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Forestry Studies in China 2008年第2期10卷 88-94页

作者： HUANG Xin ZHENG Guo-sheng DAI Si-lan GAI Shu-peng College of Landscape Architecture Beijing Forestry University Beijing 100083 P. R. China College of Life Science Qingdao Agricultural University Qingdao 266109 P. R. China

A subtractive cDNA library was developed to study genes associated with bud dormancy release in tree peonies. In order to identify genes that are highly expressed in buds released from dormancy, 588 clones were examined by differential screening. Of these, 185 clones were selected to be sequenced. A total of 37 unique sequences were obtained of which only 31 sequences have matches in the NCBI database or the Arabidopsis thaliana protein database. Semi-quantitative RT-PCR was used to confirm further the expression profiles for 12 transcripts identified within the subtractive cDNA library. Gene ontology analyses indicated that many of the different genes identified have unknown or hypothetical functions while it is speculated that other genes play different mo- lecular roles. In our study, genes involved in bud dormancy release were growth-related or stress-responsive, while low-temperature-induced ribosomal proteins may also play a role in bud dormancy release. Our results provide interesting information for further understanding of the molecular mechanism of bud dormancy release in tree peonies.

关键词： cDNA clone dormancy subtractive hybridization tree peony （Paeonia suffruticosa）

subtractive suppression hybridization combined with cDNA arrayScreening identifies differentially expressed genes between Micro- nucleates and amicronucleates of Pseudourostyla cristata

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Subtractive suppression hybridization combined with cDNA arr...

The ethanol response gene Cab45 can modulate the impairment elicited by ethanol and ultraviolet in PC12 cells

中国动物学会原生动物学分会第十二次学术讨论会

作者： Xinying Liu, Junjian Wang and Lipei Jin (Life School , Zhongshan University, Guangzhou, PRC 510275)

＜正＞ Based on morphologically comparative studies on amicronucleates and micronucleates of Pseudourostyla cristata, we supposed that the micronucleus absence may result in abnormal development of the subsurface cytoskeleton and/or other supportive elements in the vicinity of the AZM. To further provide insights into how micronucleus of P. cristata to regulate oral development, we employed suppression subtractive hybridization （SSH） and cDNA array to identify genes which express in micronucleate cells but not in amicronucleate cells.

关键词： subtractive hybridization Amicronucleates Micronucleates cDNA array

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Journal of Genetics and Genomics 2008年第3期35卷 153-161页

作者： Yunfeng Zhu Quanli Wang Wangru Xu Sha Li Department of Oncology in Beijing 307 Hospital Fengtai District Beijing 100071 China Department of Transfusion in Beijing 307 Hospital Fengtai District Beijing 100071 China

High consumption of ethanolic beverages facilitates neurodegeneration, but the mechanism of this process still remained elusive. Suppression subtractive hybridization （SSH） is a technique for detection of rare transcripts. With SSH approach, we identified one ethanol response gene Cab45, which was down-regulated by ethanol with time-dependent manner in B104 cells. The full-length sequence of Cab45 gene was obtained by 5＇-RACE （5＇Rapid Amplification of cDNA Ends） for the first time in rat. Based on the sequence of deduced amino acid of rat Cab45, the alignment was conducted with its counterparts in different species and displayed a high conservation. Using different tissues in rat and cell lines, Cab45 was characterized by a ubiquitous expression and differentiation dependent down-regulation. Given that ethanol facilitates some cell differentiation, we hypothesize that Cab45 is involved in ethanol-mediated differentiation. With transient transfection, the function of Cab45 was investigated by up-regulation and down-regulation in PC12 cells. Ethanol treatment and UV exposure were conducted subsequently and cell proliferations were detected by MTT （Methyl Thiazolyl Tetrazolium） approach. It revealed that the up-regulation of Cab45 modulated the impairment elicited by ethanol and UV in transfected cells. As a member of new calcium binding protein family, the exact role of Cab45 still remains unclear.

关键词： ethanol Cab45 calcium-binding protein gene identification subtractive hybridization

Cloning the full-length cDNA of a gene responsible for vascular smooth muscle cell proliferation in atherogenesis and study of function

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Chinese Medical Journal 2003年第2期116卷 166-170页

作者：赵高峰高鹏张华娄黎明乔绘红佘铭鹏 Department of Chest Surgery The First Hospital of Henan Medical University Department of Pathology Institute of Basic Medical SciencesChinese Academy of Medical SciencesPeking Union Medical College

To clone the full length cDNA of a gene responsible for vascular smooth muscle cell (v SMC) proliferation in atherogenesis, and study its function Methods Oxidized low density lipoprotein (ox LDL) at optimal concentration was used as the stimulant to induce v SMC proliferation in culture medium A cDNA subtractive library of v SMC proliferation specific to ox LDL stimulation was established using subtractive hybridization technique Methods, including blotting, Northern hybridization and gene sequencing, were used to clone new gene fragments By using full length cDNA screening and protein expression techniques, one full length cDNA was cloned and its function was studied Results One full length cDNA was cloned The new gene (Genbank AF 174647) expressed a 44 kDa protein, which might be associated with the activity of ox LDL Conclusion The new gene cloned may be associated with SMC proliferation in atherogenesis

关键词： subtractive hybridization associated gene cDNA whole length functional study

An Integrated Technique for Identification of Differential Genes Expressed in Patients with Cancer

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2004年第6期24卷 578-580页

作者：李守新刘尚勤雷小妹 Department of Internal Medicine Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan 430030 China

Summary: To develop a method for identification of differential gene expression between different cell populations, several convenient techniques of molecular biology, including subtractive hybridization, suppression PCR, T/A cloning and sequencing, were used to identify genes expressed differentially in CD45+ and CD45-cells isolated from U266 cell line of multiple myeloma. Our results showed that the levels of abundant genes scale down 20 times through subtractive hybridization. Plasmid DNA from CD45+ cell clones was hybridized with forward or backward cDNA probes synthesized from CD45+ and CD45-cells, respectively. A few of differentially expressed genes reconfirmed by RT-PCR were identified from 500 expressed clones of CD45+ cells. It is concluded that a strategy for gene expression identification developed from conventional molecular biological methods can be used in different laboratories.

关键词： gene expression subtractive hybridization differential display

Transcriptional Analysis of Normal Human Fibroblast Responses to Microgravity Stress

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Genomics, Proteomics & Bioinformatics 2008年第1期6卷 29-41页

作者： Yongqing Liu Eugenia Wang Department of Biochemistry and Molecular Biology University of Louisville School of Medicine Louisville KY 40202 USA. Current address. James Graham Brown Cancer Center Department of Ophthalmology and Visual SciencesUniversity of Louisville School of Medicine Louisville KY 40202 USA.

To understand the molecular mechanism（s） of how spaceflight affects cellular signaling pathways, quiescent normal human WI-38 fibroblasts were flown on the STS-93 space shuttle mission. Subsequently, RNA samples from the spaceflown and ground-control cells were used to construct two cDNA libraries, which were then processed for suppression subtractive hybridization （SSH） to identify spaceflight-specific gene expression. The SSH data show that key genes related to oxidative stress, DNA repair, and fatty acid oxidation are activated by spaceflight, suggesting the induction of cellular oxidative stress. This is further substantiated by the up-regulation of neuregulin 1 and the calcium-binding protein calmodulin 2. Another obvious stress sign is that spaceflight evokes the Ras/mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling pathways, along with up-regulating several Gl-phase cell cycle traverse genes. Other genes showing upregulation of expression are involved in protein synthesis and pro-apoptosis, as well as pro-survival. Interactome analysis of functionally related genes shows that c-Myc is the ＂hub＂ for those genes showing significant changes. Hence, our results suggest that microgravity travel may impact changes in gene expression mostly associated with cellular stress signaling, directing cells to either apoptotic death or premature senescence.

关键词： microgravity apoptosis premature senescence oxidative stress subtractive hybridization