基于位点保守性参量和位置权重矩阵预测酵母转录因子结合位点

目的:改进转录因子结合位点的理论预测方法。方法:构建转录因子结合位点位置权重矩阵,以转录因子结合位点每一位置的碱基保守性指数Mi为参量,利用位置权重打分函数算法(PWMSA)对酵母五种转录因子结合位点进行预测。结果:利用self-consistency和cross-validation两种方法对此算法进行检验,均获得了较高的预测成功率,结果表明5种转录因子结合位点的预测成功率均超过80%。结论:与已有的三种预测转录因子结合位点的软件进行比较,PWMSA算法明显优于其他三种算法,核苷酸水平上的关联系数和结合位点水平上的关联系数分别提高了0.25和0.22。     

转录因子结合位点的计算预测方法研究进展

转录因子结合位点的计算预测是研究基因转录调控的重要环节,但现有算法的预测特异性偏低.在深入分析转录因子结合位点生物特征的基础上,对当前基于保守模体和基于比较基因组学的两类计算预测方法进行了综述,指出了方法各自的优点和不足,并探讨了可能的改进方向.     

遗传算法在转录因子结合位点识别中的应用

遗传算法是模拟生物进化过程的计算模型,是一种全局优化搜索算法。将遗传算法与转录因子结合位点识别问题相结合的新方法,以一致性序列模型作为保守motif的描述模型,通过对motif序列与待测序列的比对问题进行编码,将其转化成搜索空间中的优化问题,利用遗传算法来搜索最优解,预测转录因子的结合位点。实验结果表明,这种新的方法是有效的,它在占用少量内存的情况下能够准确地识别出待测转录因子结合位点。     

利用序列保守模体和局部构象信息预测转录因子结合位点

转录因子结合位点的计算预测是研究基因转录调控的重要环节,但常用的位置特异得分矩阵方法预测特异性偏低.通过深入分析结合位点的生物特征,提出了一种综合利用序列保守模体和局部构象信息的结合位点预测方法,以极大相关得分矩阵作为保守模体的描述模型,并根据二苷参数模型计算位点序列的局部构象,将两类信息得分组合为多维特征向量,在二次判别分析的框架下进行训练和滑动预测.预测过程中还引入了位置信息量以优化似然得分和过滤备选结果.针对大肠杆菌CRP和Fis结合位点数据的留一法测试结果表明,描述模型的改进和多种信息的融合能有效地改善预测方法的性能,大幅度提高特异性.     

Inactivation of Benzodiazepine Binding Sites by N-Ethylmaleimide

Abstract: The benzodiazepine receptors of bovine brain membranes have been identified by the specific binding of radiolabeled [³H]diazepam. Pretreatment of membranes with N -ethylmdleimide causes a dose- and time-dependent decrease of 45 to 60% in the number of binding sites. No decrease occurs when membranes are pretreated with N -ethylmaleimide before administration, or in the presence, of diazepam. Binding of [³H]diazepam to the remaining sites occurs with the same characteristics as binding to the untreated receptor population.     

转录因子X-box结合蛋白1相互作用蛋白的筛选和鉴定

X-box 结合蛋白 1 是一种重要的转录因子,参与体内多项信号转导过程. 为进一步研究 XBP1 的生物学功能,运用酵母双杂交技术在肝细胞文库中筛选 XBP1 的结合蛋白. 首先运用 PCR 技术扩增获得 XBP1 的编码序列,克隆至 pGEM-T 载体,经测序鉴定后,亚克隆至诱饵载体 pGBKT7 中,转化酵母 AH109(a type). 免疫印迹检测诱饵质粒 pGBKT7-XBP1 在AH109 酵母中的表达之后,含有诱饵质粒的酵母 AH109 与含有肝细胞 cDNA 文库质粒 pACT2 的酵母 Y187(αtype)配合,配合后的二倍体酵母生长在含有 X-α-gal 的营养缺陷型培养基上 (SD/-Trp-Leu-His-Ade) 进行选择和筛选,经测序和序列比对确定阳性克隆的开放读码框 ORF,得到 7 种不同的蛋白质. 为了进一步验证这些筛选蛋白质与 XBP1 的相互作用,克隆其中一种蛋白质 MT1E,并运用 GST pulldown 和免疫共沉淀技术成功检测了 MT1E 和 XBP1 的相互作用(体外 / 体内),结果提示,MT1E 可能是 XBP1 的一个新的调节蛋白. 通过酵母双杂交技术筛选得到的 7 种蛋白质分别与肝细胞基础代谢、蛋白质的合成与运输、细胞的增殖与凋亡密切相关. 上述结果有助于揭示 XBP1 的生物学功能,为进一步探讨 XBP1 的表达和调控机制提供新线索.     

基于碱基关联二联体位置权重矩阵预测酵母转录因子结合位点

基于已知的酵母转录因子结合位点数据资料,构建转录因子结合位点碱基关联二联体位置权重矩阵,整合碱基关联二联体位置权重矩阵和碱基保守性参量M2i,提出一种新的预测转录因子结合位点的方法(PWMSA).利用self-consistency和cross-validation两种方法对此算法进行检验,均获得了较高的预测成功率,结果表明9种转录因子结合位点的总体预测成功率超过81%,明显高于单碱基位置权重矩阵,同时与已有预测转录因子结合位点的软件进行比较,核苷酸水平上的关联系数和结合位点水平上的关联系数分别达到0.42和0.52,优于现有预测方法.     

Identification of Multiple Binding Sites for [3H]5-Hydroxytryptamine in the Rat CNS

Recent studies indicate that there may be multiple subtypes of [3H]5-hydroxytryptamine ([3H]5-HT) binding sites. Mianserin and spiperone inhibited the specific binding of [3H]5-HT (2-3 nM) to rat brain cortical membranes with shallow displacement curves. The displacement data for spiperone were best described by the presence of three independent binding sites, for which spiperone had high, medium, and low affinities. The displacement data for mianserin were best fitted by two independent, high- and low-affinity sites. The inclusion of mianserin (250 nM) to inhibit [3H]5-HT binding to the mianserin-sensitive site selectively blocked one of the sites discriminated by spiperone. These results suggest the presence of three binding sites for [3H]5-HT, one blocked by low concentrations of spiperone (5-HT1A), one blocked by low concentrations of mianserin (5-HT1C), and one blocked only by high concentrations of both mianserin and spiperone (5-HT1B). Regional differences in the relative densities of the three sites were observed. The hippocampus was rich in 5-HT1A sites, whereas the striatum contained mainly 5-HT1B and 5-HT1C sites. Selective degeneration of 5-HT-containing nerve terminals induced by the neurotoxin 5,7-dihydroxytryptamine increased binding to all three sites in the cerebral cortex. Binding of [3H]5-HT to the three sites was differentially modulated by CaCl2 and guanylimidodiphosphate. The present data suggest the presence of three independent 5-HT1 binding sites having different affinities for mianserin and spiperone and having different regional distributions.     

Subnuclear Distribution of the S-100 Protein Specific Binding Sites in Rat Brain

Abstract: Fractionation of isolated brain nuclei previously reacted with ¹²⁵I-labelled S-100 showed that most of the specifically bound radioactivity associated with the nuclear membranes and the nucleoli. Labelling of nucleoli, which indicates the entrance of ¹²⁵I-labelled S-100 into the nucleus, was observed at 37°C, but not at 0–4°C. When tested separately for ¹²⁵I-labelled S-100 specific binding, both the nuclear membranes and the nucleoli were found to bind ¹²⁵I-labelled S-100 in a biphasic manner, the binding displaying a high affinity and a low affinity component, as observed with intact nuclei. However, the binding to nuclear membranes was largely irreversible, while that to nucleoli was fully reversible after any association time.     

Specific Binding Sites for S-100 Protein in Isolated Brain Nuclei

Abstract: Isolated brain nuclei possess binding sites for S-100 protein. The interaction of S-100 with these sites is specific and time-, temperature-, and Ca⁺-dependent. The profile of the ¹²⁵I-labelled S-100 binding inhibition is biphasic, displaying a high-affinity component and a low-affinity component. The S-100 binding to brain nuclei is largely irreversible, probably owing to the formation of a tight complex between the protein and its nuclear binding sites. The S-100 binding to brain nuclei is in most aspects similar to that to synaptosomal membranes. Several lines of evidence indicate, however, that the S-100 binding to nuclei is not due to contamination of these structures with plasma membranes. Isolated liver nuclei do not possess the high-affinity component of S-100 binding.     

γ-Aminobutyric Acid and Benzodiazepine Binding Sites in Audiogenic Seizure-Susceptible Mice

Abstract: The properties of γ-aminobutyric acid recognition sites, benzodiazepine binding sites and the effect of exogeneous γ-aminobutyric acid on benzodiazepine binding were determined in crude membrane fractions prepared from the brains of DBN/2 mice at ages before (8-9 and 17-18 days), during (22-23 and 28-29 days) and after (40-43 days) the age of high susceptibility to audiogenic seizures. These have been compared with data from age- matched mice of a strain (TO) with lower audiogenic seizure susceptibility. The number of high-affinity [³H]γ-aminobutyric acid binding sites was lower at all ages in DBN/2 mice compared with TO mice, but the affinity was higher in DBN/2 mice. The number of low-affinity [³H]y-aminobutyric acid binding sites was lower at 8-9 days and 40-43 days in DBN/2 mice, but was not significantly different from TO mice at other ages. For [³H]flunitrazepam binding, the only difference found was a slight reduction in the number of binding sites at 28-29 days of age in DBN/2 mice. γ-Aminobutyric acid stimulation of [³H]-flunitrazepam binding was not significantly different up to 22-23 days of age, but was higher in DBN/2 mice at 28-29 days and lower at 40-43 days. Impairment of γ-aminobutyric acid function is a possible permissive factor in the age-dependent audiogenic seizure susceptibility in DBN/2 mice.