Abidi F E, Roh H, Keath E J
Center for Molecular Studies, J. C. Self Research Institute, Greenwood Genetics Center, Greenwood, South Carolina 29646, USA.
Infect Immun. 1998 Aug;66(8):3867-73. doi: 10.1128/IAI.66.8.3867-3873.1998.
Genes expressed in the parasitic yeast (Y) phase of the dimorphic fungal pathogen Histoplasma capsulatum which are transcriptionally silent in the mycelial (M) phase have recently been cloned and analyzed. To understand the molecular regulation of genes involved in the transition to and maintenance of the Y phase, the presumptive 5' regulatory regions of two Y phase-specific genes (yps-3 and yps 21:E-9) were PCR amplified as labelled probes to identify nuclear DNA binding proteins which may influence phase-specific gene transcription. Protein-DNA interactions were assessed by Southwestern blot analysis in which sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated protein extracts from Y and M phases of the virulent G217B strain of H. capsulatum were visualized by their capability for in situ binding to the labelled 517-bp (G217B yps-3) or the 395-bp (G217B yps 21:E-9) putative 5' regulatory regions. A 30-kDa nuclear protein unique to the M-phase extracts of the highly virulent G217B strain, but absent in the Y phase of the same organism, was identified. In contrast, the low-virulence, thermal-sensitive Downs strain of H. capsulatum lacked detectable p30 binding activity in either yeast- or mycelial phase extracts, regardless of the source of labelled probe (395-bp G217B yps 21:E-9 probe or 512-bp HindIII-EcoRI-labelled Downs yps21:E-9). A decanucleotide motif, TCCTTTTTTT, was identified in the upstream regulatory regions of these yps genes, as well as in the putative alpha-tubulin promoter, and was conserved with 70 to 100% homology. This recognition sequence was sufficient for p30M binding with 32P-labelled ligated oligonucleotides when used in the Southwestern assay. These findings describe the first nuclear DNA binding factor identified in H. capsulatum which binds to target sequences in a phase-specific manner, suggesting that p30M may govern aspects of gene transcription in this pathogenic fungus, in which a temperature-sensitive switch influences morphology and virulence.
在双态真菌病原体荚膜组织胞浆菌的寄生酵母（Y）阶段表达而在菌丝体（M）阶段转录沉默的基因最近已被克隆和分析。为了了解参与向Y阶段转变和维持Y阶段的基因的分子调控，两个Y阶段特异性基因（yps - 3和yps 21:E - 9）的假定5'调控区域被PCR扩增为标记探针，以鉴定可能影响阶段特异性基因转录的核DNA结合蛋白。通过蛋白质印迹分析评估蛋白质 - DNA相互作用，其中来自荚膜组织胞浆菌强毒株G217B的Y和M阶段的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分离的蛋白质提取物通过其原位结合标记的517bp（G217B yps - 3）或395bp（G217B yps 21:E - 9）假定5'调控区域的能力而可视化。鉴定出一种30kDa的核蛋白，它是高毒力G217B菌株M阶段提取物所特有的，但在同一生物体的Y阶段不存在。相反，荚膜组织胞浆菌的低毒力、温度敏感的唐斯菌株在酵母或菌丝体阶段提取物中均缺乏可检测到的p30结合活性，无论标记探针的来源如何（395bp G217B yps 21:E - 9探针或512bp HindIII - EcoRI标记的唐斯yps21:E - 9）。在这些yps基因的上游调控区域以及假定的α - 微管蛋白启动子中鉴定出一个十核苷酸基序TCCTTTTTTT，其同源性为70%至100%。当用于蛋白质印迹分析时，该识别序列足以使p30M与32P标记的连接寡核苷酸结合。这些发现描述了在荚膜组织胞浆菌中鉴定出的第一个以阶段特异性方式与靶序列结合的核DNA结合因子，表明p30M可能控制这种致病真菌中的基因转录方面，其中温度敏感开关影响形态和毒力。
