胡杨甜菜碱醛脱氢酶基因的功能分化

甜菜碱醛脱氢酶(BADH)在植物抗逆反应中发挥着重要作用。文中从胡杨cDNA克隆到2个甜菜碱醛脱氢酶基因,分别命名为PeBADH1和PeBADH2。PeBADH1和PeBADH2均编码503个氨基酸的蛋白质,预测分子量分别是54.93 kDa和54.90 kDa。组织表达模式分析发现这2个基因在正常生长、盐和H2O2胁迫下,在不同组织中的表达模式有较大差异。在大肠杆菌中表达并纯化了2个基因的重组蛋白。酶活性分析显示PeBADH1和PeBADH2蛋白对底物的活性分别是0.073μmol/(min.mg)和0.107μmol/(min.mg)。热力学稳定性分析显示这2个蛋白的热力学稳定性具有明显差异。因此,基因表达模式差异与蛋白质酶学性质的不同预示着这2个基因可能存在功能上的分化。     

Generation and Analysis of ESTs from the Grass Carp,Ctenopharyngodon idellus

Grass carp, Ctenopharyngodon idellus (Valenciennes, 1844), is an economically important species widely cultured in the world, but its genome research resources are largely lacking. The objectives of this study were to construct normalized cDNA libraries for efficient EST analysis, to generate ESTs from these libraries, and to identify EST-related molecular markers such as microsatellites and single nucleotide polymorphisms (SNPs) for genetic analysis of this species. A total of 6,269 ESTs were generated representing 4,815 unique sequences, from which 105 putative microsatellites and 5,228 SNPs were identified. These genome resources provide the material basis for future genetic and functional analyses in this species.     

Clinical manifestation, imaging, and genotype analysis of two pedigrees with spinocerebellar ataxia

The objective of this study was to analyze the clinical manifestation, imaging characteristics, genotype, and the relationship between the three aforementioned parameters in two pedigrees suffering from spinocerebellar ataxia. To evaluate the clinical manifestation of the two pedigrees and to compare the characteristics, we performed the MRI analysis of some patients from both pedigrees, while 2 ml of the peripheral blood sample was collected for gene analysis. The gene analysis data showed that pedigree 1 was certified spinocerebellar ataxia type-2 (SCA2); the CAG repeats in the proband, proband’s mother, and proband’s brother were 44, 36, and 38, respectively. The MRI revealed brainstem cerebellar atrophy and “cross sign” and “ordinate sign” of pons. Pedigree 2 was certified SCA1; the CAG repeats of the proband, proband’s aunt, and proband’s asymptomatic cousin were 60, 51, and 52, respectively. The MRI revealed cerebellar atrophy in these individuals. We, therefore, concluded that it was difficult to diagnose the SCA subset solely through the clinical manifestation. The imaging characteristics analysis and final diagnosis depended basically on gene analysis data.     

A cytoplasmic protein, NfrC, is required for bacteriophage N4 adsorption.

At least four genes are required for irreversible adsorption of bacteriophage N4. nfrA and nfrB have been characterized previously and encode an outer membrane protein and inner membrane protein, respectively. The nfrC gene product is characterized in detail in this study. We have mapped the nfrD locus to min 52 on the Escherichia coli linkage map. Maxicell analysis of nfrC and a null allele (nfrC2) cloned into a high-copy-number plasmid shows its gene product to be 42 kDa in size. We determined the nfrC nucleotide sequence which predicts a gene product of 42 kDa. Western blots (immunoblots) of Escherichia coli proteins after cellular fractionation show NfrC to be a cytoplasmic protein which is required for irreversible bacteriophage N4 adsorption, an event occurring at the cell surface.     

cⅠ857基因的体外定位同义突变

 ｃⅠ８５７基因的体外定位同义突变陈南春,高辉,陈苏民,杨萍,刘新平（西安第四军医大学分子生物学研究所,西安７１００３２）外源基因要在大肠杆菌中获得高表达,需要合适的ＳＤ序列和可调控的强启动子［１］。ＰＬ启动子在原核启动子中属强启动子,它受ｃⅠ基因产物...     

Molecular diffusion into ferritin: pathways, temperature dependence, incubation time, and concentration effects

The detailed kinetics of permeation and effusion of small nitroxide spin probe radicals with the protein shells of horse spleen ferritin (HoSF) and human H-chain ferritin (HuHF) and a 3-fold channel variant D131H+E134H of HuHF were studied by electron paramagnetic resonance spectroscopy and gel permeation chromatography under a variety of experimental conditions. The results confirm that the permeation of molecular species of 7-9-A diameter into ferritin is a charge selective process and that the threefold channels are the likely pathways for entry into the protein. Studies with holoHoSF show that increased temperature increases the rates of penetration and effusion and also increases the concentration of positively charged spin probe accumulated within the protein in excess of that in the external solution. The interior of HoSF is much more accessible to small molecules at physiological temperature of approximately 40 degrees C than at room temperature. The large activation energy of 63-67 kJ/mol measured for the effusion/penetration and the small diffusion coefficient, D approximately 5 x 10(-22) m(2)/s at 20 degrees C, corresponding to a time of approximately 60 min for traversing the protein shell, is consistent with the kinetics of diffusion being largely controlled by the restrictive porosity of the protein itself. An inverse dependence of the first-order rate constant for effusion out of the protein channel on the incubation time used for radical penetration into the protein is attributed to increased binding of the radical within the funnel-shaped channel.     

A simple and sensitive assay for 1-aminocyclopropane-1-carboxylic acid

A simple, rapid, and sensitive method for the quantitative determination of 1-amino-cyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene in plant tissues, is described. The assay is based on the liberation of ethylene from ACC with NaOCl in the presence of Hg²⁺; ethylene is assayed by gas chromatography. The yield is normally 80% and can be determined by internal standards. The method is quite specific and can detect as little as 5 pmol of ACC.     

The C termini of Arabidopsis cryptochromes mediate a constitutive light response

Cryptochrome blue light photoreceptors share sequence similarity to photolyases, flavoproteins that mediate light-dependent DNA repair. However, cryptochromes lack photolyase activity and are characterized by distinguishing C-terminal domains. Here we show that the signaling mechanism of Arabidopsis cryptochrome is mediated through the C terminus. On fusion with beta-glucuronidase (GUS), both the Arabidopsis CRY1 C-terminal domain (CCT1) and the CRY2 C-terminal domain (CCT2) mediate a constitutive light response. This constitutive photomorphogenic (COP) phenotype was not observed for mutants of cct1 corresponding to previously described cry1 alleles. We propose that the C-terminal domain of Arabidopsis cryptochrome is maintained in an inactive state in the dark. Irradiation with blue light relieves this repression, presumably through an intra- or intermolecular redox reaction mediated through the flavin bound to the N-terminal photolyase-like domain.     

ArlR反应调节蛋白在表皮葡萄球菌不同生长期表达的检测

表皮葡萄球菌(Staphylococcus epidermidis,SE)是寄居在人体和黏膜表面的条件致病菌,因可在医疗植入材料表面形成生物膜(biofilm)而具有致病性。细菌双组分信号转导系统可调控生物膜形成,但其调控机制在SE中研究甚少。本课题对arlRS双组分信号转导系统的反应蛋白ArlR在细菌不同生长期的表达情况进行初步研究。首先构建ArlR原核表达质粒,用纯化重组ArlR免疫小鼠,获得多克隆抗-ArlR抗体,免疫Dot方法检测结果显示小鼠抗-ArlR血清效价>1∶100000。进一步采用蛋白免疫印迹法检测ArlR在SE1457野生株不同生长期中的表达水平,结果显示,ArlR在2h表达量较低,到4h达高峰,6～10h表达量较4h降低。利用反转录实时荧光定量聚合酶链反应检测arlR基因在不同生长期的转录水平,结果显示相应时间点ArlR蛋白表达水平与arlR基因转录水平一致。本研究结果为后期研究双组分信号转导系统arlRS对SE生物膜形成的影响奠定基础。