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We have developed a simple PCR strategy, termed vector-hexamer PCR, that is unique in its ability to easily recover every insert end from large insert clones in YAC and BAC vectors. We used this method to amplify and isolate all insert ends from a YAC contig covering the mouse Igh locus. Seventy-seven ends were amplified and sequenced from 36 YAC clones from four libraries in the pYAC4 vector. Unexpectedly, 40% of the insert ends of these YACs were LINE1 repeats. Nonrepetitive ends were suitable for use as probes on Southern blots of digested YACs to identify overlaps and construct a contig. The same strategy was used successfully to amplify insert ends from YACs in the pRML vector from the Whitehead Institute/MIT-820 mouse YAC library and from BACs in pBeloBAC11. The simplicity of this technique and its ability to isolate every end from large insert clones are of great utility in genomic investigation. [The nucleotide sequence data reported in this paper are accessible in GenBank under accession nos. B07512-B07598.]

The mouse T cell alloantigen, Tsud, is expressed on a minority of mature, Lyt-2+ cells, and its expression is controlled by a gene linked to the immunoglobulin heavy chain gene cluster, Igh. Tsud can be assayed by immunofluorescence staining with an antiserum made in BALB/c mice against C.AL-20 concanavalin A blasts. This antiserum can also be used to induced T suppressor cells in mice expressing Tsu(d). Both of these assays were used to type several panels of recombinant inbred strains and Igh recombinant strains to accurately map the Tsu(d) locus. The Tsu(d) gene is located very near the heavy chain constant-region genes, Igh-C, on the side toward the prealbumin gene, Pre-1. Tsu(d) is not among the heavy chain variable-region genes, Igh-V, and thus is not a variable-region framework allotype, subgroup determinant, or idiotype. The map position suggest that the Tsu(d) antigen is a constant region allotypic determinant on the as yet uncharacterized T cell receptor.

Blot-hybridization and DNA sequence analyses reveal the particular evolutionary conservation of a group of immunoglobulin heavy-chain variable-region (VH) genes in all mammalian species examined. These particular genes are group III genes--the VH7183 family in the mouse and the homologous VH III family in human. This conservation is localized to sequences encoding framework regions 1 and 3 of the antibody variable region and is exerted at the nucleotide level. Because selection acting at the amino acid level alone cannot explain the conservation of these sequences, these sequences must have a noncoding function. The preferential rearrangement of VH7183 and VH III genes, together with the similarity of the conserved sequences to elements implicated in recombination in other systems, suggest that these sequences function to target the series of rearrangements that assemble complete immunoglobulin genes.

DNA replication in mammalian cells is a precisely controlled physical and temporal process, likely involving cis-acting elements that control the region(s) from which replication initiates. In B cells, previous studies showed replication timing to be early throughout the immunoglobulin heavy chain (Igh) locus. The implication from replication timing studies in the B-cell line MPC11 was that early replication of the Igh locus was regulated by sequences downstream of the C alpha gene. A potential candidate for these replication control sequences was the 3' regulatory region of the Igh locus. Our results demonstrate, however, that the Igh locus maintains early replication in a B-cell line in which the 3' regulatory region has been deleted from one allele, thus indicating that replication timing of the locus is independent of this region. In non-B cells (murine erythroleukemia cells [MEL]), previous studies of segments within the mouse Igh locus demonstrated that DNA replication likely initiated downstream of the Igh gene cluster. Here we use recently cloned DNA to demonstrate that segments located sequentially downstream of the Igh 3' regulatory region continue to replicate progressively earlier in S phase in MEL. Furthermore, analysis by two-dimensional gel electrophoresis indicates that replication forks proceed exclusively in the 3'-to-5' direction through the region 3' of the Igh locus. Extrapolation from these data predicts that initiation of DNA replication occurs in MEL at one or more sites within a 90-kb interval located between 40 and 130 kb downstream of the 3' regulatory region.

Analysis of mouse IgG binding to Fc receptors on mouse B cells indicates that the IgG1, IgG2a, and IgGb subclasses bind to the same receptor. No differences in affinity were detected among subclass or between mouse strains. This same receptor bound rat IgG with an affinity that differed between mouse strains. This polymorphism in affinity for rat IgG maps to chromosome 12 distal to the Igh locus.

C.B-20 ( Ighb ) mice challenged with BALB/c ( Igha ) spleen cells (or vice-versa) generate cytotoxic T lymphocytes (CTL) that recognize an antigen, H-40, controlled by an Igh-linked gene. The gene maps to the Igh-C region end of the Igh complex, telomeric to Tsu in the region of Pre-1. At least three alleles, a, b, and c, can be defined. Using a cold target competition assay, no polymorphism of the a allele was detected. Both surface Igh-5a positive and negative spleen cells from (C.B-20 X BALB/c)F1 animals express the a allele of the antigen, indicating that this gene is not allelically excluded. Recognition of the target antigen by CTL is restricted by the D-end of H-2d. The tissue distribution of H-40 was explored using both bulk-cultured and cloned CTL. The antigen is expressed on surface immunoglobulin positive (sIg+) cells and correlates with the expression of sIgM. This was determined by analysis of several B lymphomas as well as of other tumors that varied in their extent of expression of sIg. Four subclones of BCL1 were analyzed. Two of the subclones are sIg+ and express H-40, while two other subclones are sIg- and H-40-. Thus, these data define an Igh-linked gene, separate from immunoglobulin structural loci, that controls an antigen expressed on sIg+ cells. Possible mechanisms to account for this finding are discussed.

Experimental anti-tubular basement membrane (anti-TBM) disease is an autoimmune interstitial nephritis elicited in susceptible rodents after immunization with renal tubular antigen. The nephritogenic antigen in the immunizing preparation is 3M-1, a 48,000 Mr noncollagenous glycoprotein. The hallmarks of the renal lesion are the presence of anti-TBM antibodies (anti-TBM-Ab) and a dense mononuclear cell infiltrate. The anti-TBM B cell repertoire in this disease was analyzed using a library of 22 anti-TBM mAbs generated in a prototypically susceptible Brown Norway rat. These anti-TBM mAbs were all demonstrated to be 3M-1 specific and their characterization formed the basis for the following observations: (a) The size of the anti-TBM B cell population is estimated at 58 distinct clones; (b) by competitive inhibition criteria, all anti-TBM mAbs recognize the same (or spatially close) epitope(s) on 3M-1. This focused recognition was maintained in spite of considerable variability in affinity. Epitopic dominance could also be demonstrated in human polyclonal anti-TBM antisera from a patient with anti-TBM disease; and (c) a crossreactive idiotype was documented, and antisera directed toward this set of variable region determinants was shown to be effective as a prophylactic regimen to abrogate disease, and as a therapeutic modality to arrest the progression of disease; (d) analysis of VH gene families suggested biased usage of Q52- and 7183-like families, although at least three gene families are used in the anti-TBM-Ab response. Thus, the anti-TBM B cell compartment in BN rats is moderately large, but is primarily focused to a single epitope on the nephritogenic antigen and is associated with a disease-modifying crossreactive idiotype.

A new target antigen for unrestricted killing was defined by NZB T lymphocytes which were immunized and restimulated with H-2-identical BALB/c spleen cells. These effector cells killed nearly all target cells tested, irrespective of their H-2 type, but did not kill NZB target cells. The response was shown to have three major components: unrestricted killing specific for Qed-1b, H-2d-restricted killing specific for minor histocompatibility antigens, and unrestricted killing specific for a new antigen, Mta. Mta is present on normal and mitogen-stimulated T and B lymphocytes and on several tumor lines. It was found on cells from 26 mouse strains tested, including two substrains of NZB, representing 9 different H-2 types and 14 different non-H-2 backgrounds. Analysis of the NX8 recombinant inbred lines (derived from Mta-NZB/Icr and Mta+C58/J parents) suggested that Mta is maternally transmitted. This was confirmed by typing of reciprocal F1 hybrids and backcrosses between positive and negative strains: Mta+ females bear Mta+ offspring and Mta- females Mta- offspring, irrespective of the phenotype of the males.

Anti-idiotypic antibodies have been used to mimic antigen in the mouse antiphosphorylcholine response in order to investigate the induction of precursors of antibody-forming cells. We have shown that interaction of anti-idiotype antibody with receptor antibody molecules induces the formation of antibodies that are specific for phosphorylcholine and carry the idiotypic determinants. This induction is dependent on the recognition of carrier determinants on the anti-idiotype antibody by helper T cells. We conclude that receptor antibody molecules on the surface of the precursors of antibody-forming cells deliver the antigenic signal for the induction of these cells.

The mechanism was investigated underlying the activity of bacterial lipopolysaccharide (LPS) as an adjuvant of antibody formation as assessed by its capacity to modulate the induction of tolerance in mice to the antigen human Ig G (HGG) into a state of immunity to HGG. The adjuvant activity of LPS was found to be closely correlated with its ability to function as a B-cell mitogen. This correlation was revealed by an analysis of the genetic control of the mitogenic and adjuvant properties of LPS utilizing the refractory state inherent in the C3H/HeJ mouse strain to these activities of LPS. Thus, mice that were the progeny of a backcross between the nonresponder C3H/JeJ parent and the responder (C3H/HeJ X CWB) F1 hybrid were individually typed for responsiveness to LPS, as an adjuvant and as a B-cell mitogen. It was found that LPS interfered with tolerance induction to HGG in vivo only in those backcross progeny whose spleen cells were also capable of responding mitogenically to LPS in vitro, demonstrating that the adjuvant and B-cell mitogenic properties of LPS are genetically linked. In contrast, these properties were observed to segregate independently from either H-2 or heavy chain allotype loci, and were not sex linked. These results are compatible with the concepts that, in this system, (a) the cellular site of action of LPS as an adjuvant is confined to B cells, and (b) the subcellular mode of action of LPS as an adjuvant may involve the delivery of a "signal" to B cells which is a stimulus for mitogenesis.

Genes encoding the T cell alloantigens Tpre, Tthy, Tind, and Tsu have been mapped to a short segment of chromosome 12 using Igh recombinant strains of mice. These loci are located in a tightly linked cluster between the immunoglobulin heavy chain constant region gene cluster, Igh-C, and the serum prealbumin locus Pre-1, in this manner: centromere--Igh-C--(Tthy, Tind)-Tsu-Tpre--Pre-1.

The mechanism of chronic allotype suppression in (SJL x BALB/c)F1 mice has been investigated by means of cell transfer studies. These mice are phenotypically negative for serum Ig-1b, the paternal allotype determinant on γG2a immunoglubulin, as a result of perinatal exposure to maternal anti-Ig-1b. When spleen or bone marrow (B) cells from suppressed mice were injected into irradiated BALB/c "indicator" hosts, detectable levels of Ig-1b were demonstrated in the sera of a majority of the recipients early after transfer. These results indicate that Ig-1b-producing cells or their precursors are present in the lymphoid tissues of suppressed mice, even though they are not expressed. Within 5–7 wk, it was no longer possible to detect Ig-1b in the sera of these hosts, although cells producing another paternal allotype (Ig-4b) were shown to persist. Control BALB/c mice, injected with spleen and B cells from normal mice, continued to produce high levels of immunoglobulin carrying this allotype. The disappearance of serum, Ig-1b occurred most frequently in the recipients of suppressed spleen cells. Similar results were obtained using a mixture of spleen cells from normal and suppressed mice. Ig-1b production in the recipient mice ceased within a few weeks, even though the majority of cells in the mixture were obtained from normal (nonsuppressed) donors. The data are interpreted as evidence that chronic allotype suppression in mice is actively maintained by cells which are resident in the lymphoid tissues, splenic cells being the most effective. These cells are capable of proliferating in a new host and exerting their suppressive influence on Ig-1b-producing cells and/or their precursors.