Genetic analysis of the yeast NUD1 endo-exonuclease: a role in the repair of DNA double-strand breaks

The deoxyribonucleases (DNases) have been shown genetically to be important in the vital processes of DNA repair and recombination. The NUD1 gene, which codes for an endo-exonuclease of Saccharomyces cerevisiae, was analyzed for its role in the DNA double-strand break (DSB) repair processes. While the nud1 strain is only slightly sensitive to ionizing radiation, expression of the HO-endonuclease to introduce a DSB at the MAT locus in that strain results in cell death. Cell survival is inversely proportional to the duration of HO-endonuclease expression. Analysis of the surviving colonies from the nud1 strain indicated that many of the survivors are sterile and that the proportion of these sterile survivors increases with the time of HO-endonuclease expression. On the other hand, the surviving colonies from the isogenic NUD1 strain are mating-proficient. Interestingly, double mutants of nud1 rad52 are more resistant to ionizing irradiation than the rad52 strain and have a cell-survival fraction of 32% for rad52-1 nud1 and 9% for rad52::URA3 nud1 following prolonged HO-endonuclease expression, indicating that nud1 has a suppressor effect on the DSB-induced lethality in rad52. Polymerase chain reaction analysis showed that many of the nud1 survivors contained small alterations within the MAT locus, suggesting that the survivors arose through the process of non-homologous end-joining. These results suggest that the endo-exonuclease acts at a DSB to promote DNA repair via the homologous recombination pathway. Received: 20 July / 20 September 1998     

Mitochondrial DNA variability detected in a single wheat regenerant involves a rare recombination event across a short repeat

The mitochondrial genome of the selfed progeny of a plant regenerated from long-term somatic tissue culture displays specific structural rearrangements characterized by the appearance of novel restriction fragments. A mitochondrial DNA library was constructed from this selfed progeny in the SalI site of cosmid pHC79 and the novel fragments were subsequently studied. They were shown to arise from reciprocal recombination events involving DNA sequences present in the parental plant. The regions of recombination were sequenced and the nucleotide sequences were aligned with those of the presumptive parental fragments. We characterized an imperfect short repeated DNA sequence, 242 bp long, within which a 7-bb DNA repeat could act as a region of recombination. The use of PCR technology allowed us to show that these fragments were present in both parental plants and tissue cultures as low-abundance sequence arrangements.     

大鼠胎胰胰岛组织培养及移植的实验观察

本实验对培养中的大鼠胎胰胰岛组织及其移植后的形态功能进行了观察。结果表明,大鼠胎胰经短期培养,不仅内分泌胰岛β细胞的活力保存良好,而且胰外分泌腺泡大大减少以致消失。21只糖尿病大鼠,同种移植经过7天培养的胎胰胰岛,未用免疫抑制剂,术后糖尿病缓解率76.2％。     

细菌内快速构建两种用于肝纤维化基因治疗的重组复制缺陷型腺病毒

目的 :在大肠杆菌内快速构建两种分别含有人肝细胞生长因子 (hepatic growth factor,HGF)和非分泌型人尿激酶型纤溶酶原激活剂 (urokinase- type plasm inogen activator,u PA) c DNA片段的重组复制缺陷型腺病毒。方法 :分别将人 HGF和 u PA c DNA片段与穿梭载体 p Track- CMV连接 ,线性化后与骨架载体 Ad Easy- 1在大肠杆菌 BJ5 183内同源重组获得腺病毒载体 p Ad HGF和 p Adu PA,经 2 93细胞包装后得到复制缺陷型重组腺病毒 Ad HGF和 Adu PA;将 Ad HGF和 Adu PA分别体外感染肝细胞株 L0 2 ,以 Northern印迹法和 Western印迹法检测两种病毒在肝细胞中的表达。 结果 :连接、重组后通过酶切和测序法筛选出病毒质粒 p Ad HGF和 p Adu PA;经 2 93细胞包装 ,3d后均观察到绿色荧光蛋白明显表达 ,Cs Cl梯度离心纯化最终分别获得 4× 10 1 0 efu/ m l滴度的重组病毒 Ad HGF和 6× 10 1 0 efu/ ml Adu PA;两种病毒体外感染肝细胞 3d后 ,HGF和u PA表达均明显增加。结论 :细菌内同源重组制备复制缺陷型腺病毒 ,纯化过程简单 ,重组腺病毒在肝细胞中均高效表达 ,为肝细胞移植基因治疗肝纤维化提供了新的手段     

The Construction fo Hybrid Plasmid pBR322:RSF1010

The plasmids pBR322 and RSF1010,extracted from E.coli HB802 and E.coliC600 respectively,were treated with restrictive endonuclease EcoRI andligated with T4 DNA Iigase.C600 strains were then transformed with thehybrid plasmid(pSMMl),and thus253transformants were obtained.Thefigures of gel electrophoresis and electromicrograph proved that pSMMlwas a pBR322:RSF1010 composite hybrid.In E.coli C600 pSMMl obtainedresistance against ampieilline(Ap),tetracycline(Tc)and streptomycin(Sm).The orientation of pBR322 and RSF1010 in hybrid pSMMl and the failureof introduction of this hybrid into P.putida AC10 were discussed.     

SARS冠状病毒刺突蛋白编码基因及氨基酸序列的同源性分析

①目的 分析严重急性呼吸综合征(SARS)冠状病毒刺突蛋白(S蛋白)编码基因及氨基酸序列的变异情况，并在GenBank中寻找其同源序列。②方法 将网上公布的具有全基因序列的12株SARS冠状病毒S蛋白的编码基因序列及氨基酸序列应用Clustal W方法对排分析变异情况，并应用GenBank的Blast对排功能寻找被GenBank收录的同源序列。③结果 SARS冠状病毒S蛋白相对比较保守，变异率较低，其氨基酸序列与鼠的肝炎病毒氨基酸序列有较高的同源性，某些核苷酸序列与人的基因组有同源性。④结论 S蛋白编码基因及氨基酸序列相对保守，为预防性疫苗的研发提供了可行性；据其同源基因及蛋白序列可以预测S蛋白的二、三级结构，对推测SARS可能的发病机制有积极作用。     

^60Co消毒与高温高压消毒胚胎骨的实验研究

在12只健康家兔下颌骨体部的两侧各造成0.7cm×0.5cm×0.3cm的骨缺损区，分别置入^60Co消毒的胚胎骨和高温高压消毒的胚胎骨，发现高温高压消毒可以替代^60Co消毒胚胎骨进行移植，为临床提供了一种新的用作同种异体移植胚胎骨的消毒方法。     

同种异体猪胰腺移植后的细胞凋亡形态学观察

目的观察胰腺移植后胰腺细胞凋亡的形态学变化。方法以6对动物猪作为实验对象，配对行胰肾联合移植（SPK），观察术后移植物细胞凋亡的形态。结果电镜检查标本可见胰腺细胞出现凋亡，观察到凋亡小体和核固缩现象，TUNEL法标记可观察到大量的凋亡细胞。结论胰腺移植早期可观察到胰腺细胞凋亡现象。     

重组人肿瘤坏死因子诱导HL－60白血病细胞分化及调控原癌基因表达的作用

采用２０～２００Ｕ／ｍｌ的重组人肿瘤坏死因子（ｒｈＴＮＦ－α）处理体外液相培养的人早幼粒白血病细胞珠ＨＬ－６０，观察到５０～２００Ｕ／ｍｌ的小剂量ＴＮＦ具有诱导其向单核－巨噬细胞途径分化及抑制其增殖的效应。采用细胞总ＲＮＡ打点杂交技术检测１～１００Ｕ／ｍｌＴＮＦ诱导ＨＬ－６０细胞早期（１２小时内）对其ｃ－ｍｙｃ，ｃ－ｆｏｓ原癌基因表达的影响，发现ＴＮＦ可抑制ｃ－ｍｙｃｍＲＮＡ水平及ｃ－ｆｏｓ短暂性表达增高。结果表明此小剂量ｒｈＴＮＦ－α具有诱导白血病细胞分化的效应及调控多癌基因表达的作用。