Biaoru Li

Regulation of Sexual Dimorphism: Mutational and Chemogenetic Analysis of the Doublesex DM Domain

ABSTRACT Doublesex (dsx) is a transcription factor in Drosophila that regulates somatic sexual differentiation. Male- and female-specific splicing isoforms of DSX share a novel DNA-binding domain, designated the DM motif. Broadly conserved among metazoan sex-determining factors, the DM domain contains a nonclassical zinc module and binds in the DNA minor groove. Here, we characterize the DM motif by site-directed and random mutagenesis using a yeast one-hybrid (Y1H) system and extend this analysis by chemogenetic complementation in vitro. The Y1H system is based on a sex-specific Drosophila enhancer element and validated through studies of intersexual dsx mutations. We demonstrate that the eight motif-specific histidines and cysteines engaged in zinc coordination are each critical and cannot be interchanged; folding also requires conserved aliphatic side chains in the hydrophobic core. Mutations that impair DNA binding tend to occur at conserved positions, whereas neutral substitutions occur at nonconserved sites. Evidence for a specific salt bridge between a conserved lysine and the DNA backbone is obtained through the synthesis of nonstandard protein and DNA analogs. Together, these results provide molecular links between the structure of the DM domain and its function in the regulation of sexual dimorphism.

Experimental and bioinformatics comparison of gene expression between T cells from TIL of liver cancer and T cells from UniGene.

Background. The major difficulty of mapping parallel gene expression obtained from solid tumors is mainly due to contaminating cells. In this study, by applying a strategy of parallel gene expression at a cell-cluster or colony level, we have identified the gene expression pattern of T cells within tumor-infiltrating lymphocytes (TILs) obtained from two liver cancer patients. Methods. Here a new method was utilized to analyze the parallel gene expression. By using bioinformatics analysis, the data were also compared with T-cell gene expression present in UniGene. Results. Our results demonstrated that 18 genes in specimen A and 13 genes in specimen B were highly expressed after the removal of a nonspecific TIL cDNA library, by pairing gene hybridization; the genes were expressed in CD3+ cells from peripheral blood mononuclear cells (PBMC). By using BlastN search, 17 of the 18, and 12 of the 13 sequences were exhibited, respectively, in Homo sapiens, with a range of BlastN E values of 0 to 4 × 10−13. The LocusLink distribution in chromosomes obtained from both specimens was not significantly different; 17 of 19 putative genes (both specimen A and specimen B) were observed in the UniGene cluster in Homo sapiens, except for dihydropyrimidinase-related protein-3 and diacyglycerol kinase alpha. Interestingly, only 4 of 19 (21%) putative genes were displayed in the T-cell UniGene database (i.e., LD-78 in Hs. 73817, IL-8 in Hs. 624, TRAIL in Hs. 83429, and Fas ligand in Hs. 2007). Conclusions. By comparison with the reported data and UniGene, the parallel gene expression of T cells obtained from TIL can provide essential new insights into T-cell activity, T-cell extravasation into tumor tissues, and T-cell cytotoxicity against tumor cells.

Genomic expression analysis by single-cell mRNA differential display of quiescent CD8 T cells from tumour-infiltrating lymphocytes obtained from in vivo liver tumours

We performed a genomic study combining single‐cell mRNA differential display and RNA subtractive hybridization to elucidate CD8 T‐cell quiescence/ignorance. By comparing actively maintained quiescent CD8 T cells from liver tumour tumour‐infiltrating lymphocytes (TILs) with quiescent T cells at the single‐cell level, we identified differentially expressed candidate genes by high‐throughput screening and comparative analysis of expressed sequence tags (ESTs). While genes for the T‐cell receptor, tumour necrosis factor (TNF) receptor, TNF‐related apoptpsis inducing ligand (TRAIL) and perforin were down‐regulated, key genes such as Tob, transforming growth factor (TGF)‐β, lung Krüpple‐like factor (LKLF), Sno‐A, Ski, Myc, Ets‐2 repressor factor (ERF) and RE1‐silencing transcription factor (REST/NRSF) complex were highly expressed in the quiescent TIL CD8 cells. Real‐time polymerase chain reaction (PCR) further confirmed these results. A regulation model is proposed for actively maintained quiescence in CD8 T cells, including three components: up‐regulation of the TGF‐β pathway, a shift in the MYC web and inhibition of the cell cycle.

Expression, crystallization and preliminary X-ray characterization of the Drosophila transcription factor Doublesex.

Doublesex (DSX) is a transcription factor in Drosophila melanogaster that regulates somatic sexual differentiation. Homologues have been found in diverse metazoans, suggesting a conserved role in development. Here, the expression, purification and crystallization of a novel 63-amino-acid dimerization domain and selenomethionine analogue are described. Native crystals belong to space group P2(1)2(1)2(1) and diffract to a resolution of 1.6 A with synchrotron radiation. Selenomethionine-containing crystals provide anomalous dispersion for phasing.