RABBIT ALLOTYPES--STRUCTURE, ORGANIZATION AND REGULATED EXPRESSION OF IG GENES

兔同种异型——IG 基因的结构、组织和调控表达

• 批准号: 2566722
• 负责人: R G MAGE
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2566722
• 关键词: B lymphocyte; animal genetic material tag; flow cytometry; gene conversion; gene expression; gene mutation; gene rearrangement; genetic polymorphism; gut associated lymphoid tissue; immunoglobulin genes; immunoglobulin structure; laboratory rabbit; leukopoiesis; monoclonal antibody; northern blottings; nucleic acid sequence; polymerase chain reaction; thymus

We studied genes of the rabbit immune system by techniques of molecular biology and immunology. We produced anti-RAG-2 antibodies and studied developing rabbit thymus and appendix tissues. We used the surface markers CD43 and IgM to distinguish two appendix cell populations from 6 to 9-week-old rabbits that expressed RAG-1 transcripts. We speculate that the appearance of CD43 during different stages of B-cell maturation may be related to the function of the appendix as a site of both B-cell development and diversification. IgM associated B cell receptor molecules were found on rabbit B cells as heteromeric structures with nonreduced molecular weights of approximately 75 kDa and 135 kDa composed of 37 and 42 kDa subunits. Immunological studies with monoclonal antibodies suggested that these proteins are the rabbit homologues of murine Ig-beta (B29) and Ig-alpha (mb-1). We investigated RAD51 in rabbit appendix because we postulated that RAD51 could play a role in the gene conversion events that occur in young rabbit appendix to diversify their antibody repertoire. We cloned and sequenced RT-PCR amplified rabbit RAD51 and found that the coding sequences of rabbit, human and mouse RAD51 are highly similar. We developed a reverse transcriptase PCR mimic assay and measured the relative abundance of RAD51 transcripts in different tissues, in FACS-sorted B and T cells and B-cell subsets. We found relatively high levels of RAD51 message in testis, thymus and appendix that may correlate with high mitotic and/or meiotic activities. Bands of three different sizes found on Northern blots could reflect distinct transcription initiation sites, alternative splicing or different poly (A) addition sites. In man and mouse, the 3'-most DH gene, DQ52, is preferentially rearranged early in B-cell development. To test whether this preference for rearranging a DH gene segment based on 3' end proximity exists in rabbit, we cloned and sequenced the rabbit DQ52 gene. The coding region sequence is identical to a mouse DQ52 but the 3' recombination signal sequence has an atypical nonamer. The DQ52 gene was utilized very infrequently, if at all. We found only one VDJ sequence from 28 day fetal liver B-cells with 8 bp that matched the germline DQ52 sequence. Instead of rearranging DQ52, another DH gene, Df, localized about 32 kb upstream of the JH was the preferred target. Thus, in contrast to man and mouse, rabbits preferentially express a DH gene located in the middle of the DH region early in B cell ontogeny. This may correlate with more frequent initial rearrangement of VH to DH in rabbit B cells.

采用分子技术对兔免疫系统基因进行了研究