Fine Structure Genetic and Molecular Characterization of a Primitive Allorecognition Locus

共生猕猴桃表现出一种明确且特征明确的异体识别反应。最近已经确定同种异体识别反应分离为单一的共同显性孟德尔特征。本实验室已开发了两个自交系和一个接近同基因的系，用于每个自交系的融合性，目前尚未获得，并将使用它们对包括位点在内的染色体区域进行遗传和分子表征。这些信息将产生足够的位置信息，从而从这个低级后生动物克隆编码同种异体识别决定因子的基因。这些近等基因动物将被用于产生融合位点上不同等位基因的纯合后代池。这些后代池将允许鉴定在1cM间隔内连接的扩增片段长度多态性（AFLP），其中包含感兴趣位点（arl）。将生成这些标记的高分辨率遗传图谱，并在大型重组测试杂交中确定最紧密的标记。将生成一个细菌人工染色体（BAC）文库，使用侧翼标记序列和生成的连续BAC集的高分辨率图筛选阳性。BAC克隆将通过反向southern法分离高度重复的片段，所得片段将用于检测cDNA文库以分离候选基因。通过在简单的粘附试验中转化赋予融合表型的能力将被进行，以确认正确位点的分离。群体动物通常表现出区分自己组织和自己物种无关成员组织的能力，识别事件最终以融合或排斥告终。这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，并且由于不相关的原因，在进化理论的问题中发挥了突出作用。尽管它们广泛存在和重要，但无脊椎动物异体识别的分子基础仍然不清楚。这一努力的成果，包括新的遗传系、来自大型重组测试杂交的DNA收集和BAC库，将向社区提供，预计需求将很高。这项研究产生的遗传图谱和大型插入文库将是任何真生动物的第一个此类资源。

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.