Structural and evolutionary comparisons of four alleles of the mouse Igk-J locus which encodes immunoglobulin kappa light chain joining (J K ) segments

The Igk-J locus of the mouse encodes the immunoglobulin light chain joining (J) segments. Four Igk-J alleles have been described on the basis of restriction enzyme length polymorphisms. The nucleotide sequences of the Igk-J ^a allele (type strain, C.C58), Igk-J ^c allele (type strain, SJL/J), and Igk-J ^d allele (type strain, SK/CamRk) have been determined and are compared with the previously reported Igk-J ^b allele sequence (type strain, BALB/c). The mouse sequences are also compared with published sequences for rat and human J _k sequences. Far more differences were found between the Igk-J ^a allele and the other mouse alleles than between any two of the latter. These result in two amino acid substitutions which distinguish the J2 and J3 ¹ segments of the Igk-J ^a allele from the other three alleles. Use of the Phylogenetic Analysis Using Parsimony program to generate a phylogenetic tree strongly indicates that after divergence from the rat ancestor, there appears to have been an early split between the Igk-J ^a allele and the evolutionary precursor of the other mouse alleles. There also appears to have been far less divergence from the ancestral condition in the Igk-J ^a allele than in the other alleles. Also, the presence of only one convergent mutation among the four mouse alleles provides strong evidence against any crossing over within the Igk-J locus during the history of these alleles. Finally, the differences in rates of evolution of the Igk-J alleles are in marked contrast to the relatively uniform rates of divergence of four alleles of a mouse V _k gene, Igk-VSer.     

Polymorphism in V k 10 genes encoding L chains of antibodies bearing the Ars-A and A48 cross-reactive idiotypes

p-azophenylarsonate-specific antibodies of A/J mice which bear the Ars-A cross-reactive idiotype utilize the V _K–Ars–A gene segment, a member of the V _K 10 family. Southern hybridization of genomic DNA from several inbred strains using a probe from the 5 flanking region of the V _K–Ars–A gene demonstrated three patterns of restrictio fragment length polymorphisms (RFLP). Six genes corresponding to hybridizing bands were obtained from DNA libraries of C.AKR, PERU and A/J mice, and nucleotide sequence comparisons revealed two allelic groups: AKRI (Igk-V10.1 ^a ), AJ1 (Igk-V10.1 ^b ) and PERU1 (Igk-V10.1 ^c ); AKR2 (Igk-V10.2 ^a ), AJ2 (Igk-V10.2 ^b ), and PERU2 (Igk-V10.2 ^c ).The Igk-V10.1 ^b gene of the A/J strain is the V _k–Ars–A gene used in Ars-A idiotype-positive antibodies. The product of the C.AKR allele (Igk-V10.1 ^a ) contained four amino acid substitutions in CDR3 as compared with Igk-V10.1 ^b . These substitutions probably explain the failure of AKR mice and other strains with the same V_K10 RFLP pattern to provide in genetic crosses a L chain which, together with the A/J V _H–ArsA gene product, form Ars-A idiotype-positive antibodies. Also, the nucleotide sequence identity between the Igk-V10.1 ^c and Igk-V10.1 ^b alleles and the Igk-V10.2 ^c and Igk-V10.2 ^b alleles is significantly greater than that seen in comparisons with the Igk-V10.1 ^a and Igk-V10.2 ^a alleles, respectively, suggesting an evolutionary pathway similar to that of the linked Igk-J locus.BALB/c antibodies bearing the A48 regulatory idiotype contain L chains encoded by the BALB/c Igk-V10.1 ^b and Igk-V10.2 ^b alleles. Strongly A48 idiotype-positive antibodies utilize the Igk-V10.1 ^b chain, and weakly A48-positive antibodies use the Igk-V10.2 ^b L chain. The possible effects of amino acid substitutions specified by the Igk-V10.1 ^a , Igk-V10.1 ^c , Igk-V10.2 ^a , and Igk-V10.2 ^c alleles on their ability to provide L chains used in A48 idiotype-positive are discussed.The locus name, Igk-V28 (D'Hoostelaere et al. 1988), will be used in this report in place of the name, Igk-VSer, used in the original publications (Goldrick et al. 1985; Boyd et al. 1986; Gottlieb et al. 1986; Ponath et al. 1988). The four alleles described at the Igk-VSer locus (Igk-VSer ^a , Igk-VSer ^b , Igk-VSer ^c , and Igk-VSer ^d ) are referred to as Igk-V28 ^a , Igk-V28 ^b , Igk-V28 ^c , and Igk-V28 ^d , respectively.The nucleotide sequence data reported in this paper have been submitted to GenBank nucleotide sequence database and have been assigned the accession numbers M54903, M54904, M54905, M54906, M54907, and M54908. Address correspondence and offprint requests to : P. D. Gottlieb.     

Moused-amino-acid oxidase gene: Restriction fragment length polymorphism among mouse strains

Summary Genomic DNA was extracted from mice of 15 strains (A/J, AKR, BALB/c, C3H/He, C57BL/6, CBA/J, CD-1, CF#1, DBA/2, ddY/DAO⁺, ddY/DAO^–, ICR, NC, NZB and NZW) for the examination of the difference in the structure of thed-amino-acid oxidase gene among the mouse strains. The DNAs were digested with restriction endonucleases and analyzed by Southern hybridization usingd-amino-acid oxidase cDNA as a probe. The 15 strains showed the same hybridization patterns in theEcoRV,BamHI orBglII digestion. In theEcoRI digestion, the DBA/2 strain showed a different hybridization pattern from the other 14 strains. In thePvuII andXbaI digestion, C3H/He, CBA/J, ddY/DAO⁺ and NC strains were different from the other 11 strains. In thePstI andHindIII digestion, restriction fragment length polymorphisms were observed, and the 15 strains were classified into four groups according to their hybridization patterns. These results indicate that the 15 strains of mice carry a structurally similard-amino-acid oxidase gene, but there is a variation in its inside sequence among the groups of the strains.     

Genetic polymorphism at the mouse immunoglobulin J ϰ locus (Igk-J) as demonstrated by southern hybridization and nucleotide sequence analysis

Comparison of the nucleotide sequences of the C.C58 M75 myeloma chain gene and the BALB/c germ-line J segments suggested that the J regions of C.C58 and BALB/c might be distinguished by restriction enzyme polymorphisms. This was shown to be the case in Southern hybridizations of Hinf I and Ace I digests of liver DNA from these and other strains with a J-specific probe. Tests of a wide variety of inbred, congenic, recombinant, and recombinant-inbred strains provided evidence for three alleles, Igk-J ^a, Igk-J ^b, and Igk-J ^c, the type strains for which are C58/J, BALB/c, and SJL/J, respectively. Analysis of the B6.PL(85NS) congenic strain suggests that the Igk-J locus lies in the neighborhood of the Lyt-2/Lyt-3 loci, approximately 0.30 cM from the V gene segment determining the Igk-VSer and Igk-Efl polymorphisms. Finally, nucleotide substitutions lead to amino acid sequence differences between the C.C58 M 75 gene and the BALB/c germ line in J2 and J4. Two of these substitutions reflect true germ-line differences, raising the possibility that idiotype differences observed among strains could reflect J as well as V differences.     

Partial amino acid sequences of marine Ia antigens of the I-ECd subregion

Partial N-terminal amino acid sequences of the Ia molecule encoded by theI-E orI-C (I-EC) subregion of theH- 2^d haplotype are presented. Several homology relationships are apparent when these sequences are compared to the gene products of the mouseI- EC^k andI-A subregions and to their human and guinea pig counterparts. The polypeptide differs from the EC ^k polypeptide in two of the seven positions at which they can be compared and shows moderate homology with its human p29 counterpart. The polypeptide is identical to the EC ^k polypeptide in the eight positions which can be compared and is highly homologous to the human p34 polypeptide. The genetic implications of these observations are discussed.Abbreviations used in this paper are SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - PTH-amino acid phenylthiohydantoin amino acid - I-EC I-E or I-C (subregion of the I region)     

Phenocopies ofBithorax mutants

Summary Exposure to ether of wild-type embryos of different strains ofDrosophila melanogaster causes phenocopies of different alleles of thebithorax system. Clonal analysis of the phenocopy spots has shown that the transformation caused by the treatment is maintained by cell heredity. Embryos heterozygous for several recessive mutant alleles ofbithorax show the same frequency of phenocopies as wild-type homozygous sib controls. The same holds for embryos heterozygous for the dominant mutant allelesCbx andUbx ¹ which are point mutants in thecis-regulatory region of the system. However, for dominant mutants which have breakpoints in this region (Ubx ⁸⁰,Ubx ¹³⁰ andHm) the frequency of phenocopies is about twice that of their sib controls. Embryos with increasing numbers of copies (from 1 to 4) of thebithorax system show a decreasing frequency of phenocopies. A model is proposed that explains bithorax phenocopies as resulting from disturbances in the distribution of positional information signals for segments (inductor molecules) which compete with the product of a regulator gene (repressor) and thecis-regulatory region of thebithorax system. On this model, the initiation of a metathoracic developmental pathway would result from the derepression of thebithorax system.     

Two closely related κ variable region pseudogenes pose an evolutionary paradox

Two pseudogenes belonging to the Igk-V1 variable region group have been isolated from BALB/c mice. The genes share >96.5% identity of nucleotide sequence in a 1800 base pair (bp) region surrounding the coding region, but deletions of 221 bp and 84 bp have removed essential sequences from the two genes. As the deletions are different in the two pseudogenes, they must have occurred independently in each gene during or subsequent to the duplication event which gave rise to the genes from a common ancestral gene. Polymerase chain reaction analysis was used to identify the pseudogenes in inbred strains of mice. BALB/c (Igk ^c) and AKR (Igk ^a), prototype strains representative of the predominant haplotypes, possess both pseudogenes but no intact copy. Only one of the pseudogenes was present in SJL (Igk ^a). Strains C58, c.C58 (Igk ^d) and NZB (Igk ^b) possessed an intact version of the gene. This distribution of haplotypes is consistent with a close linkage of the pseudogenes with other Igk-V1 genes on chromosome 6. The translated amino acid sequence of the pseudogenes indicates that prior to their acquiring deletions they encoded typical Igk-V1 variable regions except for an unusual FR2 region, in which the conserved proline at position 44 is replaced by leucine and the normally hydrophobic position 36 was occupied by histidine. Possible mechanisms to explain the occurrence of deletions in both of the pseudogenes in the recent evolution of BALB/c are discussed. One explanation would be that the two genes were already nonfunctional at the time of the duplication so that the subsequent deletions represent neutral events which became fixed in the inbred strains by a process of genetic drift. Alternatively, if the genes were functional at the time of duplication, their rapid loss due to deletion events suggests that negative selection may have acted to eliminate the genes from the V-region repertoire. Address correspondence and offprint requests to: D. M. Gibson.     

Soybean seed protein electrophoresis profiles from 15 Asian countries or regions: Hypotheses on paths of dissemination of soybeans from China

Soybean [Glycine max (L.) Merr.] seed protein extracts from 1,603 accessions obtained from 15 Asian countries or regions (not including Japan) were analyzed for the presence of alleles of 2 proteins. Three alleles of the Kunitz. trypsin inhibitor orSBTI-A ₂ designated asTi ^a,Ti ^b andTi ^c are electrophoretically distinguishable from one another by their different Rf values of 0.79, 0.75 and 0.83, respectively. The Sp₁ seed protein or β-amylase has 2 alleles designatedSp1 ^a andSp ₁ ^b which are electrophoretically distinguishable from one another by their Rf values 0.36 and 0.42. About 94 percent of the soybean accessions had theTi ^a allele. Two accessions from Korea,P.I. 157440 andP.I. 196168, do not have theSBTI-A2 protein(ti). Two accessions, one from Pakistan and the other from Korea, were identified as having theTi ^c allele. Only the Korean and central Indian soybean populations have a high frequency for theTi ^b allele. Within Korea, the soybeans from those districts that lie closest to Japan have a high frequency for theTi ^b allele whereas the soybeans from those districts that lie closest to China have a low frequency for theTi ^b allele. TheTi ^b allele is not present in soybeans from the Philippines, Vietnam, Thailand, Malaysia, Burma, Nepal, Pakistan, and Afghanistan. Only 1 accession each from Taiwan and Indonesia have theTi ^b allele. TheSp ₁ ^a allele is not present in soybeans from Taiwan, Vietnam, Thailand, Malaysia, Indonesia, Burma, Pakistan and Afghanistan. The highest frequency for theSp1 ^a allele occurs in soybean germ plasm from northern India and Nepal. The soybeans from Asia (including Japan) were divided into 3 gene centers— primary, secondary, and tertiary—containing 7 germ plasm pools. Paths of dissemination of the soybean from China to the rest of Asia were developed based upon a combination of electrophoretic data and available historical, agronomic, and biogeographical literature.     

Construction of a promoter-probe vector with thePHO5 gene encoding repressible acid phosphatase inSaccharomyces cerevisiae

Summary A YCp type promoter-probe vector, pVC701, replicable inSaccharomyces cerevisiae andEscherichia coli hosts was constructed. pVC701 has a DNA fragment bearing thePHO5 gene encoding repressible acid phosphatase (rA-Pase; EC 3.1.3.2.) without its promoter region. The clonedPHO5 gene can be expressed by insertion of a DNA fragment having promoter function at theEcoRI site on the 5-flanking region ofPHO5. rAPase activity caused by thePHO5 expression is easily detected by staining the transformant colonies with diazo-coupling reagent. These were confirmed by insertion of aHIS5 DNA fragment ofS. cerevisiae having promoter function at theEcoRI cloning site in conditions of histidine starvation. Numerous DNA fragments exhibiting promoter function were isolated by employing pVC701. Most of them expressed thePHO5 gene constitutively, while one of them conferred galactose-inducible and glucose-repressible expression.     

Esterase-29 (ES-29): Biochemical characterization and control by two independent gene loci of a testosterone-dependent mouse serum esterase

Biochemistry and genetics of a testosterone-dependent murine serum esterase designated esterase-29 (ES-29) are described. The enzyme was identified after disc electrophoresis and subsequent staining for esterase using -naphthyl acetate as the substrate. It was inhibited by bis-p-nitrophenyl phosphate and was resistant top-chlorophenylsulphonate and hence was classified as carboxylesterase EC 3.1.1.1. The molecular mass was estimated to be about 130 kDa. It was shown that ES-29 is under the control of two independent genes. The first, termed Es-29, is suggested to be a structural locus, linked to the cluster-2 esterase loci on chromosome 8. Three alleles atEs-29, Es-29 ^a, Es-29^b, andEs-29 ^care distinguished, which determine absence (SEG/1), strong activity (BALB/cJ), and low activity (MOLH/Fre), respectively. The second locus, termedMse-1 (serum esterase modifying factor), was found to be closely linked toPre-2 on chromosome 12 and is suggested to be a modifying or regulatory gene. Two alleles were distinguished,Mse-1 ^a(BALB/cJ) andMse-1 ^m(MOL3/JA, CasBgr), which determine whether ES-29 appears as a single band or a double band, respectively.Mse-1 ^mis dominant toMse-1 ^a.This work was supported by the Deutsche Forschungsgemeinschaft. This is communication No. 70 of a research program devoted to the cellular distribution, genetics, and regulation of nonspecific esterases.     

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.     

Multigene control of Mls c

Festenstein originally described the Mls locus as a single dominant autosomal gene with four alleles which mapped in the 13th linkage group of chromosome 1. We subsequently presented evidence which indicated that the mixed leukocyte reaction stimulatory products of DBA/2 and CBA/J were controlled by two independently segregating Mls loci. Recently, Mls ^d of CBA/J was shown to be composed of Mls ^a of AKR and Mls ^c of C3H. In the present report, classic segregation data is presented which indicates that Mls ^c of C3H is controlled by three independently segregating loci. As defined by stimulatory patterns of numerous cell lines, we postulate the following: either one of the loci is shared with BALB.K, CE, C58, and partially with MA/MyJ, one is shared with CBA/H and CBA/J, and one is shared with BALB.K, CBA/J, and partially with CE; or the groups of shared determinants are controlled by different alleles of unique loci (or locus). In any event, Mls ^c appears to be composed of at least three independently segregating loci; the number of alleles/locus is being investigated. In addition, C3H was stimulated by BALB.K (both were recently postulated to be Mls ^c ); this epitope was shared with CBA/J, CBA/H, AKR/Cum, Ma/MyJ, and C58/J.     

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.     

Construction and properties of new Lyt-congenic strains and anti-Lyt-2.2 and anti-Lyt-3.1 monoclonal antibodies

Hybridomas producing mouse monoclonal IgM antibodies specific for Lyt-2.2 and Lyt-3.1 T-cell surface alloantigens have been constructed. Cytotoxic titers of ascites fluids were found to be 10^–6 or greater and no lysis of thymocytes of congenic strains bearing the alternative allele was observed at the lowest dilutions tested (12). The anti-Lyt-2.2 monoclonal antibody (HO-2.2) specifically precipiated from extracts of Lyt-2.2-positive thymocytes molecular species indistinguishable from those precipitated by conventional anti-Lyt-2.2 sera. However, by immunoprecipitation criteria (though not by cytotoxicity), the anti-Lyt-3.1 antibody (HO-3.1) demonstrated some cross-reactivity with similar molecular species from Lyt-3.1-negative thymocytes.In addition, three new strains of mice differing from existing strains in the region of theLyt-2 and4Lyt-3 loci have been constructed. They are: C.C58-Lyt-2^a, Lyt-3^a and C.AKR-Lyt-2^a, Lyt-3^a, congenic with Balb/cAn and bearingLyt-2 ^a andLyt-3 ^a alleles of C58/J and AKR/J, respectively; and AKR.C-Lyt-2^b, Lyt-3^b, congenic with AKR/J and bearing theLyt-2 ^b andLyt-3 ^b alleles of Balb/cJ.Abbreviations used in this paper DMSO dimethylsulfoxide - NP40 Nonidet P-40 detergent - SaCI Staphylococcus aureus, Cowan I strain - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoreses - NP-NET buffer 0.15 M NaCl, 0.005 M EDTA, 0.05 M Tris, 0.02% sodium azide, pH 7.4, containing 0.5% or 0.05% NP40 as stated in text     

Genetic and functional analyses of the primary in vitro CTL: Response of NZB lymphocytes toH-2-compatible cells

Spleen cells from NZB mice make an unexpected primary cytotoxic T lymphocyte (CTL) response to BALB/c cells in vitro. In this study, it is shown that this response is comprised of at least three independent components. These include a response to antigens recognized in association with H-2^d products, a response to Qa-1^b-associated antigens which is notH-2-restricted and a response directed toward antigens not associated with either H-2^d- or Qa-1^b-coded determinants. The last response appears to be the weakest of the three. In addition, cells from NZB F₁ mice which were either homozygous (Qa-1 ^a /Qa-1 ^a ) or heterozygous (Qa-1 ^a /Qa-1 ^b ) forQa-1 alleles, all responded to BALB/c cells. These data suggest that the NZB CTL response to BALB/c cells is not solely dependent on antigens coded for by genes in theH-2D-Tla region for either the sensitization or effector phases of the response. The ontogeny of the NZB anti-BALB/c CTL response coincides with that of a number of B-cell abnormalities but is shown in experiments with-suppressed NZB mice to be independent of B-cell dysfunction. Studies with (NZB x B10.D2)F₁ + B10.D2 mice demonstrated that the anti-BALB/cCTL response to antigens coded for outside ofQa-1 is governed by at least two genes. Finally, it is shown that another conventionallyH-2-restricted response, that to TNP-modified isologous cells, is neither significantly cross-reactive nor markedly elevated in NZB mice. — The foregoing observations suggest that some subsets of NZB T lymphocytes are intrinsically abnormal. The possibilities that the apparent hyperreactivity of NZB CTL precursors, evidenced in the response to BALB/c cells, is primary or results from the secondary effects of excess T-cell help are discussed.     

The multi-locus H-2D w16 region has an organization distinct from the D d region

Genomic DNA blot analyses using probes derived from the BALB/c 3 flanking region of the L ^d gene (L ^d 3 fl-C) and from near the BALB/c D3 ^d gene (50.2A) indicate that the B10.GAA37 mouse strain has a multi-locus D (D ^w16) region distinct from the five-gene organization observed in the D ^d and D ^q regions. To isolate the D ^w16 region class I genes, a genomic B10. GAA37-EMBL3 library was generated and screened with probes that preferentially hybridize to K and D region class I genes. Hybridization analyses of the isolated clones with L ^d derived oligonucleotide probes suggested that one of the clones contained the L ^w16 gene, whereas several other clones contained the L ^w16 gene. The sequence of the D ^w16 gene is most similar to that of the D ^p gene, particularly in the 3 half. Furthermore, the L ^w16 gene is quite similar in the 5 half and virtually identical in the 3 half to the L ^d gene, indicating that L ^w16, but not D ^w16, is a member of the L ^d gene family. Collectively, these data suggest that, through a D region recombination event, the novel D ^w16 region may have been assembled from primordial counterparts of the D ^p and L ^d genes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M60774-M60776, and M62759.     

Mating preference tests with the recombinant congenic strain BALB.HTG

Tests of MHC-associated mating preference were conducted with the congenic mouse strains BALE (H-2 ^d), BALB.B (H-2 ^b), and BALB.HTG (recombinant ofH-2 ^d andH-2)^b. The results conform to a hypothesis that anRi gene (Ri-1), the expression of which influences mating preference in females, is situated to the right of theS region; and that anotherRi gene (Ri-2), the expression of which influences mating preference in males, is situated elsewhere, probably to the left ofH-2D. This hypothesis is consistent with conclusions previously reached from study of the mating preferences of B6 and B6-Tla^a congenic mice.Abbreviations according to Yamazaki et al. (1978) Ri recognition of identity     

Analysis of D2 d : a D-region class I gene

The mouse major histocompatibility complex is composed of several genes arranged into the K, D, Qa, and Tla regions. The D region of the BALB/c mouse includes genes D2 ^d , D3 ^d , and D4 ^d , in addition to H-2D ^d and H-2L ^d . We have determined the DNA sequence of the D2 ^d gene and compared it with the known sequences of several class I genes. The exon/intron structure of the D2 ^d gene is similar to other class I genes. It also contains similar 5 regulatory elements. A frameshift occurs in exon seven, resulting in a gene product with a truncated cytoplasmic tail. To examine the surface expression of the D2^d molecule, we generated an exon-shuffled construct containing the promoter and exons 1–3, encoding the signal peptide, 1, and 2 external domains of the D2 ^d gene linked to exons 4–8, encoding the 3, transmembrane and cytoplasmic domains, of the H-2D ^d gene. The construct was transfected into mouse L cells, and a protein was detected at the cell surface by a monoclonal antibody (mAb) specific for the 3 domain of H-2D^d, as well as by other class I-specific mAbs. Although D2^d is expressed at low levels, it may be a functional class I gene that most probably evolved from a Qa region gene.     

Identification of self-incompatibility genotypes of almond by allele-specific PCR analysis

In almond, gametophytic self-incompatibility is controlled by a single multiallelic locus (S-locus). In styles, the products of S-alleles are ribonucleases, the S-RNases. Cultivated almond in California have four predominant S-alleles (S ^a, S ^b, S ^c, S ^d). We previously reported the cDNA cloning of three of these alleles, namely S ^b, S ^c and S ^d. In this paper we report the cloning and DNA sequence analysis of the S ^a allele. The S^a-RNase displays approximately 55% similarity at the amino-acid level with other almond S-RNases (S^b, S^c, and S^d) and this similarity was lower than that observed among the S^b, S^c and S^d-RNases. Using the cDNA sequence, a PCR-based identification system using genomic DNA was developed for each of the S-RNase alleles. Five almond cultivars with known self-incompatibility (SI) geno-types were analyzed. Common sequences among four S-alleles were used to create four primers, which, when used as sets, amplify DNA bands of unique size that corresponded to each of the four almond S-alleles; S ^a (602 bp), S ^b (1083 bp), S ^c (221 bp) and S ^d (343 bp). All PCR products obtained from genomic DNA isolated from the five almond cultivars were cloned and their DNA sequence obtained. The nucleotide sequence of these genomic DNA fragments matched the corresponding S-allele cDNA sequence in every case. The amplified products obtained for the S ^a- and S ^b-alleles were both longer than that expected for the coding region, revealing the presence of an intron of 84 bp in the S ^a-allele and 556 bp in the S ^b-allele. Both introns are present within the site of the hypervariable region common in S-RNases from the Rosaceae family and which may be important for S specificity. The exon portions of the genomic DNA sequences were completely consistent with the cDNA sequence of the corresponding S-allele. A useful application of these primers would be to identify the S-genotype of progeny in a breeding program, new varieties in an almond nursery, or new grower selections at the seedling stage. Received: 21 June 1999 / Accepted: 15 November 1999     

Detection of two allotype-(Ig-1)-linked minor histocompatibility loci by the use ofH-2-restricted cytotoxic lymphocytes in congenic mice

Cytotoxic lymphocytes (CTL) were generated betweenIg-1-congenic strains BALB/c(H-2^d,Ig-1^a) andC.B-17(H-2^d,Ig-1^b) by cross-immunization in both directions and rechallenge in vitro. The effector cell populations specifically lysed target cells sharing both theH-2 haplotype and theIg-1 allele of the sensitizing strain. B- and T-cell blasts were equally good targets, suggesting thatH-2-restricted cytotoxic lymphocytes are not directed against serologically defined conventional allotypic determinants, but probably against minor histocompatibility antigens controlled by genes linked to theIg-1 complex. Competition experiments using cold target cells from a series ofIg-1^b-congenic strains of the BALB/c background (BAB-14, C.B-17, C.B-26) revealed two not yet described minor histocompatibility loci linked to theIg-1 complex: We could demonstrate that BALB/c anti-C.B-17 effector cells recognize at least two distinct antigenic determinants on C.B-17 target cells, but only one on target cells from BAB-14, which carries a recombinantIg-1 complex. From these results we conclude that one of the minor histocompatibility antigens, designated as H(C_H), is encoded by a gene linked to the heavy-chain constant-region (C_H) genes, whereas the second minor histocompatibility antigen, designated as H(V_H), is coded for by a gene linked to the heavy-chain variable-region (V_H) genes. These two new genetic markers may be useful for further analysis of the mouseIg-1 complex because the analysis of the H(C_H) and H(V_H) genes may facilitate the search for recombinants in that chromosomal region.