H2AX phosphorylation screen of cells from radiosensitive cancer patients reveals a novel DNA double-strand break repair cellular phenotype

BACKGROUND: About 1-5% of cancer patients suffer from significant normal tissue reactions as a result of radiotherapy (RT). It is not possible at this time to predict how most patients' normal tissues will respond to RT. DNA repair dysfunction is implicated in sensitivity to RT particularly in genes that mediate the repair of DNA double-strand breaks (DSBs). Phosphorylation of histone H2AX (phosphorylated molecules are known as gammaH2AX) occurs rapidly in response to DNA DSBs, and, among its other roles, contributes to repair protein recruitment to these damaged sites. Mammalian cell lines have also been crucial in facilitating the successful cloning of many DNA DSB repair genes; yet, very few mutant cell lines exist for non-syndromic clinical radiosensitivity (RS). METHODS: Here, we survey DNA DSB induction and repair in whole cells from RS patients, as revealed by gammaH2AX foci assays, as potential predictive markers of clinical radiation response. RESULTS: With one exception, both DNA focus induction and repair in cell lines from RS patients were comparable with controls. Using gammaH2AX foci assays, we identified a RS cancer patient cell line with a novel ionising radiation-induced DNA DSB repair defect; these data were confirmed by an independent DNA DSB repair assay. CONCLUSION: gammaH2AX focus measurement has limited scope as a pre-RT predictive assay in lymphoblast cell lines from RT patients; however, the assay can successfully identify novel DNA DSB repair-defective patient cell lines, thus potentially facilitating the discovery of novel constitutional contributions to clinical RS.

Tissue culture of forest trees: Clonal multiplication of Eucalyptus grandis L

Axillary shoot proliferation was obtained using explants of Eucalyptus grandis L. juvenile and mature stages on a defined medium. Murashige and Skoog medium (MS) supplemented with benzyladenine (BA), naphthalene acetic acid (NAA) and additional thiamine. Excised shoots were induced to root on a sequence of three media: (1) White's medium containing indoleacetic acid (IAA), NAA and indole butyric acid; (IBA), (2) half-strength MS medium with charcoal and (3) half-strength MS liquid medium. The two types of explants differed in rooting response, with juvenile-derived shoots giving 60% rooting and adult-derived ones only 35%. Thus, the factors limiting cloning of selected trees in vitro are determined to be those controlling rooting of shoots in E. grandis.

Biodiversity and Phylogeny of Planktic Cyanobacteria in Temperate Freshwater Lakes

Currently, the classification used for cyanobacteria is based mainly on morphology. In many cases the classification is known to be incongruent with the phylogeny of cyanobacteria. The evaluation of this classification is complicated by the fact that numerous strains are only described morphologically and have not been isolated. Moreover, the phenotype of many cyanobacterial strains alters during prolonged laboratory cultivation. In this thesis, cyanobacterial strains were isolated from lakes (mainly Lake Tuusulanjärvi) and both morphology and phylogeny of the isolates were investigated. The cyanobacterial community composition in Lake Tuusulanjärvi was followed for two years in order to relate the success of cyanobacterial phenotypes and genotypes to environmental conditions. In addition, molecular biological methods were compared with traditional microscopic enumeration and their ability and usefulness in describing the cyanobacterial diversity was evaluated. The Anabaena, Aphanizomenon, and Trichormus strains were genetically heterogeneous and polyphyletic. The phylogenetic relationships of the heterocytous cyanobacteria were not congruent with their classification. In contrast to heterocytous cyanobacteria, the phylogenetic relationships of the Snowella and Woronichinia strains, which had not been studied before this thesis, reflected the morphology of strains and followed their current classification. The Snowella strains formed a monophyletic cluster, which was most closely related to the Woronichinia strain. In addition, a new cluster of thin, filamentous cyanobacterial strains identified as Limnothrix redekei was revealed. This cluster was not closely related to any other known cyanobacteria. The cyanobacterial community composition in Lake Tuusulanjärvi was studied with molecular methods [denaturant gradient gel electrophoresis (DGGE) and cloning of the 16S rRNA gene], through enumerations of cyanobacteria under microscope, and by strain isolations. Microcystis, Anabaena/Aphanizomenon, and Synechococcus were the major groups in the cyanobacterial community in Lake Tuusulanjärvi during the two-year monitoring period. These groups showed seasonal succession, and their success was related to different environmental conditions. The major groups of the cyanobacterial community were detected by all used methods. However, cloning gave higher estimates than microscopy for the proportions of heterocytous cyanobacteria and Synechococcus. The differences were probably caused by the high 16S rRNA gene copy numbers in heterotrophic cyanobacteria and by problems in the identification and detection of unicellular cyanobacteria.

An in vitro transposon system for highly regulated gene expression: construction of Escherichia coli strains with arabinose-dependent growth at low temperatures.

Placing a gene of interest under the control of an inducible promoter greatly aids the purification, localization and functional analysis of proteins but usually requires the sub-cloning of the gene of interest into an appropriate expression vector. Here, we describe an alternative approach employing in vitro transposition of Tn Omega P(BAD) to place the highly regulable, arabinose inducible P(BAD) promoter upstream of the gene to be expressed. The method is rapid, simple and facilitates the optimization of expression by producing constructs with variable distances between the P(BAD) promoter and the gene. To illustrate the use of this approach, we describe the construction of a strain of Escherichia coli in which growth at low temperatures on solid media is dependent on threshold levels of arabinose. Other uses of the transposable promoter are also discussed.

The Loss of Functional Caspase-12 in Europe Is a Pre-Neolithic Event

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谷子和甘蔗C4型磷酸烯醇式丙酮酸羧化酶基因的克隆及其转基因水稻光合效率的提高

水稻是我国重要的粮食作物之一，它是一种典型的C3植物。与其它C3作物不一样的是，水稻的生长需要相对较高的温度和充足的阳光照射。然而高温和高光强的生长环境更加适合于C4植物的生长，更加有利于发挥C4植物高光合效率的特点。因此本论文希望将C4植物中固定CO2的酶磷酸烯醇式丙酮酸羧化酶基因导入水稻，获得一种更加适合高温和高光强生活环境的“C4型”水稻，这对于提高水稻的产量，满足人口增长对粮食需求具有重大意义。 本论文从C4植物谷子和甘蔗中克隆了其C4型磷酸烯醇式丙酮酸羧化酶cDNA基因，获得了具有自主知识产权的基因克隆，并将它们导入粳稻品种中花8号，进而对转基因材料的光合生理特性进行了研究。结果如下： 首次从谷子中得到了ppc基因两个cDNA克隆，分别命名为Mppc1和Mppc2。前者是一个C3型的ppc基因，它可能属于在根中特异表达的C3-2型ppc基因;后者是在绿色叶片中大量表达的C4型ppc基因。它们所编码的蛋白的氨基酸残基数分别为961和964，序列同源性为82.5%。C4型PEPC多出的3个氨基酸位于N末端。利用RACE的方法我们得到了谷子C4型ppc基因完整的cDNA序列，包括63bp的5'非编码区，2895bp的编码区和256bp的3'非编码区。 首次获得了甘蔗C4型ppc基因完整的cDNA序列的克隆，命名为Sppc。它包括95bp的5'非编码区、2886bp的编码区，和224bp的3'非编码区。 利用所克隆的基因，分别连上强组成型启动子Ubiquitin启动子和强光调控启动子Rubisco小亚基启动子后，再插入两个标记基因不同的表达载体pCB和pPCB的多克隆位点中，构建了八个含有外源ppc基因的植物表达载体pCB-Pubi-Mppc、pCB-Pubi-Sppc、pCB-PrbcS-Mppc、pCB-PrbcS-Sppc、pPCB-Pubi-Mppc、pPCB-Pubi-Sppc、pPCB-PrbcS-Mppc和pPCB-PrbcS-Sppc。再加上含有玉米完整的C4型ppc 核基因的载体pCB-ZMppc，共有9个载体。利用农杆菌介导法进行了水稻的转化，各个载体都获得了大量的转基因植株。对标记基因潮霉素磷酸转移酶基因hpt和磷酸甘露糖异构酶基因pmi以及导入的目的ppc基因的PCR扩增检测，结果显示绝大多数转基因植株都能扩增出目的片段，而未转化的植株则没有扩增产物。对部分转基因水稻的Southern和Western杂交以及RT-PCR分析都表明，无论从DNA水平、mRNA水平，还是从蛋白质水平上都证明外源ppc基因都成功地导入了水稻，并获得了正确的表达。 对各载体转基因植株PEPC活性大规模的测定表明，转入玉米完整C4型PEPC核基因（有内含子）的水稻表现出极大的表达效率，大多数转基因材料的PEPC活性为对照的10-20倍，其活性最高可达到对照的44倍。转入谷子和甘蔗PEPC基因cDNA的水稻，表达的效率很低，多数材料活性增加仅为对照的2-5倍，但也有极少数材料活性增加了10倍以上。用Rubisco小亚基启动子控制的ppc基因在水稻的表达活性要略高于Ubiquitin启动子控制的ppc基因。以上结果说明ppc基因的内含子在其转录或mRNA的稳定上起着重要作用。 对部分转基因材料气体交换特征的研究发现，随着转基因水稻PEPC活性的增加，净光合速率也有逐渐增加的趋势。其中PEPC活性最大的ZM24株系的三个单株净光合速率比对照增加了39.8%、13.7%和28.6%，而它们的PEPC活性比对照分别增加了21.2、21.9和23.6倍。 转PEPC水稻的净光合速率与气孔导度具有显著的相关性。这说明表达的外源ppc 基因产物PEPC参与了转基因水稻的气孔运动，使气孔开放程度增加。更有意义的是过表达PEPC的水稻具有更高的水分利用效率，这就增加了其耐旱能力。在光抑制条件下转基因水稻也具有更高的光合能力。这些特征表明转ppc基因的水稻比对照更加适合于水稻高温高光强和干旱的原生环境。

Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor

A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains. B. maxima serum albumin was subsequently purified, and its coding cDNA was further obtained by PCR-based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1: 1 molar ratio. The equilibrium dissociation constants (K-D) obtained for the skin and the serum proteins were 1.92 x 10(-9) M and 1.55 x 10(-9) M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys(53)-Cys(62)), which comprises the scissile bond Arg(58)(P-1)-His(59)(P-1'). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.

山溪鲵皮肤β-microseminoprotein样蛋白的分离纯化及其cDNA克隆

从山溪鲵(Batrachuperus pachunii)皮肤匀浆液中经过Sephadex G-50凝胶过滤、AKTA(R)Resource Q阴离子柱和反向高压液相C4柱分离纯化得到相对分子质量为12 000的蛋白.利用其N端氨基酸序列设计引物,从山溪鲵皮肤的cDNA中克隆并筛选到该蛋白的cDNA序列.该cDNA序列的开放阅读框为339 bp,编码113个氨基酸残基组成的蛋白.在BLAST数据库搜寻比对分析表明,该蛋白的氨基酸序列与来自人类及其他哺乳动物β-microseminoprotein蛋白具有约40%的序列同源性.这也是首次在两栖类动物皮肤中确认β-microseminoprotein.初级结构分析表明,该蛋白属于亲水性蛋白;多重序列比较显示,其氨基酸序列中的10个半胱氨酸位点及15个其他氨基酸位点与高等脊椎动物β-microseminoprotein中同种氨基酸有相同的位点.由此推测该蛋白属于β-microseminoprotein家族.

Identification of a novel cathelicidin gene in the rainbow trout, Oncorhynchus mykiss

We report the cloning of a novel antimicrobial peptide gene, termed rtCATH_1, found in the rainbow trout, Oncorhynchus mykiss. The predicted 216-residue rtCATH_1 prepropeptide consists of three domains: a 22-residue signal peptide, a 128-residue cathelin-like region containing two identifiable cathelicidin family signatures, and a predicted 66-residue C-terminal cationic antimicrobial peptide. This predicted mature peptide was unique in possessing features of different known (mammalian) cathelicidin subgroups, such as the cysteine-bridged family and the specific amino-acid-rich family. The rtCATH_1 gene comprises four exons, as seen in all known mammalian cathelicidin genes, and several transcription factor binding sites known to be of relevance to host defenses were identified in the 5' flanking region. By Northern blot analysis, the expression of rtCATH_1 was detected in gill, head kidney, and spleen of bacterially challenged fish. Primary cultures of head kidney leukocytes from rainbow trout stimulated with lipopolysaccharide or poly(I (.) C) also expressed riCATH_1. A 36-residue peptide corresponding to the core part of the fish cathelicidin was chemically synthesized and shown to exhibit potent antimicrobial activity and a low hemolytic effect. Thus, rtCATH_1 represents a novel antimicrobial peptide gene belonging to the cathelicidin family and may play an important role in the innate immunity of rainbow trout.

Construction and characterization of a fosmid library for pathogenic bacterium Vibrio anguillarum

Vibrio anguillarum is a common bacterial pathogen in fish. However, little is known about its pathogenic mechanism, in part, because the entire genome has not been completely sequenced. We constructed a fosmid library for V. anguillarum containing 960 clones with an average insert size of 37.7 kb and 8.6-fold genome coverage. We characterized the library by end-sequencing 50 randomly selected clones. This generated 93 sequences with a total length of 57 485 by covering 1.4% of the whole genome. Of these sequences, 58 (62.4%) were homologous to known genes, 30 (32.3%) were genes with hypothetical functions, and the remaining 5 (5.3%) were unknown genes. We demonstrated the utility of this library by PCR screening of 10 genes. This resulted in an average of 6.2 fosmid clones per screening. This fosmid library offers a new tool for gene screening and cloning of V. anguillarum, and for comparative genomic studies among Vibrio species.

A thioredoxin with antioxidant activity identified from Eriocheir sinensis

Thioredoxin, with a redox-active disulfide/dithiol in the active site, is the major ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state. In the present study, the cDNA encoding thioredoxin-1 (designated EsTrx1) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsTrx1 was of 641 bp, containing a 51 untranslated region (UTR) of 17 bp, a 3' UTR of 306 bp with a poly (A) tail, and an open reading frame (ORF) of 318 bp encoding a polypeptide of 105 amino acids. The high similarity of EsTrx1 with Trx1s from other animals indicated that EsTrx1 should be a new member of the Trx1 sub-family. Quantitative real-time PCR analysis revealed the presence of EsTrx1 transcripts in gill, gonad, hepato-pancreas, muscle, heart and haemocytes. The expression of EsTrx1 mRNA in haemocytes was up-regulated after Listonella anguillarum challenge, reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. In order to elucidate its biological functions, EsTrx1 was recombined and expressed in E. coli BL21 (DE3). The rEsTrx1 was demonstrated to possess the expected redox activity in enzymatic analysis, and to be more potent than GSH in antioxidant capacity. These results together indicated that EsTrx1 could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to bacterial challenge. (C) 2009 Elsevier Ltd. All rights reserved.

雨生红球藻Rubisco在虾青素积累过程中的表达分析

虾青素因其具有极强的抗氧化性及优越的着色作用，被广泛应用于营养保健和水产养殖中，备受国内外研究者的关注。红球藻是目前虾青素的生物来源中较有优势的一条途径。 我们选取雨生红球藻作为研究材料，收集其生长过程中四个不同的阶段，分别为绿色游动细胞阶段，绿色不动细胞阶段，绿褐色不动细胞阶段（开始积累虾青素），以及红色不动孢子阶段。本研究中提取了核酮糖-1,5-二磷酸羧化/加氧酶（Rubisco）的粗提液，并测定了酶活。其编码基因rbcL的mRNA表达量也被测定。另外，光合速率与呼吸速率的比值（P/R）通过测定与计算得出，各个阶段藻细胞中虾青素的含量由分析得出。本研究还应用了叶绿素荧光测定方法，确定了光系统II潜在最大活性（Fv / Fm），光系统II实际活性（ΦPSII），电子传递速率（ETR）和非光化学淬灭参数（NPQ）。 结果表明，绿色游动细胞的生长状态最佳，其P/R、Fv / Fm、ΦPSII均为最大，NPQ为最小。这说明在此状态的细胞中，光系统II的活性最强；但是其Rubisco活性与rbcL表达量均为最小。相比之下，在绿褐色不动细胞中，P/R和NPQ的值较低，Fv / Fm、ΦPSII和ETR值都最小，但Rubisco活性与rbcL表达量均为最高。 结合工业生产虾青素的方法，我们认为，Rubisco或许参与了虾青素的合成，而非Calvin循环为色素合成提供前体和能量。因此，在生产过程中适当加入碳源，比如CO2，可以有效增大虾青素的产量。

Regulacja karboksylazy/oksygenazy rybulozo-1,5-bisfosforanu (Rubisco) u Arabidopsis thaliana w odpowiedzi na zmianę natężenia światła z umiarkowanego na niskie

Wydział Biologii

The receptor kinase family: primary structure of rhodopsin kinase reveals similarities to the beta-adrenergic receptor kinase.

Light-dependent deactivation of rhodopsin as well as homologous desensitization of beta-adrenergic receptors involves receptor phosphorylation that is mediated by the highly specific protein kinases rhodopsin kinase (RK) and beta-adrenergic receptor kinase (beta ARK), respectively. We report here the cloning of a complementary DNA for RK. The deduced amino acid sequence shows a high degree of homology to beta ARK. In a phylogenetic tree constructed by comparing the catalytic domains of several protein kinases, RK and beta ARK are located on a branch close to, but separate from the cyclic nucleotide-dependent protein kinase and protein kinase C subfamilies. From the common structural features we conclude that both RK and beta ARK are members of a newly delineated gene family of guanine nucleotide-binding protein (G protein)-coupled receptor kinases that may function in diverse pathways to regulate the function of such receptors.

Kassinakinin S: A novel histamine-releasing heptadecapeptide from frog (Kassina senegalensis) skin secretion

Amphibian defensive skin secretions remain a largely untapped resource for the peptide biochemist with an interest in the identification, structural characterization, and precursor cDNA cloning of novel bioactive peptides. Here we report the isolation, structural characterization, functional profiling, and nucleotide sequence of precursor cDNA of a novel histamine-releasing heptadecapeptide, FIPVTLLALHKIKEKLN-amide, from the defensive skin secretion of the African running frog, Kassina senegalensis. This peptide was found to be a potent histamine secretagogue (EC[5][0]=6 µM; maximal release = 25 µM) in a rat peritoneal mast cell model system and was accordingly named kassinakinin S. The open-reading frame of the cDNA encoding prepro-kassinakinin S was found to consist of 71 amino acid residues containing a single copy of kassinakinin S and its glycyl residue amide donor at the C-terminus. Kassinakinin S can thus be added to the growing list of amphibian skin bioactive peptide prototypes.