Characterization of newly isolated monoclonal antibodies against MHC of a Japanese wild mouse

We have already developed nine B10.MOL congenic strains carrying H-2 haplotypes derived from Japanese wild mice, Mus musculus molossinus, with the C57BL/10 genetic background. To obtain monoclonal antibodies against the H-2 antigen of the Japanese wild mouse, we carried out cell fusion using spleen cells from the animal immunized with one of the B10.MOL strains, B10.MOL-SGR (H-2 ^wm7). As a result, 19 hybridomas producing monoclonal antibodies were produced. Analysis with the intro-H-2 recombinants derived from B10.MOL-SGR indicated that 8 of them reacted with the class I and II with the class II molecule. The class I antibodies were tested for their cross -reactivities on wild mice and on the panels of standard inbred and B10.MOL strains. Most of the antibodies reacted with both the Japanese wild mice and the other subspecies, including standard inbred, while two antibodies highly specific for the donor H-2K region reacted with only three wild-derived mice, two M. m. molossinus from Anj o and Shizuoka, Japan, and one M. m. domesticus from Pigeon, Canada. In addition, all of the other four antibodies reactive with the K antigen of B10.MOL-SGR also reacted with the same three wild mice. The wild mice belonging to different subspecies might share very similar H-2K antigenic determinants in spite of their genetic and geographical remoteness.     

A soluble class I molecule analogous to mouse Q10 in the horse and related species

Horse serum is shown to contain a soluble class I molecule analogous to the secreted Q10 molecule in the mouse. This molecule has several similarities to the recently described mouse Q10 molecule: (1) it is smaller than membrane-bound equine class I molecules; (2) it occurs in a high molecular mass complex of 200–300 kd in serum; and (3) the serum levels of the equine molecule are similar to that of the Q 10 molecule (about 30 g/ml). A soluble molecule is also detected in the sera of species related to the horse; it has in fact been found in all the wild members of the order Perissodactyla so far tested. However, it was not detected in the serum of members of the orders Carnivora, Sirenia, Proboscidea, Artiodactyla, and Primates that were tested, nor in the serum of members of the order Rodentia other than in that of the genus Mus. Abbreviations used in this paper ₂m beta-2 microglobulin - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Secretion of a soluble class I molecule encoded by the Q10 gene of the C57BL/10 mouse

The DNA sequence of the Q10 genes appears to be highly conserved amongst strains of mice and has only been found to be transcribed in the liver. An examination of the nucleotide sequence of the exon that normally encodes the transmembrane domain of class I molecules suggested that the Q10 gene encodes a secreted protein. We have established this by showing that L cells transformed with an expression vector containing the Q10 gene secrete a class I molecule which was identified with an antiserum raised against a peptide predicted by the Q10 transmembrane exon. Both the L cell-derived Q10 molecule and a class I protein immunoprecipitated from serum with this anti-peptide antiserum have mol. wts. of approximately 38 000; the Q10 molecule secreted by L cells is heterogeneous in mol. wt. This heterogeneity was drastically reduced after endoglycosidase F treatment, suggesting that Q10 molecules secreted into the serum by the liver may be glycosylated differently from those secreted by L cells. Endoglycosidase F treatment of both the L cell and serum forms of the soluble molecule yielded two products with mol. wts. of approximately 32 000 and 35 000; this is consistent with the observation that the predicted Q10 protein sequence has two potential glycosylation sites. In contrast to previous published results, the Q10 molecule reacted with rabbit anti-H-2 antisera which is consistent with its greater than 80% homology to the classical transplantation antigens.     

Restriction fragment polymorphism in the sex-determining region of the Y chromosomal DNA of European wild mice

Summary Using ³²P-labeled probe consisting mainly of (GATA)_n we have shown that a male specific Alu1 DNA blot pattern which defines the Y chromosome sex-determining locus in inbred mice is highly polymorphic in wild mice, indicating substantial sequence evolution in this region under field conditions. In all cases examined by in situ hybridization, the region concerned is paracentromeric. In contrast, the blot pattern of another probe (M 34) which detects repeated sequences specific to the mouse Y chromosome but outside the sex-determining locus, remains constant between different isolates.Deceased     

Allelic variants of Ly-5 in inbred and natural populations of mice

Allelic variants of Ly-5 in inbred commensal and other natural populations of mice were analyzed by patterns of restriction fragment length polymorphisms (RFLP) and Southern hybridization using an Ly-5 cDNA probe and by cell-surface staining with a panel of antibodies directed against polymorphic and nonpolymorphic Ly-5 determinants. New Ly-5 alleles were defined by RFLPs generated by both Eco RI and Bam HI restriction enzyme digests. The Mus musculus subspecies and other species within the genus Mus showed a strong correlation between allelic variants defined by restriction enzymes and serologic specificities. The data also suggest the conservation of the Ly-5 gene throughout the genus Mus.     

Tracing the evolution of H-2 D region genes using sequences associated with a repetitive element

The class I genes in the murine MHC are genetically divided into the K, D, Qa, and T1a region subfamilies. These genes presumably arose by duplication from a common class I ancestor. Oligonucleotide probes specific for sequences associated with a moderately repetitive B2 SINE element, which is inserted into the 3' untranslated region of the H-2D and H-2L genes, were used to examine the evolutionary relationship between these classically defined D region genes (H-2D and H-2L) and the other members of the class I gene family. Hybridization analyses of recombinant cosmid and genomic DNA indicated that the D region genes separated genetically from the other members of the class I gene family 12 to 14 million years ago. The evidence suggests that during this time frame the chromosomal segment harboring the characteristic insertion became fixed in the ancestral population which gave rise to Mus domesticus. Previous studies have shown that the number of genes present in the Qa and T1a regions varies among inbred strains and among laboratory stocks of wild mice derived from more distant species on the genus Mus. No evidence was found in this study to support the hypothesis that variation in class I gene number is the result of recent duplications of the functionally defined class I genes of the D region, H-2D and H-2L.     

New evidence fortrans-species evolution of theH-2 class I polymorphism

A serological survey using alloantisera specific for the H-2 class I antigens in Japanese wild mice,Mus musculus molossinus, revealed a high frequency of the H-2K^f antigen. This antigen has also been found in European wild mice,M. m. domesticus andM. m. musculus. In this survey, the H-2K^f antigen was characterized through the use of ten newly isolated monoclonal antibodies raised against cells of a Japanese wild mouse, and by Southern blot analysis using anH-2K locus-specific probe which hybridizes with the 3′ end of the gene. The serologically identified H-2K^f antigens revealed several minor variations in reactivities to the monoclonal antibodies. However, all the antigens examined could be clearly separated into two types with respect to the restriction fragment length polymorphism (RFLP) pattern. The first type, found together with a single, characteristic RFLP pattern, was always associated with the presence of reactivity to one particular monoclonal antibody, MS54. The second type, found to represent different RFLP patterns, is associated with the absence of reactivity to MS54. This concordance between the presence of an antigenic determinant and a particular RFLP was observed not only withinMus musculus subspecies but also in a different species:M. spretus, carrying the same antigenic determinant, gave an identical RFLP to that of the other MS54-positiveMus musculus subspecies. The data suggest that the antigenic determinant specific for MS54 is an ancient polymorphic structure which has survived the long period of diversification ofMus species (approximately 2–3 million years) without alteration, and is associated with a stable DNA structure at the 3′ end of theH-2K gene.     

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.     

DNA insertions distinguish the duplicated renin genes of DBA/2 andM. hortulanus mice

In a survey of inbred and wild mouse DNAs for genetic variation at the duplicate renin loci,Ren-1 andRen-2, a variantNot I hybridization pattern was observed in the wild mouseM. hortulanus. To determine the basis for this variation, the structure of theM. hortulanus renin loci has been examined in detail and compared to that of the inbred strain DBA/2. Overall, the gross features of structure in this chromosomal region are conserved in bothMus species. In particular, the sequence at the recombination site between the linkedRen-1 andRen-2 loci was found to be identical in both DBA/2 andM. hortulanus, indicating that the renin gene duplication occurred prior to the divergence of ancestors of these mice. Renin flanking sequences inM. hortulanus, however, were found to lack four DNA insertions totaling approximately 10.5 kb which reside near the DBA/2 loci. The postduplication evolution of the mouse renin genes in thus characterized by a number of insertion and/or deletion events within nearby flanking sequences. Analysis of renin expression showed little or no difference between these mice in steady state renin RNA levels in most tissues examined, suggesting that these insertions do not influence expression at those sites. A notable exception is the adrenal gland, in which DBA/2 andM. hortulanus mice exhibit different patterns of developmentally regulated renin expression.     

Cytotoxic T lymphocytes from mice with soluble class I Q10 molecules in their serum are not tolerant to membrane-bound Q10

Q10 is a class I Qa-2 region-encoded molecule that is secreted by the liver and present in serum at high concentrations (about 10 to 60 micrograms/ml) in most strains of mice. The amino terminal portion of this molecule can also be expressed as an integral membrane protein by splicing the 5' end of the Q10 gene to the 3' end of H-2Ld and transfecting the hybrid gene into murine L cells. Because CTL primarily recognize polymorphic determinants controlled by the alpha 1 and alpha 2 domains of class I molecules and because the Q10d/Ld product expressed by transfected L cells includes the alpha 1 and alpha 2 domains of Q10d, we could address whether mice bearing serum Q10 were tolerant to this molecule at the CTL level. The results of these experiments demonstrate that Q10+ mice are able to generate H-2-unrestricted CTL activity against Q10d expressed on transfected L cells, and this response was not inhibitable by the addition of Q10-containing normal mouse serum. It is unlikely that this CTL activity is due to possible polymorphic differences in Q10 alleles, since semisyngeneic BALB/c (H-2d) mice, from which the Q10d hybrid gene construct was derived, are able to generate anti-Q10d effector cells. The Q10d molecule was shown to cross-react with H-2Ld, lending support to the concept that Qa genes can serve as donors for polymorphic sequences found in H-2K, -D, and -L. That mice can generate anti-Q10 CTL activity suggests that this soluble class I protein does not act as a toleragen for these cells. The implications of these findings for an understanding of self-tolerance are discussed.     

Variability of 18S rDNA locus among Symphysodon fishes: chromosomal rearrangements

Three species of cichlids belonging to the genus Symphysodon have demonstrated interspecific and intraspecific variation in nucleolus organizer regions (NOR) detected with silver nitrate. In order to understand the evolution of this marker in the genus, the structural variability of these sequences in mitotic chromosomes from Symphysodon aequifasciatus, Symphysodon discus and Symphysodon haraldi was investigated using both silver nitrate impregnation and hybridization of the 18S rRNA gene probe. For the three species, the two markers were intraspecifically and interspecifically variable both in the number and in the size of the sites. This polymorphism may stem from duplications and translocations, which suggests that structural chromosome rearrangements effectively act in the karyoevolution of wild Symphysodon species and may have favoured the adaptability of these fishes to diverse aquatic environments in the Amazon.     

Chromosomal polymorphism of ribosomal genes in the genus Oryza

The genes encoding for 18S–5.8S–28S ribosomal RNA (rDNA) are both conserved and diversified. We used rDNA as probe in the fluorescent in situ hybridization (rDNA-FISH) to localized rDNAs on chromosomes of 15 accessions representing ten Oryza species. These included cultivated and wild species of rice, and four of them are tetraploids. Our results reveal polymorphism in the number of rDNA loci, in the number of rDNA repeats, and in their chromosomal positions among Oryza species. The numbers of rDNA loci varies from one to eight among Oryza species. The rDNA locus located at the end of the short arm of chromosome 9 is conserved among the genus Oryza. The rDNA locus at the end of the short arm of chromosome 10 was lost in some of the accessions. In this study, we report two genome specific rDNA loci in the genus Oryza. One is specific to the BB genome, which was localized at the end of the short arm of chromosome 4. Another may be specific to the CC genome, which was localized in the proximal region of the short arm of chromosome 5. A particular rDNA locus was detected as stretched chromatin with bright signals at the proximal region of the short arm of chromosome 4 in O. grandiglumis by rDNA-FISH. We suggest that chromosomal inversion and the amplification and transposition of rDNA might occur during Oryza species evolution. The possible mechanisms of cyto-evolution in tetraploid Oryza species are discussed.     

Sequence of the mouse Q4 class I gene and characterization of the gene product

The Q4 class I gene has been shown to participate in gene conversion events within the mouse major histocompatibility complex. Its complete genomic nucleotide sequence has been determined. The 5 half of Q4 resembles H-2 genes more strongly than other Q genes. Its 3 end, in contrast, is Q-like and contains a translational stop signal in exon 5 which predicts a polypeptide with an incomplete membrane spanning segment. The presence of two inverted B1 repeats suggests that part of the Q4 gene may be mobile within the genome. Gene transfer experiments have shown that the Q4 gene encodes a ²-microglobulin associated polypeptide of M_r 41 000. A similar protein was found in activated mouse spleen cells. The Q4 polypeptide was found to be secreted both by spleen cells and by transfected fibroblasts and was not detectable on the cell surface. Antibody binding and two-dimensional gel electrophoresis indicate that the Q4 molecule is identical to a mouse class I polypeptide, Qb-1, which has been previously described.     

Restriction fragment length polymorphism of DQB and DRB class II genes of the ovine major histocompatibility complex

The ovine major histocompatibility complex (MhcOvar) class II region was investigated by Southern blot hybridizations using ovine probes specific for the second exons of Ovar-DRB and Ovar-DQB genes. Multiple bands were revealed when genomic DNA was digested with each of five restriction enzymes (Bam HI, Eco RI, Hin dIII, PvuII and TaqI), and successively hybridized with the two radiolabeled ovine probes. Restriction fragment length polymorphisms (RFLPs) were analysed in 89 sheep originating from six inbred families and the inheritance of the fragment patterns was determined. Forty-one fragments were recorded with the DQB probe; 32 were detected with the DRB probe. They constituted 9 DQB and 10 DRB allelic patterns. Twelve DQB-DRB haplotypes were resolved in this study.     

Restriction fragment length polymorphisms of the mouse T-cell receptor gene families

We have studied the restriction fragment length polymorphisms (RFLPs) found in the germline T-cell receptor genes of 25 inbred Mus musculus strains and 8 wild Mus species. Included in the inbred mice tested were several strains which spontaneously develop systemic autoimmune disease. Extensive polymorphism was evident for the variable (V) gene segments of the gene family for both the inbred strains and wild mouse species. Changes in the total number of bands hybridizing with probes for V gene segments suggest that members of a V gene segment subfamily are not closely linked, but are interspersed with members of other subfamilies; that expansion and contraction of the multimembered subfamilies may be an important diversifying factor. Our data obtained with gene probes revealed genomic diversity that is much more limited than that seen for the locus. Analysis of inbred mice with probes for the gene locus revealed some RFLPs, but little evidence of expansion or contraction in the numbers of gene segments. Among the autoimmune mice, NZW, NZB, and BXSB/MpJ all display distinctive differences with gene probes. NZW mice have a large deletion of the gene family, which has been reported previously. We found no differences to distinguish the MRL/MpJ lpr/lpr mice from non-autoimmune strains.     

Identification of a short rDNA spacer sequence highly specific of a tomato line containing Tm-1 gene introgressed from Lycopersicon hirsutum

Summary We studied rDNA restriction fragment length polymorphism between two tomato lines used for F₁ hybrid seed production: line A, containing the Tm-1 gene responsible for tobacco mosaic virus tolerance introgressed from the wild species Lycopersicon hirsutum, and line B, a tobacco mosaic virus sensitive line. Hybridization patterns led to distinct rDNA maps with two size classes, 10.4 and 10.7 kb, in line A and a single, 8.9-kb class in line B. Size differences were located in the intergenie sequence (IGS). A highly specific 54-bp TaqI fragment was cloned from the line A IGS and used in dot blot experiments to probe total DNA from line A, line B, and their F₁ hybrid. It proved capable of discriminating B from A and the hybrid. This probe could thus serve to screen inbreds in commercial seed lots where line A is used as male. This fragment showed 80–90% sequence homology with the 53-bp subrepeats previously characterized in a region of the tomato IGS close to the 25S rRNA gene. Preliminary comparison of rDNA in line A and several wild related species indicated that the L. hirsutum H₂ genotype was the closest to line A. rDNA variations between line A and this wild genotype could be explained by recombination during the introgression process involving numerous backcrosses or by an important intraspecific polymorphism. Our results strongly suggest that Tm-1 and the rDNA were introgressed together into tomato from L. hirsutum through linkage drag.     

Molecular phylogeny of Fv1

Alleles at the Fv1 gene of inbred mice confer resistance to infection and spread of vertically or horizontally transmitted murine leukemia viruses (MuLV). The nucleotide sequence of Fv1 bears similarity to the gag of a human endogenous retrovirus, HERV-L, but is more closely related to the gag-coding sequence of a newly described class of HERV-L-related mouse endogenous retroviruses designated MuERV-L. Both observations suggest an origin of Fv1 from endogenous gag sequences. The molecular definition of Fv1 provided an opportunity to determine the phylogeny of the gene among wild mice and its relation to MuERV-L. PCR primers, chosen to include most of the coding region of Fv1 for both the n and b alleles, were used to amplify sequences from animals of the genus Mus, which were then sequenced. Closely related products were obtained from almost all animals examined that evolved after the separation from Rattus, in which the homologous gene was shown to be absent. A phylogenetic tree generated with Fv1 sequence data differs noticeably from that developed with sequence data from other genes. In addition, non-synonymous changes were found to be present twice as frequently as synonymous changes, a fact that departs from the standard behavior of a structural gene. These observations suggest that the Fv1 gene may have been subjected to possible horizontal transfers as well as to positive Darwinian selection. Received: 9 January 1998 / Accepted: 17 August 1998     

Preimplantation embryo development (<Emphasis Type="Italic">Ped</Emphasis>) gene copy number varies from 0 to 85 in a population of wild mice identified as <Emphasis Type="Italic">Mus musculus domesticus</Emphasis>

The preimplantation embryo development (Ped) gene regulates the rate of preimplantation embryonic cleavage division and subsequent embryo survival. In the mouse, the Ped gene product is Qa-2 protein, a nonclassical MHC class I molecule encoded by four tandem genes, Q6/Q7/Q8/Q9. Most inbred strains of mice have all four genes on each allelic chromosome, making a total of eight Qa-2 encoding genes, but there are a few strains that are missing all eight genes, defining a null allele. Mouse strains with the presence of the Qa-2 encoding genes express Qa-2 protein and produce embryos with a faster rate of preimplantation embryonic development and a greater chance of embryo survival compared to mouse strains with the null allele. There is extensive evidence that the human homolog of Qa-2 is HLA-G. HLA-G in humans, like Qa-2 in mice, is associated with enhanced reproductive success. The human population is an outbred population. Therefore, for a better comparison to the human population, we undertook an investigation of the presence of the genes encoding Qa-2 in an outbred population of mice. We used Real-Time Quantitative PCR to quantify the number of Qa-2 encoding genes in a population of 32 wild mice identified as Mus musculus domesticus both by morphologic assessment and by PCR analysis of their DNA. We found great variability in the number of Qa-2 encoding genes in the wild mice tested. The wild mouse with the highest number of Qa-2 encoding genes had 85 such genes, whereas we discovered one wild mouse without any Qa-2 encoding genes. Evolutionary implications of a range of Qa-2 encoding gene numbers in the wild mouse population are discussed, as well as the relevance of our findings to humans.