Gene content of the 750-kb critical region for mouse embryonic ectoderm lethal <Emphasis Type="Italic">tcl-w5</Emphasis>

Mice homozygous for the t^w5 allele arrest at gastrulation from defects associated with embryonic ectoderm development. The mutated gene has been genetically closely linked to the H-2K locus in the mouse MHC region, flanked by markers H-2Pb and D17Mit147. Aiming at the positional cloning of the mutated gene, we constructed a BAC contig spanning about 1 Mb of the genomic region. On the basis of our mapping and sequencing analysis of the BACs combined with public genome data, EST database searches, and gene prediction programs, we delimit the 1.06 cM of the t^w5 critical region to 750 kb, and infer 36 genes (1/20 kb) encoded in the interval. All of the 33 genes tested were confirmed as expressed in embryonic tissues by RT-PCR analyses, and in many cases by EST expression profiles as well. Thus, this highly gene-rich region is essentially totally transcribed during early development and provides priority candidates to be screened for the t^w5 embryonic lesion.     

Structural and genetic properties of the Eb recombinational hotspot in the mouse

The Eb gene of the mouse contains a recombinational hotspot which plays a predominant role in meiotic crossing-over within the I region of the mouse major histocompatibility complex (MHC). The nucleotide sequences of five recombinants derived from H-2 ^k /H-2 ^b heterozygotes at the Eb locus placed the sites of recombination in each recombinant haplotype within a 2.9 kilobase (kb) segment located fully within the second intron of the Eb gene. Further resolution of the crossover sites was not possible since the nucleotide sequences of the parental and recombinant haplotypes are identical within this segment. The molecular characterization of these five recombinants considered in conjunction with three previously reported intra-Eb recombinants indicates that there are at least two distinct sites of recombination within the Eb recombinational hotspot. In a related study, an examination of the nucleotide sequence of the H-2 ^p allele of the Eb gene revealed a major genetic rearrangement in the 5' half of the intron in this haplotype. A 597 base pair (bp) nucleotide sequence found in the H-2 ^p haplotype is replaced by a 1634 bp segment found in the H-2 ^b and H-2 ^k haplotypes. Sequence analysis of this 1634 bp segment shows strong nucleotide sequence similarity to retroposon long terminal repeat (LTR), env, and pol genes indicating that this segment of the second intron has evolved through retroposon insertion. The location of these retroposon sequences within the 2.9 kb recombination segment defined by the five H-2 ^k /H-2 ^b recombinant haplotypes suggests a possible relationship between these retroviral elements and site-specific recombination within the second intron of the Eb gene. Offprint requests to: H. C. Passmore     

An H-2K gene of the t w32mutant at the T/t complex is a close parent of an H-2K qgene

Two recombinant mice have been recovered from the progeny of Ttf/t ^w32+ animals. They have lost the t^w32 lethality factor(s) and gained tufted, presumably from the T chromosome. Southern blot analysis of class I genes of these two new partial t ^PA027 and t ^PA286 haplotypes indicates that they have retained at least part of the major histocompatibility complex of the t ^w32 chromosome (H-2 haplotype H-2 ^w28). We have prepared a phage library of Eco RI-digested DNA from homozygous t ^PA027 animals. Upon screening the library with a cDNA probe specific for H-2K genes, we isolated a class I gene displaying all of the distinctive features of a genuine H-2K gene, and which could thus be defined as an H-2K ^w28 gene. The H-2K ^w28 gene is 92–95% homologous to H-2K ^band H-2K ^dgenes and differs significantly from the other class I genes sequenced so far. Homology with the H-2K ^bsequence reaches nearly 100% in the 3 part of the H-2K ^w28 gene. Moreover, the homology with an H-2K ^qcDNA sequence reaches 99.8%. Several hypotheses can account for the near identity of H-2K ^b, H-2K ^q,and H-2K ^w28 gene sequences: either recombination between H-2 ^w28 and H-2 ^band H-2 ^qsequences occurred before or at the.time the strain was established, or the class I genes of the t ^w32 chromosome and the H-2 ^band H-2 ^qgenes found in inbred strains of mice have separated from each other rather recently.     

Intra-H-2 recombination inH-2b/H-2t1 heterozygotes in the mouse

Four cases of intra-H-2 recombination were detected during serological screening of 1066 backcross animals produced fromH-2^b/H-2^t1 heterozygous mice. Three of the intra-H-2 recombinants received theK region fromH-2^t1 and theD region from theH-2^b parental chromosome. The remaining recombinant received theK region from theH-2^b parental chromosome and theD region fromH-2^t1. Three of the four recombinants have been developed into inbred lines TBR2, TBR3, and TBR4 and were assigned the haplotype designations at², at³, and at⁴. Ss typing revealed that TBR2 and TBR3 originated fromK- S interval crossover events, while the remaining two recombinants resulted from crossing over in theS- D interval.     

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.     

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.     

Molecular organization of theD-Qa region oft-haplotypes suggests that recombination is an important mechanism for generating genetic diversity of the major histocompatibility complex

We have determined the molecular maps of theH-2D andQa regions of thet-complex haplotypest ^l2 andt ^w5 by chromosomal walking. Analysis with class I probes and other probes unique to theH-2D:Qa subregion indicates that the class I gene organization oft ¹² is:D1-D2-Q1-Q2-Q3-Qx-Q4-Q5-Q10, while that oft ^w5 is:D1-D2-Q1-Q2-Q4-Q5-Q10. Thus, the absence of theQ6-Q9 genes suggested previously int-haplotypes was confirmed. A comparison of the molecular maps of thet ¹² andt ^w5 chromosomes revealed an extremely mosaic pattern of diversity: The regions betweenD1 andD2, and betweenQ4 andQ10, are very similar in both chromosomes. However, theirQ1 toQ3 regions are strikingly different. Further comparisons of wild-type chromosomes and additionalt-haplotypes by molecular mapping and genomic Southern blot hybridization with probes to theQ1-Q3 region showed a high level of polymorphism among both wild-type chromosomes and amongt-haplotypes. The characteristics of the polymorphisms suggest that recombination may play an important role in generating this genetic diversity. Furthermore, recombination between wild-type andt-haplotype chromosomes may be involved.     

The histocompatibility-2 system in wild mice

The I-region gene products of 29 wild-derivedH-2 haplotypes on a B10 background (B10.W congenic lines) were typed with alloantisera which detect 17 inbred I-region antigens. Five new I-region antigens were defined by expanding the inbred line panel ofH-2 haplotypes to includeH-2 ^u , H-2^v, andH-2 ^j . Based on serological analyses of the inbred and B10.W lines, the polymorphism of theIA gene (or genes) is estimated to be at a minimum of 15 alleles and theIE gene (or genes) at a minimum of 4 alleles. These results indicate that theIA subregion is more polymorphic than theIE subregion. By combining the I-region typing data with theH-2K andH-2D region typing data reported previously, a total of 11 new natural recombinants of inbredH-2 alleles were detected among the B10.W lines.     

Molecular characterization of meiotic recombination within the major histocompatibility complex of the mouse: Mapping of crossover sites within theI region

The molecular analysis of crossing-over within the mouse major histocompatibility complex provides a useful approach for the study of the structural characteristics of meiotic recombination. In this study five intra-I-region recombinants, each derived fromI ^k/I ^b heterozygotes, were characterized for restriction-fragment length polymorphisms (RFLPs) characteristic of theI region of the two parental strains. Southern blot analysis of intra-I recombinant strains A.TBR2, A.TBR3, A.TBR5, A.TBR13, and A.TBR17 using sixI-region DNA probes revealed that the point of crossing-over in all five recombinants occurred within a 6.2-kbKpnI-EcoRI segment located within theE gene. The segments of DNA containing the crossover point from each of the recombinant chromosomes were cloned by screening partial genomic libraries constructed in gt7 bacteriophage. Construction of partial restriction maps of the cloned segments from the parental and recombinant chromosomes permitted the boundaries of the area containing the crossover site to be narrowed to a 4.0-kb segment located almost entirely within an intron of theE gene. The recognition that the points of crossing-over in all five recombinants studied are clustered in a relatively small area of theI region provides further evidence for a hot spot of recombination associated with theE _ß gene.This work was supported by Grants AI14424 and AI20317 from the National Institutes of Health. J. Kobori was supported by a postdoctoral fellowship from the Arthritis Foundation. E. Zimmerer was supported by a postdoctoral fellowship from the Charles and Johanna Busch Fund of the Bureau of Biological Research. D. Spinella was supported by a predoctoral fellowship from the Charles and Johanna Busch Fund.     

Search for MHC-associated genes in human: five new genes centromeric to HLA-DP with yet unknown functions

Taking advantage of five mouse genomic or cDNA probes [KE5(probe 14), KE4 (probe11), KE3 (probe7), KE2 (probe5), and SET] mapped on the H-2K region in mouse, we have identified and localized homologues of these five genes in the human major histocompatibility complex region (HKE5, HKE4, HKE3, HKE2, and HSET, respectively). Cosmid cloning and pulsed field gel electrophoresis analyses indicated that a human homologous gene, HKE5, is located 10 kilobases (kb) centromeric of the 2(XI) collagen (COL11A2) gene followed by HKE4. HKE3, closely linked to HKE2, is located 170 kb centromeric of HKE4. Furthermore, HSET is located 50 kb centromeric of HKE2. This gene organization outside the DP subregion is completely identical to that of the mouse H-2K region centromeric of I-Pb 3, a mouse homologue of the DPB gene, except the lack of genes corresponding to the H-2K and -K2 genes in human.     

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.     

Somatic cell variants of H-2k: b: A point mutation in the first extracellular domain results in altered immune recognition

A cell-surface-associated variant H-2K product was expressed by an Abelson virus-induced pre-B-cell line after chemical mutagenesis with ethyl methane sulfonate. The variant cell line (R8.313) was previously demonstrated to have altered allodeterminants in K^b as demonstrated by both K^b-specific monoclonal antibody binding and alloreactive cytotoxic T lymphocyte (CTL) cytolysis. The mutant H-2K ^b gene from R8.313 was cloned and characterized in detail. DNA sequence analysis of the region of the gene corresponding to the three extracellular domains identified a single point mutation resulting in a leucine-to-phenylalanine substitution at amino acid residue 82. The site of mutation within the 1 domain was confirmed by oligonucleotide hybridization analysis. Mouse L-cell fibroblasts transfected with the mutant gene were recognized with the same monoclonal antibody binding and CTL lytic pattern as the R8.313 cell line, confirming that the altered phenotype of the mutant cell line was due to a point mutation in the H-2K ^b gene. These data further extend the hypothesis that the region of amino acid residues 70–90 in the 1 domain is important in the formation of both antibody and CTL-defined recognition structures on major histocompatibility complex class I molecules.     

A new H-2.1-PositiveD region allele,D dx,controlling two molecules,H-2Ddx and H-2Ldx

The inbred strains GRS/A and LIS/A carry the haplotypeH-2 ^dx , which had earlier been shown to have theK ^d ,I ^f ,S ^f , andG ^f alleles and a previously unknownD region allele,D ^dx . We show here that theD ^dx allele determines a new private specificity, H-2.63, is H-2.28 negative, and determines at least one public specificity of the H-2.1 family. It is thus a second example (afterD ^k ) of a H-2.1-positive H-2.28-negativeD region allele. Capping experiments show that the D^dx product comprises two molecules: H-2D^dx bearing the private specificity H-2.63, and H-2L^dx, which is H-2.63-negative but reacts with sera against the H-2.1 family of specificities. SDS gel electrophoresis of detergent-solubilized immunoprecipitated D^dx products shows that the H-2L^dx antigen has a molecular weight of approximately 45,000 daltons and is associated with a smaller polypeptide (mol. wt. 12,000).     

Genetic analysis of the organic cation transporter genes Orct2/Slc22a2 and Orct3/Slc22a3 reduces the critical region for the t haplotype mutant t w73 to 200 kb

Here we report an analysis of two candidate genes for the t ^w73 implantation mutation. The t ^w73 gene maps to a 20-cM region of mouse Chromosome (Chr) 17 known as the t-complex, which exists in a wild-type and t haplotype form in present-day mice. The t haplotype variants contain several mutant alleles affecting male fertility and embryonic viability and offer the opportunity to identify genes critical for these processes. t ^w73 homozygous embryos are defective in trophoblast production and fail to implant adequately, with death occurring at approximately 7.5 days post coitum (pc). Two recently described organic cation transporter genes, Slc22a2 (Orct2) and Slc22a3 (Orct3), fulfill criteria predicted for t ^w73 candidate genes, since both map to the previously defined 500-kb t ^w73 minimal region and both are also expressed in 7.5 days pc post-implantation embryos. The genomic locus of the Orct2 gene appears similar in wild-type and t ^w73 chromosomes. In contrast, the genomic locus of Orct3 is amplified and displays an altered expression profile in all t haplotype variant chromosomes tested. In addition, Orct3 shows a t ^w73 specific polymorphism. To test whether either Orct2 or Orct3 is involved in the t ^w73 phenotype, we have performed a genetic rescue experiment using YAC transgenes overexpressing Orct2, and genetic complementation with an allele in which the Orct3 gene was inactivated by homologous recombination. The results eliminate both Orct2 and Orct3 as candidates and further reduce the critical region containing the t ^w73 mutant from 500 kb to 200 kb. Received: 8 February 2001 / Accepted: 1 May 2001     

Identification of specificityH-2.7 as an erythrocyte antigen: Control by an independent locus,H-2G,between theS andD regions

SpecificityH-2.7 is expressed predominantly on erythrocytes and controlled by a gene that maps within theH-2 gene complex at a locus, designated asH-2G, which apparently lies between regionsS andD. Three phenotypes have been observed with respect to this antigen: a) positive by direct test and absorption (haplotypesH-2 ^f ,H-2 ^j ,H-2 ^p ,H-2 ^s ); b) positive only by absorption (H-2 ^k ); and c) negative (H-2 ^b ,H-2 ^d ,H-2 ^q ). New crossover positions have been established for severalH-2 recombinants based on classifications for theH-2G locus.     

Cell-mediated cytotoxicity to non-MHC alloantigens on mouse epidermal cells

Mice of the C3H/He and A non-H-2 backgrounds are disparate from mice of the B10 background for the tissue-restricted, non-H-2 alloantigen of epidermal cells (EC), Epa-1, that is expressed by EC but not by lymphocytes (LC), as well as for a number of other alloantigens of the B10 background that are expressed by both EC and LC, generically referred to as lymphocyte/epidermal alloantigens (LEA). In this study, we compared the ability of various H-2 congenic strains on the C3H or A backgrounds to mount cytotoxic T-lymphocyte (CTL) responses to EC from H-2 compatible mice of the B10 background. High responses to Epa-1 were detected only in the H-2 ^aand H-2 ^khaplotypes; H-2 ^b, H-2 ^o1, H-2 ^s, H-2 ^t1, and H-2 ^t2 haplotypes were nonresponders to Epa-1. High responses to LEA were detected in H-2 ^a, H-2 ^b, H-2 ^s, H-2 ^t1, and H-2 ^t2 haplotypes; H-2 ^kand H-2 ^o1 were nonresponsive to LEA. Analysis of the H-2K, I and D region alleles of responders indicates that H-2K ^kis essential for anti-Epa CTL responses, whereas D ^d, D ^b, or K ^swere all permissive for strong anti-LEA responses. The ability to mount a given CTL response was not associated with differences in I-region alleles. These results are discussed in terms of K/D region products serving as Ir-gene products for CTL and in determining the apparent tissue-specificity of CTL.     

A novel H-2K splice form: predictions for other alternative H-2 splicing events

A large number of H-2K and H-2D cDNA clones from a C3HfB/HeN spleen cDNA library were extensively characterized. All H-2D^k cDNAs were shown to exhibit the short form of exon 8, consistent with the presence of a single lariat branchpoint site within intron 7. Twenty-five H-2K^km2 cDNAs were found to bear a short exon 8, whereas only two clones were shown to carry the longer form of this exon. In one of the H-2K^km2 cDNAs, a novel pattern of H-2 splicing was identified, in which an extra 15 nucleotides, derived from the 3′ end of intron 5, were inserted between the intact and unaltered exon 5 and exon 6 sequences. Resulting from the apparent use of a cryptic splice acceptor site in place of the canonical intron 5 site, this insertion is predicted to generate an in-frame insertion of five nonpolar amino acid residues within a highly polar region of the intracytoplasmic domain of the H-2K polypeptide. The features of this novel splice form served as the basis for predicting additional rare, alternative H-2 pre-mRNA splicing events that might produce functionally relevant microheterogeneity in the encoded H-2 gene products.     

Cloning and identification of the H-2D p gene

We have cloned six different class I genes from a B10.P sperm library. After cotransfection with the herpes simplex tk gene, one L-cell line was found to react with six H-2D^p-specific monoclonal antibodies. The cell line L12a did not react with K^p-specific monoclonal antibodies. This identification was confirmed by mapping a 2.5 kb Bam H 1 restriction fragment present in the 12a DNA clone to the D-TL region of H-2 ^p. Only a single 8.8 kb Barn H1 fragment can be assigned to K ^p by restriction fragment length polymorphism, while many others map to the D-TL interval. A restriction map of 12a is presented.     

The Mouse H-2A Region Influences the Envelope Gene Structure of Tumor-Associated Murine Leukemia Viruses

C57BL/10 (B10) strains congenic at the mouse major histocompatibility locus (H-2) were injected with a modified ecotropic SL3-3 murine leukemia virus (MuLV) to determine the effect of the H-2 genes on the envelope gene structure of recombinant MuLVs. All tested strains rapidly developed T-cell lymphomas, and recombinant proviruses were detected in the tumor DNAs by Southern blot. The B10.D2 (H-2^d), B10.Br (H-2^k), B10.Q (H-2^q), and B10.RIII (H-2^r) strains exhibited a TI phenotype in which almost all tumors contained type I recombinants. These recombinants characteristically acquire envelope gene sequences from the endogenous polytropic viruses but retain the 5′ p15E (TM) gene sequences from the ecotropic virus. The parental B10 (H-2^b) strain, however, had a novel phenotype that was designated NS for nonselective. Only 30% of the B10 tumors had detectable type I recombinants, whereas a proportion of the others appeared to contain type II recombinants that lacked the type I-specific ecotropic p15E gene sequences. Studies of other B10 congenic strains with hybrid H-2 loci and selected F₁ animals revealed that the NS phenotype was regulated by a dominant gene(s) that mapped to the A region of H-2^b. These results demonstrate that a host gene within the major histocompatibility complex can influence the genetic evolution of pathogenic retroviruses in vivo.     

Delimiting a rice wide-compatibility gene <Emphasis Type="Italic">S</Emphasis><Stack><Subscript>5</Subscript><Superscript><Emphasis Type="Italic">n</Emphasis></Superscript></Stack> to a 50 kb region

Wide-compatibility (WC) is one of the most important traits in rice, which can overcome the fertility barrier in the indica/japonica hybrids, and hence to make it possible to utilize the higher yield potential of inter-subspecific hybrids. The S ₅ ⁿ gene located on chromosome 6 has been previously reported to be responsible for the wide-compatibility in rice. Here we report the precise location of the S ₅ ⁿ gene. In the first-pass mapping, the S ₅ ⁿ gene was restricted within a 200 kb region by using a population of 242 isogenic lines in combination with high-density markers developed in the S ₅ region. In the fine mapping, the S ₅ region was further saturated with newly developed markers and more isogenic lines (549 in total) were investigated. Eventually, the S ₅ ⁿ gene was mapped within a 50 kb region delimited by the left marker J13 and the right marker J17. One BAC clone screened from the BAC library of the WC rice variety 02428 covered the whole S ₅ region. Sequence analysis of the 50 kb region revealed two candidate genes, coding an aspartyl protease and a hypothetical protein. This result would greatly accelerate both cloning and marker-assisted selection of this important S ₅ ⁿ gene. Qing Ji and Jufei Lu have contributed equally to this paper.