The nuclear vitamin D receptor (VDR) mediates the actions of its 1,25-dihydroxyvitamin D(3) ligand to control gene expression in terrestrial vertebrates. Prominent functions of VDR-regulated genes are to promote intestinal absorption of calcium and phosphate for bone mineralization and to potentiate the hair cycle in mammals. We report the cloning of VDR from Petromyzon marinus, an unexpected finding because lampreys lack mineralized tissues and hair. Lamprey VDR (lampVDR) clones were obtained via RT-PCR from larval protospleen tissue and skin and mouth of juveniles. LampVDR expressed in transfected mammalian COS-7 cells bound 1,25-dihydroxyvitamin D(3) with high affinity, and transactivated a reporter gene linked to a vitamin D-responsive element from the human CYP3A4 gene, which encodes a P450 enzyme involved in xenobiotic detoxification. In tests with other vitamin D responsive elements, such as that from the rat osteocalcin gene, lampVDR showed little or no activity. Phylogenetic comparisons with nuclear receptors from other vertebrates revealed that lampVDR is a basal member of the VDR grouping, also closely related to the pregnane X receptors and constitutive androstane receptors. We propose that, in this evolutionarily ancient vertebrate, VDR may function in part, like pregnane X receptors and constitutive androstane receptors, to induce P450 enzymes for xenobiotic detoxification.

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The rearrangement of antibody and T-cell receptor gene segments is indispensable to the vertebrate immune response. All extant jawed vertebrates can rearrange these gene segments. This ability is conferred by the recombination activating genes I and II (RAG I and RAG II). To elucidate their origin and function, the cDNA encoding RAG I from a member of the most ancient class of extant gnathostomes, the Carcharhine sharks, was characterized. Homology domains identified within shark RAG I prompted sequence comparison analyses that suggested similarity of the RAG I and II genes, respectively, to the integrase family genes and integration host factor genes of the bacterial site-specific recombination system. Thus, the apparent explosive evolution (or "big bang") of the ancestral immune system may have been initiated by a transfer of microbial site-specific recombinases.The characteristic immune response of vertebrates involves the production of immunoglobulin light and heavy chains, the capacity to generate diverse variable region sequences, and mechanisms to rearrange gene segments during development. These multiple mechanisms generate an immune response with the ability to recognize a number of potential antigens vastly greater than the number of genomic genes in an individual organism (1-5). Immunoglobulin light chains (6-8), heavy chains (6, 9-13), and homologs of T-cell receptors (14) occur in the most primitive extant gnathostomes-i.e., the chondrichthyes, an anciently evolved group that includes sharks, rays, and chimeras. These essential molecules of the vertebrate immune system have not yet been detected in vertebrate species more primitive than chondrichthyes, or in lower deuterostomes or protostomes (3-5, 13, 15). The recombination activating genes (RAG I and RAG II) are both critical for the rearrangement of immunoglobulin gene segments in T and B cells (16)(17)(18). To determine whether the mechanism for gene rearrangement in a primitive gnathastome, the bull shark, Carcharhinus leucas, was comparable to that seen in higher vertebrates, we isolated and analyzed a cDNA clone specifying the complete shark homolog of the RAG I gene. The mechanism of RAG function has not yet been established in any organism or in vitro system. What is known, however, is that both RAG I and RAG II are required for recognition and manipulation of DNA segments involved in generating immune diversity (16,17,19). We hypothesized that the comparison between the RAG I genes of two different species having an ancestral divergence of >400 million years would provide insight into the relationships of the functional domains of the RAG I protein and functional domains of other proteins known to recognize DNA sequences and modify DNA structure.Here we report that the shark RAG I homolog is strongly related to the mammalian prototype and consists of segments showing various degrees of identity to the mammalian gene product. Moreover, detection of homology to enzymes known to affect DNA processing provides a bridge between the vert...

The combinatorial immune response is restricted to jawed vertebrates with cartilaginous fishes being the lowest extant species to have the mechanism for diversification and an extensive panoply of immunoglobulins, T-cell receptors and MHC products. Here, we review the molecular events of the "big bang" or rapid evolutionary appearance of the functionally complete combinatorial immune system coincident with the appearance of ancestral jawed vertebrates, suggesting that this event was catalyzed by horizontal transfer of DNA processing systems. We analyze the nature and extent of variable and constant domain diversity among the distinct immunoglobulin sets of carcharhine sharks focusing upon the lambda-like light chains and the mu and omega heavy chains. The detection and isolation of natural antibodies from the blood of unimmunized sharks illustrates a surprising range of recognition specificities and the existence of polyspecificity suggests that the antibody-forming system of sharks offers unique opportunities for studies of immunological regulation. Although the homologies between shark and mammalian immunoglobulins are unequivocal, major differences in segmental gene organization present challenges to our understanding of basic immunological phenomena such as clonal restriction.

The 3-megabase Igκ locus undergoes differentially controlled nuclear positioning events and chromatin structural changes during the course of B cell development. The temporal association of chromatin structural changes, transcription, and recombination at the Igκ locus was determined in a murine pre-B cell line that can be induced to recombine at the Igκ locus and in ex vivo-cultured murine pre-B cells. Additionally, the timing of nuclear positioning relative to the temporal order of chromatin structural changes and recombination and transcription was determined. We demonstrate that before induction, the Igκ locus was poised for recombination; both alleles were in a contracted state, and the enrichment of histone modifications and germline transcripts of specific Vκ genes were observed. Histone modifications of the Vκ genes did not vary upon induction but the levels of modifications correlated with the levels of germline Vκ gene transcripts and recombination. Upon induction, but before VκJκ recombination, centromeric recruitment of single Igκ alleles occurred. DNase I sensitivity of the entire locus increased gradually over the course of differentiation while the enrichment of histone modifications downstream of the Vκ genes was increased in the silencer regions upstream of Jκ1, within the Igκ sterile transcript, the κ constant region, the Eκi and Eκ3′ enhancers, and the recombining sequence. The ex vivo pre-B cells showed similar patterns of histone modifications across the locus except at the Vκ genes. In this study, H3 acetylation correlated with levels of germline transcripts while H3 methylation correlated with levels of recombination.