DIFFERENT RATES OF MITOCHONDRIAL-DNA SEQUENCE EVOLUTION IN KIRK DIK-DIK (MADOQUA-KIRKII) POPULATIONS

We have investigated evolutionary rates of the mitochondrial genome among individuals of Madoqua kirkii using the relative rate test. Our results demonstrate that individuals of two chromosome races, East African cytotype A and Southwest African cytotype D, evolve about 2.3 times faster than East African cytotype B. Cytogenetic changes, DNA repair efficiency, mutagens, and more likely, hitherto unrecognized factors will account for the rate difference we have observed. Our results suggest additional caution when using molecular clocks in the estimation of divergence time, even within lineages of closely related taxa. Rate heterogeneity in microevolutionary timescales represents a potentially important aspect of basic evolutionary processes and may provide additional insights into factors which affect genome evolution. (C) 1995 Academic Press, Inc.

Radiosensitivity to high energy iron ions is influenced by heterozygosity for Atm, Rad9 and Brca1

Loss of function of DNA repair genes has been implicated in the development of many types of cancer. In the last several years, heterozygosity leading to haploinsufficiency for proteins involved in DNA repair was shown to play a role in genomic instability and carcinogenesis after DNA damage is induced, for example by ionizing radiation. Since the effect of heterozygosity for one gene is relatively small, we hypothesize that predisposition to cancer could be a result of the additive effect of heterozygosity for two or more genes critical to pathways that control DNA damage signaling, repair or apoptosis. We investigated the role of heterozygosity for Aim, Rad9 and Brad on cell oncogenic transformation and cell survival induced by 1 GeV/n Fe-56 ions. Our results show that cells heterozygous for both Aim and Rad9 or A tin and Brca1 have high survival rates and are more sensitive to transformation by high energy iron ions when compared with wild-type controls or cells haploinsufficient for only one of these proteins. Since mutations or polymorphisms for similar genes exist in a small percentage of the human population, we have identified a radiosensitive sub-population. This finding has several implications. First, the existence of a radiosensitive sub-population may distort the shape of the dose response relationship. Second, it would not be ethical to put exceptionally radiosensitive individuals into a setting where they may potentially be exposed to substantial doses of radiation. (C) 2010 COSPAR. Published by Elsevier Ltd. All rights reserved.

Hemizygosity for Atm and Brca1 influence the balance between cell transformation and apoptosis

Background: In recent years data from both mouse models and human tumors suggest that loss of one allele of genes involved in DNA repair pathways may play a central role in genomic instability and carcinogenesis. Additionally several examples in mouse models confirmed that loss of one allele of two functionally related genes may have an additive effect on tumor development. To understand some of the mechanisms involved, we examined the role of monoallelic loss or Atm and Brca1 on cell transformation and apoptosis induced by radiation. Methods: Cell transformation and apoptosis were measured in mouse embryo fibroblasts (MEF) and thymocytes respectively. Combinations of wild type and hemizygous genotypes for ATM and BRCA1 were tested in various comparisons. Results: Haploinsufficiency of either ATM or BRCA1 resulted in an increase in the incidence of radiation-induced transformation of MEF and a corresponding decrease in the proportion of thymocytes dying an apoptotic death, compared with cells from wild-type animals. Combined haploinsufficiency for both genes resulted in an even larger effect on apoptosis. Conclusions: Under stress, the efficiency and capacity for DNA repair mediated by the ATM/BRCA1 cell signalling network depends on the expression levels of both proteins.

DNA交联损伤修复与范可尼贫血症通路

由于各种内源和外在的因素,DNA会受到各种损伤,不过生物体已经进化出了一套保守的DNA修复系统,对损伤的DNA进行修复,确保基因组的稳定性及完整性。交联损伤是一种常见的DNA损伤,其修复过程相对复杂,主要是通过范可尼贫血症(Fanconi anemia,FA)通路来实现。FA是一种呈现基因组不稳定性的遗传综合征,其内在的病因就是FA通路的失活,因此不能有效修复交联损伤,导致DNA复制异常。

胸苷酸合成酶和二氢叶酸还原酶mRNA亲和肽的发现及mRNA体外展示技术的优化

胸苷酸合成酶（thymidylate synthase，简称TS）和二氢叶酸还原酶（dihydrofolate reductase, 简称DHFR）都是叶酸依赖性酶，在维持DNA合成和DNA修复上发挥关键作用，并且多年来一直是肿瘤研究和化疗的重要靶点。我们前期的研究发现，TS和DHFR在翻译水平上存在负反馈调控机制。人TS和DHFR可以与其自身的mRNA结合，从而抑制mRNA的表达，化疗药物可以与TS或者DHFR相互作用，形成的复合物不能与TS mRNA结合， 使负反馈机制丧失。因此深入研究TS和DHFR的翻译调控机理，对阐明肿瘤抗药性机制，对发现新的抗肿瘤药物和肿瘤的治疗都具有十分重要的意义。 本论文利用mRNA体外展示技术，构建多肽库（约10万亿种多肽分子），利用多种实验手段将mRNA体外展示技术进行优化，提高了多肽库融合肽的产量，提高了mRNA体外展示技术筛选的特异性。将TS mRNA分子上的顺式因子TS30 RNA固定于磁珠上，将融合肽库与顺式因子作用，经过6轮循环，由多肽库中获得了与TS mRNA高度亲和的多肽序列，体外结合实验证明亲和肽可以与TS全长mRNA结合，体外翻译实验证明多肽可以抑制TS mRNA的翻译。并且利用phage display技术由噬菌体肽库（12个氨基酸随机肽库）经过四轮筛选，分别筛选到TS和DHFR的亲和肽，凝胶阻滞实验证明它们分别能与TS和DHFR mRNA结合。 本论文利用的展示技术可以广泛应用于特异靶点的蛋白质筛选，并且本论文筛选到的TS和DHFR亲和肽可以作为TS和DHFR的抑制剂，从而为获得新型的抗肿瘤药物奠定基础。

HIV-1感染对T和B细胞核内UNG2 mRNA表达的影响

目的:探讨HIV-1感染是否影响细胞中UNG2的表达.方法:采用四步法SYBR green Ⅰ实时定量RT-PCR,对HIV-1感染者的T和B淋巴细胞,以及HIV-1感染的C8166细胞核内UNG2 mRNA的表达进行测定.结果:UNG2 mRNA的表达在HIV-1感染者的T细胞和HIV-1感染的C8166细胞中被明显上调,分别是对照的8.76倍和8.14倍,而在HIV-1感染者的B细胞中却没有被上调.结论:HIV-1感染导致的UNG2表达上调,可能通过减少TCR的多样性削弱Th的功能,另一方面可能有利于病毒对UNG2的包装.

BRCA1在人乳腺癌细胞MCF-7辐射抗性中作用的初步研究

<正>MCF-7细胞是被广泛用以研究乳腺癌的一株模式细胞,该细胞拥有野生型p53基因,但其辐射敏感性与p53基因表达状态无关,这提示可能存在其他基因参与调节其辐

ATM与BRCA1的双杂合性通过影响若干通路增加肿瘤发生风险

<正>目的:流行病学统计数据表明杂合性与肿瘤发生相关。ATM和BRCA1都是DNA损伤修复通路中的多功能基因,其单杂合性对于肿瘤易感性的影响已见报道,但双杂合

扇贝大防御素和G型溶菌酶的基因克隆与重组表达

扇贝养殖是我国传统的海水养殖产业，但自1997 年以来，养殖扇贝陆续爆发的大规模死亡，不但造成了巨大的经济损失，而且严重影响了该产业的健康发展。扇贝病害的不断爆发以及病因的多样性迫切要求制定新的疾病防治措施和开发新型的抗菌物质。 从扇贝自身的免疫防御因子入手，筛选和克隆参与免疫防御的功能基因，一方面可以研究抗病功能基因在病原感染或环境胁迫条件下的表达规律，深入探讨扇贝的免疫防御机制，并可作为抗病良种选育的分子标记，指导扇贝的遗传改良和抗病品系的培育；另一方面，可对抗菌效应物实现重组表达，开发新型的病害预防治疗制剂，取代目前普遍使用的抗生素和化学药物。抗菌效应物是机体在免疫应答过程中产生的多肽类物质，对侵入生物体内的细菌、病毒具有很强的免疫杀灭作用，对抗菌效应物的研究有助于深入了解机体先天性免疫防御的机制。 本研究采用大规模EST测序方法，结合cDNA末端快速扩增（RACE）技术，从海湾扇贝血淋巴中克隆到了大防御素基因（big defensin, AiBD）的全长cDNA序列，该cDNA全长为531 bp，其中5' 非编码区（UTR）为24 bp，开放阅读框（Open Reading Frame, ORF）含有369 bp，编码122 个氨基酸残基；随后为138-bp 的3' UTR，包括一个多聚腺苷酸信号序列（AATAAA）和ploy A尾巴。分析表明，海湾扇贝大防御素是以前体的形式合成，前体分子包括信号肽、前域和成熟肽三部分。采用Northern blot方法，以DIG标记的DNA探针检测了 AiBD mRNA在不同组织中的表达。结果发现，AiBD 基因的转录本主要在血淋巴中表达，在鳃中也有微量的表达，而在外套膜、闭壳肌、性腺及肝胰腺中检测不到杂交信号。采用QRT-PCR（quantitative real time PCR）对鳗弧菌感染后海湾扇贝血淋巴中AiBD mRNA 的表达量进行了检测，结果发现在感染后8 h 内， AiBD mRNA 的相对表达量平缓升高；随着刺激时间的增长，AiBD基因的mRNA表达量急剧增加，在刺激后16 h 和32 h 分别达到了空白组的72.3 倍和131.1 倍。为了研究海湾扇贝大防御素的抗菌活性，将其成熟肽编码区克隆到毕赤酵母表达载体pPIC9K并实现了重组表达。抑菌实验表明，重组AiBD具有广谱的抗菌活性，其对供试的三株革兰氏阳性菌（藤黄微球菌、溶壁微球菌、金黄色葡萄球菌）都表现出显著的抗菌活性，而对革兰氏阴性菌（鳗弧菌、亮弧菌）的抑菌活性则相对较弱；此外，重组AiBD对表达宿主也表现出杀菌活性，证明其具有抗真菌活性。 根据栉孔扇贝G 型溶菌酶基因的cDNA序列，利用构建的Genome Walking 文库获得了栉孔扇贝G 型溶菌酶基因的全长序列，该基因序列全长为8131 bp，由六个外显子和五个内含子组成。六个外显子长度分别为55 bp，60 bp，90 bp，113 bp，145 bp 和140 bp；五个内含子的长度分别为1126 bp，2161 bp，2744 bp，750 bp和592 bp；内含子的两侧都具有RNA正确剪接所必需的识别位点（GT/AG）。利用TRANSFAC 软件对栉孔扇贝G 型溶菌酶基因的5' 侧翼序列分析发现，该基因的5' 侧翼具有 TATA box 和 CAAT box 的共有序列；此外，在该基因的5' 侧翼发现了C/EBP、NF-κB、OCT-1 和 NF-IL6 等参与免疫基因激活的转录因子潜在结合位点。采用Northern blot方法，以生物素标记的RNA 探针检测了栉孔扇贝G 型溶菌酶基因在不同组织中的表达。结果发现，该基因的转录本主要在鳃、性腺及肝胰腺中表达，在血细胞和外套膜中也有微量的的表达，而在闭壳肌中检测不到杂交信号，这表明栉孔扇贝G 型溶菌酶可能兼备参与机体免疫防御和消化的功能。为了研究栉孔扇贝G 型溶菌酶的抗菌活性，将其成熟肽编码区克隆到毕赤酵母表达载体pPIC9K并实现了重组表达。抑菌实验表明，重组产物具有显著的抗阳性菌活性，其对供试的藤黄微球菌、溶壁微球菌表现出明显的抑制作用，对金黄色葡萄球菌未检测到抑制活性；而对革兰氏阴性菌仅表现出微弱的抑菌活性（亮弧菌和鳗弧菌），对大肠杆菌则基本无抑制活性。

RecA-mediated, targeted mutagenesis in zebrafish

We have evaluated the efficacy of RecA, a prokaryotic protein involved with homologous recombination, to direct site-specific mutagenesis in zebrafish embryos. For this we coinjected a vector containing a mutated enhanced green fluorescent protein (EGFP) gene plus 236-nucleotide corrective single-stranded DNAs coated with RecA into I-cell zebrafish embryos. Twenty-hours after fertilization, about 5% to 20% of injected embryos showed EGFP expression in I or more cells when RecA-coated corrective DNAs were used, but not when RecA was omitted. Mutated EGFP genes with 1-bp insertions or deletions were inefficiently activated, whereas those with 7-bp insertions were activated about 4-fold more efficiently. RecA-coated template strand had a higher efficiency than its complementary strand in activation of EGFP expression. Prior irradiation of the embryos with UV light enhanced RecA-mediated restoration of gene activity, suggesting that the effects we observed were augmented by one or more factors of zebrafish DNA repair systems.

Atomic force microscopic study of low temperature induced disassembly of RecA-dsDNA filaments

The assembly and disassembly of RecA-DNA nucleoprotein filaments on double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) are important steps for homologous recombination and DNA repair. The assembly and disassembly of the nucleoprotein filaments are sensitive to the reaction conditions. In this work, we investigated different morphologies of the formed nucleoprotein filaments at low temperature under different solution conditions by atomic force microscopy (AFM). We found that low temperature and long keeping time could induce the incomplete disassembly of the formed nucleoprotein filaments.

Laser conditioning of ZrO2 : Y2O3/SiO2 mirror coatings prepared by E-beam evaporation

Laser conditioning effects of the dielectric mirror coatings with different designs were investigated. Simple quarter-wave ZrO2:Y2O3/SiO2 mirrors and half-wave SiO2 over-coated ZrO2:Y2O3/SiO2 mirror coatings were fabricated by E-beam evaporation (EBE). The absorbance of the samples before and after laser conditioning was measured by surface thermal lensing (STL) technology and the defects density was detected under Nomarski microscope. The enhancement of the laser damage resistance was found after laser conditioning. The dependence of the laser conditioning on the coating design was also observed and the over-coated sample obtained greatest enhancement, whereas the absorbance of the samples did not change obviously. During the sub-threshold fluence raster scanning, the minor damage about defects size was found and the assumption of pre-damage mechanism, based on the functional damage concept, was put forward. The improvement of the laser induced damage threshold (LIDT) was attributed to the benign damage of the defects and the dependence on the coating design owed to the damage growth behavior of different coating designs. (C) 2004 Elsevier B.V. All rights reserved.

A mechanistic model of algal photoinhibition induced by photodamage to Photosystem-II

Photoinhibition is a central problem for the understanding of plasticity in photosynthesis vs. irradiance response. It effectively reduces the photosynthetic rate. In this contribution, we present a mechanistic model of algal photoinhibition induced by photodamage to photosystem-II. Photosystem-IIs (PSIIs) are assumed to exist in three states: open, closed and inhibited. Photosynthesis is closely associated with the transitions between the three states. The present model is defined by four parameters: effective cross section of PSII, number of PSIIs, turnover time of electron transfer chains and the ratio of rate constant of damage to that of repair of D1 proteins in PSIIs. It gives a photosynthetic response curve of phytoplankton to irradiance (PI-curve). Without photoinhibition, the PI-curve is in hyperbola with the first three parameters. The PI-curve with photoinhibition can be simplified to the same form as the hyperbola by replacing either the number of PSIIs with the number of functional PSIIs or the turnover time of electron transfer chains with the average turnover time.

PURIFICATION AND CHARACTERIZATION OF A KINASE SPECIFIC FOR THE SERINE-RICH AND ARGININE-RICH PRE-MESSENGER-RNA SPLICING FACTORS

Members of the SR family of pre-mRNA splicing factors are phosphoproteins that share a phosphoepitope specifically recognized by monoclonal antibody (mAb) 104. Recent studies have indicated that phosphorylation may regulate the activity and the intracellular localization of these splicing factors. Here, we report the purification and kinetic properties of SR protein kinase 1 (SRPK1), a kinase specific for SR family members. We demonstrate that the kinase specifically recognizes the SR domain, which contains serine/arginine repeats. Previous studies have shown that dephosphorylated SR proteins did not react with mAb 104 and migrated faster in SDS gels than SR proteins from mammalian cells. We show that SRPK1 restores both mobility and mAB 104 reactivity to a SR protein SF2/ASF (splicing factor 2/alternative splicing factor) produced in bacteria, suggesting that SRPK1 is responsible for the generation of the mAb 104-specific phosphoepitope in vivo. Finally, we have correlated the effects of mutagenesis in the SR domain of SF2/ASF on splicing with those on phosphorylation of the protein by SRPK1, suggesting that phosphorylation of SR proteins is required for splicing.