Gras Diana E, Silveira Henrique C S, Martinez-Rossi Nilce M, Rossi Antonio
Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil.
FEMS Microbiol Lett. 2007 Apr;269(2):196-200. doi: 10.1111/j.1574-6968.2006.00613.x. Epub 2007 Jan 11.
Subtractive hybridization was used to isolate transcripts up-regulated in the nuc-2 mutant strain of Neurospora crassa grown under phosphate starvation. Following differential screening, 66 cDNA clones of the total enriched were screened in a second round by reverse Northern hybridization. The 17 cDNA candidates displaying visual positive differential expression were sequenced, and functional grouping identified putative proteins possibly involved in diverse cellular processes as, for example, protein synthesis, signal transduction mechanisms, and transport facilitation. Four of them, confirmed by both virtual and Northern blot analyses, revealed genes involved in the initiation of mRNA translation that are significantly up-regulated in the nuc-2 mutant strain, which may be relevant to a further understanding of the molecular events involved in the phosphorus sensing in N. crassa.
采用消减杂交技术分离在磷酸饥饿条件下生长的粗糙脉孢菌nuc-2突变菌株中上调的转录本。经过差异筛选后，对总共富集得到的66个cDNA克隆进行第二轮反向Northern杂交筛选。对显示出明显阳性差异表达的17个cDNA候选克隆进行测序，并通过功能分组鉴定出可能参与多种细胞过程的推定蛋白，例如蛋白质合成、信号转导机制和运输促进。其中四个经虚拟分析和Northern印迹分析均得到证实，揭示了在nuc-2突变菌株中显著上调的参与mRNA翻译起始的基因，这可能有助于进一步了解粗糙脉孢菌中磷感知所涉及的分子事件。
