Using microarrays to facilitate positional cloning: identification of tomosyn as an inhibitor of neurosecretion.
Using microarrays to facilitate positional cloning: identification of tomosyn as an inhibitor of neurosecretion.
复制标题
DOI：
10.1371/journal.pgen.0010002
复制
发表时间：
2005-07
期刊：
影响因子：
4.5
通讯作者：
Kaplan JM
中科院分区：
文献类型：
--
作者：
Dybbs M;Ngai J;Kaplan JM
在许多模型系统中，正向遗传筛选已被用作剖析复杂生物通路的有力策略。这种方法的一个重要限制是对这些筛查中分离的突变进行定位克隆和分子表征的过程既耗时又昂贵。在这里，作者描述了一种使用微阵列杂交来促进位置克隆的策略。这种方法依赖于这样一个事实，即过早停止密码子(即无义突变)构成了筛选中分离的一类频繁的突变，并且无义突变信使RNA被保守的无义介导的衰变途径有效地降解。他们通过鉴定两个以前未确定的突变来验证这一策略：(1)tom-1突变，这是在秀丽线虫乙酰胆碱分泌增加的正向遗传筛查中发现的突变，以及(2)HIF-1转录因子基因明显的自发突变。他们进一步证明了这一策略在线虫、拟南芥和小鼠中使用其他已知突变体的广泛适用性。对TOM-1突变体的研究表明，线虫TOM-1是哺乳动物Tomosyn的同源基因，是神经递质分泌的内源性抑制因子。这些结果还表明，微阵列杂交有可能显著减少位置克隆所需的时间和精力。基因筛查通常被用来找出哪些基因参与了生物过程。基因筛查的第一步是分离在研究过程中有缺陷的突变动物。下一步是找出数千个基因中哪一个有导致观察到的缺陷的突变。定位克隆是一种久经考验的定位突变的方法，速度慢，成本高。作者建议使用微阵列杂交来加速这一过程。他们的方法依赖于这样一个事实，即在筛查中发现的大部分突变是过早终止密码子的结果，这是一种特别严重的突变类型。在细胞中，含有提前终止密码子的信息会被一种被称为无义介导的衰变的保护性途径迅速破坏，从而使它们可以通过微阵列杂交直接检测到。作者将这一策略应用于秀丽线虫、拟南芥和小鼠的已知突变体。他们在线虫中发现了两个未表征的突变，其中一个是tom-1，它是在神经传递增强剂的正向遗传筛查中发现的。有趣的是，他们对tom-1突变体的特征表明，高度保守的蛋白tomosyn抑制了神经元中的神经传递。这项研究表明，微阵列杂交将有助于减少定位克隆所需的时间和精力。
Forward genetic screens have been used as a powerful strategy to dissect complex biological pathways in many model systems. A significant limitation of this approach has been the time-consuming and costly process of positional cloning and molecular characterization of the mutations isolated in these screens. Here, the authors describe a strategy using microarray hybridizations to facilitate positional cloning. This method relies on the fact that premature stop codons (i.e., nonsense mutations) constitute a frequent class of mutations isolated in screens and that nonsense mutant messenger RNAs are efficiently degraded by the conserved nonsense-mediated decay pathway. They validate this strategy by identifying two previously uncharacterized mutations: (1) tom-1, a mutation found in a forward genetic screen for enhanced acetylcholine secretion in Caenorhabditis elegans, and (2) an apparently spontaneous mutation in the hif-1 transcription factor gene. They further demonstrate the broad applicability of this strategy using other known mutants in C. elegans, Arabidopsis, and mouse. Characterization of tom-1 mutants suggests that TOM-1, the C. elegans ortholog of mammalian tomosyn, functions as an endogenous inhibitor of neurotransmitter secretion. These results also suggest that microarray hybridizations have the potential to significantly reduce the time and effort required for positional cloning. Genetic screens are commonly used to figure out which genes are involved in a biological process. The first step in a genetic screen is to isolate mutant animals that are defective in the process being studied. The next step is to find which of the thousands of genes has the mutation that causes the observed defect. Positional cloning, the tried-and-true method for locating mutations, is slow and expensive. The authors propose using microarray hybridizations to speed the process. Their approach relies on the fact that a large fraction of the mutations found in screens are the results of premature stop codons, a particularly severe type of mutation. In cells, messages containing premature stop codons are rapidly destroyed by a protective pathway, called nonsense-mediated decay, thus making them directly detectable by microarray hybridization. The authors apply this strategy retrospectively to known mutants in Caenorhabditis elegans, Arabidopsis, and mouse. They identify two uncharacterized mutations in C. elegans, including one, tom-1, found in a forward genetic screen for enhancers of neurotransmission. Interestingly, their characterization of tom-1 mutants suggests that the highly conserved protein tomosyn inhibits neurotransmission in neurons. This study shows that microarray hybridizations will help reduce the time and effort required for positional cloning.
登录
查看更多内容
影响因子：
56.9
作者：
Jaakkola, P;Mole, DR;Ratcliffe, PJ
通讯作者：
Ratcliffe, PJ
影响因子：
56.9
作者：
COOLEY, L;KELLEY, R;SPRADLING, A
通讯作者：
SPRADLING, A
影响因子：
16.2
作者：
Fujita, Y;Shirataki, H;Takai, Y
通讯作者：
Takai, Y
影响因子：
56.9
作者：
Ivan, M;Kondo, K;Kaelin, WG
通讯作者：
Kaelin, WG
影响因子：
12.3
作者：
Cui X;Churchill GA
通讯作者：
Churchill GA