Packaging of transducing DNA by bacteriophage P1

Summary P1 transduces bacterial chromosomal markers with widely differing frequencies. We use quantitative Southern hybridisations here to show that, despite this, most markers are packaged at similar levels. Exceptions are a group of markers near 2 min and another at 90 min which seem to be packaged at levels two-to threefold higher. We thus conclude that certain marker frequency variations in transduction can be explained by differences in packaging level, but that most cannot. The limited range in packaging levels suggests that P1 can initiate the packaging of chromosomal DNA from many sites. This idea is supported by our failure to find any chromosomal sequences with homology to the phage pac site and by the occurrence of hybridising bands which seem to suggest sequential packaging from a large number of specific sites. We eliminate the possibility that chromosomal DNA packaging is the result of endonucleolytic cutting by the P1 res enzyme.     

In vivo transfer of chromosomal mutations onto multicopy plasmids by transduction with bacteriophage P1

A technique is presented by which chromosomal mutations may be efficiently transferred onto chimeric multicopy plasmids in vivo. The technique employs the transduction of plasmids using bacteriophage P1 as vector. The utility of this method was demonstrated by cloning a chromosomal ompR mutation of Escherichia coli K-12. The high-frequency transduction of the chimeric plasmid appeared to be dependent on its integration into the chromosome by homologous recombination. The results also suggest that the plasmid was transduced as part of the chromosome and resolved from its integrated state in the recipient cell, resulting in a high yield of mutant plasmid segregants.     

Characterization of Saccharomyces cerevisiae strains expressing ira1 mutant alleles modeled after disease-causing mutations in NF1

The 2818 amino acids of neurofibromin, the product of the human NF1 gene, include a 230 amino acid Ras-GAP related domain (GRD). Functions which may be associated with the rest of the protein remain unknown. However, many NF1 mutations in neurofibromatosis 1 patients are found downstream of the GRD, suggesting that the C-terminal region of the protein is also functionally important. Since the C-terminal region of neurofibromin encompassing these mutations is homologous with the corresponding regions in the two Saccharomyces cerevisiae Ras-GAPs, Ira1p and Ira2p, we chose yeast as a model system for functional exploration of this region (Ira-C region). Three missense mutations that affect the Ira-C region of NF1 were used as a model for the mutagenesis of IRA1. The yeast phenotypes of heat shock sensitivity, iodine staining, sporulation efficiency, pseudohyphae formation, and GAP activity were scored. Even though none of the mutations directly affected the Ira1p-GRD, mutations at two of the three sites resulted in a decrease in the GAP activity present in ira1 cells. The third mutation appeared to disassociate the phenotypes of sporulation ability and GAP activity. This and other evidence suggest an effector function for Ira1p.     

Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains

Summary A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD ^- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.     

Site-specific recombination events mediated by the DNA invertase Cin of bacteriophage P1 during transformation

Abstract Bacteriophage P1 encodes the site-specific recombinase Cin which promotes inversion of the C segment, thus controlling the P1 host range. Cin can also mediate inefficient inversion between the normal crossover site cixL and a quasi-crossover site cixQ 1 in inverted orientation. Inversion between cixL and cixQ 1 occurs more frequently in a short period of time after transformation with a plasmid carrying the cin gene, cixL and cixQ 1 than in an established transformant of the plasmid. This is also the case for Cin-mediated deletion on a plasmid containing the cin gene and directly repeated cix sites.     

口蹄疫病毒P1+2A基因在BHK-21细胞中的表达

本研究将已构建好的、包含口蹄疫病毒P1+2A基因的重组表达质粒pQE-Tri/P1+2A经质脂体2000转染哺乳动物细胞BHK-21,转染后一定时间进行检测.通过SDS-PAGE、Western-blotting、荧光抗体染色、ELISA等检测方法表明,FMDV P1+2A基因片段在BHK-21细胞中成功表达,表达的蛋白能被口蹄疫阳性血清所识别而且具有良好的生物学活性.这一结果的取得,为进一步研制新型口蹄疫基因工疫苗及其配套诊断试剂奠定了基础.     

P fimbriae on uropathogenic Escherichia coli O16:K1 and O18 strains

Abstract The fimbrial composition of 12 P-fimbriate uropathogenic Escherichia coli O16 and O18 strains was analysed by immunoprecipitation with 14 fimbria-specific antisera. All the O16 strains possessed a P fimbrial serovariant with an apparent M _r of 17500. One strain had an additional, serologically closely related P fimbria with an apparent M _r of 19 800. Two groups were found among the O18 strains; one possessing a type 1C fimbria and a 19800-Da P fimbria, the other lacking type 1C fimbriae and possessing a P-fimbrial variant with an apparent M _r of 17 800. Fimbriae on strains within the groups were serologically similar by immunoprecipitation assays. Also, the fimbriae on the O16 and O18 strains were mutually cross-reactive. The grouping of the O18 strains by fimbrial serology corresponded to the previous clonal grouping based on other phenotypic characters.     

Involvement of transposable elements in the formation of hybrid phages between bacteriophage P1 and the R plasmid NR1

Homologous recombination between IS1 elements present on both replicons, P1 and NR1, resulted in P1-NR1 cointegrates and P1-RTF and P1-r-det phages. Cointegration between P1 and NR1-B, and NR1 derivative with multiple DNA rearrangements including insertion of the transposable element γδ, was also mediated by reciprocal recombination in IS1 sequences. However, all 4 hybrids studied carried deletions promoted by γδ residing on NR1-B. Further IS1-mediated deletions on the hybrid genomes resulted in plaque-forming P1Cm phages.     

Cooperation of NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferases reduces menadione cytotoxicity in HEK293 cells

Previous studies have shown that NAD(P)H:quinone oxidoreductase 1 (NQO1) plays an important role in the detoxification of menadione (2-methyl-1,4-naphthoquinone, also known as vitamin K3). However, menadiol (2-methyl-1,4-naphthalenediol) formed from menadione by NQO1-mediated reduction continues to be an unstable substance, which undergoes the reformation of menadione with concomitant formation of reactive oxygen species (ROS). Hence, we focused on the roles of phase II enzymes, with particular attention to UDP-glucuronosyltransferases (UGTs), in the detoxification process of menadione. In this study, we established an HEK293 cell line stably expressing NQO1 (HEK293/NQO1) and HEK293/NQO1 cell lines with doxycycline (DOX)-regulated expression of UGT1A6 (HEK293/NQO1/UGT1A6) and UGT1A10 (HEK293/NQO1/UGT1A10), and evaluated the role of NQO1 and UGTs against menadione-induced cytotoxicity. Our results differed from those of previous studies. HEK293/NQO1 was the most sensitive cell line to menadione cytotoxicity among cell lines established in this study. These phenomena were also observed in HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells in which the expression of UGT was suppressed by DOX treatment. On the contrary, HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells without DOX treatment were resistant to menadione-induced cytotoxicity. These results demonstrated that NQO1 is not a detoxification enzyme for menadione and that UGT-mediated glucuronidation of menadiol is the most important detoxification process.     

A mutational analysis of the bacteriophage P1 cin recombinase gene: intragenic complementation

Summary Bacteriophage P1 encodes a site-specific recombinase, Cin, which regulates the alternate expression of tail fibre genes by inverting a DNA segment. To define regions of Cin important for the recombination process, we have isolated and characterised 24 different mutations of the cin gene. Most of these mutations affected amino acids that are highly conserved in other related recombinases. Some of these mutants complement each other in vivo. This intragenic complementation could be due to the assembly of heteromers containing both mutant proteins, suggesting that the active enzyme is at least a dimer.     

Genetic analysis of the P1 region of human enterovirus 71 strains and expression of the 55 F strainVP1 protein

Enterovirus 71 (EV71) is a member of the Entero-virus genus of the Picornaviridae family and is the major cause of Hand,foot,and mouth disease (HFMD) in children.Different strains from Gansu were clone...     

用P300评价连续工作记忆能力差异的研究

目的：评价长时间连续工作记忆的行为学表现与事件相关电位（Event-related potential,ERP）的关系。方法：对16位健康男性青年（平均年龄21.4岁）,连续进行1h的3-back数字倒背实验,记录ERP。结果：受试者的行为学表现明显差异,记录的ERP的P300波幅存在显著性差异（P〈0.01）。结论：ERP的P300成分可以客观评价行为学上的差异。     

Mechanisms involved in the formation of plaque-forming derivatives from over-sized hybrid phages between bacteriophage P1 and the R plasmid NR1

Three case histories document how subsequent events of genomic rearrangements and selection interplay in the evolution of infectious bacteriophage genomes carrying acquired genes. Two of the phages studied were plaque-forming P1CmTc recombinants derived from P1Cm1 and P1Tc1, both of which are hybrids between phage P1 and the R plasmid NR1. In the formation of the P1CmTc4 genome a postulated intermediate underwent IS1-mediated deletion formation. From the same intermediate P1CmTc1 must have evolved by IS1-mediated inversion followed by homologous recombination with a parental phage DNA. The third case documents formation of the P1Cm2 genome by “illegitimate” intramolecular recombination in the genome of P1-r-det, a hybrid between P1 and NR1.     

Discovery of S1P agonists with a dihydronaphthalene scaffold

Structure-activity relationship of sphingosine-1-phosphate receptor agonists was examined. Cinnamyl derivative 1 was modified to improve S1P₁ agonistic activity as well as selectivity over S1P₃ agonistic activity. Dihydronaphthalene derivative 10d was identified as a potent S1P₁ receptor agonist with high selectivity against S1P₃ and enhanced efficacy in lowering peripheral lymphocyte counts in mice.     

Analysis of point mutations of the BRCA1 gene by hybridization with hydrogel microarrays

BRCA1 mutations are associated with a higher risk of breast (BC) and ovarian cancer in women. Testing for such mutations allows BC prognosis, selection of an individual treatment strategy, and prevention of disease recurrence. Hybridization on a hydrogel microarray was developed for identifying point mutations in BRCA1. The microarray was designed to detect five-point mutations: 185delAG, 300T→G, 4153delA, 4158A→G, and 5382insC. The microarray was tested with 36 control specimens with known genotypes and used to examine 65 BC patients. The results demonstrated the advantage of employing the microarray in analyzing BRCA1 mutations.     

An in vitro transposon system for highly regulated gene expression: construction of Escherichia coli strains with arabinose-dependent growth at low temperatures.

Placing a gene of interest under the control of an inducible promoter greatly aids the purification, localization and functional analysis of proteins but usually requires the sub-cloning of the gene of interest into an appropriate expression vector. Here, we describe an alternative approach employing in vitro transposition of TnΩP_BAD to place the highly regulable, arabinose inducible P_BAD promoter upstream of the gene to be expressed. The method is rapid, simple and facilitates the optimization of expression by producing constructs with variable distances between the P_BAD promoter and the gene. To illustrate the use of this approach, we describe the construction of a strain of Escherichia coli in which growth at low temperatures on solid media is dependent on threshold levels of arabinose. Other uses of the transposable promoter are also discussed.     

胃癌中P16、nm23-h1、P53表达及其与患者预后的关系

目的:探讨胃癌组织中P16,nm23-h1和P53表达情况及其与患者预后的关系。方法:应用免疫组化SABC法检测91例手术切除胃癌组织中P16,nm 23-h1和P53表达。结果:胃癌中P16,nm23-h1和P53表达阳性率分别33．0%(30/91),38．5%(35/91), 53．8%(49/91)。p16,nm23-h 1和P53在胃癌中表达与淋巴结有无转移,不同的浸润深度,TNM分期,不同组织学类型均有显著差异(P＜0．05)。而它们与肿瘤患者年龄,性别差异无统计学意义(P＞0．05)。等级相关分析结果显示P16与P53表达呈负相关(P＜0．05),P16与nm23-h1表达呈正相关(P＜0．05),P53与nm23-h1表达呈负相关(P＜0．05)。生存曲线研究结果显示P16,nm23-h1和P53表达与患者术后生存时间显著相关(P＜0．05)。结论:P16和nm23-h1在胃癌组织中低表达及P53高表达能客观反映胃癌组织分化程度,侵袭转移能力,且其与患者预后密切相关。故检测三种指标可作为正确判断胃癌患者预后,在指导临床治疗方面有一定价值。