人同源异型框Csx及其变体的克隆、在大肠杆菌中的表达及其DNA结合性能研究

人心脏同源异型框基因Csx是同源异型框基因家族NK2的成员之一，也是心脏早期发育必须的转录因子之一。虽然目前关于Csx基因在发育中的作用及其与先天性心脏病的关系得到了广泛的关注，并研究得较为深入，然而Csx本身的结构与功能的关系仍不十分清楚，Csx包含了3个保守的结构域：TN结构域、同源异型框结构域和NK2-SD结构域。通过选择合适的酶切位点，构建了Csx的5个变体，分别缺失了不同的结构域，并将Csx及这5个变体克隆到pFT32系列载体中，将这6个构建好的质粒转化E．coli BL21(EE3)，在IPTG的诱导下，得到了Csx基因及5个变体在大肠杆菌中的可溶性表达．凝胶阻滞实验显示：同源异型框是Gx与DNA相互作用的区域，缺失了同源异型框结构域，Csx就无法和靶序列结合．其它结构域的缺失对Csx和其靶DNA序列的结合影响不大。     

Direct homeodomain-DNA interaction in the autoregulation of the fushi tarazu gene.

A major problem in the elucidation of the molecular mechanisms governing development is the distinction between direct and indirect regulatory interactions among developmental control genes. In vivo studies have indicated that the Drosophila segmentation gene fushi tarazu (ftz) directly or indirectly autoregulates its expression. Here we describe a generally applicable experimental approach which establishes a direct in vivo interaction of the homeodomain protein ftz with the ftz cis-autoregulatory control region. In vitro studies have shown that the DNA-binding specificity of the ftz homeodomain can be changed by a single amino-acid substitution in the recognition helix (Gln 50----Lys). Whereas wild-type ftz homeodomain binds preferentially to a CCATTA motif, the mutant homeodomain (ftzQ50K) recognizes a GGATTA motif. We now find that the in vivo activity of an ftz autoregulatory enhancer element is reduced by mutations of putative ftz-binding sites to GGATTA. This down-regulatory effect is specifically suppressed in vivo by the DNA-binding specificity mutant ftzQ50K. These results establish a direct positive autoregulatory feedback mechanism in the regulation of this homeobox gene.     

High expression of a rice homeobox inE. coli

Homeotic genes share a characteristic DNA segment, the homeobox, which encodes a defined domain of the homeotic protein-homeodomain which seems to mediate the binding to specific DNA sequences, whereby the homeotic protein exerts a gene regulatory function. In this study, the homeodomain encoded by the OSIH-1 (designated fromOryza sativa-Indica homeobox-1) homeobox of rice was overproduced in a expression vector inE. coli, as a form of inclusion body and analyzed by Western blotting and crossed-immunoreaction. Crossed-immunoreaction studies among OSIH-1, Kn-1 and Quox-1 indicate all three are homologous at the protein level. The OSIH-1 homeodomain is then to identify the homeotic target genes. Foundation item: Supported by the National Natural Science Foundation of China (#39570370) Biography: CHENG Xing-guo (1973-), male, Graduate student.     

Evidence from mosaic analysis of the masculinizing gene her-1 for cell interactions in C. elegans sex determination.

Sex in Caenorhabditis elegans is determined by a regulatory cascade of seven interacting autosomal genes controlled by three X-linked genes in response to the X chromosome-to-autosome (X/A) ratio. XX animals (high X/A) develop as self-fertile hermaphrodites, and XO animals (low X/A) develop as males. The activity of the first gene in the sex-determining cascade, her-1, is required for male sexual development. XO her-1 loss-of-function mutants develop as self-fertile hermaphrodites, whereas XX her-1 gain-of-function mutants develop as masculinized intersexes. By genetic mosaic analysis using a fused free duplication linking her-1 to a cell-autonomous marker gene, we show here that her-1 expression in a sexually dimorphic cell is neither necessary nor sufficient for that cell to adopt a male fate. Our results suggest that her-1 is expressed in many, possibly all, cells and that its gene product can function non-autonomously through cell interactions to determine male sexual development.