The use of yellow fluorescent hybrids to indicate mating in Trypanosoma brucei
The use of yellow fluorescent hybrids to indicate mating in Trypanosoma brucei
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DOI：
10.1186/1756-3305-1-4
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发表时间：
2008-02-25
影响因子：
3.2
通讯作者：
Bailey, Mick
中科院分区：
文献类型：
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作者：
Gibson, Wendy;Peacock, Lori;Bailey, Mick
背景：布氏锥虫通过未知的机制在其昆虫载体采采蝇中进行遗传交换。使用这种遗传交换实验系统的困难阻碍了研究，特别是因为所涉及的锥虫生命周期阶段无法在体外培养，因此必须在昆虫中进行检查。通过掺入荧光报告基因，直接在果蝇中寻找少量杂交锥虫已成为可能，并且我们之前已经使用报告基因-阻遏基因策略进行了成功的杂交。然而，我们不能确定在该杂交中观察到的所有荧光锥虫都是杂交体，因为阻遏物的突变会导致自发荧光，因此我们开发了一种替代策略。结果：为了可视化果蝇中杂交体的产生，用编码绿色荧光蛋白（GFP）或红色荧光蛋白的基因转染亲本锥虫克隆 （征求建议书）。果蝇与红色和绿色荧光亲代锥虫的共同感染产生了黄色荧光杂交体，这些杂交体在果蝇唾液腺中很容易观察到。黄色锥虫在中肠或前室样本中未见，早在果蝇感染后 13 天就首次以上鞭毛体形式出现在腺体中。来自个体唾液腺的克隆后代具有黄色、红色、绿色或无荧光，并通过微卫星、分子核型和动质体（线粒体）DNA 分析证实为杂种。杂交克隆显示出核基因组和动质体基因组的双亲遗传。虽然报告基因和微卫星等位基因的分离和重配与孟德尔遗传一致，但 DNA 含量的流式细胞术测量揭示了杂交后代克隆中的二倍体和多倍体锥虫。 结论：利用黄色杂交体的产生来指示锥虫交配的策略为锥虫提供了一个强大且明确的系统 遗传交换分析。在这些实验杂交中，交配发生频率很高，仅受亲本锥虫侵入唾液腺的能力的限制。一旦锥虫侵入唾液腺，黄色杂交体就会出现，这表明短的、未附着的上鞭毛体是有性阶段。在这些杂交中二倍体、三倍体和四倍体杂种的恢复是令人惊讶的，因为遗传标记似乎是根据孟德尔规则遗传的。由于多倍体杂种可能是由未减数配子融合产生的，因此与涉及减数分裂的遗传交换模型没有根本冲突。
Background: Trypanosoma brucei undergoes genetic exchange in its insect vector, the tsetse fly, by an unknown mechanism. The difficulties of working with this experimental system of genetic exchange have hampered investigation, particularly because the trypanosome life cycle stages involved cannot be cultured in vitro and therefore must be examined in the insect. Searching for small numbers of hybrid trypanosomes directly in the fly has become possible through the incorporation of fluorescent reporter genes, and we have previously carried out a successful cross using a reporter-repressor strategy. However, we could not be certain that all fluorescent trypanosomes observed in that cross were hybrids, due to mutations of the repressor leading to spontaneous fluorescence, and we have therefore developed an alternative strategy.Results: To visualize the production of hybrids in the fly, parental trypanosome clones were transfected with a gene encoding Green Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP). Co-infection of flies with red and green fluorescent parental trypanosomes produced yellow fluorescent hybrids, which were easily visualized in the fly salivary glands. Yellow trypanosomes were not seen in midgut or proventricular samples and first appeared in the glands as epimastigotes as early as 13 days after fly infection. Cloned progeny originating from individual salivary glands had yellow, red, green or no fluorescence and were confirmed as hybrids by microsatellite, molecular karyotype and kinetoplast (mitochondrial) DNA analyses. Hybrid clones showed biparental inheritance of both nuclear and kinetoplast genomes. While segregation and reassortment of the reporter genes and microsatellite alleles were consistent with Mendelian inheritance, flow cytometry measurement of DNA content revealed both diploid and polyploid trypanosomes among the hybrid progeny clones.Conclusion: The strategy of using production of yellow hybrids to indicate mating in trypanosomes provides a robust and unequivocal system for analysis of genetic exchange. Mating occurred with high frequency in these experimental crosses, limited only by the ability of both parental trypanosomes to invade the salivary glands. Yellow hybrids appeared as soon as trypanosomes invaded the salivary glands, implicating the short, unattached epimastigote as the sexual stage. The recovery of diploid, triploid and tetraploid hybrids in these crosses was surprising as genetic markers appeared to have been inherited according to Mendelian rules. As the polyploid hybrids could have been produced from fusion of unreduced gametes, there is no fundamental conflict with a model of genetic exchange involving meiosis.