人脑中脱碘酶相互作用蛋白的筛选与验证

阐述人脑内硒蛋白在维护细胞正常功能、抵御氧化损伤和预防脑疾病方面的重要作用,指出脱碘酶3是人脑中一种重要的硒蛋白.克隆了人脱碘酶3的开放读码框,将其编码区中编码硒代半胱氨酸的TGA码突变为编码半胱氨酸的密码,以脱碘酶3突变体为诱饵,利用酵母双杂交系统从人胎脑cD-NA文库中筛选一个能与脱碘酶3相互作用的蛋白,即人丝氨酸蛋白酶抑制剂A族蛋白3.采用荧光共振能量转移技术中的敏化发射法和荧光寿命法,验证了人脑中脱碘酶的相互作用.     

The paternally expressed gene Peg3 regulates sexual experience-dependent preferences for estrous odors.

Sexual experience has marked and long-lasting effects on male behavior in mammals, regulating traits such as the anticipation and display of sexual behavior, aggression, and olfaction. The authors conducted urine preference, habituation-dishabituation, and partner choice tests with sexually experienced and naive male mice and found that wild-type males acquire adaptively significant preferences for the odors of receptive, estrous females with sexual experience, and that these preferences are matched by changes in main olfactory system responses involving the piriform cortex, as indicated by c-Fos expression. The authors also report that these experiential effects are disrupted in male mice carrying a knockout of the imprinted gene Peg3. This paternally expressed gene regulates maternal care and offspring development, but the authors here report that Peg3 mutant males suffer a complex olfactory deficit that affects estrous odor preferences and the responses of the main olfactory system to such odors. Peg3 appears to have evolved to regulate the experience-dependent preference for receptive females, an adaptive trait that would enhance male reproductive success and so potentially increase paternal transmission of this paternally expressed gene. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

干扰ATM基因表达增强胃癌细胞AGS的放射敏感性

为探讨RNA干扰共济失调毛细血管扩张性突变(Ataxia-telangiectasia mutated,ATM)基因表达后对胃癌细胞AGS放射敏感性的影响,通过构建ATM基因RNA干扰质粒转染AGS细胞,获得干扰效率高的克隆细胞AGS-Ri-ATM,并采用RT-PCR和Western blot分别检测m RNA和蛋白表达水平,平板克隆形成实验观察细胞经放射后对细胞生长的影响,流式细胞术检测细胞周期和凋亡情况。检测结果显示其ATM基因表达被干扰下调。经X射线照射至不同吸收剂量,AGS-Ri-ATM平板克隆形成数量/率远低于AGS-Vector的平板克隆形成数量/率,差异显著(p0.05),具有统计学意义。流式细胞术检测显示当吸收剂量为4 Gy时,AGS-Ri-ATM被阻滞在G1期,同时在12 h后出现明显的凋亡想象。结果表明,ATM基因被干扰后可以诱导细胞周期的阻滞,以及凋亡的增加而增强AGS细胞的放射敏感性。     

Using archaeal histones for precise DNA fragmentation

The fragmentation of DNA is a useful procedure for many molecular biology procedures. However, most methods used to fragment DNA are poorly controllable, and cannot be used to create small fragments. We describe a method to generate random DNA fragments of a predictable size to be cloned in expression vectors for the construction of display libraries. The DNA is allowed to form complexes with archaeal histones from Methanothermus fervidus (HMf) and the HMf/DNA core complex is naturally protected from nuclease DNaseI activity, giving rise to DNA fragments of approximately 60 bp and multiples thereof. We found that by varying the wt/wt ratio between DNA and HMf, the concentration of DNA and the incubation time with DNaseI, DNA fragments of desired size can be obtained. This approach should be applicable to the efficient fragmentation of DNA for the construction of phage display polypeptide libraries, as well as any other molecular biology procedures in which small DNA fragments of defined size are required.     

An experimental system for the study of mutations in the HMR locus of Saccharomyces cerevisiae: the insertion of Ty into HMRa vs. the conversion of HMRa to HMRalpha

A cross between a sir4-11 strain (sir4-11 HMLalpha MATalpha HMRa, non-mating type) and an a-mating strain (SIR(+) HMLalpha MATa HMRa) of Saccharomyces cerevisiae forms diploid clones at a frequency of 5 x 10(-6), but the obtained diploid clones often (>70%) have altered forms of the HMRa-containing restriction fragment, designated @ HMRa'. We previously found that some HMRa's are associated with the conversion of HMRa to HMRalpha. In this report, we present evidence that another @ HMRa' associates with the insertion of Ty into Ya of HMR. We also found that the sir4-11 strain increased mating frequency by UV irradiation to a level of 9 x 10(-4), and that generation of HMRa' was completely prevented by disruption of RAD52 of the sir4-11 strain. Hence, we conclude that the mutations that cause generation of HMRa' occur in the sir4-11 strain prior to mating. Due to these mutations, the sir4-11 strain converts to alpha-mating type and readily mates with the a-mating strain. We discuss the usefulness of the sir4-11 strain for the study of mutations in the HMR locus of S. cerevisiae.     

A single-step photolithographic interface for cell-free gene expression and active biochips

We have developed a biochip platform technology suitable for controlled cell-free gene expression at the micrometer scale. A new hybrid molecule, "Daisy", was designed and synthesized to form in a single step a biocompatible lithographic interface on silicon dioxide. A protocol is described for the immobilization of linear DNA molecules thousands of base pairs long on Daisy-coated surfaces with submicrometer spatial resolution and up to high densities. On-chip protein synthesis can be obtained with a dynamic range of up to four orders of magnitude and minimal nonspecific activity. En route to on-chip artificial gene circuits, a simple two-stage gene cascade was built, in which the protein synthesized at the first location diffuses to regulate the synthesis of another protein at a second location. We demonstrate the capture of proteins from crude extract onto micrometer-scale designated traps, an important step for the formation of miniaturized self-assembled protein chips. Our biochip platform can be combined with elastomeric microfluidic devices, thereby opening possibilities for isolated and confined reaction chambers and artificial cells in which the transport of products and reagents is done by diffusion and flow. The Daisy molecule and described approach enables groups not proficient in surface chemistry to construct active biochips based on cell-free gene expression.