Fine Structure Genetic and Molecular Characterization of a Primitive Allorecognition Locus

原始同种异体识别基因座的精细结构遗传和分子特征

• 批准号: 9817380
• 负责人: Leo Buss
• 金额: $ 46.06万
• 依托单位: Yale University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9817380&HistoricalAwards=false
• 关键词: Fine; Structure; Genetic; Molecular; Characterization

Hydractinia symbiolongicarpus displays an unambiguous and well-characterized allorecognition response. It has recently been established that the allorecognition response segregates as a single co-dominant Mendelian trait. Two inbred lines and a line near-isogeneic for fusibility for each inbred line, heretofor unavailable, have been developed in this laboratory and using them, a genetic and molecular characterization of the chromosomal region that includes the locus will be done. That information will yield sufficient positional information to clone the gene encoding allorecognition determinants from this lower metazoan. The near-isogenic animals will be used to generate pools of offspring homozygous for different alleles at the fusibility locus. These offspring pools will allow identification of amplified fragment length polymorphisms (AFLP) linked within a 1cM interval containing the locus of interest (arl). A high resolution genetic map of these markers will be generated and the markers most tightly flanking arl will be determined in a large recombinant test cross. A bacterial artificial chromosome (BAC) library will be generated, screened for positives using flanking marker sequences and a high resolution map of the set of contiguous BAC's generated. BAC clones will be shed of the highly repetitive fraction by reverse Southerns and resulting fragments will be used to probe a cDNA library to isolate candidate gene(s). The ability to confer fusibility phenotype by transformation in a simple adhesion assay will be preformed to confirm the isolation of the correct locus.Colonial animals typically display the capacity to discriminate between their own tissues and those of unrelated members of their own species, with the recognition event culminating in either fusion or rejection. Such allorecognition systems are often claimed to reflect the evolutionary origins of the vertebrate immune response and, for unrelated reasons have played a prominent role in problems in evolution theory. Despite their widespread occurrence and importance, the molecular basis of invertebrate allorecognition remains obscure. The products of this effort, novel genetic lines, a DNA collection derived from a large recombinant test cross and a BAC library, will be available to the community and the demand is expected to be high. The genetic map and large insert library emanating from this research will be the first such resources for any eumetazoan animal.

共生果水藻表现出明确的同种异体识别反应。最近已经证实，同种异体识别反应分离为单一的共显性孟德尔性状。本实验室已培育出两个自交系和一个近等基因系，每个自交系的融合性迄今尚不存在，并将利用它们对包括该基因座的染色体区域进行遗传和分子鉴定。这些信息将产生足够的位置信息，从这种较低的后生动物中克隆编码同种识别决定因素的基因。这些近等基因的动物将被用来产生融合基因座上不同等位基因的纯合子后代池。这些后代池将允许鉴定在包含感兴趣基因座(ARL)的1 cM间隔内连锁的扩增片段长度多态(AFLP)。这些标记的高分辨率遗传图谱将被生成，最紧密位于ARL两侧的标记将在一个大型重组测试杂交中确定。将生成细菌人工染色体(BAC)文库，使用侧翼标记序列和生成的一组连续BAC的高分辨率图谱来筛选阳性。BAC克隆将被反向Southerns去掉高度重复的片段，得到的片段将被用来探测cDNA库以分离候选基因(S)。通过在简单的黏附试验中进行转化来赋予融合表型的能力将被执行，以确认正确的位点的分离。集群动物通常表现出区分自己的组织和与其物种无关的成员的能力，识别事件最终导致融合或排斥。这种同种异体识别系统通常被认为反映了脊椎动物免疫反应的进化起源，并且出于无关的原因，在进化论中发挥了突出的作用。尽管无脊椎动物同种异体识别的广泛存在和重要性，但其分子基础仍然不清楚。这项工作的产品、新的基因系、来自大型重组测试杂交的DNA收集和BAC文库将提供给社区，预计需求将很高。这项研究产生的遗传图谱和大插入库将是任何有尾类动物的第一个这样的资源。