the major histocompatibility complex

Previous studies from our laboratory have described two endogenous provirus-like sequences in a series of cosmids spanning theTL region of the major histocompatibility complex (MHC) of normal C57BL/10 mice. At least one of these viruses shares similarities withVL30 elements. To determine if additionalVL30-like retroviral elements are integrated in the MHC, we constructed a cosmid library using DNA from a radiation leukemia virus (RadLV)-transformed cell line derived from C57BL/6 mice. The library was first screened using theH-2III (5) probe, which detects Class I genes of theH-2 complex. In the primary screening 163H-2III positives were isolated. TheH-2III-positive isolates were then hybridized with an AKR-derived virus probe,EcoB/S, which contains sequences from both thepol and theenv genes of the virus. Nine virus-positive isolates were detected. Localization of these cosmid isolates containing viral sequences within theH-2 complex was done utilizing low-copy probes and confirmed using previously mapped cosmid isolates from other laboratories. We report here the isolation and characterization ofVL30-like elements from theQa andD regions of theMHC of several inbred mouse strains.     

Use ofH-2 mutations to quantitate alloreactivity: Alloreactivity is strongest against H-2 antigens which are closest to self

Lymph-node cells fromH-2 allogeneic, intra-H-2 recombinant andH-2 mutant congenic strains were sensitized in limiting dilution cultures to quantitate the cytotoxic T-lymphocyte precursor frequencies (CTL.Pf) against antigens encoded by different regions of theH-2 complex. When fourH-2K ^b mutants of C57BL/6 (B6) were tested, we observed anti-B6 CTL.Pf that were as high or higher than those of recombinant strains which differ from B6 at theK end of theH-2 complex. Relative to strains completelyH–2 allogeneic to B6, the CTL.Pf inH-2 ^bm1,H-2 ^bm3 andH-2 ^bm5 averaged 40–50 percent, andH-2 ^bm8 averaged 140 percent. Recombinant strains B10.A (4R) and B10.D2 (R103), which differ from B6 at theK end of theH-2 complex, averaged 60 percent of the completelyH-2 allogeneic value. Since the mutant and wild-type gene products have no serological and minimal structural differences relative to other alleles atH-2K, these results indicate that the CTL.Pf does not increase with increasing H-2 antigenic disparity between any two strains. Rather, the data suggests that the T-cell receptor repertoire recognizes those H-2 molecules or determinants closest to self.     

Genetic mapping of thet-complex region on mouse chromosome 17 including theHybrid sterility-1 gene

t-haplotypes occupy a region on chromosome (Chr) 17 which slightly overlaps the ends of theT-H-2 interval. The wild-type form of this 14 centi-Morgan (cM) region was mapped in a multilocus backcross (C57BL/10-T×C3H)F₁×C57BL/10 using 15 DNA probes on Southern blots of the DNA extracted from 53 animals which were recombinants in theT-H-2 interval. Each recombinant was also progenytested to ascertain itsHybrid sterility-1 (Hst-1) genotype by crossing to PWB/Ph, aMus musculus-derived inbred strain. The limit of resolution of the cross was 0.27 cM. The map distances have been determined for the DNA loci in theT-H-2 interval and theHst-1 gene was mapped in close vicinity to theD17Rp17 locus.     

Intra-H-2 recombination in the mouse

Serological characterization of threeK-S interval recombinant strains, TBR2 (H-2 ^at2 ), TBR3 (H-2 ^at3 ) and AIR1 (H-2 ^a2 ) was performed using anti-H-2, Ia, Ss and Slp antisera. The data presented here reveal that the crossover events in both TBR2 and TBR3 occurred between theI-A andI-E subregions. In both cases, theH-2K andI-A subregions were derived from theH-2 ^t1 chromosome, while theI-E, S andH-2D regions were derived from theH-2 ^b chromosome (K ^s A ^k E ^b S ^b D ^b ). TheH-2 ^a2 chromosome resulted from a crossover event between theH-2 ^a1 andH-2 ⁱ⁹ chromosomes. Ia and Ss typing of AIR1 suggested that theK toI-E regions originated fromH-2 ^a1 and theS andD regions originated fromH-2 ⁱ⁹ (K ^k A ^k E ^k S ^b D ^d ).     

Topographical relationships among H-2 specificities controlled by theD region

In the present work, we used the differential redistribution method to study the molecular expression of several H-2 specificities controlled by theD region of theH-2 ^a haplotype. We observed that: capping of the private specificity H-2.4 induced capping of the public specificities H-2.3, H-2.35, and H-2.36, and vice versa; capping of any one of these specificities did not induce capping of the public specificity H-2.28, controlled by the same region. By contrast, capping of the H-2.28 specificity induced capping of these specificities; redistribution of H-2K and H-2D private specificities or redistribution of H-2D private specificity and Ia specificities did not induce capping of the H-2.28 specificity. These data indicate that a part of a molecule carrying the H-2.28 specificity is linked to a molecule carrying H-2.4, H-2.3, H-2.35, and H-2.36 specificities and that a part of a polypeptide chain bearing the H-2.28 specificity is independent from that bearing other specificities controlled either by theD region (i.e., H-2.4, H-2.3, H-2.35, and H-2.36) or by theK andI regions. These results further strengthened the hypothesis of the existence of at least two genes controlling theD-region H-2 antigenic specificities.     

Genetic control of contact sensitivity in mice: Effect ofH-2 and nonH-2 loci

The genetic control of delayed-type hypersensitivity in mice was investigated by contact sensitization with picryl chloride. Distribution patterns of contact sensitivity in 11 inbred strains of mice showed significant differences among strains. Comparison of levels of response between congenic-resistant lines and their inbred partners, at 9 to 11 weeks of age, revealed a clear association betweenH-2 haplotype and the magnitude of response. Testing ofH-2 recombinants further suggested the influence of two genes mapping at either end of theH-2 complex. While theH-2K ^d andH-2D ^k alleles were associated with a high response, theH-2K ^k ,H-2K ^b ,H-2D ^d , andH-2D ^b alleles were associated with a low response. Analysis of the ontogeny of response suggested that theH-2 haplotype manifests its effect through the maturation of contact sensitivity. On both the C57BL/6By and C57BL/10Sn backgrounds, theH-2 ^d haplotype was associated with early maturation of response, while theH-2 ^b haplotype was associated with late maturation. Analysis of the response of congenic lines with different genetic backgrounds and of CXB recombinant-inbred lines further revealed the marked effects of yet other genes on this trait.     

A new locus (H-2T) at theD end of theH-2 complex

A.TH (H-2 ^t2) anti-A.TL (H-2 ^t1) effectors, obtained after in vitro restimulation of in vivo sensitized cells, react in the CML assay not only withH-2 ^t1, but also with a number of other targets carrying unrelatedH-2 haplotypes. The broad cross-reactivity can be explained by postulating the presence among the effectors of at least two populations of cells, one reacting with antigens controlled by theI region, and the other directed against antigens controlled by a locus at theD end, outside theH-2 complex. The existence of the two cell populations is also supported by cold-target inhibition data. The locus coding for the D-end CML antigens maps betweenQa-2 andTla. The locus is assigned the symbolH-2T. TheH-2T-locus CML is seen only after in vivo presensitization, but the killing is not K/D-restricted.     

TheD17Tu5 locus in thet complex: implications for the origin oft haplotypes and inbred strains

The mouse × Chinese hamster cell line R4 4-1 contains only one mouse chromosome, the bulk of which corresponds toMus musculus chromosomes 17 and 18 (MMU17 and MMU18, respectively). A genomic library was prepared from the R4 4-1 DNA, and a mouse clone was isolated from the library, which—with the help of somatic cell hybrids-could be mapped to the MMU17. A locus defined by a 2.7-kb longBam HI probe from this clone was designatedD17Tu5 (Tu for Tübingen). The locus proved to be polymorphic among inbred strains and wild mice. By testing of recombinant inbred strains and partialt haplotypes, theD17Tu5 locus could be mapped to a position between theD17Leh66E andD17Rp17 loci within thet complex. Two alleles were found at this locus,D17Tu5 ^a andD17Tu5 ^b , defined byTaq I restriction fragment length polymorphism. Both alleles are present among inbred strains and wild mice of the speciesM. domesticus. All completet haplotypes tested carry theD17Tu5 ^a allele and all tested wild mice of the speciesM. musculus, with the exception of those bearingt haplotypes, carry theD17Tu5 ^b allele. Additional alleles are found in some populations of wild mice and in other species of the genusMus. The distribution of the two alleles among the inbred strains correlates well with their known or postulated genealogy. Their distribution between the two species ofMus and among the mice withT haplotypes suggests a relatively recent origin of thet haplotypes.     

Serological identification of sperm antigens specified by lethalt-alleles in the mouse

Cytotoxic antisera were prepared by immunizing wild-type recipient mice with sperm from donors carrying different recessive lethal alleles at theT locus (T/t ⁰,T/t ^{w 1} ,T/t ^{w 5} , andT/t ^{w 32} ). After removal of sperm autoantibody by absorption with sperm of recipient type, each antiserum reacted only with sperm from males whose genotype contained at least one of the immunizing alleles. Cytotoxicity was high against sperm populations in which both immunizing alleles were represented and was lower when only one was present. Thus, each allele at theT locus which has so far been tested serologically is recognizable as a discrete antigen on the surface of sperm.This paper is dedicated to Professor Hans Grüneberg, F.R.S. on the occasion of his retirement from the chair of Animal Genetics at University College London, in appreciation of the fact that it was he who first suggested that surface properties played an important role in the origin of abnormalities associated with mutants at theT-locus.     

Genetic Characterization of the Chromosomal Rearrangements That Accompany the Transgene Insertion in theChakragatiMouse Mutant

We have previously reported that the circling phenotype of thechakragatimouse segregates with the transgene integration event as an autosomal recessive trait. It was unclear, however, whether the phenotype was linked to the transgene integration point nearD16Ros1or to a potential disruption atD16Ros2,10 cM away. We report here that animals recombinant betweenD16Ros1andD16Ros2,homozygous for the transgene insertion atD16Ros1,but wildtype forD16Ros2,do indeed show the phenotype. We conclude that any potential disruption at theD16Ros2locus is not responsible for the circling phenotype. We further show that recombination betweenD16Ros1andD16Ros2occurs at a greatly reduced level in thechakragatimouse compared to wildtype strains. Detailed genetic analysis of recombinants indicates that the proximal-most 4.5 cM shows no recombination in over 1400 meioses. We propose that this is due to an inversion in this region, and we genetically define the proposed distal inversion break point to a 1.3-cM region betweenD16Mit63andD16Mit169.     

H-2 effects on cell-cell interactions in the response to single non-H-2 alloantigens

Immune response (Ir) genes mapping in theI region of the mouseH-2 complex appear to regulate specifically the presentation of a number of antigens by macrophages to proliferating T cells. We have investigated the possibility that similarIr genes mapping in theH-2K andH-2D regions specifically regulate the presentation of target antigens to cytotoxic effector T cells. We report that the susceptibility of targets expressing specific non-H-2 H alloantigens to lysis by H-2-compatible, H-antigen-specific cytotoxic effector T cells is controlled by polymorphicH-2K/D genes. This control of susceptibility to lysis is accomplished through what we have defined operationally as antigen-specific regulation of non-H-2 H antigen immunogenicity. High immunogenicity of the H-4.2 alloantigen is determined by a gene mapping in theH-2K region ofH-2 ^b . However, high immunogenicity of H-7.1 is determined by a gene mapping in theH-2D region ofH-2 ^b . High immunogenicity of the H-3.1 alloantigen is determined by genes mapping in both theH-2K andH-2D regions ofH-2 ^b . Therefore, genes mapping in theH-2K andH-2D regions serve a function in presenting antigen to cytotoxic effector T cells. This function is analogous to that played byI-regionIr genes expressed in macrophages which present antigen to proliferating T cells. We present arguments for classification of theseH-2K/D genes as a second system ofIr genes and discuss the implications of twoH-2-linkedIr-gene systems, their possible functions, and their evolution.     

Serological characterization of previously unknown H-2 molecules identified in the products of theK d andD k region

The molecular relationship between H-2 private and some public specificities was studied in C3H.OH (H-2 ⁰² ) mice using surface-antigen re-distribution methods. Besides the K^d- and D^k-region antigens, which can be capped by antisera against the private and public specificities characteristic for a given allele, a previously unknown type of molecule was found in the products of both theK ^d andD ^k regions. These can be capped by the respective anti-private serum but not by antisera against some public specificities. The two K^d-region molecules are provisionally named H-2K1^d and H-2K2^d. We detected them onH-2 ⁰² (K ^d ,I ^d ,S ^d ,D ^k ) and also onH-2 ^dx (K ^d ,I ^f ,S ^f ,D ^dx ) T lymphocytes. Similarly, the two types of molecules detected on the products of theD ^k region are provisionally named H-2D1^k and H-2D2^k. The serological characteristics of these molecules are described. When compared with the products of theD ^d region, in which we previously described three different molecules (H-2D^d, H-2M^d, and H-2L^d), the mutual relationship between H-2K1^d and H-2K2^d as well as between H-2D1^k and H-2D2^k appears to be similar to that between H-2D^d and H-2M^d. In the absence of relevant recombinants or informative biochemical data, it is, however, difficult to establish homology between molecules produced by differentK- andD-region alleles.     

Mapping of theHox-3.1 andMyc-1.2 genes on chromosome 15 of the mouse by restriction fragment length variations

Restriction endonuclease fragment length variations (RFLV) were detected by use of the cDNA probeHox-3.1 for the homeo box-3.1 gene and also thec-myc oncogene probe for exon 2. RFLV ofHox-3.1 were found inHindIII restriction patterns, and RFLV of theMyc-1.2 gene inEcoRV patterns. From the RFLV, theHox-3.1 andMyc-1.2 genes were mapped on chromosome 15. Three-point cross test data showed that the frequency of recombination is 26.4% betweenMyc-1.2 andGpt-1, 30.2% betweenGpt-1 andGdc-1, and 9.4% betweenGdc-1 andHox-3.1. The following order of these genes is proposed,Myc-1.2—Gpt-1—Gdc-1—Hox-3.1. All laboratory strains carry theHox-3.1 ^a andMyc-1.2 ^a alleles. Among strains of wild origin,domesticus strains carry only theHox-3.1 ^a andMyc-1.2 ^a alleles, as do the laboratory strains. One strain ofbrevirostris carries theHox-3.1 ^a andMyc-1.2 ^b alleles. Other wild subspecies from Europe and Asia,M. m. musculus, M. m. castaneus, M. m. molossinus, Chinese mice of wild origin, andM. m. yamashinai carry theHox-3.1 ^b andMyc-1.2 ^b alleles.     

The use of the S haplotype-specific F-box protein gene,SFB, as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot (Prunus mume)

Japanese apricot (Prunus mume) exhibits the S-RNase-based gametophytic self-incompatibility system as do other self-incompatible Prunus species. This report identifies the S haplotype-specific F-box protein gene (SFB), a candidate gene for pollen-S, of Japanese apricot, which leads to the development of a molecular typing system for S-haplotype in this fruit species. Both 5- and 3-RACE (rapid amplification of cDNA ends) were performed with SFB gene-specific oligonucleotide primers to clone Pm-SFB¹ and Pm-SFB⁷ of 'Nanko (S¹S⁷)'. As in the case of SFB of other Prunus species, Pm-SFB¹ and Pm-SFB⁷ showed a high level of S-haplotype-specific sequence polymorphism and their expression was specific to pollen. Genomic DNA-blot analyses of 11 Japanese apricot cultivars with the Pm-SFB probes under low stringency conditions yielded RFLP bands specific to the S¹- to S⁸-haplotypes as well as a self-compatible S^f-haplotype. A practical usage of SFB as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot is discussed.Communicated by H.F. LinskensThe nucleotide sequences reported in this paper have been submitted to the EMBL/GenBank/DDBJ database under accession numbers, AB101440 and AB101441, for SFB¹ and SFB⁷, respectively     

Characterization of wheat/Aegilops ventricosa introgression and addition lines with respect to the Mv genome

Summary Stable wheat-Aegilops introgression lines with 42 chromosomes (H-93), derived by repeated selfing from a cross (Triticum turgidum x Aegilops ventricosa) x T. aestivum, have been characterized using the following DNA probes and isozyme markers: (1) single or low-copy DNA fragments from Ae. ventricosa; (2) known cDNA probes corresponding to 1-thionin, monomeric -amylase inhibitor, the CM3 subunit of tetrameric -amylase inhibitor, and sucrose synthase from wheat; (3) anonymous cDNA probes from wheat that have been mapped by Sharp et al. (1989); (4) isozyme markers corresponding to aconitase, shikimate dehydrogenase, adenylate kinase, and endopeptidase. Meiotic metaphases of appropriate hybrids involving selected H-93 lines have been investigated by the Giemsa C-banding technique. The substitution of whole chromosomes [(5A) 5M^v; (4D) 4M^v; (5D) 5M^v; (7D) 7M^v] and chromosomal segments (1M^v; 3M^v; 5M^v; 7M^v) from the M^v genome of Aegilops ventricosa has been demonstrated. The distribution of selected markers among putative wheat-Ae. ventricosa addition lines has also been investigated. The 7M^v addition has been characterized for the first time, while the identity of the previously reported 5M^v and 6M^v additions has been confirmed.     

The genetic control of the immune response to murine histocompatibility antigens

The genetic control of the immune response to H-4 histocompatibility alloantigens is described. The rejection of H-4.2-incompatible skin grafts is regulated by anH-2-linkedIr gene. Fast responsiveness is determined by a dominant allele at theIrH-4.2 locus. TheH-2 ^b ,H-2 ^d , andH-2 ^s haplotypes share the fast response allele;H-2 ^a has the slow response allele. Through the use of intra-H-2 recombinants, we have mapped theIrH-4.2 locus to theI-B subregion of theH-2 complex; theH-2 ^h4 ,H-2 ¹⁵, andH-2 ^t4 haplotypes are fast responder haplotypes. These observations suggest that the strength of non-H-2 histocompatibility antigens is ultimately determined by the antigen-specific recipient responsiveness.     

Genetic and physical characterization of theLR1 leaf rust resistance locus in wheat (Triticum aestivum L.)

The objective of this study was to characterize the leaf rust resistance locusLr1 in wheat. Restriction fragment length polymorphism (RELP) analysis was performed on the resistant lineLr1/6^*Thatcher and the susceptible varieties Thatcher and Frisal, as well as on the segregating F₂ populations. Seventeen out of 37 RFLP probes mapping to group 5 chromosomes showed polymorphism betweenLr1/6^*Thatcher and Frisal, whereas 11 probes were polymorphic between the near-isogenic lines (NILs)Lr1/6^*Thatcher and Thatcher. Three of these probes were linked to the resistance gene in the segregating F₂ populations. One probe (pTAG621) showed very tight linkage toLr1 and mapped to a single-copy region on chromosome 5D. The map location of pTAG621 at the end of the long arm of chromosome 5D was confirmed by the absence of the band in the nulli-tetrasomic line N5DT5B of Chinese Spring and a set of deletion lines of Chinese Spring lacking the distal part of 5DL. Twenty-seven breeding lines containing theLr1 resistance gene in different genetic backgrounds showed the same band asLr1/6^*Thatcher when hybridized with pTAG621. The RFLP marker was converted to a sequence-tagged-site marker using polymerase chain reaction (PCR) amplification. Sequencing of the specific fragment amplified from both NILs revealed point mutations as well as small insertion/deletion events. These were used to design primers that allowed amplification of a specific product only from the resistant lineLr1/6^*Thatcher. This STS, specific for theLr1 resistance gene, will allow efficient selection for the disease resistance gene in wheat breeding programmes. In addition, the identification of a D-genome-specific probe tightly linked toLr1 should ultimately provide the basis for positional cloning of the gene.