Restriction endonuclease resistant chromatin in human chromosomes

Summary The recent addition of restriction endonucleases in obtaining selective bands in the human genome has added a new dimension to molecular genetics. However, a considerable discrepancy exists in banding patterns produced by AluI in chromosomes 19 and 20, by MboI in chromosomes 4, 5, 8, 21 and 22 and by RsaI in chromosomes 12, 21 and 22. The principal causes of these differences are highlighted.     

培养抗性植物的细胞/组织培养途径

抗性育种在作物品种改良中已日益显得重要。近10多年来,随着细胞/组织培养、遗传操作等生物技术的迅速发展,以及由于植物具有全能性,在人工培养条件下,从单细胞原生质体可以再生成一个完整的个体成功以来,人们试图利用细胞/组织培养等生物技术将野生种的抗性基因引入到作物中来,或是人工诱发抗性变异,从而筛选出抗性愈     

受白粉菌诱导大麦抗感等基因系蛋白质变化的双向电泳分析

分别对接种与否的大麦抗—感白粉病等基因系—叶期幼苗取材进行蛋白质双向电泳分析。结果表明,病原的侵入使抗—感两系在30Kd以下的低分子量区域的蛋白质发生了明显变化。接种48小时之后,抗病系在pH5.5、6.0、6.8及8.8附近出现了对照中所没有的蛋白质,而在pH6.0和8.8附近的蛋白质则较对照有减小的趋势;感病系在pH6.0附近蛋白质明显增多,在pH8.8处不仅在量上有大幅度提高,而且种类也有增加。结果还表明,抗—感系间在未接种的情况下双向电泳图谱也有差异,接种之后由于感病系在pH8.8处蛋白质的特异性合成,使抗—感两系间的差异缩小。     

高浓度Cd,Pb污染水域中的微生物生态

用选择性培养基和滤纸片法研究了Cd、Pb污染水域中的微生物生态分布、对Cd、Pb的抗性及富集力。结果表明,污水中微生物量的变化与Cd、Pb浓度不相关。抗性微生物生物量受环境温度影响较大。微生物对Cd、Pb的抗性是霉菌>酵母和细菌,Pb>Cd。霉菌最高抗Cd、Pb浓度可达2×10~4mg·L~(-1)。酵母和细菌最高抗Cd浓度为5000mg·L~(-1)、Pb浓度为1×10~4mg·L~(-1),抗性微生物对Cd、Pb的富集力与其抗性及Cd、Pb的毒性密切相关。含Cd、Pb污水中的优势菌有欧文氏菌、产碱杆菌、节细菌、假丝酵母、曲霉和枝孢霉。     

用细胞融合的方法选育耐高酒精度的酿酒酵母

选择耐高渗透压、耐高酒精度和发酵终了产酒精量较高的黄酒酵母H—1和目前酒精工业生产用菌K号酒精酵母K—1,运用细胞融合技术选育发酵速度快、产酒精量高的工业用酒精酵母.双倍体黄酒酵母H—1和双倍酒精酵母K—1经过产孢前预培养和产孢培养后,蜗牛酶水解子囊孢子壁,离心收集单倍体子囊孢子,培养后得到单倍体黄酒酵母H—2和单倍体酒精酵母K—2.用硫酸二乙醇诱变处理单倍体细胞,得到单倍体黄酒酵母的维生素缺陷型H—3和单倍体酒精酵母的氨塞骏营养缺陷型K—3通过正交试验找出了H—3和K—3原生质体形成及再生的较优条件是:对数生长后期的细胞33℃、0.2%的β—巯基乙醇预处理15分钟,然后4%蜗牛酶作用2小时.用35%聚乙二醇和10mMCaC1_2诱导融合40分钟,于再生基本夹层培养基上培养获得营养互补融合子,并且考查了融合子的遗传稳定性.通过耐酒精度、一发酵速度和最终产酒精量的测定,筛选出融合子HK—6.HK—6与生产用K号酒精酵母相比,发酵速度相接近,而最终产酒精量提高了11%.可耐受16%的酒精度.     

Metabolism of p-fluorophenylalanine in p-fluorophenylalanine sensitive and resistant tobacco cell cultures

The metabolism of D- and L-p-fluorophenylalanine (PFP) in DL-PFP resistant and sensitive tobacco cell cultures (Nicotiana tabacum), cell lines TX4 and TX1, respectively, has been compared. The amino acid analogue was taken up at a lower rate by the resistant cell line TX4. Incorporation of PFP into protein was also considerably reduced in TX4 cells, compared to TX1 cells. This, however, resulted mainly from a diminished availability of PFP due to a more rapid conversion of PFP by TX4 cells. TX1 cells and TX4 cells converted PFP qualitatively in the same way. The only detectable metabolite of D-PFP was N-malonyl-D-PFP, while all metabolites of L-PFP were identified as sequent products of the initial deamination of L-PFP by the enzyme phenylalanine ammonia-lyase (PAL). As TX4 cells were endowed with higher PAL-activity than TX1 cells, the resistant cells were able to metabolize L-PFP more rapidly to give, e.g., p-fluorocinnamoyl glucose ester and p-fluorocinnamoyl putrescine. In the presence of the specific PAL-inhibitor -aminooxy--phenylpropionic acid TX4 cells were slightly more sensitive to PFP. This suggests that the better detoxification contributes to the acquired resistance. The use of PFP as specific indicator for cell lines with increased PAL-activity, and hence increased levels of phenolic compounds, is discussed.Abbreviations AOPP -aminooxy--phenylpropionic acid - MCW methanol:chloroform:water - PAL phenylalanine ammonia-lyase - PFP p-fluorophenylalanine - Phe phenylalanine     

Phylogenetic diversity and mutational analysis of New Delhi Metallo-β-lactamase (NDM) producing E. coli strains from pediatric patients in Pakistan

The evolution of NDM genes (bla_NDM) in E. coli is accounted for expansive multidrug resistance (MDR), causing severe infections and morbidities in the pediatric population. This study aimed to analyze the phylogeny and mutations in NDM variants of E. coli recovered from the pediatric population. Carbapenem-resistant clinical strains of E. coli were identified using microbiological phenotypic techniques. PCR technique used to amplify the bla_NDM genes, identified on agarose gel, and analyzed by DNA sequencing. The amino acid substitutions were examined for mutations after aligning with wild types. Mutational and phylogenetic analysis was performed using Lasergene, NCBI blastn, Clustal Omega, and MEGA software, whereas PHYRE2 software was used for the protein structure predictions. PCR amplification of the bla_NDM genes detected 113 clinical strains of E. coli with the contribution of bla_NDM-1 (46%), bla_NDM-4 (3.5%), and bla_NDM-5 (50%) variants. DNA sequencing of bla_NDM variants showed homology to the previously described bla_NDM-1, bla_NDM-4, and bla_NDM-5 genes available at GenBank and NCBI database. In addition, the mutational analysis revealed in frame substitutions of Pro60Ala and Pro59Ala in bla_NDM-4 and bla_NDM-5, respectively. The bla_NDM-1 was ortholog with related sequences of E. coli available at GenBank. The phylogenetic analysis indicated that the NDM gene variants resemble other microbes reported globally with some new mutational sites.     

Cardiovascular Risk of Resistant Hypertension: Dependence on Treatment-Time Regimen of Blood Pressure–Lowering Medications

In resistant hypertension, ingesting one or more blood pressure (BP)-lowering medications at bedtime is associated with significant reduction of sleep-time BP, a sensitive prognostic marker of cardiovascular disease (CVD) risk. This randomized trial investigated if bedtime therapy with at least one hypertension medication exerts better BP control and CVD risk reduction than conventional, morning-time therapy with all medications. We conducted a prospective, open-label, blinded-endpoint trial on 776 patients (387 men/389 women) with resistant hypertension, 61.6?±?11.2 (mean?±?SD) yrs of age. Patients were randomized to ingest all their prescribed hypertension medications upon awakening or ≥1 of them at bedtime. BP was measured by ambulatory monitoring for 48?h at baseline, and again annually or more frequently (quarterly) if treatment adjustment was required. After a median follow-up of 5.4 yrs (range, .5–8.5 yrs), participants ingesting ≥1 hypertension medications at bedtime showed a significantly lower hazard ratio (HR) of total CVD events (adjusted by age, sex, and diabetes) than those ingesting all medications upon awakening (.38 [95% CI: .27–.55]; number of events 102 vs. 41; p?<?.001). The difference between groups in the adjusted HR of major CVD events (a composite of CVD death, myocardial infarction, ischemic stroke, and hemorrhagic stroke) was also statistically significant (.35 [95% CI: .18–.68]; number of events 32 vs. 12; p?=?.002). At the last evaluation, patients treated with the bedtime versus awakening-time-treatment regimen showed significantly lower sleep-time systolic/diastolic BP mean values (121.6/65.4 vs. 113.0/61.1?mm Hg; p?<?.001) and higher prevalence of controlled ambulatory BP (61% vs. 46%; p?<?.001). The progressive decrease in the sleep-time systolic BP mean during follow-up was the most significant predictor of event-free survival (15% risk reduction per 5?mm Hg decreased asleep systolic BP mean). Among patients with resistant hypertension, ingestion of at least one hypertension medication at bedtime, compared with all medications upon waking, resulted in improved ambulatory BP control and fewer hard and soft CVD events. (Author correspondence: rhermida@uvigo.es)     

抗性淀粉对HFA小鼠肠道菌群的影响

目的 以人源菌群(HFA)小鼠为研究模型,观察抗性淀粉(RS)对高脂饮食诱导的肥胖小鼠肠道菌群的多样性的影响.方法 将30只无菌小鼠接种健康人志愿者的粪便悬液构建HFA小鼠模型后,随机分成3组,一组喂养含20％的抗性淀粉的高脂饲料(RS组),一组喂养纯高脂饲料(CK组),一组喂养普通饲料(CONV组),取第0周和第8周的小鼠新鲜粪便,用PCR-DGGE分析3组小鼠的肠道菌群的相似性和多样性.结果 3组小鼠在第0周时肠道菌群多样性的相似度达到79％～87％,与人的肠道菌群相似性达到39％,说明构建HFA小鼠模型成功,第8周时,3组之间的均匀度(E)和Shannon指数差异无统计学意义(P＞0.05),而丰富度(S)在高脂组(CK)与普通饲料组(CONV)和抗性淀粉组(RS)之间差异都有统计学意义(P＜0.05),说明高脂饮食引起肠道菌群多样性增加,而抗性淀粉则能降低这种多样性.结论 抗性淀粉可以显著影响HFA小鼠的肠道菌群多样性.     

一种高效构建同源重组DNA片段的方法——融合PCR

融合PCR技术（fusion PCR）采用具有互补末端的引物,形成具有重叠链的PCR产物,通过PCR产物重叠链的延伸,从而将不同来源的任意DNA片段连接起来,此技术在不需要内切酶消化和连接酶处理的条件下实现DNA片段的体外连接,为同源重组片段的构建提供了快速简捷的途径。对原有的融合PCR技术进行改进,以三个同源重组线性DNA片段的构建为例,详细论述了改进的融合PCR技术的反应过程及技术体系。结果表明,改进的融合PCR技术可以同时进行三个片段及四个片段的融合反应,产物长度均在4.5kb以上,各同源重组片段在扩增过程中均无突变发生,获得的片段可以用于后续实验分析。