Chung J D, Conner S, Stephanopoulos G
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, USA.
Cytometry. 1995 Aug 1;20(4):324-33. doi: 10.1002/cyto.990200408.
We report on 1) the development of a flow cytometry-based technique for detecting beta-galactosidase in differentiating cultures of Bacillus subtilis and 2) the application of this technique in the study of early developmental gene expression. The problems associated with generating detectable signals (despite the small size of B. subtilis cells) have been overcome using the fluorogenic substrate 5-octanolyaminofluorescein di-beta-D-galactopyranoside (C8-FDG). Additionally, to control for background fluorescence during the staining process, we included a control population in the C8-FDG staining mixture that consists of cells devoid of the lacZ gene prestained with another dye, PKH26. The distinct emission spectra of C8-fluorescein and PKH26 allow nonspecific C8-FDG staining in this control population to be monitored using two-color analysis. This technique has been applied in the study of developmental gene expression in sporulating cultures of B. subtilis, and it has been found that such cultures are heterogeneous, comprising two cell populations. One population is induced for expression of early sporulation genes, which is determined using lacZ fusions, whereas the other remains uninduced. These results have allowed us to understand better the patterns of gene expression exhibited by wild-type and mutant cultures early during the development process of spore formation.
1）一种基于流式细胞术的技术的开发，用于在枯草芽孢杆菌的分化培养物中检测β-半乳糖苷酶；2）该技术在早期发育基因表达研究中的应用。使用荧光底物5-辛酰氨基荧光素二-β-D-吡喃半乳糖苷（C8-FDG）克服了产生可检测信号的相关问题（尽管枯草芽孢杆菌细胞体积小）。此外，为了在染色过程中控制背景荧光，我们在C8-FDG染色混合物中加入了一个对照群体，该群体由用另一种染料PKH26预染的缺乏lacZ基因的细胞组成。C8-荧光素和PKH26的不同发射光谱使得可以使用双色分析监测该对照群体中的非特异性C8-FDG染色。该技术已应用于枯草芽孢杆菌芽孢形成培养物中的发育基因表达研究，并且发现此类培养物是异质的，由两个细胞群体组成。一个群体被诱导表达早期芽孢形成基因，这是通过使用lacZ融合来确定的，而另一个群体仍未被诱导。这些结果使我们能够更好地理解野生型和突变型培养物在孢子形成发育过程早期所表现出的基因表达模式。
