The interactions of basic proteins and DNA

I. ELECTROPHORESIS OF THE NUCLEIC ACIDS

A zone electrophoresis apparatus using ultraviolet optics has been constructed to study nucleic acids at concentrations less than 0.004%. Native DNA has a mobility about 15% higher than denatured DNA over a range of conditions. Otherwise, the electrophoretic mobility is independent of molecular weight, base composition or source. DNA mobilities change in the expected way with pH but the fractional change in mobility is less than the calculated change in charge. A small decrease in mobility accompanies an increase in ionic strength. RNA’s from various sources have mobilities slightly lower than denatured DNA except for s-RNA which travels slightly faster. The important considerations governing the mobility of nucleic acids appear to be the nature of the hydrodynamic segment, and the binding of counterions. The differences between electrophoresis and sedimentation stem from the fact that all random coil polyelectrolytes are fundamentally free draining in electrophoresis.

II. THE CYTOCHROME C/DNA COMPLEX

The basic protein, cytochrome c, has been complexed to DNA. Up to a cytochrome:DNA mass ratio of 2, a single type of complex is formed. Dissociation of this complex occurs between 0.05F and 0.1F NaCl. The complexing of cytochrome to DNA causes a slight increase in the melting temperature of the DNA, and a reduction of the electrophoretic mobility proportional to the decrease in net charge. Above a cytochrome:DNA mass ratio of 2.5, a different type of complex is formed. The results suggest that complexes such as are formed in the Kleinschmidt technique of electron microscopy would not exist in bulk solution and are exclusively film phenomena.

III. STUDIES OF THE ELECTROPHORESIS AND MELTING BEHAVIOUR OF NUCLEOHISTONES

Electrophoresis studies on reconstituted nucleohistones indicate that the electrophoretic mobility for these complexes is a function of the net charge of the complex. The mobility is therefore dependent on the charge density of the histone complexing the DNA, as well as on the histone/DNA ratio. It is found that the different histones affect the transition from native to denatured DNA in different ways. It appears that histone I is exchanging quite rapidly between DNA molecules in 0.01 F salt, while histone II is irreversibly bound. Histone III-IV enhances the capacity of non-strand separated denatured DNA to reanneal. Studies on native nucleoproteins indicate that there are no gene-sized uncomplexed DNA regions in any preparations studied.

IV. THE DISSOCIATION OF HISTONE FROM CALF THYMUS CROMATIN

Calf thymus nucleoprotein was treated with varying concentrations of NaCl. The identity of the histones associated and dissociated from the DNA at each salt concentration was determined by gel electrophoresis. It was found that there is no appreciable histone dissociation below 0.4 F NaCl. The lysine rich histones dissociate between 0.4 and 0.5 F NaCl. Their dissociation is accompanies by a marked increase in the solubility of the chromatin. The moderately lysine rich histones dissociate mainly between 0.8 and 1.1 F NaCl. There are two arginine rich histone components: the first dissociates between 0.8 F and 1.1 F NaCl, but the second class is the very last to be dissociated from the DNA (dissociation beginning at 1.0 F NaCl). By 2.0 F NaCl, essentially all the histones are dissociated.

The properties of the extracted nucleoprotein were studied. The electrophoretic mobility increases and the melting temperature decreases as more histones are dissociated from the DNA. A comparison with the dissociation of histones from DNA in NaClO₄ shows that to dissociate the same class of histones, the concentration of NaCl required is twice that of NaClO₄.

A mobilidade eletroforética e o perfil de potencial da membrana celular

O objetivo do presente trabalho foi estudar o comportamento dos potenciais superficiais e do perfil de potencial atraves da membrana de eritr ocito em func ao da forca i onica e das cargas superficiais, usando um modelo que leva em conta as cargas el etricas do glicoc alix e das proteınas citoplasm aticas, al em das cargas superficiais da bicamada lipıdica e os efeitos dos eletr olitos divalentes. Programas especıficos em linguagem C foram elaborados para o c alculo desses potenciais, tomando como dados num ericos resultados experimentais de medidas de mobilidade eletrofor etica de eritr ocitos para diferentes valores de forca i onica. Neste c alculo, o metodo para tratamento dos dados eletrofor eticos indicado por Hsu et al.[57] foi incluıdo em nosso modelo. A equac ao de Poisson-Boltzmann nao linear foi resolvida por computac ao num erica, usando o metodo de Runge-Kutta de quarta ordem, obtendo-se os perfis de potencial. Os resultados mostraram que a estimativa da densidade de carga el etrica na superfıcie de c elulas usando a equac ao cl assica de Helmholtz-Smoluchowski conduz a valores que nao conseguem refletir as forcas que regem o comportamento eletrofor etico das mesmas. O presente modelo gerou valores de potenciais superficiais e perfis de potencial para a membrana do eritr ocito bem distintos daqueles obtidos anteriormente para um modelo descrito por uma equac ao de Poisson-Boltzmann linear. Nossos resultados confirmam que a avaliac ao de parametros el etricos superficiais da membrana de eritr ocito, envolvendo dados oriundos de eletroforese, deve incluir c alculos hidrodin amicos al em de eletroest aticos, como sugerido por Hsu et al. [57].

I. Studies on the activation of Drosophila phenol oxidase. II. Studies on serum insulin in normal subjects and diabetics

Part I

Phenol oxidase is the enzyme responsible for hardening and pigmentation of the insect cuticle. In Drosophila, phenol oxidase is a latent enzyme. Enzyme activity is produced by the interaction of a number of protein components. A minimal activation scheme consisting of six protein components, designated Pre S, S activator, S, P. P' and Ʌ₁ is described. Quantitative assays have been developed for the S activator, S, P and P' proteins and these components have been partially purified. Experiments describing the interactions of the six components have been conducted and a model for the activation of phenol oxidase in a minimal system is proposed. Possible mechanisms of the reactions between the constituents of the activating system and potential regulatory mechanisms involved in phenol oxidase production and function are discussed.

Part II

A method has been developed for the partial purification of insulin from human serum. A procedure for the determination of the electrophoretic mobility of serum insulin on polyacrylamide gels is described. An electrophoretic analysis of insulin isolated from a normal subject is reported and in addition to a major band, the existence of a number of minor bands of immunoreactive insulin is described. A comparison of the electrophoretic patterns of insulin isolated from normal and diabetic subjects was carried out and indications that differences between them may occur are reported.

Efeito de ExoU na ativação de NF-κB e na secreção de IL-8 por células humanas infectadas por Pseudomonas aeruginosa

ExoU, uma citotoxina produzida pelo patógeno oportunista Pseudomonas aeruginosa e translocada para o citossol de células hospedeiras via sistema de secreção do tipo III, é associada à gravidade de infecções agudas. Estudos anteriores realizados em nosso laboratório relataram a potente atividade pró-inflamatória de ExoU, responsável por um intenso recrutamento de neutrófilos para o sítio de infecção. No presente trabalho, o efeito de ExoU na modulação da ativação do fator transcricional NF-κB e na regulação da expressão e da secreção da quimiocina para neutrófilos IL-8 foi avaliado em culturas de células epiteliais respiratórias e endoteliais humanas infectadas com a cepa PA103 de P. aeruginosa (produtora de ExoU) ou com a mutante deletada no gene exoU, PA103κexoU. Análises por RT-PCR semi-quantitativo mostraram que a infecção pela cepa produtora de ExoU levou ao aumento dos níveis de mRNA de IL-8, enquanto ensaios de alteração da mobilidade eletroforética (EMSA), supershift e com gene repórter mostraram que ExoU induziu a translocação nuclear do heterodímero transativador p65/p50 de NF-κB e a ativação da transcrição de genes dependente deste fator transcricional. Adicionalmente, o tratamento das culturas celulares com um inibidor de NF-κB antes da infecção bacteriana reduziu significativamente os níveis de mRNA de IL-8 e da secreção desta quimiocina. Em conjunto, estes resultados mostram que ExoU ativa NF-κB e, consequentemente, estimula a expressão e a secreção de IL-8 por células epiteliais respiratórias e células endoteliais infectadas com P. aeruginosa

Estudo da cromatina nos sítios de fixação à matriz nuclear no domínio do gene TP53 e das modificações epigenéticas e no modelo de progressão tumoral mamária 21T

Dentre os diversos tipos de câncer agressivos, o câncer de mama é o mais comum em mulheres. Mutações hereditárias e adquiridas, assim como alterações epigenéticas atuam em sinergia na carcinogênese mamária e na progressão tumoral. A proteína P53 é uma supressora de tumor e possui uma atuação fundamental na integridade genômica. Apesar do vasto conhecimento sobre o controle da P53 a nível de proteína, ainda pouco se sabe sobre o controle transcricional do gene TP53. A série 21T, uma série de 4 linhagens celulares originadas da mama da mesma paciente, representando diferentes estágios de progressão tumoral mamária, é um eficiente modelo para investigação das alterações epigenéticas e suas influências na expressão gênica ao longo da progressão do câncer de mama. Nós analisamos a organização do domínio do gene TP53 através da técnica de arranjo de DNA, em diversas linhagens celulares de câncer de mama e linhagens controle, e realizamos uma tentativa de caracterizar estes elementos de DNA nas linhagens controle não-tumorais HB2 e MCF10A e nas tumorais MCF-7, MDA-MB-231, T47D, através dos marcadores epigenéticos de eucromatina, H4Ac, e heterocromatina, H3K9me3. Ainda analisamos a ligação de proteínas à região associada à matriz nuclear (MAR), denominada MAR 2, e a possível ligação da proteína ligante à matriz nuclear (MARBP), PARP-1, através de ensaios de gel shift (EMSA). Detectamos que na linhagem controle epitelial mamária, HB2, o gene TP53 está posicionado num domínio de DNA relativamente pequeno, aproximadamente 50 kb, delimitado por dois sítios de fixação à matriz nuclear. Interessantemente, esta estrutura de domínio se apresentou radicalmente diferente nas linhagens de câncer de mama estudadas, MCF7, T47D, MDA-MB-231 e BT474, nos quais o tamanho do domínio estudado estava aumentado e a transcrição do TP53 diminuída. Os enriquecimentos com os marcadores epigenéticos de cromatina H4Ac e H3K9me3 estão diferentemente distribuídos nas MARs nas linhagens celulares. Surpreendentemente, a MAR 2 apresentou uma ligação altamente específica, o que poderia representar a atuação de fatores transcricionais envolvidos na organização da cromatina. Através de programas de bioinformática, detectamos putativos sítios para interessantes fatores de transcrição, tais como o c/EBP-beta e c-myb, que poderiam atuar em cis regulando a expressão do gene TP53 e outros flanqueadores. Nós propusemos um modelo para a organização da cromatina na região de domínio do gene TP53 com os genes flanqueadores. Através da série 21T, detectamos uma hipometilação global genômica, nas células cancerosas 21NT e 21MT1. Uma importante diminuição da expressão global do marcador H4Ac nas células metastáticas 21MT1, foi detectada em relação às outras linhagens. Os níveis de RNAm das principais enzimas relacionadas as modificações epigenéticas são consistentes com as observadas hipometilação genômica e hipoacetilação. Através de microscopia confocal, verificamos que o marcador H4Ac está localizado, na maior parte na periferia e o marcador H3K9me3, pericêntrico nos núcleos tumorais. Por fim, verificamos que o promotor P1 do gene TP53 apresenta um estado de cromatina aberta, e a expressão do gene TP53 é similar em todas as células da série 21T.

Tunable hydrodynamic chromatography of microparticles localized in short microchannels.

This paper describes a new way to perform hydrodynamic chromatography (HDC) for the size separation of particles based on a unique recirculating flow pattern. Pressure-driven (PF) and electro-osmotic flows (EOF) are opposed in narrow glass microchannels that expand at both ends. The resulting bidirectional flow turns into recirculating flow because of nonuniform microchannel dimensions. This hydrodynamic effect, combined with the electrokinetic migration of the particles themselves, results in a trapping phenomenon, which we have termed flow-induced electrokinetic trapping (FIET). In this paper, we exploit recirculating flow and FIET to perform a size-based separation of samples of microparticles trapped in a short separation channel using a HDC approach. Because these particles have the same charge (same zeta potential), they exhibit the same electrophoretic mobility, but they can be separated according to size in the recirculating flow. While trapped, particles have a net drift velocity toward the low-pressure end of the channel. When, because of a change in the externally applied PF or electric field, the sign of the net drift velocity reverses, particles can escape the separation channel in the direction of EOF. Larger particles exhibit a larger net drift velocity opposing EOF, so that the smaller particles escape the separation channel first. In the example presented here, a sample plug containing 2.33 and 2.82 microm polymer particles was introduced from the inlet into a 3-mm-long separation channel and trapped. Through tuning of the electric field with respect to the applied PF, the particles could be separated, with the advantage that larger particles remained trapped. The separation of particles with less than 500 nm differences in diameter was performed with an analytical resolution comparable to that of baseline separation in chromatography. When the sample was not trapped in the separation channel but located further downstream, separations could be carried out continuously rather than in batch. Smaller particles could successfully pass through the separation channel, and particles were separated by size. One of the main advantages of exploiting FIET for HDC is that this method can be applied in quite short (a few millimeters) channel geometries. This is in great contrast to examples published to date for the separation of nanoparticles in much longer micro- and nanochannels.

Molecular basis of transferrin polymorphism in goldfish (Carassius auratus)

Transferrin (TF) polymorphism was investigated in a color variety of goldfish (Carassius auratus), and its molecular basis analyzed. Three TF variants (A(1), A(2) and B-1) were identified from an inbred strain of the goldfish, of which A(1) and B-1 displayed a large electrophoretic difference on both native and SDS-PAGE gels. The TF cDNAs corresponding to variants A(1) and B-1 were cloned and sequenced from A(1)A(1), A(1)B(1) and B1B1 individuals, and their deduced amino acid sequences were analyzed. Substantial amino acid variation occurred between variants A(1) and B-1, with significant differences in peptide length, theoretical molecular weight (Mw) and isoelectric point (pI). No potential glycosylation sites were observed in the two amino acid sequences, which excluded the possibility that carbohydrate difference might cause electrophoretic variation among the TF variants. Further analysis suggested that the distinct electrophoretic mobility of the two variants A(1) and B-1 by SDS-PAGE resulted from their Mw difference, while the difference by the native PAGE could be explained by their pI variation. Furthermore, genomic DNA fragments containing the transferrin alleles were amplified and subjected to RFLP analysis in A(1)A(1), A(1)B(1) and B1B1 individuals. The data revealed characteristic banding patterns for each TF genotype, and demonstrated that the TF alleles A(1) and B-1 could be used as a co-dominant marker system. The initial work relating to the goldfish TF variants will benefit the understanding of the evolutionary and functional significance of TF polymorphism in fish.

青藏高原栽培青稞遗传多样性研究

青稞，是我国藏区居民对裸大麦的称谓，它不仅是藏民的主要食粮、燃料和牲畜饲料，而且也是啤酒、医药和保健品生产的原料；青稞不仅为藏区人民的健康和经济发展做出了很大的贡献，而且对人类健康和社会经济的可持续发展都有重要的意义。青藏高原是我国及世界上青稞分布和种植面积最大的地区，资源极其丰富。虽然从经典遗传直到分子标记对我国大麦遗传多样性都有研究，但研究手段、数量仍然不够深入，对我国大麦资源遗传多样性研究的信息非常有限，不能很好地满足大麦遗传研究和育种应用的需要，尤其是对西藏栽培大麦的遗传多样性的研究还只是刚刚开始，关于栽培青稞多态性的研究报道很少。本研究采用SSR标记和蛋白质电泳两类技术，从SSR标记位点、单体醇溶蛋白、B组醇溶蛋白和淀粉粒结合蛋白（SGP）等四个方面对我国青藏高原栽培青稞的遗传多样性进行了综合评价。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究采用SSR标记分析了64份青藏高原栽培青稞的遗传多样性，同时评估SSR标记在我国大麦育种和品种鉴定中的应用潜力。选择了30个已知作图位点SSR标记，其中25个标记与重要性状的控制位点连锁紧密。选择的30个SSR标记，5个未得到很好的扩增产物，3个无多态性。22个多态性SSR标记位点中，每位点检测出等位基因2~15个，共检测出等位基因132个，平均每位点6.0 个。各多态位点检测出基因型为2~11种，位点HVM33的基因型最多。各多态位点的多态信息指数为0.16~0.91, 平均为0.65。根据PIC值选择了13个SSR标记用于我国青藏高原栽培青稞基因型鉴定，这些标记的PIC值为0.6以上。结合PIC值和基因型差异，选择了8个多态信息含量高的SSR标记，构建了高效指纹图谱，此图谱能把64份材料完全区分。 贮藏蛋白电泳分析是研究相关编码蛋白基因多态性的非常有效的方法。大麦单体蛋白与小麦醇溶蛋白相对应，具有丰富的多态性，可用于大麦遗传多样性、品种鉴定和群体进化等研究。本研究通过A-PAGE电泳技术研究了84份青藏高原栽培青稞的单体醇溶蛋白多态性。大麦单体醇溶蛋白图谱与小麦醇溶蛋白电泳图谱类似，所分离的蛋白清晰地分为ω－，γ－，β－和α－四个部分。青藏高原栽培青稞单体醇溶蛋白具有丰富的多态性，84份青稞材料中存在43条不同的蛋白带，75种组合带谱；其中67种为单一材料所独有，另8种则分别包含了2-3份材料。每份材料中拥有醇溶蛋白带为6－16条，含有6-10条单体醇溶蛋白带材料较多。西藏和四川材料群体单体醇溶蛋白多态性不同，具有区域特异性。西藏材料中发现了40条不同蛋白带，3条特异带，46 种蛋白组合；四川材料中出现了40种不同蛋白带，26种条带组合, 3条特异带。基于单体蛋白多态性的聚类与材料的来源有一定的相关性。A-PAGE单体蛋白具有丰富的多态性，可作为遗传研究和品种鉴定的标记。 大麦醇溶蛋白（hordein）是大麦籽粒的主要贮藏蛋白，与大麦的营养品质和加工品质密切相关，而且具有丰富的多态性，广泛用于品种鉴定、种质筛选、遗传多样性和亲缘关系研究。B组醇溶蛋白是主要的醇溶蛋白组份，约占总醇溶蛋白的80%，而且具有丰富的多态性。本研究采用SDS-PAGE分析了72份青藏高原栽培青稞B组醇溶蛋白的遗传多样性。青藏高原栽培青稞B组醇溶蛋白具有丰富的多态性，72份青稞材料中存在15种蛋白带，30种组合带谱，其中15种为单一材料所独有，另15种则分别包含了2-10份材料。每份材料中B组醇溶蛋白条带数为4-8条，含5、6条的材料较常见。不同来源的群体材料间B组醇溶蛋白组成存在差异，西藏青稞含有26种蛋白组合带谱，其中有19种特异带谱；四川群体中共发现11种蛋白组合带型，其中有4种特有带谱。两群体中都存在稀有条带。聚类分析将材料分成三组，材料聚类与材料来源地没有明显的相关性。 淀粉粒蛋白（Starch granule proteins, SGPs）是一类与淀粉粒结合的微量蛋白，一些淀粉粒蛋白具有淀粉生化合成中主要的酶蛋白功能，其变异会影响淀粉含量和特性，从而影响淀粉的应用。关于我国大麦淀粉粒组成研究还未见报道。本实验首次开创了我国大麦淀粉粒结合蛋白的研究工作。采用SDS-PAGE电泳技术研究了青藏高原栽培青稞的SGP组成，并分析了不同SGP组合间淀粉含量的差异，初步探索了所分离的SGP蛋白与淀粉合成的关系。66份青稞材料中分离了10种主要的SGP，其表观分子量为40-100KD，低于60KD的SGP带有7条，共有16种组合带谱；各SGP蛋白和组合带谱出现的频率存在差异，青藏高原青稞的SGP组成存在多态性。西藏青稞和四川青稞的SGP组成有很大差异，SGP组成具有地域差异性，西藏青稞含有12种蛋白组合带谱，其中有9种特异带谱；四川群体中共发现7种蛋白组合带型，其中有4种特有带谱；两群体中仅有3种共同的蛋白组合带谱。SGP蛋白特性将66份青稞分为三组, 即Ⅰ、Ⅱ、Ⅲ，材料聚类与材料来源具有一定的相关性。不同组合带谱材料间淀粉含量差异显著性检验结果显示，不同带谱间材料的总淀粉含量、直链淀粉含量和支链淀粉含量有差异，带谱2（SGP1+3+7+9+10）和8（SGP1+2+4+6+8）的总淀粉含量及支链淀粉含量显著大于组合带谱3（SGP1+3+7+10）的总淀粉含量。组合带谱7（SGP1+2+6+8）的直链淀粉含量显著低于带谱11（SGP1+5+8）的直链淀粉。带谱SGP2、3、4、5、6、7、8、9、10可能参与淀粉合成，SGP9可能与高支链淀粉的合成相关。 SSR标记位点、单体醇溶蛋白、B组醇溶蛋白、淀粉结合蛋白等四个方面的研究结果表明青藏高原SSR标记多态性、单体醇溶蛋白多态性、B组醇溶蛋白多态性和SGP多态性都非常丰富，与青藏高原是栽培青稞的多样性分布中心的观点一致。 青藏高原栽培青稞的SSR标记、单体醇溶蛋白、B组醇溶蛋白和SGP多态性表现出很大差异。SSR标记覆盖了整个基因组，多态性非常高。单体蛋白、B组醇溶蛋白、SGP蛋白是育种中非常关注的性状，他们只是代表基因组中的某一区域或位点，多态性相对较低。但单体蛋白多态性很高，84份材料中检测出43条不同蛋白带，75种不同的组合带谱。SSR标记技术和单体蛋白技术都是遗传多样性研究的有力工具，但单体蛋白技术不仅多态性高，而且经济、操作简便，是种质鉴定的理想方法。 对不同标记的多态性材料数据进行聚类，聚类图能为我们提供各材料间的遗传相似信息，为材料选择提供参考。但材料聚类与材料来源的地理区域的相关性表现不一致。SSR聚类和B组醇溶蛋白聚类与材料的来源地无相关性，而单体醇溶蛋白和SGP聚类与材料来源地有一定相关性，即西藏群体和四川群体分别有集中类群，这可能是人为选择的附加效应。 不同来源的群体材料的遗传多样性不同，具有区域特异稀有基因，加强不同地区间资源的交换和配合使用，有利于增加群体遗传多样性和新品种培育。 青藏高原栽培青稞的麦芽浸提性状、淀粉性状、病虫及裸粒等重要农艺性状控制位点存在丰富的变异，遗传基础宽广，可能蕴藏着多种不同的等位基因，是研究重要性状遗传特性、基因资源挖掘和遗传育种的宝贵资源库。 Hulless barley, due to its favorable attributes such as high feed value, good human nutrition，rich dietary fiber and ease processing, attracts people,s attention . Hulless barley plays a very important role in Tibetan life, used as essential food crop, main animal feed and important fuel. In addition to tsampa (roasted barley flour), a main food for Tibetan, hulless barley is also made into cake, soup, porridge, recent naked barley liquor and cornmeal. Qinghai-Tibet Plateau is one of a few areas which plant naked barley widely in the world and also has a long growing history. Genetic diversity of the cultivated hulless barley in this region , however, has not been documented. The study of genetic diversity existing within this population is of particular interest in germplasm identification, preservation, and new cultivar development. This study analyzed the genetic diversity of the cultivated naked barley from Qinghai-Tibet plateau through the study of SSR marker loci and monomeric prolamins, B-horden and starch granule proteins. SSRs are present abundantly in genomes of higher organisms and have become a popular marker system in plant studies. SSRs offer a number of advantages, such as the high level of polymorphisms, locus specificity, co-dominance, reproducibility, ease of use through PCRand random distribution throughout the genome. In barley, several hundred SSRs have been developed and genetically mapped and can therefore be selected from specific genomic regions. The genetic diversity of 64 cultivated naked barley from Tibet and Sichuan was studied with 30 SSRs of known map location.Among the selected SSR markers, PCR products of 5 SSR markers were not obtained and 3 SSR marker loci were monomeric. A total of 132 alleles were identified at 22 polyomeric SSR loci. The number of alleles per locus ranged from 2 to 15, with an average of 6.0. The polymorphism information content values for the SSRs ranged from 0.08 to 0.94, with an average of 0.65. 13 SSR markers with the PIC value >0.6 have been selected for discrimination of Qinghai-Tibet naked barley genotypews. A finger Print map was developed through 7 SSR markers with the high PIC value. It could be used as an efficient tool for gene discovery and identification of gernplasm. Hordeins, the main storage proteins of the barley seed, are composed of momomeric and polymeric prolamins and divided into -A, B, C and D groups in order of decreasing electrophoretic mobility. Hordeins show high inter-genotypic variation and have been extensively used as markers for cultivar identification and analyzing the genetic diversity. This study analyzed the genetic diversity of B-hordein in 72 naked barley from Qinqhai-Tibet Plateau. Extensive diversity was observed. A total of 15 different bands and 30 distinct patterns were found. Jaccard's coefficient of similarity was calculated, and the accessions were divided into three　main groups by cluster analysis using UPGMA. Differentiation among the populations from different collecting regions based on the polymorphism of B-hordein was investigated. Monomeric prolamins show high inter-genotypic variation and have been used as molecular markers for cultivar identification, analyzing the genetic diversity in collections and investigating the evolution processes and structure of populations However, the cultivated hulless accessions from Qinghai-Tibet Pateau in China have never been examined with respect to monomeric prolamins. This study analyzed the genetic diversity of monomeric prolamins (protein fraction corresponding to wheat gliadins) using the Acid -PAGE technique in eighty-four cultivated hulless barley from Qinqhai-Tibet Plateau in China. Extensive diversity was observed. A total of 43 different bands were found, of which 21 different bands were in the region of ω group, 8 in the region of γ, 8 in the region of β, and 6 in the region of α group. Among the 86 accessions, 75 distinct patterns were identified. The number of bands ranged from 6 to 16, depending on the variety. Jaccard’s coefficient of similarity was calculated, and the lines were grouped by cluster analysis using UPGMA. A dendrogram was obtained from the analysis of the groups and five main clusters were identified. No relationship between the distribution in the dendrogram and growth habits and origins of the cultivars could be detected. Starch is the major constituent of the cereal endosperm, comprising approximately 65% of the dry weight of the mature wheat grain. The starch formed in all organs of plants is packaged into starch granules, which vary widely between species and cultivars in size and shape. Wheat endosperm starch granules contain about corresponding to the main biosynthase of starch. This report firstly dealed with intraspecific variation of the major SGPs in cultivated naked barley from Qinghai-Tibet plateau. A total of 10 major SGPs were observed in the range of 40KD-100KD and 16 types of patterns were found. Based on the variation of SGPs, accessions studied were classified into 3 groups. A geographical cline of electrophoregram was observed. In addition, significance test of the difference of starch content among groups and types of patterns were done, and the results indicated those SGPs could be related to the content of starch. Diagram obtained through cluster analysis exhibited a structuration of diversity and genetic relationship among cultivated hulless accessions. In breeding program, parents with genetically distant relationship for hybridization will increase genetic diversity of progenies. In conclusion, cultivated naked barley from Qinghai-Tibet Plateau in China presents a high variability with respect to monomeric prolamins，SSR markers , B- hordeins and SGPs. The result of this study supports Qinghai-Tibet Plateau is the center of cultivated hulless barley and the cultivated naked barley is considered to be a gene pool with large diversity and could be applied to breeding for cereal.

Glutathione-Mediated Release of Functional Plasmid DNA from Positively Charged Quantum Dots

DNA was efficiently bound to water-soluble positively charged CdTe quantum dots (QDs) through complementary electrostatic interaction. These QDs-DNA complexes were disrupted and DNA was released by glutathione (GSH) at intracellular concentrations. Interestingly, there was almost no detectable DNA released by extracellular concentration of GSH. The formation of QDs-DNA complexes and GSH-mediated DNA release from the complexes were confirmed by dye displacement assay, electrophoretic mobility shift assay (EMSA), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) experiments.

Surface modification of poly(dimethylsiloxane) microchips using a double-chained cationic surfactant for efficiently resolving fluorescent dye adsorption

This paper described a double-chained cationic surfactant, didodecyldimethylammonium bromide (DDAB). for dynamic surface modification of poly(dimethylsiloxane) (PDMS) microchips to reduce the fluorescent dyes adsorption onto the microchannel. When DDAB with a high concentration was present as the dynamic modification reagent in the running and sample buffer, it not only reversed the direction of electroosmotic flow, but also efficiently suppressed fluorescent dyes pyronine Y (PY) or rhodamine 8 (RB) adsorption onto the chip surface. In addition, vesicles formed by DDAB in the buffer with higher surface charge density and electrophoretic mobility could provide wider migration window and potential for the separation of compounds with similar hydrophobicity. Factors affecting modification, such as pH and concentrations of the buffer, DDAB concentration in the buffer were investigated. Compared with commonly used single-chained cetyltrimethylammonium bromide, DDAB provided a better modification performance.

Zebrafish thymidylate synthase binds to its own mRNA in vitro and in vivo

Thymidylate synthase (TS), an essential enzyme for DNA de novo synthesis, is a critical therapeutic target in cancer therapy. Previous study has shown that TS was able to bind to its own mRNA in human and E.coli, resulting in translational repression. Zebrafish is the best animal model for vertebrate study. In order to study the regulatory mechanism of zebrafish TS, the enzyme were expressed in E. coli BL21 (DE3) and it was purified to homogeneity. Electrophoretic mobility shift assay (EMSA) was used to detect the interaction of zebrafish TS protein and its own TS transcript in vitro and the results showed that zebrafish TS could bound with its own mRNA specifically. Further study revealed that zebrafish TS was able to interact with its own mRNA in vivo using immunoprecipitation : RT-PCR technique. The results provide evidence that zebrafish may be developed as an useful model for studying the anti-metabolism agents.

Isolation and characterization of a novel variant of HMW glutenin subunit gene from the St genome of Pseudoroegneria stipifolia

The x- and y-type high molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe investigations on the HMW glutenin subunits from several Pseudoroegneria accessions. The electrophoretic mobilities of the HMW glutenin subunits from Pd. stipifolia, Pd tauri and Pd strigosa were much faster than those of orthologous wheat subunits, indicating that their protein size may be smaller than that of wheat subunits. The coding sequence of the Glu-1St1 subunit (encoded by the Pseudoroegneria stipifolia accession PI325181) was isolated, and found to represent the native open reading frame (ORF) by in vitro expression. The deduced amino acid sequence of Glu-1St1 matched with that determined from the native subunit by mass spectrometric analysis. The domain organization in Glu-1St1 showed high similarity with that of typical HMW glutenin subunits. However, Glu-1St1 exhibited several distinct characteristics. First, the length of its repetitive domain was substantially smaller than that of conventional subunits, which explains its much faster electrophoretic mobility in SDS-PAGE. Second, although the N-terminal domain of Glu-1St1 resembled that of y-type subunit, its C-terminal domain was more similar to that of x-type subunit. Third, the N- and C-terminat domains of Glu-1St1 shared conserved features with those of barley D-hordein, but the repeat motifs and the organization of its repetitive domain were more similar to those of HMW glutenin subunits than to D-hordein. We conclude that Glu-1St1 is a novel variant of HMW glutenin subunits. The analysis of Glu-1St1 may provide new insight into the evolution of HMW glutenin subunits in Triticeae species. (C) 2007 Elsevier Ltd. All rights reserved.

Effects of organic modifiers on solute retention and electrokinetic migrations in micellar electrokinetic capillary chromatography

Influences of seven organic modifiers, including urea, methanol (MeOH), dioxane (DIO), tetrahydrofuran (THF), acetonitrile (ACN), 1-propanol (1-PrOH) and 2-propanol (2-PrOH), on the solute retention and the electrokinetic migrations in micellar electrokinetic capillary chromatography (MEKC) are investigated with sodium dodecyl sulfate (SDS) micelle as pseudostationary phase. It is observed that in the limited concentration ranges used in the MEKC systems the effect of organic modifier concentration on the retention can be described by the equation logk'=logk'(w)-SC for most binary aqueous-organic buffer, but deviations from this retention equation are observed at ACN and particularly THF as organic modifiers. With parameter S as a measure of the elutropic strength, the elutropic strength of the organic modifiers is found to follow a general order urea mobility decreases linearly with increasing modifier concentrations. The strength of modifiers to suppress the electroosmotic mobility follows the order urea < MeOH, ACN < DIO, THF, 1-PrOH, 2-PrOH. The addition of ACN affects the electrophoretic mobility of the SDS micelle in different patterns depending on the buffer composition used, while the addition of the other modifiers leads to a rough linear decrease of the electrophoretic mobility of the SDS micelle. The suppressing strength of THF on the electrophoretic mobility of the SDS micelle is strongly related to the buffer composition, whereas the suppressing strength for the other modifiers follows the sequence urea < MeOH < DIO, 1-PrOH, 2-PrOH. The migration time window is expanded by the use of organic modifiers. Urea and MeOH have the least expanding strength while ACN, DIO, THF, 1-PrOH and 2-PrOH have the highest expanding strength on the migration time window.

Co-regulation of nuclear respiratory factor-1 by NFkappaB and CREB links LPS-induced inflammation to mitochondrial biogenesis.

The nuclear respiratory factor-1 (NRF1) gene is activated by lipopolysaccharide (LPS), which might reflect TLR4-mediated mitigation of cellular inflammatory damage via initiation of mitochondrial biogenesis. To test this hypothesis, we examined NRF1 promoter regulation by NFκB, and identified interspecies-conserved κB-responsive promoter and intronic elements in the NRF1 locus. In mice, activation of Nrf1 and its downstream target, Tfam, by Escherichia coli was contingent on NFκB, and in LPS-treated hepatocytes, NFκB served as an NRF1 enhancer element in conjunction with NFκB promoter binding. Unexpectedly, optimal NRF1 promoter activity after LPS also required binding by the energy-state-dependent transcription factor CREB. EMSA and ChIP assays confirmed p65 and CREB binding to the NRF1 promoter and p65 binding to intron 1. Functionality for both transcription factors was validated by gene-knockdown studies. LPS regulation of NRF1 led to mtDNA-encoded gene expression and expansion of mtDNA copy number. In cells expressing plasmid constructs containing the NRF-1 promoter and GFP, LPS-dependent reporter activity was abolished by cis-acting κB-element mutations, and nuclear accumulation of NFκB and CREB demonstrated dependence on mitochondrial H(2)O(2). These findings indicate that TLR4-dependent NFκB and CREB activation co-regulate the NRF1 promoter with NFκB intronic enhancement and redox-regulated nuclear translocation, leading to downstream target-gene expression, and identify NRF-1 as an early-phase component of the host antibacterial defenses.

The NRF-1/α-PAL transcription factor regulates human E2F6 promoter activity

E2F6 is widely expressed in human tissues and cell lines. Recent studies have demonstrated its involvement in developmental patterning and in the regulation of various genes implicated in chromatin remodelling. Despite a growing number of studies, nothing is really known concerning the E2F6 expression regulation. To understand how cells control E2F6 expression, we analysed the activity of the previously cloned promoter region of the human E2F6 gene. DNase I footprinting, gel electrophoretic-mobility shift, transient transfection and site-directed mutagenesis experiments allowed the identification of two functional NRF-1/α-PAL (nuclear respiratory factor-1/α-palindrome-binding protein)-binding sites within the human E2F6 core promoter region, which are conserved in the mouse and rat E2F6 promoter region. Moreover, ChIP (chromatin immunoprecipitation) analysis demonstrated that overexpressed NRF-1/α-PAL is associated in vivo with the E2F6 promoter. Furthermore, overexpression of full-length NRF-1/α-PAL enhanced E2F6 promoter activity, whereas expression of its dominant-negative form reduced the promoter activity. Our results indicate that NRF-1/α-PAL is implicated in the regulation of basal E2F6 gene expression.