GENETIC MAPPING OF NOVEL ISLET GENES

胰岛细胞功能障碍伴外周胰岛素 抵抗，是非胰岛素发病机制的原始特征 依赖型糖尿病(NIDDM)。因此，胰岛基因很可能是 与这种复杂疾病的遗传易感性有关。我们的 有关的知识。在人胰岛中表达的基因是 然而非常有限，这主要是由于 这种组织的分离。这项资助的目的是识别、克隆、测序 并在遗传连锁图谱中放置大量特定的基因 在纯化的人类胰岛中表达。使用人的差异显示 胰岛和腺泡mRNA我们将鉴定和测序.500胰岛表达 序列标签(EST)。我们的初步数据显示，与之相反 从人胰岛cdna文库中随机测序，这是一种便利的方法。 识别新的胰岛特异基因的策略，同时避免常见的 家族性和污染性腺泡特异性mRNAs。半定量 逆转录聚合酶链式反应(RT-PCR)分析将被用来评估 新的EST在人体多个组织中的分布。[据估计有300人 新的胰岛EST将被绘制在人类/啮齿动物体细胞杂交面板上 染色体定位。当基因组的区域被发现是 与NIDDM连锁，然后通过各种手段进行亚染色体定位 将为映射到该对象的小岛EST子集完成 染色体。示意性地，以下战略层次结构将是 以下是：a)将使用已有的 合成的聚合酶链式反应和胰岛EST进一步定位 荧光原位杂交(FISH)，b)选定的EST、YAC 将从CEPH文库中分离出来，以及c)必要时，P1 基因组克隆也可用于微卫星标记的开发 并在CEPH家系中进行连锁定位。此外，EST还可以 被用来发现新的基因，这将增强我们对 胰岛细胞功能障碍的分子基础。在这个项目中，我们 因此将选择胰岛特定EST的一个较小子集(10-15)进行分离 并鉴定全长的人类胰岛cDNA。序列和映射 数据将存放在公共数据库中，从而可供使用 对于整个研究界来说。总之，这个项目的目的是 根据选定的一组数据生成序列和遗传连锁数据 新的人类胰岛基因，从长远来看将被证明是无价的 了解NIDDM的病因和遗传学的目标，并将 促进持续努力，将表达、遗传和 实物地图。

Pancreatic islet-cell dysfunction, coupled with peripheral insulin resistance, is a primordial feature of the pathogenesis of non-insulin dependent diabetes mellitus (NIDDM). Hence, islet genes are likely to be involved in the genetic susceptibility to this complex disorder. Our knowledge concerning. genes expressed in human pancreatic islets is however very limited, largely due to the difficulties involved in the isolation of this tissue. This grant aims to identify, clone, sequence and place in the genetic linkage map a large number of genes specifically expressed in purified human islets. Using differential display of human islet and acinar mRNA we will identify and sequence .500 islet expressed sequence tags (ESTs). Our preliminary data has shown that, contrary to random sequencing from a human islet cDNA library, this is an expedient strategy to identify novel islet-specific genes, while avoiding common household and contaminating acinar-specific mRNAs. Semiquantitative reverse transcription PCR (RT-PCR) analysis will be used to assess the distribution of novel ESTs in multiple human tissues. [An estimated 300 novel islet ESTs will be mapped on human/rodent somatic hybrid panels for chromosomal localization. When regions of the genome are found to be linked to NIDDM, then subchromosomal localization by a variety of means will be completed for the subset of islet ESTs which map to that chromosome. Schematically, the following strategic hierarchy will be followed: a) P1 genomic clones will be isolated using the already synthesized PCR primers, and the islet ESTs further localized by fluorescence in situ hybridization (FISH), b) for selected ESTs, YACs will be isolated from CEPH libraries, and c) when necessary, the P1 genomic clones can also be used for development of microsatellite markers and linkage mapping in CEPH pedigrees.] In addition, the ESTs can be employed to discover new genes which will enhance our understanding of the molecular basis of islet-cell dysfunction. During this project we will thus select a minor subset (10-15) of islet-specific ESTs to isolate and characterize full-length human islet cDNAs. Sequences and mapping data will be deposited in public databases, and will be thus available for the entire research community. In summary, this project is intended to generate sequence and genetic linkage data from a selected group of novel human islet genes, which will prove invaluable in the long term goal of understanding the etiology and genetics of NIDDM, and will contribute to ongoing efforts to integrate expression, genetic and physical maps.