Fine Structure Genetic and Molecular Characterization of a Primitive Allorecognition Locus

Hydractinia symbiolongapus显示出明确的和良好的特点异源识别反应。 最近已经确定，异源识别反应分离作为一个单一的共显性孟德尔性状。本实验室已开发了两个近交系和一个近等基因的可融合性系（迄今为止尚未获得），并将使用它们对包括该基因座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，以便从这种低等后生动物中克隆编码同种识别决定簇的基因。近等基因动物将用于产生融合性基因座上不同等位基因纯合的后代库。这些后代池将允许鉴定在1cM间隔内连锁的扩增片段长度多态性（AFLP），其包含感兴趣的基因座（arl）。将生成这些标记的高分辨率遗传图谱，并且将在大重组测试杂交中确定最紧密侧翼的标记。将生成细菌人工染色体（BAC）文库，使用侧翼标记序列筛选阳性，并生成一组连续BAC的高分辨率图谱。BAC克隆将通过反向Southern去除高度重复部分，所得片段将用于探测cDNA文库以分离候选基因。 在简单的粘附试验中，通过转化赋予融合性表型的能力将被执行，以确认正确的基因座的分离。群体动物通常显示出区分自身组织和其自身物种的无关成员的组织的能力，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，并且由于不相关的原因，在进化理论的问题中发挥了突出的作用。尽管它们的广泛发生和重要性，无脊椎动物的同种异体识别的分子基础仍然模糊。这一努力的成果，即新的遗传品系、从大型重组测试杂交中获得的DNA集合和BAC文库，将提供给社区，预计需求量很大。这项研究产生的遗传图谱和大型插入文库将是任何真后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.