肿瘤坏死因子-α基因启动子-850位多态性与强直性脊柱炎相关性研究(病人和13个家族调查)

目的探讨肿瘤坏死因子-α(TNF-α)的基因启动子-850位点多态性对强直性脊柱炎(AS)易感性和临床表现的影响。方法采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法,对33名AS患者、13个家庭的AS患者一级亲属39名以及187名正常对照者的TNF-α基因启动子-850位点进行基因分型,并分析基因型与疾病临床表现之间的关系。结果经病例-对照研究,TT基因型在AS组中的分布显著高于正常对照组(15.2%vs2.1%,P<0.01);T等位基因携带者在AS组与正常对照组间分布存在显著差异(75.8%vs21.4%,P<0.01);家庭调查也发现在AS一级亲属中T等位基因携带者显著高于正常对照组(64.1%vs21.4%,P<0.01);TX(CT+TT)基因型与CC基因型的AS患者在病程、临床症状、B27阳性率方面无明显差异;CT分级方面,TX基因型AS患者的骶髂关节CT分级≥Ⅲ级者的比例较CC基因型AS患者高(60%vs37.5%,P>0.05);但在ESR、CRP方面,TX基因型AS患者较CC基因型AS患者明显升高(分别为80%vs37.5%;56%vs12.5%。P<0.05)。结论TNF-α基因启动子-850C→T的突变可能是AS发生的新易感基因。与AS的炎症严重程度也可能存在关联。

胰岛素启动子因子1基因P239Q突变与中国汉族人2型糖尿病易感性的关系

【目的】了解胰岛素启动子因子 1基因P2 39Q突变在中国汉族人中的分布情况 ,以探讨其在 2型糖尿病的发生发展中的作用。【方法】用PCR RFLP对 2 33例 2型糖尿病患者及 89例非糖尿病健康志愿者进行该点突变的筛查 ,对检出阳性者进行家系调查。【结果】病人组中仅发现 1例突变基因携带者 ,对照组均为阴性 ,家系研究显示该点突变不与糖尿病共分离。【结论】该点突变在中国汉族人 (包括 2型糖尿病患者 )中相当少见。该突变不与糖尿病共分离 ,且在非糖尿病者中亦可被检出 ,推测其可能作为一种修饰基因 ,与其它位点的突变共同作用 ,促进糖尿病发生。

十二指肠溃疡启动因子基因A的研究进展

幽门螺杆菌（Hp）是一种革兰阴性螺旋形细菌,可以通过感染胃黏膜,介导炎症反应,引起慢性胃炎、消化性溃疡、胃癌等多种消化道疾病.流行病学调查显示,虽然胃癌发病率不断下降,但它仍是全球第四常见的癌症.然而,在南非和南亚这两个Hp高感染区,其胃癌发病率却远远低于其他国家,这种现象被称为非洲谜和亚洲谜[1].现在普遍认为,该现象除与饮食、环境因素有关外,Hp的毒力因子也发挥了重要作用.Hp是一种高度异质性的细菌,约有1 600个基因,其中5％ ～ 10％是种属特异性的.关键词：十二指肠溃疡;胃癌;十二指肠溃疡启动因子基因A;免疫分类号：R378.3

溃疡性结肠炎患者TNF-α基因启动子-308G/A单核苷酸多态性观察

目的分析溃疡性结肠炎(UC)患者肿瘤坏死因子-α(TNF-α)基因启动子-308G/A单核苷酸多态性。方法 UC患者750例(观察组),健康体检者750例(对照组),采用聚合酶链反应-限制性片段长度多态性(PCRRFLP)技术分析两组TNF-α基因启动子-308G/A单核苷酸多态性;ELISA法检测两组血浆TNF-α。结果观察组GG、GA、AA基因型频率及G、A等位基因频率分别为14.93%、42.27%、42.80%、36.07、63.93%,对照组分别为67.73%、16.40%、15.87%、75.93、24.07%,两组比较,P均<0.05。观察组及对照组血浆TNF-α水平分别为(31.48±11.52)、(14.82±5.61)pg/L,两组比较,P<0.05。观察组GG、GA、AA基因型携带者TNF-α水平分别为(17.95±7.82)、(34.32±0.75)、(34.64±13.83)pg/L,对照组分别为(10.63±3.29)、(21.25±10.47)、(21.91±8.51)pg/L,GA、AA基因型与同组GG基因型携带者比较,两组GG、GA、AA基因型携带者比较,P均<0.05。结论UC患者TNF-α基因启动子-308G/A存在单核苷酸多态性,GA、AA基因型是UC的易患因素,这可能与两基因型可提高患者血浆TNF-α水平密切相关。

IFN-γ基因多态性与小儿哮喘的相关性

目的探讨IFN-γ基因多态性、等位基因在儿童哮喘发病机理中的意义。方法采用PCR-SSP方法对30例小儿哮喘病儿及26例非哮喘呼吸道疾病患儿作IFN-γ基因多态性分析。结果30例哮喘组IFN-γ基因型中,T/T1例,T/A7例,A/A22例,26例非哮喘组IFN-γ基因型中,T/T7例,T/A11例,A/A8例。χ2=11.696,P<0.005,两组IFN-γ基因型有明显差异。哮喘组IFN-γ等位基因T和A的频率为15%和85%,非哮喘组IFN-γ等位基因T和A的频率为51.9%和48.1%,哮喘组IFN-γ等位基因A的频率比非哮喘组IFN-γ等位基因A高,相对危险度0.191,95%可信度区间0.078-0.466,χ2=14.416,P<0.005,两组IFN-γ等位基因频率有明显差异。结论本研究证实小儿哮喘易感性与IFN-γ基因启动子多态性相关联。

猪源转录因子AP-2δ通过增强rep基因启动子活性促进猪圆环病毒2型的复制

本研究旨在探究猪圆环病毒2型(Porcine circovirus type 2, PCV2)能否借助rep基因启动子区的转录因子结合位点调控自身基因转录,并寻找参与这一调控过程的宿主因子。凝胶迁移实验证实rep基因启动子具有核蛋白结合活性。DNA-pull down联合液相-串联质谱分析鉴定到了可与rep基因启动子结合的猪源转录因子AP-2δ(Porcine transcription factor AP-2δ, poTFAP2δ)。双荧光素酶报告基因试验、实时荧光定量PCR、免疫印迹及间接免疫荧光等试验证明poTFAP2δ不仅可以特异性地增强rep基因启动子活性,而且可以在PCV2感染过程中促进rep和cap基因的转录、翻译及PCV2病毒滴度。文中揭示了PCV2利用宿主蛋白促进自身增殖的分子机制,为进一步从病毒与宿主互作角度阐明PCV2的致病机制提供新的研究思路,亦为PCV2高效疫苗的研制提供理论基础。

Isolation and Analysis of Rubber Hevein Gene and Its Promoter Sequence

Hevein, a lectin_like protein, is a major factor of lutoids in the latex of rubber trees ( Hevea brasiliensis Muell._Arg.). This factor is involved in coagulation of the latex and has the ability to bind chitin. The hevein gene with a length of 680 bp was cloned by the method of RT_PCR. Its promoter region with 1 306 bp of this gene was also isolated by genome walking, and its sequence included the typical TATA and CAAT boxes as well as the homologous sequence of abscisic acid (ABA) response elements. Expression of the hevein gene in the latex and leaves was detected by Northern blot. After treatment of the trees with ethylene and ABA, the results showed that the hevein gene was expressed principally in latex, and the expression could be induced by ethylene and ABA.

Isolation of a Genomic DNA for Gastrodia Antifungal Protein and Analyses of Its Promoter in Transgenic Tobacco

A genomic DNA containing 5'-upstream region and complete open reading frame of a Gastrodia antifungal protein was isolated by screening of a genomic library from Gastrodia elata B1. To investigate the promoter activity, the 5'-flanking region - 1 157 lip upstream from the putative transcription start site was fused to the coding sequence of beta-glucuronidase (GUS) gene and transformed into Nicotiana tabacum. The strongest GUS activity was detected in the roots of transgenic tobacco, followed by stems. The leaves only showed a low GUS activity. Furthermore, the promoter established inducible expression pattern in transgenic tobacco upon fungus Trichoderma viride inoculation and jasmonic acid and salicylic acid treatments.

PEG-mediated Transformation of Lentinus edodes

Expression vector p301-bG1 contains a Sw gene and a bialaphos resistance gene both driven by glyceraldehydes-3-phosphate dehydrogenase (GPD) gene promoter isolated from Lentinus edodes ( Berk.) Sing. Using p301-bG1, PEG-mediated transformation of protoplast of L. edodes was studied. Mixed with PEG-purified plasmid DNA, the protoplasts of L. edodes were treated with PEG solution and cultured on CYM regeneration plate containing 40 mug/mL bialaphos. Bialaphos-resistant and GUS-positive transformants were obtained using this transformation system. Although the transformation efficiency was relatively low, the protocols release large expenses on expensive instrument and restriction enzymes, providing a simple and economical method for mushroom breeding at the molecular level.

Gilbert's syndrome: High frequency of the (TA)_7 TAA allele in India and its interaction with a novel CAT insertion in promoter of the gene for bilirubin UDP-glucuronosyltransferase 1 gene

AIM： To identify the variants in U rase 1 （UGT1A1） gene in Gilbert＇s syndrome （GS） and to estimate the association between homozygosity for TA insertion and GS in India, as well as the frequency of TA insertion and its impact among normal controls in India. METHODS： Ninety-five GS cases and 95 normal controls were selected. Liver function and other tests were done. The promoter and all 5 exons of UGT1A1 gene were resequenced. Functional assessment of a novel trinucleotide insertion was done by in silico analysis and by estimating UGT1A1 promoter activity carried out by ludferase reporter assay of appropriate constructs in Hep G2 cell line. RESULTS： Among the GS patients, 80% were homozygous for the TA insertion, which was several-fold higher than reports from other ethnic groups. The mean UCB level was elevated among individuals with only one copy of this insertion, which was not significantly different from those with two copies. Many new DNA variants in UGT1A1 gene were discovered, including a trinucleotide （CAT） insertion in the promoter found in a subset （10%） of GS patients, but not among normal controls. In-silico analysis showed marked changes in the DNA-folding of the promoter and functional analysis showed a 20-fold reduction in transcription efficiency of UGT1A1 gene resulting from this insertion, thereby significantly elevating the UCB level. CONCLUSION： The genetic epidemiology of GS is variable across ethnic interactions among UGT1A1 groups and the epistatic promoter variants modulate bilirubin glucuronidation.

PHF8 is a histone H3K9me2 demethylase regulating rRNA synthesis

Dimethylation of histone H3 lysine 9 （H3K9me2） is an important epigenetic mark associated with transcription repression. Here, we identified PHF8, a JmjC-domain-containing protein, as a histone demethylase specific for this repressing mark. Recombinant full-length wild type protein could remove methylation from H3K9me2, but mutation of a conserved histidine to alanine H247A abolished the demethylase activity. Overexpressed exogenous PHF8 was colocalized with B23 staining. Endogenous PHF8 was also colocalized with B23 and fibrillarin, two well-established nucleolus proteins, suggesting that PHF8 is localized in the nucleolus and may regulate rRNA transcription. Indeed, PHF8 bound to the promoter region of the rDNA gene. Knockdown of PHF8 reduced the expression of rRNA, and overexpression of the gene resulted in upregulation of rRNA transcript. Concomitantly, H3K9me2 level was elevated in the promoter region of the rDNA gene in PHF8 knockdown cells and reduced significantly when the wild type but not the catalytically inactive H247A mutant PHF8 was overexpressed. Thus, our study identified a histone demethylase for H3K9me2 that regulates rRNA transcription.

Genetic diagnosis strategy of hereditary non-polyposis colorectal cancer

AIM: To study the characteristics of mismatch repair gene mutation of Chinese hereditary non-polyposis colorectal cancer (HNPCC) and hMLH1 gene promoter methylation, and to improve the screening strategy and explore the pertinent test methods. METHODS: A systematic analysis of 30 probands from HNPCC families in the north of China was performed by immunohistochemistry, microsatellite instability (MSI), gene mutation and methylation detection. RESULTS: High frequency microsatellite instability occurred in 25 probands (83.3%) of HNPCC family. Loss of hMLH1 and hMSH2 protein expression accounted for 88% of all microsatellite instability. Pathogenic muta-tion occurred in 14 samples and 3 novel mutational sites were discovered. Deletion of exons 1-6, 1-7 and 8 of hMSH2 was detected in 3 samples and no large fragment deletion was found in hMLH1. Of the 30 probands, hMLH1 gene promoter methylation occurred in 3 probands. The rate of gene micromutation detection combined with large fragment deletion detection was 46.7%-56.7%. The rate of the two methods in combination with methylation detection was 63.3%. CONCLUSION: Scientific and rational detection strategy can improve the detection rate of HNPCC. Based on traditional molecular genetics and combined with epigenetics, multiple detection methods can accurately diagnose HNPCC.

Regulated Gene Expression with Promoters Responding to Inducers

Genetically engineered transgenic animals and plants have proven to be extremely useful for analyzing biochemical and developmental processes.Promoters responding to chemical inducers will be powerful tools for basic research in molecular biology and biotechnological applications.Various chemical inducible systems based on activation and inactivation of the target gene had been described.The transfer of regulatory elements from prokaryotes,insects,and mammals has opened new avenues to construct chemically inducible promoters that differ in their ability to regulate the temporal and spatial expression patterns,and this will dramatically increase the application of transgenic technology.This review provides an overview on regulation of gene expression,promoter activating systems,promoter inactivation systems,inducible gene over expression,and inducible anti suppression.

Reactive protein, plasminogen activator inhibitor type-1 （PAI-1） levels, PAI-1 promoter 4G/5G polymorphism and acute myocardial infarction

Objective To investigate the relationship between CRP, plasminogen activator inhibitor type 1 （PAI-1） levels, PAI-1 gene promoter 4G/5G polymorphism and the type of acute myocardial infarction （ST elevation myocardial infarction, STEMI vs the non-ST elevation Myocardial infarction, NSTEMI）. Methods One hundred seventy-six consecutive patients with AMI were included for the study, of whom 60 had STEMI and 56 had NSTEMI, and 60 adults without cardiovascular and cerebrovascular disease were selected as controls. Blood samples were obtained from patients within 6 h of AMI and the plasma PAI-1, CRP, and the gene polymorphism were measured. Results Plasma levels of PAI- 1 and CRP were higher in AMI groups, compared those in the control group, and plasma levels of PAI-1 were significantly higher in patients with STEMI compared to those with NSTEMI （80.12ng/ml VS.73.01ng/ml, P 〈0.01）, while CRP levels were not significantly different between patient with STEMI and NSTEMI （3.87 ± 0.79 mg/ml VS.4.01 ±0.69mg/ml, P〉0.05）. PAI-1 levels presented a significant correlation with CRP levels in the NSTEMI subjects. However, PAI-1 and CRP levels could explain the lack of a significant relationship between them in control and STEMI subjects.The frequencies of 4G/4G genotype in the AMI group were higher than those in the control group and higher in patient with STEMI than in patient with NSTEMI. Plasma levels of PAI-1 in subjects with 4G/4G genotype were significantly increased as compared to those in subjects with 4G/5G and 5G/5G genotype. Conclusions Plasma PAI-1 levels were associated with different myocardial infarction type, and PAI-1 promoter 4G/5G polymorphisms and CRP may be related to plasma PAI-1 levels

Pharmarcogenetic Mechanism of ACE liD Polymorphism Adversely Responding to ACE Inhibitors in Regulating the ACE Promoter Activity in Neurons

The ACE （angiotensin converting enzyme） inhibitors are not only drugs widely prescribed drugs in cardiovascular diseases, but also potentially therapeutic agents in dementia. Based on the findings that the ACE inhibitors could activate the c-Jun N-terminal kinase signal to increase the ACE gene expression and that the Alu element of the human ACE gene involved in regulating ACE promoter activity, we aimed to investigate whether there are different pharmacogenetic responses of ACE I/D polymorphism to the ACE inhibitors in neurons. The three reporter vectors, pACEpro（0-SEAP, p-I-ACEpro-SEAP, and p-D-ACEpro-SEAP were used to examine the transcriptional activity of the vectors responding to the lisinopril treatment using a transient-transfection method in SH-SY5Y cells. Our results showed that lisinopril increased the promoter activity of an ACE gene by 16.7%. Additionally, we found the lisinopril enhanced the ACE promoter activity of the I-form vector by 17.2%, but adversely reduced that of the D-form vector by 16.8%, as compared with the respective control without the lisinopril treatment. Firstly, our findings had proved that the UD polymorphism of ACE gene contrarily responds to the ACE inhibitors in regulating the ACE expression in neurons, which provide a novel insight suggesting genetic testing to tailor the treatment regimens in AD （Alzheimer＇s disease） patients.

Cloning and Identification of promoter of Pseudomonas Pseudoaligenes

Promoter-probe vector pSUPV4 is used to clone the promoter of Pseudomonas pseudoalcaligenes directly in E. coli, and the recombinant pPA7, which has the highest kanamycin resistance, is obtained. Sequencing the PA7 fragment discloses several motifs similar to the conservative domains of prokaryotic promoters, including -10 box, -35 box, parallel SD fragment essential to transcription initiation, and the translation initiation site ATG. Southern blotting of PA7 indicates that the PA7 fragment comes from P. pseudoalcaligenes genome and has probably one copy. The PA7 fragment is subcloned by PCR, and the result shows that the 5’-flanking fragment from 889 to 1120 bp has promoter activity, which can be enhanced by the 0.7Kb fragment at 5’ end. The fragments of pPA7 and pPA7-2 are transferred into pseudomonas pseudoaligenes by electroporation, and the significant higher kanamycin resistance of transformants than that of control indicates that the PA7 fragment has the promoter activity in P. pseudoaligene.

Specific activation of 2'-5'oligoadenylate synthetase gene promoter by hepatitis C virus-core protein:A potential for developing hepatitis C virus targeting gene therapy

AIM： TO examine whether 2＇-5＇oligoadenylate synthetase （OAS） gene promoter can be specifically activated by hepatitis C virus （HCV）-core protein. METHODS： Human embryo hepatic cell line L02 was transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence was amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luci plasmid was transiently transfected into L02/core cells and luciferase activity was assayed. I^ESULTS： L02/core cell line stably expressing HCV- core protein was established. The pGL3-OAS-Luci construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells. CONCLUSION： HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.

MODY4的研究、认识现状及处理

在研究糖尿病时,人们总不会忘记英年早逝的美国高年级医学生Paul Langerhans,是他在1869年发表了胰腺组织学的论文,将其描述为“细胞团”,在他逝后不久的1888年被Morrison等将这“细胞团”命名为Langerhans胰岛[1],以纪念他的历史贡献,

Molecular cloning of promoter in human fibrinogenlike protein 2 (hfgl2) gene and functional analysis of its sequence

The aim of this study is to investigate the important regulative elements region which plays an important role on the activation of transcription exerted by the 5' noncoding region of hfgl2 gene in response to HBc and HBx. A series of promoter luciferase report plasmids, in which the hfgl2 gene has been deleted of the 5' and retained the common 3', were constructed. All the plasmids constructed were subjected to electrophoretic analysis and DNA sequencing. A eukaryotic construct expressing HBc or HBx, a luciferase reporter construct containing hfgl2 promoter and aβ-galactosidase (β-gal) plasmid were co-transfected into Chinese hamster ovary (CHO) cells and hepG2 cells, respectively. Luciferase report plasmids containing hfgl2 promoter were successfully constructed, and a serial assays of deletion of hfgl2 gene promoter showed that a strong regulatory region from -817 to -467 (relative to the transcription start site) was responsible for transcription and expression regulation of hfgl2 gene. The important regulative elements region in the promoter of hfgl2 gene was in response to HBc and HBx. which contributes to further pursuit of cis-acting elements and transcriptional factors involved in the transcription of hfgl2 gene.