辐射诱导基因LRIGx的细胞周期特异性表达及编码产物同源性分析

低剂量辐射诱导表达新基因LRIGx被克隆 .Northern印迹杂交结果表明 ,在 0 2Gyγ射线照射后 2～ 4h ,人A5 4 9细胞中该基因mRNA表达水平显著上调 .当照射剂量增加到 2Gy时 ,其诱导表达水平明显低于 0 2Gy照射 .通过细胞周期同步化 ,观察到LRIGx基因表达高峰在G2 M期 .同源性比较和功能保守域分析结果显示 ,该基因编码产物与DNA修复和重组蛋白RAD5 4、ERCC 6 ,染色质重构和转录调节功能蛋白SWI2 SNF2等有同源性 ,其N端具有与染色质重构、基因转录调控和DNA修复有关的 3个功能结构域 ,即CHROMO、SNF2N和解旋酶C端结构域     

酵母DNA修复基因RAD16温度敏感突变株的分离及人类互补基因的鉴定

用PCR介导的基因重组法敲除酵母RAD16基因, 用羟胺处理酵母RAD16基因表达质粒, 获得RAD16基因突变库,并将其转化RAD16基因敲除的酵母细胞. 用平皿复制法获得温度敏感突变株 rad16-ts2,经互补试验证实,该突变表型为RAD16基因突变所致.将人cDNA表达文库转化rad16-ts2后筛选温度敏感拯救(rescue)表型, 回收拯救表型酵母细胞中的质粒. 测序结果表明,所获得的人cDNA克隆为HLTF/Zbu1/SMACA3基因.比较分析显示,人类该基因编码蛋白质氨基酸序列与酵母Rad16的同一性为32%,相似性则达50%.人HLTF/Zbu1/MACA3基因在HeLa细胞中过表达,可显著抑制过氧化氢损伤和UV照射诱导的细胞凋亡.     

578.2 nm激光照射对兔视网膜的损伤效应研究

研究578.2 nm激光照射对兔视网膜的作用特点,以新西兰白兔5只10眼为实验对象,铜蒸汽激光(578.2 nm)通过裂隙灯照射兔视网膜后极部,照射时间为100 s,光斑直径为2 mm,照射剂量分别为60 J/cm2、80 J/cm2、100 J/cm2、120 J/cm2、160 J/cm2、200 J/cm2,每组4个光斑。照后1 h及24 h进行眼底照相及光镜观察。照光后可见,随激光功率密度的增加,兔视网膜的损伤也逐渐加重,并且照后24 h的损伤要重于照后1h。80 J/cm2和60 J/cm2在照后1 h和24 h均未发现明显改变。578.2 nm激光照射白兔后的主要病理学改变位于脉络膜。因此,以578.2 nm激光作为光动力治疗眼底疾病的光源时,照射剂量不宜超过80 J/cm2。     

荧光假单胞杆菌2P24中gacA、dsbA和phoP/phoQ基因对小RNA因子RsmZ的转录调控

Pseudomonas fluorescens 2P24是分离自麦田的植物病害生物防治菌株,产生抗生素2, 4-二乙酰基间苯三酚（2,4-diacetylphloroglucinol;2,4-DAPG）是其主要防病机制。菌株2P24中小RNA基因rsmZ正调控抗生素2,4-DAPG的产量。【目的】本文研究上游调控因子对RsmZ转录表达的影响,以进一步理解抗生素产生机制。【方法】构建了rsmZ: : lacZ的转录融合结构,将含有该结构的报告载体转入2P24的多个调控基因缺失突变体中,检测相应的缺失基因对rsmZ转录水平的调控作用。【结果】结果表明,反应调控因子GacA对rsmZ基因的转录具有正调控作用,二硫键合成蛋白DsbA对其负调控;双因子调控系统PhoP/PhoQ突变后,rsmZ基因的转录明显滞后。【结论】小RNA基因rsmZ在菌株2P24中受到多个基因的调控,并在信号传递网络中起到重要作用。     

658 nm低能量激光照射对人牙周膜细胞生物学效应的影响

目的:探讨658 nm低能量激光照射对人牙周膜细胞增殖、碱性磷酸酶活性及纤维连接蛋白合成的影响.方法:改良组织块法体外培养人牙周膜细胞.通过658 nm激光照射人牙周膜细胞,观察能量密度为1.86 J/cm2和3.72 J/cm2激光照射后不同时间点细胞增殖效应、碱性磷酸酶活性和纤维连接蛋白的变化.结果:1.86 J/cm2和3.72 J/cm2能量密度的激光照射人牙周膜细胞,可显著促进细胞增殖效应.3.72 J/cm2能量密度的激光照射可提高人牙周膜细胞碱性磷酸酶活性;能量密度为1.86 J/cm2的激光照射人牙周膜细胞72 h后,细胞中纤维连接蛋白分泌量增加.结论:658 nm低剂量激光照射可促进人牙周膜细胞增殖;适量的低剂量激光照射人牙周膜细胞可促进其碱性磷酸酶活性及纤维连接蛋白的分泌.     

芸香苷对家蚕谷胱甘肽-S-转移酶部分基因的诱导表达

谷胱甘肽-S-转移酶(glutathione-S-transferases, GSTs) 是对一种机体的解毒代谢起重要作用并可以被诱导的酶系。为了从分子水平上探究家蚕Bombyx mori GSTs 基因与植物次生物质芸香苷代谢的关系, 本实验采用双跟踪标定定量PCR (dual-spike-in qPCR)方法, 用 5×10^-1, 5×10^-2, 5×10^-3 ng/μL 3 个浓度芸香苷溶液处理家蚕5龄幼虫, 并对各组织中 GSTs Epsilon 家族不同基因的转录水平进行检测。结果显示, 浓度为 5×10^-2 ng/μL 的芸香苷溶液能诱导GSTs 基因的转录水平发生显著变化。在中肠中, BmGSTe1, BmGSTe2 和 BmGSTe6的诱导转录水平较高, 在诱导后24 h达到最大值; 在脂肪体中BmGSTe1, BmGSTe6和BmGSTe7的转录水平相对较高且分别在诱导后 2 h, 2 h和4 h 达到最大值; 而GSTs基因在马氏管中表达量均很低或者检测不到表达。与 5×10^-2 ng/μL 浓度相比, 5×10^-1 ng/μL 的芸香苷溶液诱导后各基因的转录水平上升的幅度较小并且达到峰值的时间不同, 而 5×10^-3 ng/μL 浓度的芸香苷溶液并不能使GSTs基因的诱导转录水平发生变化。结果提示, 家蚕 GSTs Epsilon 家族基因对芸香苷的代谢具有重要作用。     

染色质改构因子BRG1降低UV照射引起的细胞凋亡

生命体的遗传物质基础是DNA分子,多种因素可以作用于细胞内的DNA分子,导致多种类型的DNA损伤。若受损的DNA得不到及时和有效的修复,细胞将走向凋亡或发生变异。染色质改构复合物(chromatin remodeling complex)在基因表达调控和DNA复制等方面扮演着重要角色。依赖ATP的染色质改构复合物SWI/SNF的核心亚基Brahma Related Gene1(BRG1)在染色质结构调整和基因转录调控等多个细胞进程中具有重要作用,仅有有限的文献报道BRG1参与到DNA的损伤修复过程。因此,进一步研究与验证BRG1在调控DNA的损伤修复进而挽救细胞凋亡中的作用十分重要。本文通过利用不同强度的UV照射检测细胞凋亡的情况,初步建立了DNA损伤修复的实验体系。将BRG1表达质粒瞬时转染到SW13(BRG1-/-)细胞系中,并利用30J/m2的UV照射,分别在0h、6h和24h检测细胞早期凋亡程度。结果表明,SW13(BRG1-/-)细胞中瞬时表达BRG1可以明显降低由UV照射引起的细胞凋亡,其中UV照射后24h的细胞表现最明显。我们进一步在HeLa细胞中通过瞬时表达BRG1验证了上述结果。由于BRG1通过染色质改构在基因的转录调控、复制和重组等方面起着重要的作用,我们推测BRG1可能通过染色质改构参与了DNA的损伤修复过程,进而影响了细胞凋亡。     

硫酸锌添加对酿酒酵母乙酸胁迫条件下全局基因转录的影响

【目的】利用转录组测序研究硫酸锌添加提高絮凝酿酒酵母SPSC01乙酸胁迫耐性的分子机理。【方法】在10.0 g/L乙酸胁迫条件下,添加0.03 g/L硫酸锌,取对数期酿酒酵母细胞,与不添加硫酸锌的对照组细胞进行比较转录组分析。【结果】添加硫酸锌的实验组与对照组相比较,50个基因转录水平上调,162个基因转录水平下调,这些转录水平变化明显的基因涉及糖代谢、甲硫氨酸合成、维生素合成等多条代谢途径,此外,转录水平变化的基因还包括抗氧化酶基因等关键胁迫响应基因。【结论】硫酸锌添加可改变酿酒酵母全局基因转录水平,提高抗氧化酶及其他胁迫耐性相关基因的表达,影响细胞氧化还原平衡和能量代谢,通过对多基因转录的调控提高酿酒酵母乙酸耐受性。     

荧光假单胞菌2P24中PcoI-PcoR群体感应系统的自体反馈调控

荧光假单胞菌2P24的PcoI-PcoR 群体感应(QS)系统信号合成基因pcoI的表达受多种因子的调控, 其中GacS-GacA双因子调控系统在转录水平正调控信号合成基因pcoI的表达。为进一步研究QS系统调控因子, 将2P24基因组文库转入gacA缺失的pcoI基因转录报告菌株PM203 (pcoI-lacZ, gacA-), 筛选可提高pcoI表达的基因。结果表明粘粒pP32-24可显著提高pcoI转录水平, 亚克隆实验证明其中的功能基因为pcoI; 外源添加标准信号分子3-氧-己酰高丝氨酸内酯(3-oxo-C8-HSL)同样可显著提高pcoI基因的表达, 表明pcoI基因的表达对自身有正调控作用。同时构建了QS系统的另外一个组分pcoR基因的缺失突变体, pcoR基因缺失后pcoI的表达和N-乙酰高丝氨酸内酯信号分子(AHL)的产量明显低于野生菌株及其互补菌株, 并显著降低该菌株的生物膜(Biofilm)形成能力。这些结果表明菌株2P24的PcoI-PcoR QS系统中, 信号合成基因pcoI的表达受自体反馈调控, pcoR基因参与pcoI基因表达的调控以及生物膜的形成。     

猪链球菌2型强毒株S.suis 05ZY和弱毒株S.suis 1940的比较转录谱分析

目的:比较猪链球菌2型强毒株S.suis 05ZY和弱毒株S.suis 1940毒力相关基因转录水平的差异,为进一步研究强毒株S.suis 05ZY毒力增强的原因提供实验基础。方法:分别提取S.suis 05ZY和S.suis 1940的RNA,反转录成cDNA并纯化,用Cy5或Cy3标记,与猪链球菌全基因组DNA芯片进行杂交,扫描芯片进行数据分析,比较二者在转录水平上的差异基因。结果:编码溶血素、精氨酸氨基肽酶的基因分别上调4.4和6.0倍,参与荚膜多糖合成的相关基因cps2H、cps2I、cps2J和一些可能的毒力相关基因ofs、dpr、SSU05₀196、SSU05₀272、SSU05₁408-1409均发生转录水平的上调。结论:溶血素、荚膜多糖、精氨酸氨基肽酶及一些可能的毒力因子在转录水平的上调很可能与S.suis 05ZY的毒力增强有关。     

啤酒酵母DNA修复基因RAD24温度敏感突变株的分离及其特性的初步研究

以ＹＣｐｌａｃ系列带Ｔｒｐ、Ｈｉｓ和Ｕｒａ标志基因的载体为骨架构建含野生型和经羟胺处理的突变型的啤酒酵母ＲＡＤ２４基因质粒,用质粒替换方法分离ＲＡＤ２４基因温度敏感突变株（ｒａｄ２４－ｔｓ３）．紫外生存试验发现,ｒａｄ２４－ｔｓ３对紫外线敏感;同位素（３Ｈ－ＴｄＲ,３Ｈ－ＵＲ,３Ｈ－Ｌｅｕ）参入试验表明,该突变株ＤＮＡ、ＲＮＡ及蛋白质合成均较野生型明显降低．     

Effects of 50 Hz magnetic field on cell cycle kinetics and the colony forming ability of budding yeast exposed to ultraviolet radiation.

To investigate the effects of extremely low frequency magnetic fields on ultraviolet radiation (UV) exposed budding yeast, haploid yeast (Saccharomyces cerevisiae) cells of the strain SEy2101a were exposed to 50 Hz sine wave magnetic field (MF) of 120 microT with simultaneous exposure to UV radiation. Most of the UV energy was in the UVB range (280-320 nm). The biologically weighted (CIE action spectrum) dose level for the UV radiation was 175 J/m2. We examined whether 50 Hz MF affected the ability of UV irradiated yeast cells to form colonies (Colony Forming Units, CFUs). In addition, the effect of coexposure on cell cycle kinetics was investigated. Although the significant effect of MF on the cell cycle phases of UV exposed yeast cells was seen only at one time point, the overall results showed that MF exposure may influence the cell cycle kinetics at the first cycle after UV irradiation. The effect of our particular MF exposure on the colony forming ability of the UV irradiated yeast cells was statistically significant 420 min after UV irradiation. Moreover, at 240, 360, and 420 min after UV irradiation, there were fewer CFUs in every experiment in (UV+MF) exposed populations than in only UV exposed yeast populations. These results could indicate that MF exposure in conjunction with UV may have some effects on yeast cell survival or growth.     

Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination.     

A yeast DNA repair gene partially complements defective excision repair in mammalian cells.

The RAD10 gene of Saccharomyces cerevisiae is required for nucleotide excision repair of DNA. Expression of RAD10 mRNA and Rad10 protein was demonstrated in Chinese hamster ovary (CHO) cells containing amplified copies of the gene, and RAD10 mRNA was also detected in stable transfectants without gene amplification. Following transfection with the RAD10 gene, three independently isolated excision repair-defective CHO cell lines from the same genetic complementation group (complementation group 2) showed partial complementation of sensitivity to killing by UV radiation and to the DNA cross-linking agent mitomycin C. These results were not observed when RAD10 was introduced into excision repair-defective CHO cell lines from other genetic complementation groups, nor when the yeast RAD3 gene was expressed in cells from genetic complementation group 2. Enhanced UV resistance in cells carrying the RAD10 gene was accompanied by partial reactivation of the plasmid-borne chloramphenicol acetyltransferase (cat) gene following its inactivation by UV radiation. The phenotype of CHO cells from genetic complementation group 2 is also specifically complemented by the human ERCC1 gene, and the ERCC1 and RAD10 genes have similar amino acid sequences. The present experiments therefore indicate that the structural homology between the yeast Rad10 and human Ercc1 polypeptides is reflected at a functional level, and suggest that nucleotide excision repair proteins are conserved in eukaryotes.