Dzitoyeva Svetlana, Dimitrijevic Nikola, Manev Hari
The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
BMC Genomics. 2003 Aug 12;4(1):33. doi: 10.1186/1471-2164-4-33.
RNA interference (RNAi) is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly) gene (corresponding to a putative gene CG5652/GM06434), that we named beltless based on an embryonic loss-of-function phenotype.
Beltless mRNA is expressed in all developmental stages except in 0-6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp) beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless) of the abdominal segments A2-A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1)RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1)RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts.
We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should elucidate the role and mechanism of action of beltless during Drosophila development and in adults, including in the adult nervous system.
RNA干扰（RNAi）是由双链RNA触发的一个过程，可导致基因表达的靶向下调/沉默，并可用于功能基因组学研究，即功能缺失研究。在此，我们报告了RNAi在鉴定一个对发育具有重要意义的新型果蝇基因（对应于一个假定基因CG5652/GM06434）中的应用，我们根据胚胎功能缺失表型将其命名为无带基因。
无带基因的mRNA在除0 - 6小时胚胎外的所有发育阶段均有表达。原位逆转录聚合酶链反应（in situ RT-PCR）将无带基因的mRNA定位在晚期胚胎的腹神经索和大脑以及成年果蝇的神经系统、卵巢和附属腺中。通过将短的（22个碱基对）无带基因双链RNA注射到胚胎或成年果蝇中诱导RNA干扰。胚胎RNA干扰改变了表皮表型，从部分形成到腹部A2 - A4节段的齿状带缺失（因此称为无带）。胚胎期无带基因的RNA干扰是致死性的。成年果蝇的RNA干扰导致卵巢缩小一半，并减少产卵数量。我们还检查了Df(1)RK4果蝇，其中的缺失删除了16个基因，包括无带基因。在一些胚胎中，我们观察到与无带基因RNA干扰的结果相似的表皮异常。在将Df(1)RK4胚胎区分为具有可见齿状带的胚胎和齿状带缺失的胚胎后，我们检测了无带基因mRNA的存在情况；在齿状带缺失的胚胎中未检测到无带基因mRNA。
我们鉴定了一个对发育具有重要意义的新型果蝇基因——无带基因，该基因已通过RNA干扰进行了功能缺失研究。假定的无带蛋白与秀丽隐杆线虫抗氟西汀的鼻子（NRF）NRF - 6基因具有同源性，也与一些未表征的秀丽隐杆线虫和黑腹果蝇基因具有同源性，其中一些基因具有显著的酰基转移酶结构域。未来的研究应阐明无带基因在果蝇发育过程中和成年期，包括在成年神经系统中的作用和作用机制。
