David M. Gibson

Recombination between kappa chain genetic markers and the Lyt-3 locus

Recombination has been detected for the first time between chromosome 6 loci controlling kappa chain expression in normal mouse serum immunoglobulin and the Lyt-3 locus. The recombination event occurred at the 26th or 27th backcross generation during the derivation of the Lyt-2a, Lyt-3a-congenic line B6.PL(85NS). The line is now homozygous for the Lyt-2a, Lyt-3a allele(s) at N30F13 and homozygous animals express the Igk-Ef1b allele derived from C57BL/6. The frequency of recombination has been estimated to be 0.30% based on the present results and previous studies in which no recombination was detected. The results rule out the hypothesis that the Lyt-3 locus itself controls the light chain phenotype observed in normal serum immunoglobulin.

Limited Clonal Diversity of Serum Immunoglobulin in Leaky Scid Mice

Mice homozygous for the scid mutation are characterized by the absence of functional B and T cells (Bosma et al. 1983; Dorshkind et al. 1984; Custer et al. 1985). Most scid mice do not possess detectable serum immunoglobulin (Bosma et al. 1983). The exact nature of the scid defect is not yet known but considerable evidence suggests that it may be due to a defective recombinase system which precludes the functional rearrangement of antigen receptor genes for B and T cells (Schuler et al. 1986; Kim et al. 1988; Malynn et al. 1988; Okazaki et al. 1988).

Reconstitution of scid mice by injection of varying numbers of normal fetal liver cells into scid neonates.

In our original report (Bosma et al. 1983) we showed that young adult C.B-17scid/scid mice (scid mice) could be successfully engrafted with bone marrow cells of normal BALB/c donors. The reconstituted recipients readily produced serum immunoglobulin heavy (Igh) chain allotype of the donor cells, indicating that they could support the differentiation of normal lymphocytes. However, the extent of reconstitution in these and other similarly reconstituted scid mice was variable and often incomplete (Custer et al. 1985). Furthermore, the extent of reconstitution did not correlate with the number of donor cells injected (1−5 × 106 bone marrow cells) nor with the elapsed time between injection and analysis. In agreement with these findings, Fulop and Phillips (1986) reported that injection of variable numbers of normal bone marrow cells into young adult scid mice seldom resulted in normal numbers of lymphoid cells, surface Ig+ cells or colony-forming B cells. To ensure complete reconstitution, they found it necessary to irradiate scid recipients prior to cell transfer (Fulop and Phillips 1986).

Recombination between kappa chain genetic markers in the mouse

In this study we report the first instance of recombination between kappa chain genetic markers in the mouse. The recombination frequency, 0.45% (95% limits, 0.12–1.61), is similar to that previously found for recombination between the kappa chain locus and the Lyt-2, 3 locus (0.3%, 95% limits, 0.05–1.6), but is relatively low in comparison with that found at the heavy chain locus (0.41–5.4%). Lyt-2, 3-typing of the recombinants permits a partial ordering of the kappa chain and Lyt-2, 3 loci as (Lyt-2, 3, Igk-Ef1) - Igk-Ef2. Light chains controlled by the two kappa markers include the Vk-(ser) subgroup (controlled by Igk-Ef1) and Vk−1 (controlled by Igk-Ef2). One of the recombinants has been recovered in a homozygous state (“NAK”) and should be suitable for Vkgene mapping studies.

Linkage of a 7S RNA sequence and Kappa light chain genes in the mouse

A mouse 7S RNA cDNA plasmid clone was employed to identify and map DNA restriction fragment variants using recombinant inbred (RI) and congenic mouse strains. More than a dozen such restriction variants were identified and mapped to different regions of the mouse genome. One such variant, designated Rn7s-6, showed close linkage to the Ly-2,3-Igk-V (T lymphocyte antigens 2 and 3, kappa immunoglobulin variable region) cluster of markers on chromosome 6. No recombinants were detected among three of these markers in 59 RI strains. On the basis of these data, the Rn7s-6 sequence may be placed within 1.3 centimorgans of Ly-3 and one of the Igk-V-region markers, Igk-Efl. Two mouse stocks with previously identified crossovers within the Ly2,3-Igk-V region were used to sublocalize Rn7s-6. The results are consistent with the gene order (Ly-2, Ly-3)-(Rn7s-6, Igk-Efl)-Igk-Ef2. Several mouse plasmacytomas, known to have various parts of the kappa chain complex deleted, retain the Rn7s-6 sequence. The Rn7s-6 variant is a plus/minus variant; no sequence allelic to Rn7s-6 is found in inbred strains that share the Ly-3a-Igk-Efla haplotype.

The Organization of the Immunoglobulin Kappa Locus in Mice

The genetic locus that codes for the immunoglobulin kappa (Igκ) light chains in the mouse is located on chromosome 6 approximately 32 centimorgans from the centromere (Hengartner et al., 1978; Gibson et al., 1979; D’Hoostelaere et al., 1985). The number of germline sequences for variable kappa (Vκ) in inbred mice has been estimated to be from 90 to 320 (Cory et al., 1981; Briles and Carroll, 1981; Potter et al., 1982; Gibson, 1984; Nishi et al., 1985). Cory et al. (1981) made their estimates on the basis of 7 cDNA probes which detected non-overlapping sets of restriction endonuclease fragments (REFs). The sum of these sets of related Vκ genes are believed to correspond to a part but not all of the Vκ groups identified by partial or complete amino acid sequencing (Potter et al., 1982). Some of the sets are clusters of nearest neighbor exons, e.g. as with Vκ21 (Heinrich et al., 1984). Clustering must be demonstrated for each Vκ exon group since human Vκ groups have been found to be interspersed (Jaenichem et al., 1984; Peck et al., 1985). All of the Igκ genes are believed to be on chromosome 6 of the mouse; however, the organizational patterns have not been examined in great detail.

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 (Igkc) and AKR (Igka), prototype strains representative of the predominant κ haplotypes, possess both pseudogenes but no intact copy. Only one of the pseudogenes was present in SJL (Igka). Strains C58, c.C58 (Igkd) and NZB (Igkb) 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.