心脏神经脊衍生物表达转录因子2(Hand2)是bHLH家族的成员,也称为dHand、Hed和Thing2,可在人以及鼠、鸡、斑马鱼、果蝇等模式动物中表达.国内外的研究显示,Hand2主要影响右心室的发育,在维持右心室祖细胞和右心室形态发生中起关键作用.为了进一步研究Hand2基因参与心脏发育的作用机制,拟通过CRISPR/Cas9基因编辑技术构建斑马鱼Hand2基因敲除品系.根据生物信息学网站的分析筛选出最优的两个靶位点,设计并合成引物,利用PCR扩增得到Hand2基因sgRNA的cDNA,再转录为mRNA,将两个靶位点sgRNA的mRNA和Cas9蛋白共同注射到斑马鱼胚胎的Ⅰ-细胞期.在斑马鱼的胚胎和成鱼时期进行基因敲除的有效性检测,发现在靶位点附近有碱基的缺失和插入,表明CRISPR/Cas9对斑马鱼Hand2基因敲除是有效的.本研究利用CRISPR/Cas9基因编辑技术构建了斑马鱼的Hand2基因敲除模型,为后期研究Hand2基因在人类疾病中的作用机制和新型治疗靶点的临床开发等奠定了基础.

TRAF2 with E3 ligase activity is an important protein in immunity and inflammation.GFP was often expressed in fusion protein for co-localizing the distribution of target proteins.We found reduced green fluorescence in pCMV-myc-TRAF2 co-transfected cells.The increased degradation of GFP by TRAF2 was detected by Western blotting,and further experiment showed that the degradation was through the proteasome pathway.By co-transfecting with pCMV-myc-TRAF2 plus pEGFP-C3-LNX,we showed a TRAF2-related reduction of GFP-LNX expression,but not LNX.These results suggested that the degradation of GFP promoted by TRAF2 appeared to be specific.As the GFP is a foreign protein to human cells,we postulated that TRAF2 might participate in the cellular resistance of pathogenic infections.

Objective To explore the relationship between the expression of gliomas amplified sequence 41(GAS41) and grading of human glioma.Methods Thirty fresh gliomas specimens of different grades were collected.The expression of GAS41 was detected by immunohistochemical staining.Results The expression of GAS41 in the high-grade gliomas specimens(grade III,grade IV)was significantly higher than that in the low-grade(grade I,grade II)(P<0.05).Conclusion The expression of GAS41 in gliomas is enhanced with the increasing degree of malignancy,and it is suggested that the expression of GAS41 may be used as a judging criterion of the high grade gliomas.

The change of precipitation in Beijing- Tianjin- Hebei Province since recent 47 years was analyzed by using linear regression, abrupt change analysis and wavelet analysis. The results showed that annual precipi-tation decreased at a climatic tendency of -15mm/10a. The 11 -year running mean curve showed that trend will continue in the future. Precipitations in summer were decreasing in most of the areas, while increasing in the other seasons. The abrupt change occurred in summer during the early 1990s. Generally, both annual precipitati-on and seasonal precipitation presented three kinds of periodic change.

Human stomatin like protein 2(SLP-2) is a novel cancer-related gene.Its protein is overexpressed in many human cancers,such as esophageal squamous cell carcinoma and lung cancer.In Breast cancer,high-level SLP-2 expression were associated with significantly decreased survival.To study the function of SLP-2 in these cancers,the coding region of human SLP-2 gene was successfully subcloned into the eukaryotic green fluorescent protein(GFP) expressing recombinant plasmids,pEGFP-C3.The vector was then transfected into MCF-7 cells for efficient expression.Moreover, results from fluorescent observation shows that GFP-SLP-2 mainly localized in cytoplasma,thus providing a useful clue for further research on the function of SLP-2 in breast cancer.

Using the 1960—2005 data of annual precipitation and evaporation,the spatial distribution of regional drought in Hu’nan Province is analyzed by linear regression,analyses of wavelet,abrupt change,clusters,and EOF and REOF.The results of an analysis of evaporation precipitation ratio data shows that there are four high-value areas and three low-value areas in Hu’nan Province.The spatial variations of Hunan Province are showing a wetness trend in all but its western and eastern areas.The most typical pattern of annual precipitation is a dry-wet consistency across the province.Other typical patterns include dry-wet differences,e.g.wet(or dry) in the north versus dry(or wet) in the south,wet(or dry) in the center and dry(or wet) in surrounding areas.The distribution of evaporation and precipitation ratio exhibits both common features as well as spatial differences,which can be classified into four types of South Hu’nan,North Hu’nan,Northeast Hu’nan and Central Hu’nan.There is an abrupt change from dry to wet in the early 1990s.Generally,the drought-flood distribution presents variations of three periods.In the late 2000s,Hu’nan Province will be in a period of drought,followed by a period of flood.

AP-2β is an important member of transcription factor AP-2 family.AP-2 is a transcription factor implicated in mammalian development,cell proliferation,apoptosis and carcinogenesis.Recent studies have shown that interaction partners can modulate the transcriptional activity of AP-2 over the downstream targets.To identify potential AP-2 β interacting partners,we screened AP-2 β immunocomplexes by mass spectrometry and identified SLP-2 as a putative AP-2 β interacting protein.It is demonstrated that both exogenously expressed and endogenous SLP-2 interacted with AP-2 β in vivo by co-immunoprecipitation assays.In addition,indirect immunofluorescent analyses demonstrated that AP-2 β and SLP-2 co-localized in the cytoplasm of the MCF-7 cell lines.Furthermore,over-expression of SLP-2 enhanced the protein expression level of AP-2 β.Thus,through protein-protein interaction,SLP-2 can stabilize AP-2 β.These results provided a new clue for further research on the AP-2β and SLP-2 function.

The change of precipitation at 84 meteorological stations in Beijing,Tianjin and Hebei in recent 47 years was analyzed by using linear regression,abrupt change analysis and wavelet analysis in this article.The results showed that annual precipitation decreased at a climatic tendency of-15 mm/10 a,and the 11-year running mean curve indicated the trend would continue in the future.Precipitation in summer was decreasing in most of the areas,while increasing in the other seasons.The abrupt change occurred in summer precipitation during the early 1990s.Generally,both annual and seasonal precipitation presented four periodic change of three,nine,fourteen and twenty-four years.

Dnd1 is an evolutionary conserved RNA binding protein(RBP) known as a mouse testicular germ cell tumor(TGCT) susceptible protein.To understand its subcellular localization in mammalian cells in connection with cell proliferation and cell cycle,we transfected the recombinant plasmid pEGFP-Dnd1 into HeLa or GC-1 cell lines and detected its subcellular localization by fluorescent microscopy.The results were compared with the predicted subcellular localization of Dnd1 using several softwares,such as Target P,Subloc V,and WOLFPSORT.The cell growth curve and cell cycle analysis of Dnd1-transfected HeLa cells were determined by MTT assay and flow cytometry,and the effect of Dnd1 on AP-1 transcription activity was also assayed.We found that EGFP-Dnd1 mainly located in the nuclear and little in the cytoplasma in both HeLa and GC-1 cells,which is consistent with the bioinformatic predictions.Dnd1 inhibited cell proliferation and led to cell cycle arrest at G_1/S phase in HeLa cells,with down-regulated transcription activity of AP-1.Our work may have important implication for further studies of Dnd1 function.

The gene ZNF256 belongs to C2H2 Zinc finger protein families in which most of the proteins are transcriptional factors,and they are involved in cell differentiation,embryonic and heart development,sperm formation and cancer etc.In this paper,the coding region of human ZNF256 was cloned,the expression profile of ZNF256 gene was studied in different cell lines and tissues by using real-time PCR.ZNF256 protein was located in the nucleus with laser confocal microscopy.Luciferase assay indicated that ZNF256 could inhibit the transcriptional activity of AP1,suggesting that ZNF256 was a transcriptional inhibitor.Our results provided a basis for further investigating the function of human ZNF256 in embryo development and the related diseases occurring.

The change of drought and flood in Dongting Lake Basin since recent 47years is analyzed using abrupt change analysis,wavelet analysis,coefficient of variance,EOF and REOF.The results showed that the 1990s were in flood periods,the 1960s and the 1980s were in drought period.There is a decreasing trend entering the 21st century,and there is an abrupt change from dry to wet 1980s.Generally,the Z Index presented variations of four periods.In the late 2000's,Dongting Lake Basin will be in a period of drought,after then will be in a period of flood.The most typical pattern of Z Index is the drought-flood consistence.The Z Index abnormal area can be divided into four:northern,the central,southern,northwestern.

To study the interaction mechanism between transcription factor AP-2α and TES protein,prokaryotic vectors for expressing GST-AP-2α and His-TES were constructed respectively.Fusion proteins were induced in E.coli BL 21 and then were analyzed with GST pull-down assay.Results show that the interaction between TES and AP-2α is due to AP-2α C-terminus binding with the first LIM domain of TES C-terminus.These results were also confirmed in yeast two-hybrid system.Furthermore,a possible self-interaction of TES protein,which could block the binding site for AP-2α in the first LIM domain of TES,is suggested,based on the fact that AP-2α interacted with TES C-terminus instead of TES full-length protein.

Objective To construct a bait vector containing mouse Dnd1 gene in yeast two-hybrid sistem in order to screen the cDNA library of mouse 10.5d embryo.Methods The DNA fragment of coding Dnd1 was amplified by PCR with designed primer from the plasmid of pcDNA3.1-Dnd1 and then cloned into pMD18-T vector.After verified with restriction endonuclease digestion of SalⅠand NotⅠ,the right gragment of Dnd1 gene determined by sequencing was subcloned into the bait vector of pDBLeu.Then right fragment of recombinant plasmid pDBLeu-Dnd1 tested by both the same endonuclease and sequence analysis was transformed into the yeast cell MaV203 and its self-activation was tested.Results The pDBLeu-Dnd1 plasmid was successfully constructed and had no autonomous activation.Conclusion The bait plasmid of pDBLeu-Dnd1 was proved to be suitable for the further research on the interactors with DND1 in the yeast two-hybrid sestem.

小鼠睾丸生殖细胞瘤(testicular germ cell tumour,TGCT)易感基因Dnd1属于RNA结合蛋白基因家族成员.Dnd1在进化中具有保守性,Dnd1缺乏导致小鼠原始生殖细胞(primordial germ cell,PGC)的丧失、TGCT的发生和部分胚胎死亡,但Dnd1作用机制还未明了.利用酵母双杂交系统,构建小鼠Dnd1基因诱饵载体pDBLeu-Dnd1,筛选10.5 d的小鼠胚胎cDNA文库,成功筛选到4个与Dnd1相互作用蛋白,其中之一为原癌基因Jun蛋白;进一步的酵母双杂交实验和GST pull down实验再次确认Dnd1与Jun蛋白之间的相互作用.研究发现Dnd1新的相互作用蛋白,这为探明其信号传导通路及Dnd1致病机制研究建立了基础.

To Clone the N terminal gene of mouse Shh protein,we extract the total RNA from the pregnant mouse embryo of 10.5 d. N terminal gene of Shh protein was amplified using RT-PCR and inserted into the prokaryotic expression vector pQEN3. The recombinant vector pQEN3-Shh-N was transformed into E.coli BL21,and induced with 1.0 mmol/L IPTG. A 20 kD protein was detected on the SDS-PAGE and identified by western blot,it showed that the 20 kD protein was the N terminal protein of Shh. The recombinant N terminal protein of Shh was up to thirty percent of the total protein,which provided a basis for studying the relationship between the hair growth and the N terminal protein of Shh.

In the post-genomic era, identifying and characterzing various DNA-protein interactions are a major challenge in the research of gene transcriptional regulation. Now, chromatin immunoprecipitation assay (ChIP) is ideally suited for investigating DNA-protein interactions in vivo. The conditions of chromatin immunoprecipitation assay was optimized and utilized to screen unknown target genes of transcription factor AP-2 alpha. The results yielded a more complete understanding of AP-2α-regulated signaling pathways and further elucidated the complex transcriptional networks.

Using 1960～2005 annual precipitation data over 86 stations of Hu′nan Province,temporal and spatial variability features are studied by applying the methods of REOF,wavelet analysis and M-K methods. The result indicates: (1) The most typical pattern of annual precipitation is the dry-wet consistency. Other typical patterns show the dry-wet difference. For example,it is wet(or dry)in the north and dry(or wet)in the south,wet(or dry)in the center and dry(or wet) in the surrounding area,wet(or dry)in the northeast and dry(or wet)in the southwest. (2) The annual precipitation distribution exhibits common features as well as spatial differences,which can be classified into six types as Southeast Hu′nan,Northwest Hu′nan,North Hu′nan,central part of Hu′nan,West Hu′nan,and Southwest Hu′nan. (3) There is an wetter trend over Hu′nan Province in the past 46 years. Generally,annual precipitation presents an upward tendency and the periods of 4a,7a,10～12a and 24a. The abrupt change occurred about in the 1990s.

Human gene HTR1E is a member of HTR gene family, and all of the members in this family are one of the factors that lead to schizophrenia, depression and suicide. But the mechanism leaded to these diseases is unclear. The expression profile of HTR1E gene was analyzed in different cell lines and tissues with Real-time PCR. The HTR1E was found to be located at plasmalemma. The luciferase assay indicated that HTR1E could inhibit the transcription activity of the promoter of erbB2. The results in the communication provided a basis for further investigating the function of HTR1E gene.

In order to express three tandem LIM domains in the C-terminus of TES individuallyin Escherichia coli BL21,three gene fragments encoding corresponding LIM domains in TESC-terminus were amplified from pCMV-HA-TES plasmid containing the full length of TES coding sequence by PCR and cloned into pGEM-T vector respectively.The target gene fragments obtained by digesting the recombinant plasmids with restriction endonuclease were subcloned into prokaryotic expression vector pQE-N1 for fusion expression.The results of restriction endonuclease digestion and sequence analysis indicated that three recombinant plasmids for expression of corresponding LIM domains in TES C-terminus were successfully constructed.The fusion proteins with the molecular weights as prospected were separated by SDS-PAGE gel after induced expression.

KCTD10 is a TNF-alpha inducible protein that can interact with the small subunit of DNA polymerase a and PCNA. In order to study the function of KCTD10, we prepared the rabbit anti-mouse KCTD10 polyclonal antibody by using the His-tagged recombinant mouse KCTD10 protein to immune New Zealand white rabbit. Mouse KCTD10 shares significant similarity with PDIP1 (polymerase delta-interacting protein 1) and TNFAIP1 (tumor necrosis factor alpha-induced protein 1) protein,and then KCTD10 polyclonal antiserum possesses cross-reactivity with PDIP1 protein and TNFAIP1 protein. The partially digested fragments of homogeneous proteins PDIP1 and TNFAIP1 were mixed and incubated with anti-KCTD10 antiserum at 4 degrees C for 3 h to deplete unspecific antibodies. Through this method, we removed successfully the cross-reactivity of anti-KCTD10 antibody with PDIP1 and TNFAIP1 and obtained specific anti-KCTD10 antibody. Then, the anti-KCTD10 antibody was used in immunohistochemistry experiments of mouse. The results of immunohistochemistry on whole-mount embryo and paraffin section demonstrated that KCTD10 is highly expressed in neuroepithelium of neural tube and dorsal root ganglion of 12.5 d embryos. These results suggest that KCTD10 may play roles in the development of neuroepithelium of neural tube and dorsal root ganglion.