Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

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Organisation of the lamprey (Lampetra fluviatilis) embryonic brain: insights from LIM-homeodomain, Pax and hedgehog genes

To investigate the embryonic development of the central nervous system of the lamprey Lampetra fluviatilis, we have isolated and analysed the expression patterns of members of the LIM-homeodomain, Pax, Hedgehog and Nkx2.1 families. Using degenerate RT-PCR, single representatives of Lhx1/Lhx5, Lhx2/Lhx9, Pax3/Pax7 and Hedgehog families could be isolated in L. fluviatilis. Expression analysis revealed that the lamprey forebrain presents a clear neuromeric pattern. We describe the existence of 4 embryonic diencephalic prosomeres whose boundaries can be identified by the combined and relative expressions of LfPax37, LfLhx15 and LfLhx29. This suggests that the embryonic lamprey and gnathostome forebrain are patterned in a highly similar manner. Moreover, analysis of the LfHh gene, which is expressed in the hypothalamus, zona limitans intrathalamica and floor plate, reveals the possible molecular origin of this neuromeric brain pattern. By contrast, LfHh and LfNkx2.1 expressions suggest major differences in patterning mechanisms of the ventral telencephalon when compared to gnathostomes. In summary, our findings highlight a neuromeric organisation of the embryonic agnathan forebrain and point to the possible origin of this organisation, which is thus a truly vertebrate character. They also suggest that Hh/Shh midline signalling might act as a driving force for forebrain evolution.     

Dok1 and Dok2 proteins regulate natural killer cell development and function

Natural killer (NK) cells are involved in immune responses against tumors and microbes. NK‐cell activation is regulated by intrinsic and extrinsic mechanisms that ensure NK tolerance and efficacy. Here, we show that the cytoplasmic signaling molecules Dok1 and Dok2 are tyrosine phosphorylated upon NK‐cell activation. Overexpression of Dok proteins in human NK cells reduces cell activation induced by NK‐cell‐activating receptors. Dok1 and Dok2 gene ablation in mice induces an NK‐cell maturation defect and leads to increased IFN‐γ production induced by activating receptors. Taken together, these results reveal that Dok1 and Dok2 proteins are involved in an intrinsic negative feedback loop downstream of NK‐cell‐activating receptors in mouse and human.     

Isolation and characterization of two pathogen- and salicylic acid-induced genes encoding WRKY DNA-binding proteins from tobacco

A pathogen- and salicylic acid (SA)-induced DNA-binding activity has been recently identified in tobacco that is related to a previously identified class of WRKY DNA-binding proteins. To identify members of the WRKY gene family associated with this DNA-binding activity, we have attempted to isolate those WRKY genes that are induced by pathogen infection. Using a domain-specific differential display procedure, we have isolated two tobacco WRKY genes, tWRKY3 and tWRKY4, that are rapidly induced in resistant tobacco plants after infection by tobacco mosaic virus (TMV). Both tWRK3 and tWRKY4 encode proteins with a single WRKY domain that contain the conserved WRKYGQK sequence. Unlike other isolated WRKY proteins that contain the Cys₂His₂ zinc motif, tWRKY3 and tWRKY4 appear to contain the Cys₂HisCys zinc motif. Nonetheless, both tWRKY3 and tWRKY4 are capable of binding DNA molecules with the W-box (TTGAC) element recognized by other WRKY proteins. Expression of the tWRKY3 and tWRKY4 genes could be rapidly induced not only by TMV infection but also by SA or its biologically active analogues that are capable of inducing pathogenesis-related genes and enhanced resistance. Interestingly, induction of both genes by TMV infection was still observed in resistant tobacco plants expressing the bacterial salicylate hydroxylase gene (nahG), although the levels of induction appeared to be reduced. Identification of pathogen- and SA-induced genes encoding WRKY DNA-binding proteins should facilitate future studies on the regulation and functions of this novel group of DNA-binding proteins.     

Dps proteins, an efficient detoxification and DNA protection machinery in the bacterial response to oxidative stress

The proteins belonging to the Dps (DNA-binding proteins from starved cells) family play an important role within the bacterial defence system against oxidative stress. They act on Fe(II) and hydrogen peroxide that are potentially toxic in the presence of air. Fe(II) forms spontaneously insoluble Fe(III) and reacts with molecular oxygen or its reduced forms to yield the highly damaging hydroxyl radicals. All Dps proteins have the distinctive capacity to annul the toxic combination of iron and hydrogen peroxide as they use the latter compound to oxidise Fe(II). In addition to this intrinsic DNA protection capacity, several members of the family, including the archetypical Escherichia coli Dps, protect DNA physically by shielding it in large Dps-DNA complexes. The structural and functional characteristics that endow Dps proteins with the chemical and physical protection mechanism are presented and discussed also in the framework of the varied situations that may be encountered in different bacterial species.      

Solution Structure and DNA-binding Properties of the Winged Helix Domain of the Meiotic Recombination HOP2 Protein

The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination.     

Global analysis of gene expression in the estrogen induced pituitary tumor of the F344 rat

The F344 rat rapidly forms large prolactinomas in response to chronic estrogen treatment. To identify genes expressed in the course of this estrogen induced pituitary tumor growth, we performed microarray analysis on the F344 rat pituitary after chronic estrogen treatment and on untreated controls. At a significance level set to minimize type I error, some 72 genes were found to be differentially expressed between estrogen treated and untreated. Of those genes, 70 have not been reported previously as being affected by estrogen in the F344 rat pituitary. Since many other investigators have studied the effect of estrogen on specific gene expression in rat pituitary, we also examined the mRNA expression of the 36 genes that have been previously reported as having their expression affected by estrogen in the rat pituitary. Of these, 13 were found to have their expression affected by estrogen treatment in the same direction as had been reported by others.     

Transfected insect cells in suspension culture rapidly yield moderate quantities of recombinant proteins in protein-free culture medium

Methodology to rapidly express milligram quantities of recombinant proteins through the Lipofectin-mediated transfection of insect cells in small-scale, protein-free suspension culture is presented. The transfection phase in suspension culture was first optimized using the green fluorescence protein coupled with FACs analysis to examine the effect of variables such as the transfection media, duration, and cell density on transfection efficiency and expression level. The recombinant protein production phase was optimized using secreted alkaline phosphatase (SEAP) as a reporter protein to evaluate the cell seeding density and harvest time. Using this method, 5 secreted, 2 intracellular, and 1 chimeric protein were expressed at levels ranging from 6 to 50 mg/L. Furthermore, the ability to purify over 2 mg of His(6)-tagged SEAP by immobilized metal affinity chromatography from 50 mL insect cell culture medium to greater than 95% purity was also demonstrated. This method is suitable for scale-up and high-throughput applications.     

雄兔垂体GnRHR的序列测定与生物信息学分析

目的探讨GnRH-A激动剂(阿拉瑞林)主动免疫对垂体GnRHR基因表达的影响和生物信息学特性。方法 30只日本大耳白兔(Oryctolagus cuniculus)随机均分为三组,在EG-Ⅰ和EG-Ⅱ皮下注射1.0 mL(100μg/mL)阿拉瑞林抗原,EG-Ⅱ于20 d以原剂量加强免疫1次;从兔垂体中提取总RNA,PCR扩增GnRHR基因,进行反转录、克隆和测序;实时荧光定量PCR分析垂体中GnRHR、FSH-β和LH-βmRNA的表达,用DNAman、Tm-pred、Signal P 3.0、Target P 1.1、Expasy、PSORT II prediction等生物信息学分析软件和在线工具,对GnRHR序列及其蛋白的理化特性、跨膜结构、信号肽和二级结构等进行分析与预测。结果①雄兔GnRHR的核苷酸为1179 bp,同源性达96%。ssDNA分子量362.66×103,dsDNA 726.78×103。②GnRHR二级结构中151(40.27%)个氨基酸组成α-螺旋,15(4.00%)个氨基酸组成β-折叠。③GnRHR由389个氨基酸组成,蛋白质的序列长度为375;理论等电点(pI)5.02,不稳定指数58.08,脂肪指数28.67,疏水性平均值(GRAVY)0.869,表明该蛋白为不稳定的疏水性蛋白。④GnRHR蛋白TM螺旋长度为17～23,有34个强跨膜螺旋区,从内到外有35个螺旋。⑤GnRHR信号肽的概率0.999,最可能的酶切部位在17和18位点。结论阿拉瑞林免疫可以明显降低垂体GnRHR、FSH-β和LH-β基因表达,加强免疫效果更佳。GnRHR是一种含有信号肽序列的不稳定的疏水性跨膜蛋白,具有明显的生物信息学特征。     

Expression of pannexin family of proteins in the retina

Expression of the Panx1 and Panx2 members of the pannexin family of gap junction proteins was studied in the retina by in situ hybridization and qRT-PCR. Both pannexins showed robust expression across the retina with predominant accumulation in the retinal ganglion cells (RGCs). In concordance, immunohistochemical analysis showed accumulation of the Panx1 protein in RGCs, amacrine, horizontal cells and their processes. Two Panx1 isoforms were detected: a ubiquitously expressed 58 kDa protein, and a 43 kDa isoform that specifically accumulated in the retina and brain. Our results indicated that Panx1 and Panx2 are abundantly expressed in the retina, and may therefore contribute to the electrical and metabolic coupling, or to signaling between retinal neurons via the secondary messengers.     

Comparative immunocytochemical demonstration of ACTH-, LH and FSH-containing cells in the pituitary of neonatal,immature and adult rats

Summary By means of immunocytochemistry, the development of ACTH-, LH- and FSH cells was examined in the anterior pituitary of 5-day-old neonatal, 15-day-old immature and adult rats. ACTH-positive cells are angular and the periphery of these cells is strongly reactive with anti-ACTH serum. In contrast, LH- and FSH-immunopositive cells are ovoid elements, ranging in cell size and intensity of staining. Angular cells, in which only the cell periphery reacted with anti-LH serum, were observed in neonatal and immature rats; however, these cells were not stained with either anti-FSH serum or anti-ACTH serum. Observation of serial semithin sections revealed that ACTH-immunopositive cells do not react with either anti-LH or anti-FSH serum. Finally, it was observed that ACTH cells and LH cells are both functionally differentiated already in 5-day-old neonatal rats.     

Temperature and androgens regulate the biosynthesis of secretory proteins from rabbit cauda epididymidis

We have compared the biosynthesis of secretory proteins in rabbit cauda epididymidis maintained for 15 days at abdominal temperature with that of the scrotal cauda. Explants from both situations were incubated in vitro in the presence of [³⁵S] methionine, and the labelled proteins released into the incubation medium were analyzed by polyacrylamide gel electrophoresis. Body temperature specifically inhibited the synthesis of at least two polypeptides of 43 kDa and 21 kDa (designated EP21), whereas the synthesis of polypeptides of 80, 39, 31, and 24 kDa was increased. These changes resembled those produced by castration, but androgen treatment was not able to reverse the effect of body temperature. To confirm these observations, poly (A)⁺ RNA from the scrotal and the abdominal cauda respectively, was translated in vitro and the synthesized products were immunoprecipi-tated with an antibody against EP21 polypeptide. Both castration and body temperature strongly decreased the concentration of EP21 mRNA. In vivo testosterone administration restored the content of EP21 mRNA in cauda from castrated animals, but not in cauda maintained at body temperature. The changes observed might be related to the adverse effect of body temperature on sperm storage in the cauda epididymidis. © 1993 Wiley-Liss, Inc.