Unequal VH gene rearrangement frequency within the large VH7183 gene family is not due to recombination signal sequence variation, and mapping of the genes shows a bias of rearrangement based on chromosomal location.

Much of the nonrandom usage of V, D, and J genes in the Ab repertoire is due to different frequencies with which gene segments undergo V(D)J rearrangement. The recombination signal sequences flanking each segment are seldom identical with consensus sequences, and this natural variation in recombination signal sequence (RSS) accounts for some differences in rearrangement frequencies in vivo. Here, we have sequenced the RSS of 19 individual V(H)7183 genes, revealing that the majority have one of two closely related RSS. One group has a consensus heptamer, and the other has a nonconsensus heptamer. In vitro recombination substrate studies show that the RSS with the nonconsensus heptamer, which include the frequently rearranging 81X, rearrange less well than the RSS with the consensus heptamer. Although 81X differs from the other 7183-I genes at three positions in the spacer, this does not significantly increase its recombination potency in vitro. The rearrangement frequency of all members of the family was determined in microMT mice, and there was no correlation between the in vitro recombination potential and V(H) gene rearrangement frequency in vivo. Furthermore, genes with identical RSS rearrange at different frequencies in vivo. This demonstrates that other factors can override differences in RSS potency in vivo. We have also determined the gene order of all V(H)7183 genes in a bacterial artificial chromosome contig and show that most of the frequently rearranging genes are in the 3' half of the region. This suggests that chromosomal location plays an important role in nonrandom rearrangement of the V(H)7183 genes.

Genetic control of hematopoietic stem cell frequency in mice is mostly cell autonomous.

Previously we reported that the size of the stem cell compartment (measured as LTC-IC) is 11-fold greater in DBA/2 than in C57BL/6 mice, and we identified genes that regulate the size of the stem cell pool. To determine whether stem cell intrinsic or extrinsic events account for these differences, we created chimeras by aggregating morulae from the strains C57BL/6 and DBA/2. In these chimeras stem cells of both genotypes are exposed to a common mixed environment. Thus, an equalization of stem cell frequencies is expected if stem cell extrinsic effects dominate. Conversely, the parental ratio of LTC-IC should be preserved if the regulation is stem cell autonomous. For each chimera, individual LTC-IC were genotyped on the clonal levels by analyzing their progeny. We found that most of the difference that regulates the size of the stem cell compartment was intrinsic.

Ig heavy chain complex-linked genes influence the immune response in a murine cryptococcal infection.

A murine pulmonary infection with Cryptococcus neoformans (Cne) has been used to determine mechanisms regulating effective T cell-mediated immunity in the lungs. In BALB/c and C.B-17 mice, following intratracheal deposition of Cne, the fungus initially grows rapidly and is then progressively cleared from the lungs. Cne clearance in C.B-17 mice requires CD4 and CD8 T cells, IFN-gamma, and NO. Clearance in congenic BALB/c mice proceeds more slowly than in C.B-17 mice, even though the only genetic difference between these strains is at the Ig H chain-containing region of chromosome 12. Examination of the pulmonary immune response in the two strains revealed that both cleared lung Cne by T cell-dependent mechanisms and generated equivalent levels of NO. Furthermore, both strains recruited equal numbers of macrophages, lymphocytes, and neutrophils to the lungs, although BALB/c mice recruited higher numbers of eosinophils. Notably, leukocytes isolated from BALB/c lungs during infection secreted lower levels of IFN-gamma and higher levels of the Th2 cytokines IL-4 and IL-5 as compared with lung leukocytes from C.B-17 mice. Furthermore, serum levels of IgM, IgG1, IgG2a, and IgG3 anti-Cne Abs generated during infection were significantly greater in BALB/c mice than C.B-17 mice. These data suggest that although both BALB/c and C.B-17 mice clear pulmonary cryptococcosis through T cell-mediated mechanisms, Ig H chain-linked genes in BALB/c mice are associated with a decreased effectiveness of the host response, which we suggest might influence the balance in Th1/Th2 T cell subset development or increase anti-Cne Abs, or both.

Sequence-ready physical map of the mouse chromosome 16 region with conserved synteny to the human velocardiofacial syndrome region on 22q11.2.

Proximal mouse Chromosome (Chr) 16 shows conserved synteny with human Chrs 16, 8, 22, and 3. The mouse Chr 16/human Chr 22 conserved synteny region includes the DiGeorge/Velocardiofacial syndrome region of human Chr 22q11.2. A physical map of the entire mouse Chr 16/human Chr 22 region of conserved synteny has been constructed to provide a substrate for gene discovery, genomic sequencing, and animal model development. A YAC contig was constructed that extends ca. 5.4 Mb from a region of conserved synteny with human Chr 8 at Prkdc through the region conserved with human Chr 3 at DVL3. Sixty-one markers including 37 genes are mapped with average marker spacing of 90 kb. Physical distance was determined across the 2.6-Mb region from D16Mit74 to Hira with YAC fragmentation. The central region from D16Jhu28 to Igl-C1 was converted into BAC and PAC clones, further refining the physical map and providing sequence-ready template. The gene content and borders of three blocks of conserved linkage between human Chr 22q11.2 mouse Chr 16 are refined.

Evidence that a single replication fork proceeds from early to late replicating domains in the IgH locus in a non-B cell line.

In non-B cell lines, like the murine erythroleukemia cell line (MEL), the most distal IgH constant region gene, C alpha, replicates early in S; other heavy chain constant region genes, joining and diversity segments, and the most proximal Vh gene replicate successively later in S in a 3' to 5' direction proportional to their distance from C alpha. In MEL, replication forks detected in the IgH locus also proceed in the same 3' to 5' direction for approximately 400 kb, beginning downstream of the IgH 3' regulatory region and continuing to the D region, as well as within the Vh81X gene. Downstream of the initiation region is an early replicating domain, and upstream of Vh81X is a late replicating domain. Hence, the gradual transition between early and late replicated domains can be achieved by a single replication fork.

Vector-hexamer PCR isolation of all insert ends from a YAC contig of the mouse Igh locus.

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.]

Regulation of the replication of the murine immunoglobulin heavy chain gene locus: evaluation of the role of the 3' regulatory region.

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.

Variation and evolution of class I Mhc in sexual and parthenogenetic geckos.

We present the first Mhc class I sequences in geckos. We compared Mhc variation in gekkonid species that reproduce sexually (Hemidactylus frenatus, Lepidodactylus aureolineatus, L. moestus, L. sp. Arno, L. sp. Takapoto) to others reproducing parthenogenetically (H. garnotii, L. lugubris). These comparisons include the known maternal (L. moestus) and paternal (L. sp. Arno) ancestors of the asexual L. lugubris. Sequences similar to other vertebrate species were obtained from both nuclear and cDNA templates indicating that these sequences are derived from expressed class I Mhc loci. Southern blot analysis using gecko class I probes, revealed that parthenogenetic clonal lineages of independent evolutionary origin have no within-clone band variation at class I loci and that no detectable recombination between restriction sites had taken place. Variability in the sexual species was similar to mammalian taxa, i.e. class I genes are highly variable in outbreeding sexual populations. Sequence analysis of the alpha-2 domain of class I genes identified point mutations in a clonal lineage of L. lugubris which led to amino acid substitutions. Potential transspecific allelic lineages were also observed. The persistence of asexual lineages with little or no class I diversification over thousands of generations seems to argue against strong selection for Mhc multi-allelism caused by pathogen-Mhc allele specificity. On the other hand, the high level of heterozygosity in the parthenogenetic species (a consequence of their hybrid origin) may provide clonal lineages with adequate antigen presenting diversity to survive and compete with sexual relatives.

Genetic control of the frequency of hematopoietic stem cells in mice: mapping of a candidate locus to chromosome 1.

The genetic elements that govern the differentiation and proliferation of hematopoietic stem cells remain to be defined. We describe here marked strain-specific differences in the frequency of long-term culture-initiating cells (LTC-IC) in the bone marrow of different strains of mice. Mice of C57Bl/6 background showed the lowest levels of stem cells in marrow, averaging 2.4 +/- .06 LTC-IC/10(5) cells, BALB/c is intermediate (9.1 +/- 4.2/10(5) cells), and DBA/2 mice contained a 11-fold higher frequency of LTC-IC (28.1 +/- 16.5/10(5) cells) than C57Bl/6 mice. The genetic factors affecting the size of the stem cell pool were analyzed in the C57Bl/6 X DBA/2 recombinant inbred strains; LTC-IC frequencies ranged widely, indicating that stem cell frequencies are controlled by multiple genes. Quantitative trait linkage analysis suggested that two loci that have major quantitative effects are located on chromosome 1 near Adprp and Acrg, respectively. The mapping of the locus near Adprp was confirmed by finding an elevated stem cell frequency in B6.C-H25, a C57Bl/6 congenic strain that carries a portion of chromosome 1 derived from BALB/c mice. We have named this gene Scfr1 (stem cell frequency regulator 1). The allelic forms of this gene may be an important predictor of stem cell number and thus would be useful for evaluating cell sources in clinical stem cell transplantation.

Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus.

The gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) has been proposed recently as a candidate gene for the mouse severe combined immune deficiency (scid) locus. We have used a partial cDNA clone for human DNA-PKcs to map the mouse homologue using a large interspecific backcross panel. We found that the mouse gene for DNA-PKcs does not recombine with scid, consistent with the hypothesis that scid is a mutation in the mouse gene for DNA-PKcs.

Phylogeny of six Sciurus aberti subspecies based on nucleotide sequences of cytochrome b.

The tassel-eared squirrel, Sciurus aberti, is separated into six subspecies which occupy restricted and comparable habitats in ponderosa pine forests in the south-western United States and Mexico. These forests and squirrel populations are currently isolated by large arid areas and, as such, S. aberti appears to offer an example of incipient speciation. We sequenced the complete mitochondrial cytochrome b gene to construct a molecular phylogeny for S. aberti and to determine whether subspecific genetic structure and geographic patterns are correlative. Twenty alleles were identified among 612 squirrels throughout the species' range. Nucleotide divergence between alleles ranged from 0.009 to 0.0233, whereas average sequence divergence between S. aberti and an outgroup species, Sciurus niger, was 0.1823. Neighbor-joining and maximum parsimony analyses identified three major genetic assemblages composed of the following subspecies groups: (1) barberi and durangi; (2) aberti and kaibabensis; and (3) ferreus. The chuscensis samples were the only population with divergent sequences; one sequence was identical to an aberti sequence and a second unique sequence clustered with the ferreus sequences. The presence of divergent sequences in the chuscensis population, coupled with its central geographic position between aberti and ferreus, suggests a relatively recent influx of aberti mtDNA. Estimates of the times separating sequences in subspecies within different groups ranged from 0.94 to 1.52 x 10(6) years, based on a rate estimate of 7.15 x 10(-9) substitutions/year/site. The limited divergence observed between (1) aberti and kaibabensis as well as (2) barberi and durangi suggests relatively recent separations of subspecies within each assemblage. In fact, populations defined morphologically and geographically as ferreus exhibited greater sequence divergence than the aforementioned groups, identifying ferreus as the subspecies with the greatest genetic substructuring. The levels of cytochrome b divergence observed for the three distinct groups argues against a significant role for late Pleistocene glaciation in dispersal of this particular species; however, the proposed intermixing of aberti and chuscensis populations may well have been associated with such glacial events. Nucleotide diversity within subspecies ranked chuscensis >> aberti > barberi approximately kaibabensis approximately ferreus subpopulations; the relatively high level of diversity of chuscensis samples likely results from the apparent introgression of an aberti haplotype. The comparative levels of diversity in the aberti, barberi, kaibabensis, and ferreus sample populations do not correlate with respective habitat size (and presumably population size), suggesting that relatively recent forces, e.g., glaciation and inconsistent timber harvests, may have influenced diversity in these populations without apparent alterations in population size.

The mouse severe combined immune deficiency (scid) mutation is closely linked to the B-cell-specific developmental genes VpreB and lambda 5.

The mouse severe combined immune deficiency (scid) phenotype is due to a recessive, autosomal mutation which results in failed development of lymphocytes. An important step during normal lymphocyte development is the germline rearrangement of DNA segments to assemble functional immunoglobulin or T cell receptor genes. scid lymphocytes fail to rearrange these genes properly, resulting in the absence of mature B and T lymphocytes. This mutation was originally mapped to chromosome 16 by linkage to the immunoglobulin lambda light chain genes (Igl-1) and the coat color mutation mahoganoid. We have typed 288 progeny from backcrosses between MOLF/Ei or CAST/Ei and C.B-17-scid for the scid phenotype and nine other loci mapped to the centromeric region of MMU16. We have established a refined map of this region which places the scid gene between Prm-2 and Igl-1. In addition, no recombinations were found between scid and three other loci, VpreB, lambda 5, and D16Mit31, providing markers useful for isolating the scid gene by positional cloning.

Restricted IgH V gene usage in the response to the ese epitope on "Hi" sheep red blood cells.

We had previously shown that the in vitro antibody response to a single epitope (ese; extra sheep E Ag) present on some sheep E but absent from others could be monitored by assay of the plaque-forming cell response on both Lo3 and Hi SRBC. We had shown also that the response was seen only in certain strains of mice and that the gene(s) controlling the response mapped to the IgH V region of the IgH chain complex. An additional feature of the response is that it is only seen in vitro and is absent and, we hypothesize, is suppressed in vivo. The strain distribution of the response to the ese determinant suggested that the response may only use one V gene (or a small set of closely related V genes) that would be present in the responder strains and absent from the nonresponder strains. To test this hypothesis, we made hybridomas with specificity for the ese determinant and for the shared determinants. cDNA from these hybridomas were sequenced. All four anti-ese hybridomas were almost identical in V region sequence, but varied considerably in D and J segment usage, thus confirming the hypothesis that the ese response would be limited at the V segment. The four anti-ese hybridomas used two Vh J558 genes that differed only by one, or possibly two, nucleotide(s). Importantly, these genes are quite different from most other published J558 sequences. The sequence is very similar to an unexpressed sequence from a C57Bl/6 perinatal mouse and slightly less similar to two other Vhb sequences. It was quite similar to two sequences from autoantibodies, one an anti-DNA hybridoma antibody, BXW-14, isolated from an NZB x NZWF1 mouse, and the other, an NZB hybridoma, G8, with specificity for a mouse E Ag. We speculate that the Ig encoded by the V ese gene react with an autoantigen, that the B cells persist in the animal, but that the secretion of Ig is somehow suppressed.