中国人雄激素受体N端转示激活区的测序及突变检测

雄激素受体（ａｎｄｒｏｇｅｎ ｒｅｃｅｐｔｏｒ,ＡＲ）Ｎ端转录激活功能区（ＡＦ１）是ＡＲ发挥转录激活所必需的。用４对引物（Ａ３－Ａ４）ＰＣＲ扩增２０例中国正常男性ＡＲ的ＡＦ１,双链ＤＮＡ循环测序以确定正常中国人ＡＲ的ＡＦ１的核苷酸顺序。在此基础上,用ＰＣＲ－ＳＳＣＰ分析和双链ＤＮＡ循环测序法对２例雄激素抵抗征（ＡＩＳ）患者外周血白细胞和１５例前列腺癌（ＰＣ）患者癌组织中ＡＲ的ＡＦ１区进行突变检测。     

用错配PCR技术检测中国人β-地中海贫血基因突变

β-地中海贫血(β-地贫)的分子基础主要为点突变,目前中国β-地贫人群中已发现16种点突变。我们在基因频率调查的基础上,发现我国南方95％以上的β-地贫由5种点突变所致,它们是:codon 41-42(-CTTT)缺失突变,IVS-2 nt 654(C→T)突变,codon 17     

雄激素非依赖性前列腺癌细胞雄激素受体靶基因的筛选与初步鉴定

采用染色质免疫共沉淀技术在全基因组水平筛选雄激素非依赖性前列腺癌细胞LNCaP-AI的雄激素受体结合位点,行高通量测序及生物信息学分析共得到2 876个peak（pvalue〈1×10–5）,peak平均长度为673 bp;将peak序列定位到Hg19基因组,共有1 865个靶基因,其中fold enrichment≥10的基因有425个。对peak相关基因进行GO分析发现,与细胞、细胞组分、细胞过程、结合、细胞器相关的基因位列前五位;对peak相关基因进行通路分析发现,与黏着斑、代谢通路、癌症中的转录错误调控、嘌呤代谢等信号通路相关的基因占大多数。筛选出7个候选AR靶基因,采用Real-time qPCR技术分析它们在LNCaP-AI细胞和雄激素依赖性前列腺癌细胞LNCaP中对DTH刺激的反应性,发现DHT刺激可改变7个候选AR靶基因在LNCaP-AI细胞中的表达,为进一步研究雄激素依赖性前列腺癌向非依赖性前列腺癌发展的过程中雄激素受体及其调控的下游靶基因功能起着至关重要的作用。     

人前列腺癌雄激素受体的变异与临床

人前列腺癌雄激素受体的变异与临床王晓慧卢建（第二军医大学病理生理教研室,上海２００４３３）关键词前列腺癌雄激素受体内分泌治疗Ｆｌｕｔａｍｉｄｅ撤药综合征前列腺癌（ＰＣ）是老年男性的常见肿瘤,在美国是造成男性癌症死亡仅次于肺癌的第二位原因,在我国随着人...     

水稻钙调蛋白基因的克隆及结构分析

钙调蛋白(Calmodulin)是生物细胞内一种重要的调控蛋白,通过其与靶酶的相互作用,控制细胞正常的生长和发育。我们以湖北光敏感核不育水稻cDNA第一条链为模板,参考大麦钙调蛋白基因序列合成5’端和3’端引物,利用多聚酶链式反应(PcR)合成了完整的水稻钙调蛋白cDNA,克隆并测定其序列。结果表明,光敏核不育水稻的钙调蛋白基因由450个桉苷酸组成,共编码148个氨基酸,且与迄今为止在植物领域里发表的大麦及苜蓿的钙调蛋白基因有很高的同源性。在核苷酸序列上与大麦有90％的同源性,与苜蓿有86％的同源性,其编码的148个氨基酸与大麦和苜蓿的差异分别只有1个。这是国际上第一次克隆水稻的钙调蛋白基因。     

人Y染色体特异DNA片段的原位PCR扩增

报道了以人类外周血白细胞为材料,利用人类Y染色体特异片段一对扩增引物Y3和 Y₄,在玻片细胞样品上进行原位 PCR获得的结果.用Dig-11-dUTP于原位PCR过程中直接掺入法(直接原位PCR)和细胞原位PCR后再作原位杂交的方法(间接原位PCR)都证明:玻片上细胞样品内的Y染色体特异DNA片段有明显的原位扩增.并对原位PCR的专一性与可重复性,细胞内PCR扩增产物的存留等方法学问题,以及原位PCR技术的价值和应用前景进行了讨论.     

人雄激素芳香化酶基因（CYP19）结构及其表达调控研究进展

人雄激素芳香化酶与乳腺癌的发生和发展密切相关.综述了编码该酶的CYP19基因的结构、独特的组织特异性表达和调控CYP19基因表达的各种因素及其有关的调控机制.     

上海某高校新校区中抗生素抗性基因污染状况分析

探究新型环境污染物—抗生素抗性基因（ARGs）在校园环境中的分布状况。通过聚合酶链式反应(PCR)对上海某高校使用5年新校区不同区域污水检查井污泥中8种四环素类、4种磺胺类、7种β-内酰胺类、4种链霉素类和5种氯霉素类ARGs进行定性研究,并利用变性梯度凝胶电泳(DGGE)技术分析污泥中细菌群落的多样性。结果显示,校园各区域中共检出19种ARGs,有8种ARGs的检出率大于50%,其中磺胺类抗性基因sulI、sulII的检出率最高,为100%。实验区及餐饮区的ARGs检出种类最多,均为14种,其次为宿舍区(12种),教学区的ARGs检出最少(8种)。通过DGGE分析细菌群落结构,证明该地区的ARGs分布与细菌多样性无明显关系。新校区使用5年但ARGs污染严重,可能是由于人类活动（尤其是科研活动）对ARGs的产生及扩散存在促进作用。此外,细菌群落多样性与ARGs种类的关系表明ARGs在环境中的迁移可能受到除细菌种类之外其他环境因素的影响。     

分子信标探针用于PCR检测对虾白斑杆状病毒

将对虾白斑杆状病毒的一段特异性DNA设计成分子信标探针,用于该病毒的PCR检测.温度与荧光强度之间的关系表明,所设计探针的发夹既可以形成也可以打开,符合PCR对分子信标探针的要求.结果表明,在PCR同时加入分子信标探针不影响PCR扩增,分子信标探针只能与目的DNA杂交,具有较高的特异性.随着PCR循环数的增加以及含目的DNA的质粒拷贝数的增加,荧光强度都随之增强.     

Simultaneous PCR amplification of two barley 5S rDNA sequence types in the same PCR reaction

Many researchers are currently using PCR technology to amplify individual members of multigene families, including 5S rDNA, for sequencing and related purposes. When members of the family differ in length, analyses would be facilitated if different units could be simultaneously and efficiently amplified. In the present paper we describe conditions that can be used to amplify simultaneously both the “long” and “short” 5S rDNA repeats found in barley (Hordeum vulgare L.).     

Application of Polymerase Chain Reaction to Detect HIV-1 DNA in Pools of Dried Blood Spots

Nucleic acid tests that detect HIV infection at an early phase are available and have been applied on individual dried blood spot (DBS). The present study was undertaken with an aim to evaluate the feasibility of performing PCR for HIV-1 DNA on pools of DBS as an alternative to individual testing. Standardization of PCR by a modified Amplicor HIV-1 DNA assay version 1.5 (Roche molecular diagnostics, USA), on pooled DBS was performed using five confirmed HIV reactive samples with known low viral load of HIV-1 and HIV non-reactive samples in pools of 5, 10 and 20 DBS. After successful standardization of pooling procedure, a total of 183 pools (of 10 DBS each) were prepared from 1,823 DBS samples, collected from a population-based study that tested negative for HIV antibodies and p24 antigen. All these pools were screened for HIV-1 DNA by the Amplicor assay. Standardization of pooling procedure indicated that pooling of 10 DBS gave an optimum result. Out of 183 pools tested, one pool of 10 samples was positive and of these ten DBS that were tested individually to identify the positive DBS, one sample was detected to be positive for HIV-1 DNA. Our study demonstrates that PCR for HIV-1 DNA can be successfully performed on pools of DBS. However, this may be needed only on specialized studies of HIV and not for routine epidemiology studies as only a very small fraction of cases would be missed if only antibody/antigen testing were done.     

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e. samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.     

Real-time PCR investigation on the expression of sboA and ituD genes in Bacillus spp

Aims: To investigate the expression of sboA and ituD genes among strains of Bacillus spp. at different pH and temperature. Methods and Results: Different Bacillus strains from the Amazon basin and Bacillus subtilis ATCC 19659 were investigated for the production of subtilosin A and iturin A by qRT‐PCR, analysing sboA and ituD gene expression under different culture conditions. Amazonian strains presented a general gene expression level lower than B. subtilis ATCC 19659 for sboA. In contrast, when analysing the expression of ituD gene, the strains from the Amazon, particularly P40 and P45B, exhibited higher levels of expression. Changes in pH (6 and 8) and temperature (37 and 42°C) caused a decrease in sboA expression, but increased ituD expression among strains from Amazonian environment. Conclusions: Temperature and pH have an important influence on the expression of genes sboA (subtilosin A) and ituD (iturin A) among Bacillus spp. The strains P40 and P45B can be useful for the production of antimicrobial peptide iturin A. Significance and Impact of the Study: Monitoring the expression of essential biosynthetic genes by qRT‐PCR is a valuable tool for optimization of the production of antimicrobial peptides.     

快速可靠的双链PCR产物直接测序法

利用T7DNA聚合酶在低温下仍具较高活性的特点,在热变性后低温下进行测序反应,使用该方法对多种PCR产物进行序列分析均取得较好的结果．     

Greater Amount of HCV-RNA in Tears Compared to Blood

Plasma, tear fluid and swabs from eye, nose and pharynx of 33 patients were examined for presence of hepatitis C virus (HCV) RNA by polymerase chain reaction (PCR). All samples from plasma, tear fluid and eyeswabs were found to show a positive reaction in HCV-RNA PCR. Remarkably, we regularly found greater amounts of amplification products in tear fluid and eyeswabs compared to plasma using the same conditions for sample preparation.     

用PCR检测细胞培养中支原体污染

细胞培养中支原体污染已经成为严重的问题.为了扩增6种支原体(精氨酸支原体,口腔支原体,人型支原体,猪鼻支原体,发酵支原体及莱氏支原体)核糖体RNA操纵子的16s和23s DNA间区,设计了三个通用PCR引物(F1,F2及R1).当以6种支原体DNA为模板时,引物F1和R1产生340到468bp的片段,引物F2和R1产生145到211bp的片段,当用Hela细胞或E.coli DNA作为模板,用引物F1和R1时,在电泳中未观察到特定区带.此法最小能检出8.5fg精氨酸支原体DNA,相当于13个精氨酸支原体.这说明,当这些支原体污染细胞培养时,能用PCR法检测出来.     

Correlation between the Presence of Virulence-Associated Genes as Determined by PCR and Actual Virulence to Mice in Various Strains of Listeria Spp.

Five chromosomal genes, prfA, plcA, hlyA, mpl and plcB, are implicated in the virulence of Listeria monocytogenes and some of these genes have been used for the identification of bacteria by polymerase chain reaction (PCR). Using 6 strains of L. monocytogenes and 3 L. innocua strains, the relationship was examined between the presence of five virulence-associated genes and actual virulence to mice in terms of 50% lethal dose (LD₅₀), bacterial viability in the organ of infected mice and the intracellular growth in cultured macrophages. None of the five genes could be amplified by PCR in all the L. innocua strains and they were actually avirulent to mice. All L. monocytogenes strains were shown to be virulent and to have intact virulence-associated genes except for the strain ATCC15313. This particular strain was revealed to be avirulent and defective in hlyA and plcA in PCR amplification. It was suggested that PCR detection of genes prfA, mpl, or plcB may not be sufficient to detect virulent strains of L. monocytogenes. It appeared that the ability to produce listeriolysin O (LLO), which is encoded by hlyA, was critical for the expression of virulence regardless of the amount of LLO produced.