Cloning and expression of mouse 60 kDa ribonucleoprotein SS-A/Ro

Autoantibodies to SS-A/Ro are among the most common found in sera of patients with systemic rheumatic diseases. These autoimmune diseases can affect various organ systems of the body and are variable in their manifestations and presentation. One of the autoimmune targets is the 60 kDa SS-A/Ro protein known to be associated with small cytoplasmic Y RNAs. To study systematically the expression of the protein, we have cloned the mouse full length 60 kDa SS-A/Ro cDNA using 5′ RACE based on a cDNA sequence reported in the mouse genome project. The recombinant protein derived from the putative full-length construct was shown to react with human prototype anti-SS-A/Ro serum Ge in western blot and immunoprecipitation and comigrated with cellular 60 kDa SS-A/Ro protein in 3T3 cells. Cellular expression, measured by RT-PCR, was highest in mouse brain, followed by lung, muscle, kindney and heart. Lower levels were found in testis, liver and spleen. Like the human 60 kDa SS-A/Ro protein, the deduced mouse homolog has 538 amino acids. Sequence analysis showed 89.9% identity and 95.0% similarity between the mouse and human proteins.     

In vivo persistence of DNA triple helices containing psoralen-conjugated oligodeoxyribonucleotides.

Triple helices represent an attractive method for modulating specific gene expression. In particular, cross-linking between a triplex-forming oligonucleotide (TFO) and its duplex DNA target, typically through the formation of psoralen photoadducts, allows efficient blocking of elongation by RNA polymerases in vitro. However, in vivo, this approach is limited by DNA repair of the photoadduct. Here we describe the use of an oligodeoxyribonucleotide 19mer psoralen-modified TFO to form covalent linkages between an oligonucleotide and both strands of the targeted duplex DNA, thereby efficiently blocking expression of a luciferase reporter gene. Most importantly, we demonstrate that both the psoralen cross-link and the purine-motif triplex remained intact for at least 72 h post-transfection, indicating that such species can persist for an extended period of time in vivo. These findings support the feasibility of an antigene approach for the therapeutic regulation of specific gene expression.     

腺苷三磷酸和环腺苷单磷酸对丝状真菌纤维素酶合成的调节

以虫荧光素酶法检验了四株丝状真菌在葡萄糖—无机盐液体培养过程中的胞内ATP含量。结果表明,只有当胞内ATP浓度低于10~(-S)mg/ml时,真菌才开始合成胞外纤维素酶(FPA)。以不同浓度的各种碳源培养时,菌体胞内ATP含量只要超过10~(-1)mg/ml,FPA的合成即发生阻遏。菌体胞内ATP含量与FPA合成呈显著负相关。以高效液相色谱(HPLC)法检测了菌体培养液中的cAMP含量。在非阻遏条件下,外源cAMP可以提高FPA的合成水平。但外源cAMP不能解除已经发生的酶合成阻遏。菌体ATP和cAMP水平是调节真菌纤维素酶合成的重要因子。     

微菌落观察法快速检测和鉴别结核杆菌培养物的研究

将取自肺结核患者的219例痰标本接种匡氏琼脂平板,共分离到112例结核菌生长物。其中培养法104例(92.8％)阳性,微菌落观察法108例(96.4％)阳性,微菌落法阳性率稍高。培养法和微菌落法阳性标本首次检出时间分别为18.6d和11d,微菌落检出时间更短(P<0.01)。常规菌型鉴别方法与微菌落法对结核杆菌菌型鉴别的符合率为99％。     

太湖16000年来沉积环境的演变

通过对太湖钻孔岩芯岩性,结构,构造的剖析及粒度,磁化率的测试,发现冰后期东太湖形成于跑今６５００年前,在距今６５００－５８００年,为一水深约２－３ｍ的,经常受到流水作用影响的浅水湖泊,距今约５８００－５７００年,东太湖曾一度干枯或接近于干枯,距今５７００年以来湖泊变浅,平均水深只有１ｍ左右,由于湖泊变浅,湖底经常遭受波浪的扰动,形成波状层理或透镜状层理。西太湖局部洼地集水成湖的时间比东太湖早,并且     

太湖全新世沉积物有机联同位素的分布及其古气侯意义

通过太湖两个钻孔岩芯有机碳同位素（１３Ｃ／１２Ｃ）的分析,发现δ１３Ｃ的垂直分布与太湖地区１６０００ａＢ．Ｐ．以来古气候波动相关。根据δ１３Ｃ垂直分布曲线可推测,大约在１１０００—６０００ａＢ．Ｐ,太湖地区处于温暖湿润期,气温高于现在平均温度。另一方面,根据太湖两岩芯沉积物的δ１３Ｃ值的对比发现,大约在１１０００—６０００ａＢ．Ｐ,西太湖（Ｗ１Ｂ）岩芯沉积物的δ１３Ｃ平均值明显高于东太湖（Ｅ２Ｂ）。据此可认为,在这一期间西太湖很可能有过海水侵入。     

Molecular basis and haplotyping of the alphaII domain polymorphisms of spectrin: application to the study of hereditary elliptocytosis and pyropoikilocytosis.

Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are inherited disorders of erythrocyte shape that are frequently associated with abnormalities in alpha-spectrin, one of the principal structural proteins of the erythrocyte membrane skeleton. Five polymorphisms of the alpha-spectrin gene, located in a 6-kb interval of genomic DNA, were identified and analyzed in normal and mutant alpha-spectrin alleles. Three of these polymorphisms are due to single nucleotide substitutions in the alpha-spectrin gene coding region that lead to changes in the amino acid sequence. In combination, these three polymorphisms are responsible for the different peptide phenotypes of the alphaII domain previously observed following limited tryptic digestion of spectrin protein. The most common haplotype, type 1, was found predominantly in Caucasians and was the only haplotype identified in Asians. Haplotypes 2, 3, and 4 were identified predominantly in individuals of African ancestry and were commonly found in patients with HE or HPP. Analysis of coinheritance of alphaII domain polymorphisms with alpha-spectrin gene mutations causing HE or HPP in African-American patients with HE and HPP suggests that, with one exception, a given HE/HPP mutation is present in an alpha-spectrin gene of only one haplotype, indicating a founder effect. The other two polymorphisms located in this region of the alpha-spectrin gene do not change the amino acid sequence of the encoded alpha-spectrin chain and are not in linkage disequilibrium with three of the four alphaII domain haplotypes. A model is proposed for the evolutionary origin of the different haplotypes.     

Incorporation of Molecular Oxygen and Water during Enzymatic Oxidation of Cyanide by Pseudomonas fluorescens NCIMB 11764

Cell extracts (high-speed [150,000 x g] supernatants) from Pseudomonas fluorescens NCIMB 11764 catalyzed the oxidation of cyanide to CO(inf2) (and NH(inf3)). Conversion was both oxygen and NADH dependent, with 1 mol of each being consumed per mol of cyanide degraded. Analysis of (sup13)CO(inf2) by mass spectrometry indicated that one atom each of isotopically labelled oxygen 18 from molecular oxygen and water were incorporated during enzymatic conversion. The results confirm earlier reports of oxygenase-mediated cyanide conversion in this organism. A reaction pathway for cyanide oxidation involving initial monooxygenation followed by hydrolysis of a hypothetical oxygenated intermediate to CO(inf2) (and NH(inf3)) is proposed.     

Evolutionally guided enzyme design

A combination of "rational" and "irrational" strategies for the creation of enzymes with novel properties is proving to be a powerful concept in the field of enzyme engineering. Guided by principles of physical organic chemistry, rational design strategies are used to identify suitable target enzymes and to choose appropriate molecular biological methods for engineering purposes. In contrast, irrational (or random) strategies are centered around the biological paradigm of stochastic molecular evolution. As illustrated in this review, such a hybrid approach is particularly useful for the design of new modular enzymes. (c) 1996 John Wiley & Sons, Inc.     

G418 Selection and stability of cloned genes integrated at chromosomal delta sequences of Saccharomyces cerevisiae

The chromosomal delta sequences of the yeast Saccharomyces cerevisiae were employed as recombination sites to integrate the bacterial neo(r) gene and the yeast SUC2 gene into the yeast genome. A dominate selection method employing the aminoglycoside antibiotic G418 was used. Transformation efficiencies and growth behaviors of the transformants were studied. Transformants were obtained with more than 40 integrations; the majority of insertions were tandem with a maximum of three different insertion sites utilized at one time. After 70-100 generations of growth in nonselective medium, the high copy number SUC2-neo(r) integrants were found to be unstable; only minor instability was observed for the neo(r) and low copy number SUC2-neo(r) integrants. (c) 1996 John Wiley & Sons, Inc.