Structure of PTB bound to RNA: specific binding and implications for splicing regulation

The polypyrimidine tract binding protein (PTB) is a 58-kilodalton RNA binding protein involved in multiple aspects of messenger RNA metabolism, including the repression of alternative exons. We have determined the solution structures of the four RNA binding domains (RBDs) of PTB, each bound to a CUCUCU oligonucleotide. Each RBD binds RNA with a different binding specificity. RBD3 and RBD4 interact, resulting in an antiparallel orientation of their bound RNAs. Thus, PTB will induce RNA looping when bound to two separated pyrimidine tracts within the same RNA. This leads to structural models for how PTB functions as an alternative-splicing repressor.     

A cytotoxic ribonuclease targeting specific transfer RNA anticodons

The carboxyl-terminal domain of colicin E5 was shown to inhibit protein synthesis of Escherichia coli. Its target, as revealed through in vivo and in vitro experiments, was not ribosomes as in the case of E3, but the transfer RNAs (tRNAs) for Tyr, His, Asn, and Asp, which contain a modified base, queuine, at the wobble position of each anticodon. The E5 carboxyl-terminal domain hydrolyzed these tRNAs just on the 3' side of this nucleotide. Tight correlation was observed between the toxicity of E5 and the cleavage of intracellular tRNAs of this group, implying that these tRNAs are the primary targets of colicin E5.     

Hemoglobin Gun Hill: deletion of five amino acid residues and impaired heme-globin binding

Hemoglobin Gun Hill, a new variant of adult hemoglobin, was found in a Caucasian and one of his three daughters. The abnormal hemoglobin had only half of the expected number of heme groups. Five amino acid residues appeared to be missing from the beta-globin chains. These residues occur in linear sequence in normal beta-chains in a region involved in heme-globin binding. A deletion of five amino acids in the beta-chains of hemoglobin Gun Hill is postulated. The most likely mechanism for the origin of such a hemoglobin variant would appear to be unequal crossing-over during meiosis.     

纳豆激酶第36位氨基酸突变对其活性及热稳定性的影响

为了解纳豆激酶分子结构与其功能的关系,研究了纳豆菌诱变株DU115的纳豆激酶基因突变对其酶活性和热稳定性的影响。从原生质体紫外诱变纳豆菌DU115和野生纳豆菌BN10基因组DNA中扩增纳豆激酶成熟肽基因nkD和nkB,构建重组表达载体pPICZα-A—nkD和pPICZα-A—nkB,在毕赤酵母X33中实现了表达,并对表达产物进行分离纯化、酶活性测定及热稳定性检测。结果表明,与nkB基因序列相比,nkD基因有两处的核苷酸发生了突变（A107G和C396T）,A107G碱基突变导致氨基酸的替代D36G（Asp→Gly）;诱变菌株纳豆激酶DNK与野生菌的BNK在65℃处理15min后,前者的热稳定性提高20％,比活力提高16．6％。由此可推断,第36位氨基酸残基的突变可能与其酶热稳定性及活性提高密切相关。     

Cyclophilin: a specific cytosolic binding protein for cyclosporin A

Cyclophilin, a specific cytosolic binding protein responsible for the concentration of the immunosuppressant cyclosporin A by lymphoid cells, was purified to homogeneity from bovine thymocytes. Cation-exchange high-performance liquid chromatography resolved a major and minor cyclophilin species that bind cyclosporin A with a dissociation constant of about 2 X 10(-7) moles per liter and specific activities of 77 and 67 micrograms per milligram of protein, respectively. Both cyclophilin species have an apparent molecular weight of 15,000, an isoelectric point of 9.6, and nearly identical amino acid compositions. A portion of the NH2-terminal amino acid sequence of the major species was determined. The cyclosporin A-binding activity of cyclophilin is sulfhydryl dependent, unstable at 56 degrees C and at pH 4 or 9.5, and sensitive to trypsin but not to chymotrypsin digestion. Cyclophilin specifically binds a series of cyclosporin analogs in proportion to their activity in a mixed lymphocyte reaction. Isolation of cyclophilin from the cytosol of thymocytes suggests that the immunosuppressive activity of cyclosporin A is mediated by an intracellular mechanism, not by a membrane-associated mechanism.     

Poliovirus host range is determined by a short amino acid sequence in neutralization antigenic site I

The mouse-adapted strain of poliovirus type 2 (Lansing) induces fatal poliomyelitis in mice after intracerebral inoculation, whereas mice inoculated with poliovirus type 1 (Mahoney) show no signs of disease. Previous work indicated that the adaptation to mouse virulence is associated with the viral capsid proteins and that mutations in neutralization antigenic site I of poliovirus reduce neurovirulence of the Lansing strain in mice. The role of antigenic site I in mouse neurovirulence was further explored by constructing an antigenic hybrid virus. Six amino acids in antigenic site I of the Mahoney strain were replaced with a sequence specific for the Lansing strain by using a mutagenesis cartridge. The hybrid virus was neutralized by polyclonal antisera elicited by the type 1 and type 2 strains of poliovirus and by neutralizing monoclonal antibodies directed against antigenic site I of type 2 virus. The hybrid virus induced paralytic disease in mice, an observation demonstrating that a short sequence of amino acids in antigenic site I is an important determinant of poliovirus host range. Antigenic site I may be involved in attachment of poliovirus to cells of the mouse central nervous system.     

氨基酸生物配比及外源核苷对雏鸡肝脏核酸含量的影响

以NRC推荐的赖Aa为基准 ,以各种Aa对应的密码子使用频率的比率来确定饲料配方中各主要Aa之间的生物配比。在添加外源核苷酸的条件下 ,雏鸡肝脏中核酸含量明显增加 ,34日龄对照组与试验Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ组鸡肝脏中总RNA含量分别为 (34 5 7.3± 146 .4)、(380 0 .7± 16 5 .6 )、(3814.8± 170 .4)、(36 15 .1± 2 17.2 )、(4 785 .4± 12 2 .9)和 (4 916 .0± 2 0 7.1) μg·g-1。结果显示 ,外源核苷酸在家禽体内能被利用 ,且在氨基酸比例趋于平衡时 ,RNA总量有所增加。     

Control of directionality in lambda site specific recombination

The simple relation between the substrates and products of site-specific recombination raises questions about the control of directionality often observed in this class of DNA transactions. For bacteriophage lambda, viral integration and excision proceed by discrete pathways, and DNA substrates with the intrinsic property of recombining in only one direction can be constructed. These pathways display an asymmetric reliance on a complex array of protein binding sites, and they respond differently to changes in the concentrations of the relevant proteins. The Escherichia coli protein integration host factor (IHF) differentially affects integrative and excisive recombination, thereby influencing directionality. A four- to eightfold increase in intracellular IHF coincides with the transition from exponential to stationary phase; this provides a mechanism for growth phase-dependent regulation of recombination that makes the cellular physiology an intrinsic part of the recombination reaction.     

A modular PIP2 binding site as a determinant of capsaicin receptor sensitivity

The capsaicin receptor (TRPV1), a heat-activated ion channel of the pain pathway, is sensitized by phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis after phospholipase C activation. We identify a site within the C-terminal domain of TRPV1 that is required for PIP2-mediated inhibition of channel gating. Mutations that weaken PIP2-TRPV1 interaction reduce thresholds for chemical or thermal stimuli, whereas TRPV1 channels in which this region is replaced with a lipid-binding domain from PIP2-activated potassium channels remain inhibited by PIP2. The PIP2-interaction domain therefore serves as a critical determinant of thermal threshold and dynamic sensitivity range, tuning TRPV1, and thus the sensory neuron, to appropriately detect heat under normal or pathophysiological conditions.     

一种新的氨基酸序列进化距离及其应用

在选择的思想下,建立了蛋白质氨基酸序列的进化动力学方程,提出了一个新的蛋白质氨基酸序列的进化距离———选择进化距离,该距离统一了前人提出的各种氨基酸序列的进化距离;将该动力学方程转换为线性模型后,利用最小二乘法可实现模型中参数b的估计;最后,通过17个物种的细胞色素b的氨基酸序列说明了该选择进化距离的计算方法,并根据自展法比较了不同进化距离得到的物种进化树。结果表明,利用选择进化距离构建的进化树与其他几种进化距离得到的进化树的拓扑结构一致。选择进化距离为估计蛋白质氨基酸序列的进化距离提供了新方法。     

Cyclosporin A binding to calmodulin: a possible site of action on T lymphocytes

Cyclosporin A, a potent immunosuppressive agent, has been widely used to treat patients with solid organ transplants. Although its precise mechanism of action is unknown, it appears to inhibit subsets of T lymphocytes at an early stage in cell activation. Fluorescent, fully active derivatives of cyclosporin A and calmodulin, a protein that binds calcium and is therefore essential to normal cell function, were utilized to demonstrate that cyclosporin A binds to calmodulin. Flow cytometry showed that the calmodulin inhibitors R24571 and W-7 competitively inhibited binding of cyclosporin A to cloned T lymphocytes. Cyclosporin A inhibited the calmodulin-dependent activation of phosphodiesterase in a dose-dependent manner. Binding of cyclosporin A to calmodulin may prevent the latter's role in the activation of the second messengers and enzymes required for effective cell proliferation and function in the immune response.     

A single amino acid mutation contributes to adaptive beach mouse color pattern

Natural populations of beach mice exhibit a characteristic color pattern, relative to their mainland conspecifics, driven by natural selection for crypsis. We identified a derived, charge-changing amino acid mutation in the melanocortin-1 receptor (Mc1r) in beach mice, which decreases receptor function. In genetic crosses, allelic variation at Mc1r explains 9.8% to 36.4% of the variation in seven pigmentation traits determining color pattern. The derived Mc1r allele is present in Florida's Gulf Coast beach mice but not in Atlantic coast mice with similar light coloration, suggesting that different molecular mechanisms are responsible for convergent phenotypic evolution. Here, we link a single mutation in the coding region of a pigmentation gene to adaptive quantitative variation in the wild.     

中国番茄黄化曲叶病毒编码的RNA沉默抑制子AC4蛋白的核酸结合特性

农杆菌共浸润试验结果表明:中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus,TYLCCNV)Y10分离物编码AC4蛋白是RNA沉默抑制子.为了研究RNA沉默抑制子AC4蛋白的作用机制,将TYLCCNV-Y10的AC4基因插入到原核表达载体pET-32a的多克隆位点上,构建重组原核表达载体pET32a-YIOACA.将重组载体导入BL21(DE3)pLysS进行ACA蛋白表达,并在自然条件下纯化蛋白.利用原核表达的ACA蛋白进行电泳迁移率变动试验(electrophoretic mobility shift assay,EMSA),分析ACA蛋白分别与单、双链siRNA和单、双链长RNA的结合情况.试验结果表明:TYLCCNV-Y10编码的ACA蛋白具有结合单链siRNA和单链长RNA特性,而不与双链siRNA和双链长RNA结合.