Characterization of new mouse V kappa groups.

A lambda gt10 BXSB spleen cDNA library was screened with a DNA probe for the C kappa region. Forty individual C kappa+ phages were tested for hybridization with DNA probes representing 11 V kappa region groups. Of the phage inserts large enough to contain V kappa region sequences, 3 were negative for hybridization with all 11 V kappa region probes. The inserts from those three were subcloned, sequenced, and compared with V kappa region sequences in the gene bank. One was identical to 87.92.6 for the region sequenced (a member of V kappa RF). The second showed 93.8% sequence similarity with AN04 and called V kappa 32. The third called V kappa 33 showed 76% sequence similarity with the human sequence V52 and 73.2% sequence similarity with the mouse sequence L6. An insert from V kappa 32 containing the 5' untranslated regions through the codon for Cys 88 of the V kappa region was used as a probe in Southern blot analysis of genomic DNA from inbred and congenic strains of mice. V kappa 32 is a four to eight member group and some of the members are retained in the B6.PL-Ly2a congenic and missing from the B6.PL (85NS) congenic consistent with a map location near V kappa 28. The same filters were hybridized with the insert from V kappa 33 containing 5' untranslated region through the codon for Ser 93 of the V kappa region. V kappa 33 is a one to three member group and using the B6.PL congenics maps with the polymorphic fragments of V kappa 32 and V kappa 28.

Restriction fragment analysis of V preB and lambda 5 within the genus Mus.

DNA from a panel of inbred strains of mice and colony bred mice, isolated from different geographical locations, was hybridized to mouse V preB and lambda 5 probes under stringent conditions, indicating sequence similarities greater than 80%. The probe for lambda 5 detects one gene and the probe for V preB detects two genes (V preB1 and V preB2) in the inbred strains of mice examined under the stringency used. No restriction endonuclease fragment length polymorphisms (RFLP) were detected with the V preB and lambda 5 DNA probes among the inbred strains of mice using Bam HI and Hind III. Very few RFLP were detected among Mus musculus subspecies, and the intensity of the hybridization did not differ significantly with either DNA probe. The number of RFLP increased slightly when different species and subgenera were examined, and the intensity of the hybridization signal began to decrease in samples from the different subgenera, suggesting a slight decrease in sequence similarity for both V preB genes with increased time of divergence. Fewer RFLP were detected with the lambda 5 DNA probe. DNA from 11 different Mus species representing 4 subgenera, genetically isolated from laboratory mice for approximately 1-12 million years, continued to hybridize under high stringency conditions using both DNA probes. A comigrating lambda 5 and V preB restriction endonuclease fragment was detected in most of the samples examined, suggesting the close physical linkage of V preB1 and lambda 5 is maintained within the genus Mus. These results suggest that V preB1, V preB2 and lambda 5 have been present for over 12 million years.

The Ig kappa L chain allelic groups among the Ig kappa haplotypes and Ig kappa crossover populations suggest a gene order.

The Ig kappa complex locus of inbred mice found on chromosome 6 contains one constant (C kappa), five joining (J kappa), and 100 to 300 variable (V kappa) exons and spans an estimated 500 to 2000 kbp of DNA. The V kappa exons are organized into groups of highly homologous coding regions (approximately 300 bp) separated by approximately 10 kbp of intervening sequence. A group contains from 1 to 30 or more exons (exon refers to uninterrupted coding region DNA which is capable of encoding all or part of V kappa gene) that can be detected with specific DNA probes in conjunction with restriction endonuclease fragments (REF) from genomic DNA. Thirteen DNA probes specific for different V kappa exon groups and one DNA probe specific for J kappa and C kappa exons were used in conjunction with 55 inbred strains in an attempt to detect RFLP that could be used to establish Ig kappa allelic groups and Ig kappa haplotypes. Each probe detected two to four different REF patterns (allelic groups) among the panel of inbred mice examined. Size estimates of the REF were made, and each probe detected 4.2 to 107.7 kbp of DNA, including faint REF, 675.6 to 723.6 kbp of DNA could be detected within a single haplotype. Based on these allelic groups, seven haplotypes were identified among the 55 inbred strains of mice. No subline differences were detected, and the distribution of allelic groups implied common ancestry among many of the inbred strains examined. The DNA probes were also used in conjunction with recombinant inbred, congenic strains and backcross populations of mice. By using the analysis of known Ig kappa r populations, and assuming a common ancestry among the inbred strains, a gene order was predicted: Centromere-Hd-(Ig kappa-V11, Ig kappa-V24, Ig kappa-V9-26)-(Ig kappa-V1, Ig kappa-V9)-(Ig kappa-V4, Ig kappa-V8, Ig kappa-V10, Ig kappa-V12, 13, Ig kappa-V19)-(Ig kappa-V28, Rn7s-6)-Ig kappa-V23-(Ig kappa-V21, Ig kappa-J, Ig kappa-C)-(Ly2, Ly3)-wa-1.

T-cell receptor VT beta genes in natural populations of mice.

The composition of 15 VT beta gene subfamilies has been examined by Southern hybridization among a broad spectrum of colony bred rat and mouse species extending phylogenetically from Rattus to Mus musculus domesticus. Most mouse species contain a similar content of VT beta genes as determined by the number of hybridizing restriction fragment (RF) bands. Furthermore, the extent of restriction fragment length polymorphism (RFLP) appears to be limited. Some VT beta gene families, however, are missing from Rattus (VT beta 7, VT beta 12) and M. shortridgei (VT beta 9, VT beta 16). Extension of the VT beta survey to a panel of 38 wild caught mice reveals that nearly a third lack specific hybridization to the VT beta 5 probe. Previous reports have established that the mouse inbred strains SJL, C57BR, C57L, and SWR lack 50% of their VT beta repertoire, including VT beta 5 (Behlke et al. 1985). This study demonstrates that natural populations of mice also carry a significantly reduced VT beta gene repertoire.

Mapping of the bcl-2 oncogene on mouse chromosome 1.

Two bcl-2 alleles have been identified in inbred strains of mice by restriction fragment length polymorphism (RFLP). Analysis of a bcl-2 RFLP in a series of bilineal congenic strains (C.D2), developed as a tool for chromosomal mapping studies, revealed linkage of bcl-2 to the Idh-1/Pep-3 region of murine chromosome 1. The co-segregation of bcl-2 alleles with allelic forms of two other chromosome 1 loci, Ren-1,2 and Spna-1, in a set of back-cross progeny, positions bcl-2 7.8 cM centromeric from Ren-1,2.

A mouse homeo box gene, Hox-1.5, and the morphological locus, Hd, map to within 1 cM on chromosome 6.

Mo-10, a homeo box-containing sequence in the Hox-1 complex of genes referred to as Hox-1.5, was found to be polymorphic in inbred and wild mice, and a strain distribution of three allelic forms of Hox-1.5 are reported. The position of Hox-1.5 was mapped in backcross experiments to within 1 cM of the hypodactyly locus on chromosome 6. This identifies the Hd mutation as a useful model for the examination of homeo box expression during mammalian development.

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.

The organization of immunoglobulin variable kappa chain genes on mouse chromosome 6.

One mouse with a known recombination (NAK) at the Igk locus on chromosome 6 and two new recombinants [B6.PL (75NS) and B6.PL (85NS)] were examined using a series of probes, each of which is specific for a set of immunoglobulin (Ig) Vk genes. Under high stringency conditions, each probe detects from 1 to 19 Bam HI restriction endonuclease fragments (REFs) in genomic DNA by Southern transfer hybridization techniques. Analysis of the REF patterns indicate that the NAK recombination event occurred within the variable region of Igk. The REF patterns of the two B6.PL congenic mice provided two additional recombination events which could be examined. Although some of the REFs had shared mobility among the parental strains, at least 1 and up to 13 polymorphic REFs were present for a given probe among the NZB and AKR parental strains. The results from the NAK mouse indicate that at least some members of Vk4, Vk8, Vk10, and Vk21 were on one side of the recombination event linked to the Lyt-2 alpha and Igk-Ef1 alpha alleles of AKR, while the Vk9, Vk11, and Vk24 REF patterns came from the NZB parental strain linked to the Igk-Ef2 beta (Vk1) allele. The two B6.PL congenics produced a refined map on the Lyt-2, Lyt-3 side of the Vk region. The B6.PL (85NS) mice retained the Vk21 REF pattern of the Lyt-2 alpha, Lyt-3 alpha donor strain PL/J, while displaying the C57BL/6 REF pattern for the other Vk gene groups tested. The B6.PL (75NS) mice retained the REF patterns of PL/J for Vk21 and Ef-1, indicating a third recombination. This indicates the Vk gene order is (Lyt-2; Vk21); Ef-1; (Vk4; Vk8; Vk10); and (Vk9; Vk11; Vk24; Ef-2).

Localization of CT beta and C kappa on mouse chromosome 6.

In the mouse three lymphocyte gene families have been positioned on the proximal region of chromosome 6. Originally the immunoglobulin kappa light chain (Igk) and the thymocyte surface antigens Lyt-2 and Lyt-3 were assigned to chromosome 6, and recently the beta chain of the T-cell receptor for antigen was positioned proximal to Igk. Molecular clones which recognize the constant (C) region of the beta chain of the T-cell receptor for antigen (CT beta) and the constant region of the immunoglobulin kappa (C kappa) chain were used to determine recombination frequencies with respect to the morphological marker hypodactyly (Hd). SJL/JL W pi mice were mated with C.B6.C3-Hd/+ mice, and the progeny expressing the Hd phenotype were mated with SJL/JL W pi mice. Backcross progeny which expressed the Hd phenotype were nephrectomized, and kidney DNA was examined by Southern hybridization for the polymorphic restriction endonuclease fragment (REF) patterns of the parental mice. Of the 88 progeny tested in this three-point cross, 3 CT beta and 4 C kappa homozygote REF patterns were detected. These homozygotes were mutually exclusive. This implies the following gene order: centromere-CT beta-Hd-Igk and CT beta 1 would be 7.95 +/- 2.88 centimorgans from C kappa.