Organization, complexity and allelic diversity of the porcine (Sus scrofa domestica) immunoglobulin lambda locus

We have characterized the organization, complexity, and expression of the porcine (Sus scrofa domestica) immunoglobulin lambda (IGL) light chain locus, which accounts for about half of antibody light chain usage in swine, yet is nearly totally unknown. Twenty-two IGL variable (IGLV) genes were identified that belong to seven subgroups. Nine genes appear to be functional. Eight possess stop codons, frameshifts, or both, and one is missing the V-EXON. Two additional genes are missing an essential cysteine residue and are classified as ORF (open reading frame). The IGLV genes are organized in two distinct clusters, a constant (C)-proximal cluster dominated by genes similar to the human IGLV3 subgroup, and a C-distal cluster dominated by genes most similar to the human IGLV8 and IGLV5 subgroups. Phylogenetic analysis reveals that the porcine IGLV8 subgroup genes have recently expanded, suggesting a particularly effective role in immunity to porcine-specific pathogens. Moreover, expression of IGLV genes is nearly exclusively restricted to the IGLV3 and IGLV8 genes. The constant locus comprises three tandem cassettes comprised of a joining (IGLJ) gene and a constant (IGLC) gene, whereas a fourth downstream IGLJ gene has no corresponding associated IGLC gene. Comparison of individual BACs generated from the same individual revealed polymorphisms in IGLC2 and several IGLV genes, indicating that allelic variation in IGLV further expands the porcine antibody light chain repertoire.     

Mx1 causes resistance against influenza A viruses in the Mus spretus-derived inbred mouse strain SPRET/Ei

Inbred SPRET/Ei mice, derived from Mus spretus, were found to be extremely resistant to infection with a mouse adapted influenza A virus. The resistance was strongly linked to distal chromosome 16, where the interferon-inducible Mx1 gene is located. This gene encodes for the Mx1 protein which stimulates innate immunity to Orthomyxoviruses. The Mx1 gene is defective in most inbred mouse strains, but PCR revealed that SPRET/Ei carries a functional allele. The Mx1 proteins of M. spretus and A2G, the other major resistant strain derived from Mus musculus, share 95.7% identity. We were interested whether the sequence variations between the two Mx1 alleles have functional significance. To address this, we used congenic mouse strains containing the Mx1 gene from M. spretus or A2G in a C57BL/6 background. Using a highly pathogenic influenza virus strain, we found that the B6.spretus-Mx1 congenic mice were better protected against infection than the B6.A2G-Mx1 mice. This effect may be due to different Mx1 induction levels, as was shown by RT-PCR and Western blot. We conclude that SPRET/Ei is a novel Mx1-positive inbred strain useful to study the biology of Mx1.     

GENETIC ANALYSIS OF SKULL SHAPE VARIATION AND MORPHOLOGICAL INTEGRATION IN THE MOUSE USING INTERSPECIFIC RECOMBINANT CONGENIC STRAINS BETWEEN C57BL/6 AND MICE OF THE MUS SPRETUS SPECIES

To assess the genetic basis of the skull shape variation and morphological integration in mice, we have used a tool based on the cross between the distantly related mouse species Mus spretus (SEG/Pas strain) and the laboratory strain C57BL/6 called interspecific recombinant congenic strains (IRCSs). The genome of each IRCS consists on average of 1.3% of SEG/Pas derived sequences, located on multiple chromosomes as small-sized, DNA segments. Quantitative trait loci (QTL) on the skull shape, separated into dorsal and ventral sides, were analyzed in 17 IRCSs by a Procrustes superimposition method using 3D landmarks. The shapes of 16 strains differed significantly from C57BL/6. Discrepancy in the QTLs effects was found between the dorsal side and the anterior region of the ventral side due to a differential effect of the SEG/Pas alleles on the skull shape. A comprehensive analysis of all allelic combinations of the BCG-66H strain showed strong epistatic interactions between SEG/Pas segment acting on both skull sides. Epistatic pleiotropy and covariation between sides were dependent in SEG/Pas alleles direction and contributed to the strong morphological integration between sides. Introduction of Mus spretus alleles in a C57BL/6 background induced strong morphological changes mostly in SEG/Pas alleles direction and provided evidence for high level of morphological integration.     

Allelic polymorphisms and RFLP in the human immunoglobulin lambda light chain locus

The organization of the human immunoglobulin lambda light chain locus (IGL) was recently described. This locus has been entirely sequenced. To evaluate the extent of the genomic variability existing inside that locus, we compiled all the available sequences of germline IGLV genes to find variants of Vλ sequences. We also looked for RFLP polymorphisms in a reputedly highly polymorphic human population from eastern Senegal, and compiled all RFLP data previously published. Analysis of these data indicates that IGLV alleles are frequent and increase the diversity of the lambda light chain repertoire in the human population. In contrast, RFLP and polymorphism by insertion and/or deletion are limited in that locus. This observation reinforces our hypothesis that the human IGL locus has undergone less evolutionary shuffling than the human kappa or heavy-chain loci. Received: 23 December 1998 / Accepted: 1 March 1999     

Structure and diversity of Mexican axolotl lambda light chains

We report here the structure of cDNA clones encoding axolotl light chains of the lambda type. A single IGLC gene and eight different potential IGLV genes belonging to four different families were detected. The axolotl Cgamma domain has several residues or stretches of residues that are typically conserved in mammalian, avian, and Xenopus Cgamma, but the KATLVCL stretch, which is well conserved in the Cgamma and T-cell receptor Cbeta domains of many vertebrate species, is not well conserved. All axolotl Vgamma sequences closely match several human and Xenopus Vgamma-like sequences and, although the axolotl Cgamma and Vgamma sequences are very like their tetrapod homologues, they are not closely related to nontetrapod L chains. Southern blot experiments suggested the presence of a single IGLC gene and of a limited number of IGLV genes, and analysis of IGLV-J junctions clearly indicated that at least three of the IGLJ segments can associate with IGLV1, IGLV2, or IGLV3 subgroup genes. The overall diversity of the axolotl Vgamma CDR3 junctions seems to be of the same order as that of mammalian Vgamma chains. However, a single IGLV4 segment was found among the 45 cDNAs analyzed. This suggests that the axolotl IGL locus may have a canonical tandem structure, like the mammalian IGK or IGH loci. Immunofluorescence, immunoblotting, and microsequencing experiments strongly suggested that most, if not all L chains are of the gamma type. This may explain in part the poor humoral response of the axolotl.     

Regulation of murine telomere length by Rtel: an essential gene encoding a helicase-like protein

Little is known about the genes that regulate telomere length diversity between mammalian species. A candidate gene locus was previously mapped to a region on distal mouse Chr 2q. Within this region, we identified a gene similar to the dog-1 DNA helicase-like gene in C. elegans. We cloned this Regulator of telomere length (Rtel) gene and inactivated its expression in mice. Rtel(-/-) mice died between days 10 and 11.5 of gestation with defects in the nervous system, heart, vasculature, and extraembryonic tissues. Rtel(-/-) embryonic stem cells showed telomere loss and displayed many chromosome breaks and fusions upon differentiation in vitro. Crosses of Rtel(+/-) mice with Mus spretus showed that Rtel from the Mus musculus parent is required for telomere elongation of M. spretus chromosomes in F1 cells. We conclude that Rtel is an essential gene that regulates telomere length and prevents genetic instability.     

Polymorphism of C lambda genes and units of duplication in the genus Mus

The number of Ig C lambda genes in nine geographically widespread species from the four subgenera in the genus Mus was estimated from the number of Bam HI and Eco RI restriction fragments that hybridize under high stringency conditions to cDNA probes of BALB/c inbred mouse origin (Mus musculus domesticus). Three closely related species in the subgenus Mus, M. musculus, M. spretus, and M. spicelegus, show considerable variation in the number of C lambda genes. Estimates of gene numbers in these animals range from two C lambda genes in M. spretus from Puerto Real, Spain to 12 C lambda genes in M. musculus musculus from Studenec, Czechoslovakia. Strains of mice carrying either six or 10 C lambda genes were derived from a single population of M. musculus domesticus from Centreville, MD. The hybridization patterns of mice exhibiting C lambda gene amplification indicate that duplications are of relatively recent origin and probably occurred by reiteration of a DNA segment closely related to the 6.5 kb [C lambda 3 - C lambda 1] unit found in BALB/c inbred mice. Three more distantly related species in the subgenus Mus, and a species representing the Nannomys subgenus all appear to carry only four C lambda genes. DNA of species representing the Coelomys and Pyromys subgenera hybridized weakly to the C lambda cDNA probes, but these animals also have no more than four C lambda genes. Thus, there may be a base number of four C lambda genes in most species in the genus Mus. All inbred strains of mice so far examined also have only four C lambda genes, but no feral M. musculus examined have fewer than six C lambda genes. One explanation of the discrepancy in the number of genes between inbred and feral M. musculus is that C lambda genes were deleted during the process of inbreeding.     

Analysis of candidate imprinted genes linked to Dlk1-Gtl2 using a congenic mouse line

The study of genomic imprinting requires the use of DNA sequence polymorphisms between interfertile mouse species or strains. Most commonly, crosses between Mus musculus domesticus and Mus musculus castaneus or Mus spretus animals are used. Difficulties arise in the maintenance of these wild-derived mice in conventional animal facilities, however, and can be overcome by the use of a congenic strain for the region under study. We describe here the generation of a new mouse line, congenic for a region on distal Chromosome (Chr) 12 that encompasses the Dlk1–Gtl2 imprinted domain. We have taken a first step towards demonstrating the utility of these animals by assaying known genes located within the congenic interval for imprinted expression. We show that the two genes located immediately proximal to Dlk1, the Yy1 and Wars genes, are expressed in a biallelic manner. In addition, we have analyzed the Dio3 gene, located distal to Gtl2. This gene displays preferential expression of the paternal allele, with approximately 75% of the total message level originating from the paternal allele and 25% originating from the maternal allele. These data delineate the position of the Wars gene as the proximal boundary of the Dlk1–Gtl2 imprinted domain, and identify Dio3 as another potentially imprinted gene within this domain.     

Variation in V lambda genes in the genus Mus

The complement of Ig V lambda genes in nine species of feral mice representing the four extant subgenera of the genus Mus was examined and compared with that of BALB/c inbred mice. Although all inbred strains examined have two V lambda genes, there is variation in the number of copies of V lambda genes in the wild mice. All feral representatives of M. musculus domesticus, from which inbred strains are derived, have at least three V lambda genes, indicating that a V lambda gene may have been lost during the inbreeding process. At least three V lambda genes are also found in representatives of three other M. musculus subspecies, including the stock of M. musculus musculus "Czech II" shown to have at least 12 C lambda genes. In comparing the complement of V lambda and C lambda genes in these animals, evidence is found that supports a mechanism of lambda gene reiteration involving duplication of a unit containing a V lambda and two C lambda genes. However, the possibility that C lambda gene amplification occurred independent of V lambda gene evolution cannot be ruled out. M. spicelegus and M. spretus, species that are semifertile with M. musculus, have one to three V lambda genes. Species more distantly related to M. musculus, such as M. cookii and M. platythrix, appear to have more (four to six) V lambda genes. Greater V lambda gene heterogeneity is also found in these animals. We propose that the ancestors of the subgenus Mus had more V lambda genes than are seen in modern species and that the paucity of V lambda genes in M. musculus, M. spicelegus, and M. spretus may be the result of V lambda gene deletion events that occurred since the divergence of the ancestor of these three species and those of the distantly related species.     

Genetic linkage analysis of the murine developmental mutant velvet coat (Ve) and the distal chromosome 15 developmental genes Hox-3.1, Rar-g, Wnt-1, and Krt-2.

We have identified restriction fragment length polymorphisms between Mus musculus and Mus spretus for the Chromosome 15 loci Hox-3, Wnt-1, Krt-2, Rar-g, and Ly-6. We followed the inheritance of these alleles in interspecific genetic test crosses between velvet coat (Ve) heterozygotes and M. spretus. The results suggest a gene order and recombination distances (in cM) of Ly-6-22-Wnt-1-2-Ve/Krt-2/Rar-g-3-Hox-3. No recombination was found between Ve, Krt-2, and Rar-g. The data also provide evidence for the hypothesis of a large-scale genomic duplication involving homologous gene pairs on mouse Chromosomes 15 and 11.     

Genetics of the alpha 1,6-dextran response: expression of the QUPC52 idiotype in different inbred and congenic strains of mice

Antibodies to dextran B512 were raised in various strains of mice and were assayed by a radioimmunoassay procedure. Idiotypic antibodies to the IgA(k) dextran B512 binding myeloma proteins QUPC52 and W3129 of BALB/c origin were prepared in rabbits. After adsorption each antiserum was specific for the immunizing myeloma protein and did not react with hundreds of other myeloma proteins; nonetheless, antibodies to dextran B512 from various strains of mice cross-reacted in these test systems. Of the 2 idiotypes tested, the W3129 idiotype was more universally expressed in different strains of mice. The QUPC52 idiotype was the predominant idiotype in BALB/c anti-dextran B512 antibodies and was found in only a few other inbred strains. Using a battery of congenic and inbred strains, it was shown that the QUPC52 idiotype was controlled by genes linked to the Igh complex locus (chromosome 12) and to the Ig kappa complex locus (chromosome 6). The W3129 idiotype was found in a number of stocks of mice in the genus Mus recently isolated from the wild. The QUPC52 idiotype thus far was found only in inbred mice.     

Long-term balancing selection at the blood group-related gene B4galnt2 in the genus Mus (Rodentia; Muridae)

Recent surveys of the human genome have highlighted the significance of balancing selection in relation to understanding the evolutionary origins of disease-associated variation. Cis-regulatory variation at the blood group-related glycosyltransferase B4galnt2 is associated with a phenotype in mice that closely resembles a common human bleeding disorder, von Willebrand disease. In this study, we have performed a survey of the 5' flanking region of the B4galnt2 gene in several Mus musculus subspecies and Mus spretus. Our results reveal a clear pattern of trans-species polymorphism and indicate that allele classes conferring alternative tissue-specific expression patterns have been maintained for >2.8 My in the genus Mus. Furthermore, analysis of B4galnt2 expression patterns revealed the presence of an additional functional class of alleles, supporting a role for gastrointestinal phenotypes in the long-term maintenance of expression variation at this gene.     

Biosystematic study of mice of the genus Mus: karyotype and electrophoretic analysis of total proteins

Two forms of sympatric mice are captured in North - Tunisia: long - tailed mouse and short - tailed mouse. They are often considered as two semi - species of genus Mus, respectively Mus musculus musculus et Mus musculus spretus. They have the same Karyotype (2n = acrocentrics). The electrophoretic study of total proteins, shows up genetics differences. These two forms of mice may be considered as two different species, as like as mices of Europe: Mus musculus at long-tailed and Mus spretus at short tailed.     

Physical performance and soleus muscle fiber composition in wild-derived and laboratory inbred mouse strains.

We compared four inbred mouse strains in their physical performance, measured as a maximal treadmill running time, characteristics of soleus muscle, anatomic character, and growth. The strains used were Mus musculus domesticus [C57BL/6 (B6) and BALB/c], Mus musculus molossinus (MSM/Ms), and Mus spretus. Maximal running time was significantly different among these four mouse strains. Running time until exhaustion was highest in MSM/Ms and lowest in M. spretus. Maximal times for the laboratory mouse strains were nearly identical. Soleus muscle fiber type and cross-sectional area also differed significantly among the species. In particular, M. spretus was significantly different from the other inbred mouse strains. Growth in the wild-derived inbred mice appeared to be complete earlier than in the laboratory mice, and the body size of the wild strains was about half that of the laboratory strains. From these results, we propose that wild-derived inbred mouse strains are useful models for enhancing phenotypic variation in physical performance and adaptability.     

Interspecific recombinant congenic strains between C57BL/6 and mice of the Mus spretus species: a powerful tool to dissect genetic control of complex traits

Complex traits are under the genetic control of multiple genes, often with weak effects and strong epistatic interactions. We developed two new collections of mouse strains to improve genetic dissection of complex traits. They are derived from several backcrosses of the Mus spretus SEG/Pas or STF/Pas strains on the C57BL/6J background. Each of the 55 interspecific recombinant congenic strains (IRCSs) carries up to eight SEG/Pas chromosomal segments with an average size of 11.7 Mb, totalizing 1.37% of the genome. The complete series covers 39.7% of the SEG/Pas genome. As a complementary resource, six partial or complete interspecific consomic strains were developed and increased genome coverage to 45.6%. To evaluate the usefulness of these strains for QTL mapping, 16 IRCSs were compared with C57BL/6J for seven hematological parameters. Strain 66H, which carries three SEG/Pas chromosomal segments, had lower red blood cell volume and higher platelet count than C57BL/6J. Each chromosomal segment was isolated in a congenic strain to evaluate individual effects. Congenic strains were combined to assess epistasis. Our data show that both traits were controlled by several genes with complex epistatic interactions. IRCSs are therefore useful to unravel QTL with small effects and gene-by-gene interactions.     

Genomic<Emphasis Type="Italic"> Eco</Emphasis>RI polymorphism and cosmid sequencing reveal an insertion/deletion and a new<Emphasis Type="Italic"> IGLV5</Emphasis> allele in the human immunoglobulin lambda variable locus (22q11.2/IGLV)

The human immunoglobulin lambda locus ( IGL) is mapped at Chromosome 22q11.2, spanning about 1 Mb of DNA, and directs the synthesis of lambda-type immunoglobulin light chains. The positions of about 73-74 germline V-lambda genes, depending on the haplotypes, are known, with 29-33 of them being functional IGLV genes. These genes were divided into 11 subgroups ( IGLV1 to IGLV11) distributed into three gene clusters ( VA, VB, and VC). We constructed a high-resolution restriction map of a 37-kb cosmid clone (cosmid 8.3) harboring genes of the IGLV1, IGLV7, and IGLV5 families and the non-coding sequences IGLV(I)-42 and IGLV(VII)-41-1, located at cluster VB of the IGL locus. These IGLV genes were associated with unique EcoRI fragments detectable in Southern blots of genomic DNA. Population RFLP has revealed new IGLV alleles and haplotypes. We used the restriction map of cosmid 8.3 and the IMGT database as a reference for RFLP studies. EcoRI Southern blot hybridizations with subgroup-specific probes of the functional and open reading frame sequences present in cosmid 8.3 revealed different frequencies of IGLV gene fragments, as well as deletions of IGLV1-50 and IGLV5-39 genes and RFLP involving IGLV5-45 and IGLV5-48 genes. All members of the IGLV7 subgroup were monomorphic. Sequencing of the genes present in cosmid 8.3 revealed a new allelic variant of the IGLV5 subgroup. These data contribute to a better understanding of the contribution of the germline IGLV genes to the human genetic background and polymorphism.     

Reduced nucleotide variability at an androgen-binding protein locus (Abpa) in house mice: evidence for positive natural selection.

Previous work has shown that the gene for the alpha subunit of androgen-binding protein, Abpa, may be involved in premating isolation between different subspecies of the house mouse, Mus musculus. We investigated patterns of DNA sequence variation at Abpa within and between species of mice to test several predictions of a model of neutral molecular evolution. Intraspecific variation among 10 Mus musculus domesticus alleles was compared with divergence between M. m. domesticus and M. caroli for Abpa and two X-linked genes, Glra2 and Amg. No variation was observed at Abpa within M. m. domesticus. The ratio of polymorphism to divergence was significantly lower at Abpa than at Glra2 and Amg, despite the fact that all three genes experience similar rates of recombination. Interspecific comparisons among M. m. domesticus, Mus musculus musculus, Mus musculus castaneus, Mus spretus, Mus spicilegus, and Mus caroli revealed that the ratio of nonsynonymous substitutions to synonymous substitutions on a per-site basis (Ka/Ks) was generally greater than one. The combined observations of no variation at Abpa within M. m: domesticus and uniformly high Ka/Ks values between species suggest that positive directional selection has acted recently at this locus.     

Mus musculus and Mus spretus homologues of the human telomere-associated protein TIN2

Telomere length is regulated by TRF1, which binds telomeric DNA, and TIN2, which binds TRF1. Laboratory mice (Mus musculus) have long telomeres, although a related mouse species, Mus spretus, has human-sized telomeres. Because differences in TIN2 might explain these differences in telomere length, we cloned cDNAs encoding murine TIN2s and compared their sequence to that of human TIN2. M. musculus (Mm) and M. spretus TIN2s were >95% identical, but shared only 67% identity with human TIN2. An N-terminal truncation, or N-terminal fragment, of MmTIN2 elongated M. spretus telomeres. These findings suggest that mouse TIN2, like human TIN2, negatively regulates telomere length, and that N-terminal perturbations have dominant-negative effects. Our findings suggest that differences in TIN2 cannot explain the telomere length differences among Homo sapiens, M. musculus, and M. spretus. Nonetheless, M. spretus cells appear be a good system for studying the function of mouse telomere-associated proteins.     

The properdin structural locus (Pfc) lies close to the locus for tissue inhibitor of metallothionine proteases (Timp) on the mouse X chromosome

We have confirmed the assignment of the structural locus of the complement factor properdin (Pfc) to the mouse X chromosome and mapped it between monoamine oxidase-A (Mao-a) and hypoxanthine phosphoribosyltransferase (Hprt) using a Mus spretus x Mus musculus interspecific backcross of 108 animals. The structural locus for murine tissue inhibitor of metallothionine proteases (Timp) could not be separated from properdin in a panel of 18 recombinant animals. By minimizing the number of double recombinants the following gene order was obtained: Otc-Mao-a-(Pfc, Timp)-Hprt-Cf-9. The implications for comparative mapping of human and mouse X chromosomes are discussed.     

Genetic dissection of X-linked interspecific hybrid placental dysplasia in congenic mouse strains.

Interspecific hybridization in the genus Mus results in male sterility and X-linked placental dysplasia. We have generated several congenic laboratory mouse lines (Mus musculus) in which different parts of the maternal X chromosome were derived from M. spretus. A strict positive correlation between placental weight and length of the M. spretus-derived part of the X chromosome was shown. Detailed analysis was carried out with one congenic strain that retained a M. spretus interval between 12.0 and 30.74 cM. This strain consistently produced hyperplastic placentas that exhibited an average weight increase of 180% over the weight of control placentas. In derived subcongenic strains, however, increased placental weight could no longer be observed. Morphometric analysis of these placentas revealed persistence of abnormal morphology. Fully developed placental hyperplasia could be reconstituted by recombination of proximal and central M. spretus intervals with an intervening M. musculus region. These results may suggest that placental dysplasia of interspecific mouse hybrids is caused by multiple loci clustered on the X chromosome that act synergistically. Alternatively, it is possible that changes in chromatin structure in interspecific hybrids that influence gene expression are dependent on the length of the alien chromosome.