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Sample records for cerevisiae plasmid libraries

  1. Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries

    Directory of Open Access Journals (Sweden)

    Gray Elizabeth C

    2009-11-01

    Full Text Available Abstract Background In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. Results It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. Conclusion These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The

  2. Optimization of ordered plasmid assembly by gap repair in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine Valerie; Pedersen, Mette Louise; Krogh, Berit Olsen;

    2012-01-01

    Combinatorial genetic libraries are powerful tools for diversifying and optimizing biomolecules. The process of library assembly is a major limiting factor for library complexity and quality. Gap repair by homologous recombination in Saccharomyces cerevisiae can facilitate in vivo assembly of DNA...... in mutants carrying a deletion of the SGS1 helicase-encoding gene. Using our experimental conditions, a gap-repair efficiency of > 10(6) plasmid-harbouring colonies/µg gapped vector DNA is obtained in a single transformation, with a recombination fidelity > 90%....

  3. Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening.

    Science.gov (United States)

    Viña-Gonzalez, Javier; Gonzalez-Perez, David; Alcalde, Miguel

    2016-01-01

    Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps. PMID:27077451

  4. A fast method to diagnose chromosome and plasmid loss in Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Hegemann, J H; Klein, S; Heck, S; Güldener, U; Niedenthal, R K; Fleig, U

    1999-07-01

    We have developed a simple, fast and reliable method for the analysis of genetic stability in budding yeast strains. The assay relies on our previous finding that cells expressing the green fluorescent protein (GFP) can be detected and counted by flow cytometric analysis (FACS) (Niedenthal et al., 1996). Expression of a gfp-carrying CEN-plasmid in a wild-type strain resulted in the emission of strong fluorescence from 80% of the cell population. Strong fluorescence and presence of the plasmid, determined by the presence of the URA3 genetic marker, was strictly correlated. Expression of this plasmid in 266 yeast strains, each carrying a complete deletion of a novel, non-essential gene identified in the S. cerevisiae sequencing project, pinpointed 12 strains with an increased level of mitotic plasmid loss. Finally we have shown that measurement of mitotic loss of artificial chromosome fragments equipped with the gfp expression cassette can be performed quantitatively using FACS.

  5. Studies on the plasmid stability, plasmid copy number and endo(1, 3)(1, 4) b-glucanase production by free and alginate immobilised recombinant saccharomyces cerevisiae cells

    OpenAIRE

    Canavan, Peter D.

    1994-01-01

    A recombinant yeast strain, Saccharomyces cerevisiae DBY746, containing the plasmid pJG317, was grown in a variety of fermentation modes including batch, serial batch and chemostat culture incorporating a wide range of media types Plasmid pJG317 consists of a 2^-denved yeast episomal plasmid containing the gene which encodes for the bacterial enzyme endo (1,3)(1,4) P-glucanase. The concentration of enzyme produced appears to be proportional to the number of plasmid copies per cell. Specific e...

  6. Telomere-Mediated Plasmid Segregation in Saccharomyces Cerevisiae Involves Gene Products Required for Transcriptional Repression at Silencers and Telomeres

    OpenAIRE

    Longtine, M. S.; Enomoto, S.; Finstad, S L; Berman, J

    1993-01-01

    Plasmids that contain Saccharomyces cerevisiae TG(1-3) telomere repeat sequences (TRS plasmids) segregate efficiently during mitosis. Mutations in histone H4 reduce the efficiency of TRS-mediated plasmid segregation, suggesting that chromatin structure is involved in this process. Sir2, Sir3 and Sir4 are required for the transcriptional repression of genes located at the silent mating type loci (HML and HMR) and at telomeres (telomere position effect) and are also involved in the segregation ...

  7. Mutations in a Partitioning Protein and Altered Chromatin Structure at the Partitioning Locus Prevent Cohesin Recruitment by the Saccharomyces cerevisiae Plasmid and Cause Plasmid Missegregation

    OpenAIRE

    Yang, Xian-Mei; Mehta, Shwetal; Uzri, Dina; Jayaram, Makkuni; Velmurugan, Soundarapandian

    2004-01-01

    The 2μm circle is a highly persistent “selfish” DNA element resident in the Saccharomyces cerevisiae nucleus whose stability approaches that of the chromosomes. The plasmid partitioning system, consisting of two plasmid-encoded proteins, Rep1p and Rep2p, and a cis-acting locus, STB, apparently feeds into the chromosome segregation pathway. The Rep proteins assist the recruitment of the yeast cohesin complex to STB during the S phase, presumably to apportion the replicated plasmid molecules eq...

  8. Reconstitution of papillomavirus E2-mediated plasmid maintenance in Saccharomyces cerevisiae by the Brd4 bromodomain protein

    OpenAIRE

    Brannon, Angela R.; Maresca, Julia A.; Boeke, Jef D.; Basrai, Munira A.; McBride, Alison A.

    2005-01-01

    The papillomavirus E2 protein functions in viral transcriptional regulation, DNA replication, and episomal genome maintenance. Viral genomes are maintained in dividing cells by attachment to mitotic chromosomes by means of the E2 protein. To investigate the chromosomal tethering function of E2, plasmid stability assays were developed in Saccharomyces cerevisiae to determine whether the E2 protein could maintain plasmids containing the yeast autonomous replication sequence replication element ...

  9. Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    CHEN; Xiangling

    2005-01-01

    [1]Brunelli, J. P., Pall, M. L., A series of yeast vectors for expression of cDNAs and other DNA sequences, Yeast, 1993, 9: 1299―1308.[2]Sikorski, R. S., Hieter, P., A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae, Genetics, 1989, 122: 19―27.[3]Bonneaud, N., Ozier-Kalogerogoulos, O., Li, G. et al., A family of low and high copy replicative, integrative and single-stranded S. cerevisiae /E. coli shuttle vector, Yeast, 1991, 7: 609―615.[4]Huo, K. K., Yu, L. L., Chen, X. J., Li, Y. Y., A stable vector for high-level expression and secretion of human interferon alpha A in yeast, Science in China, Ser. B, 1993, 36(5): 557―567.[5]Zhou, Z. X., Yuan, H. Y., He, W. et al., Expression of the modified HBsAg gene SA-28 directed by a constitutive promoter, Journal of Fudan university (Natural Science), 2000, 39(3): 264―268.[6]Paques, F., Haber, J. E., Multiple pathways of recombination induces by double-strand breaks in Saccharomyces cerevisiae, Microbiology and Molecular Biology Reviews, 1999, 63(2): 349―404.[7]Martin, K., Damage-induced recombination in the yeast Saccharomyces cerevisiae, Mutation Research, 2000, 451: 91―105.[8]Alira, S., Tomoko, O., Homologous recombination and the roles of double-strand breaks, TIBS, 1995, 20: 387―391.[9]Patrick, S., Kelly, M. T., Stephen, V. K., Recombination factor of Saccharomyces cerevisiae, Mutation Research, 2000, 451: 257―275.[10]Manivasakam, P., Weber, S. C., McElver, J., Schiestl, R. H., Micro-homology mediated PCR targeting in Saccharomyces cerevisiae, Nucleic Acids Res., 1995, 23(14): 2799―2800.[11]Baudin, A., Lacroute, F., Cullin, C., A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae, Nucleic Acids Res., 1993, 21(14): 3329―3330.[12]Hua, S. B., Qiu, M., Chan, E., Zhu, L., Luo, Y., Minimum length of sequence homology required for in vivo cloning by homolo-gous recombination in yeast, Plasmid, 1997, 38

  10. A novel plasmid-based microarray screen identifies suppressors of rrp6Delta in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jensen, Torben Heick

    2007-01-01

    temperature sensitivity or a novel microarray enhancer/suppressor screening (MES) strategy. MES combines DNA microarray technology with high-copy-number plasmid expression in liquid media. The plate screen and MES identified overlapping, but also different, suppressor genes. Only MES identified the novel m......Genetic screens in Saccharomyces cerevisiae provide novel information about interacting genes and pathways. We screened for high-copy-number suppressors of a strain with the gene encoding the nuclear exosome component Rrp6p deleted, with either a traditional plate screen for suppressors of rrp6Δ......RNP protein Nab6p and the tRNA transporter Los1p, which could not have been identified in a traditional plate screen; both genes are toxic when overexpressed in rrp6Δ strains at 37°C. Nab6p binds poly(A)+ RNA, and the functions of Nab6p and Los1p suggest that mRNA metabolism and/or protein synthesis...

  11. A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae.

    OpenAIRE

    Houtteman, S W; Elder, R T

    1993-01-01

    Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis. While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell. A screen was designed to isolate mutations that suppress this segregation bias. A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed. When cells were grown under selection for the wARS plasmid, the resulting...

  12. A novel plasmid-based microarray screen identifies suppressors of ∆rrp6 in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jensen, Torben Heick

    Genetic screens can provide novel information about interacting genes and pathways in S. cerevisiae.  Conventional approaches are limited, however, because only strong suppressors or enhancers are usually identified.  We describe here a novel Microarray-based Enhancer and Suppressor screening (MES......) strategy that is capable of identifying a large number of genes that exert more modest effects on the mutant phenotype.  MES combines DNA microarray technology with high-copy plasmid expression in liquid media.  We conducted MES on a strain deleted for Rrp6p, a nuclear exosome component involved...

  13. High-Throughput Plasmid cDNA Library Screening

    OpenAIRE

    Wan, Kenneth H.; Yu, Charles; George, Reed A; Carlson, Joseph W.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, Susan E.

    2006-01-01

    Libraries of cDNA clones are valuable resources for analysing the expression, structure, and regulation of genes, as well as for studying protein functions and interactions. Full-length cDNA clones provide information about intron and exon structures, splice junctions and 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs) derived from cDNA clones can be used to generate constructs allowing expression of native proteins and N- or C-terminally tagged proteins. Thus, obtaini...

  14. High-Throughput Plasmid cDNA Library Screening

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Kenneth H.; Yu, Charles; George, Reed A.; Carlson, JosephW.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, SusanE.

    2006-05-24

    Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins.Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions.Expressed sequence tag (EST) sequencing samples cDNA libraries at random,which is most useful at the beginning of large-scale screening projects.However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.

  15. Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiangling; YUAN Hanying; HE Wei; HU Xianghua; LU Hong; LI Yuyang

    2005-01-01

    Based on a previously used plasmid pHC11, a new plasmid pHC11R was constructed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R' was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R- IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.

  16. MEGAWHOP cloning: a method of creating random mutagenesis libraries via megaprimer PCR of whole plasmids.

    Science.gov (United States)

    Miyazaki, Kentaro

    2011-01-01

    MEGAWHOP allows for the cloning of DNA fragments into a vector and is used for conventional restriction digestion/ligation-based procedures. In MEGAWHOP, the DNA fragment to be cloned is used as a set of complementary primers that replace a homologous region in a template vector through whole-plasmid PCR. After synthesis of a nicked circular plasmid, the mixture is treated with DpnI, a dam-methylated DNA-specific restriction enzyme, to digest the template plasmid. The DpnI-treated mixture is then introduced into competent Escherichia coli cells to yield plasmids carrying replaced insert fragments. Plasmids produced by the MEGAWHOP method are virtually free of contamination by species without any inserts or with multiple inserts, and also the parent. Because the fragment is usually long enough to not interfere with hybridization to the template, various types of fragments can be used with mutations at any site (either known or unknown, random, or specific). By using fragments having homologous sequences at the ends (e.g., adaptor sequence), MEGAWHOP can also be used to recombine nonhomologous sequences mediated by the adaptors, allowing rapid creation of novel constructs and chimeric genes. PMID:21601687

  17. Direct cloning of specific genomic DNA sequences in plasmid libraries following fragment enrichment.

    OpenAIRE

    Nicholls, R D; Hill, A V; Clegg, J B; Higgs, D R

    1985-01-01

    We describe a simple method to directly clone any DNA fragment for which a flanking restriction enzyme map is known. Genomic DNA is digested with multiple enzymes cutting outside the fragment to be cloned, selected by electroelution from an agarose gel, and cloned directly into a plasmid vector. It is only necessary to screen 10-1000 colonies and recombinant DNA is ready for immediate molecular analysis without further subcloning. The use of this technique is demonstrated for the cloning of a...

  18. Reverse transcription-polymerase chain reaction construction of plasmid-based, full-length cDNA libraries from Leishmania infantum for in vitro expression screening

    Directory of Open Access Journals (Sweden)

    Bernard Couvreur

    2003-06-01

    Full Text Available We describe a streamlined reverse transcription-polymerase chain reaction methodology for constructing full-length cDNA libraries of trypanosomatids on the basis of conserved sequences located at the 5' and 3'ends of trans-spliced mRNAs. The amplified cDNA corresponded to full-length messengers and was amenable to in vitro expression. Fractionated libraries could be rapidly constructed in a plasmid vector by the TA cloning method (Invitrogen. We believe this is useful when there are concerns over the use of restriction enzymes and phage technology as well as in cases where expression of proteins in their native conformation is desired.

  19. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

    Science.gov (United States)

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J; Fox, Catherine A

    2016-01-01

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  20. Acetic acid inhibits nutrient uptake in Saccharomyces cerevisiae: auxotrophy confounds the use of yeast deletion libraries for strain improvement.

    Science.gov (United States)

    Ding, Jun; Bierma, Jan; Smith, Mark R; Poliner, Eric; Wolfe, Carole; Hadduck, Alex N; Zara, Severino; Jirikovic, Mallori; van Zee, Kari; Penner, Michael H; Patton-Vogt, Jana; Bakalinsky, Alan T

    2013-08-01

    Acetic acid inhibition of yeast fermentation has a negative impact in several industrial processes. As an initial step in the construction of a Saccharomyces cerevisiae strain with increased tolerance for acetic acid, mutations conferring resistance were identified by screening a library of deletion mutants in a multiply auxotrophic genetic background. Of the 23 identified mutations, 11 were then introduced into a prototrophic laboratory strain for further evaluation. Because none of the 11 mutations was found to increase resistance in the prototrophic strain, potential interference by the auxotrophic mutations themselves was investigated. Mutants carrying single auxotrophic mutations were constructed and found to be more sensitive to growth inhibition by acetic acid than an otherwise isogenic prototrophic strain. At a concentration of 80 mM acetic acid at pH 4.8, the initial uptake of uracil, leucine, lysine, histidine, tryptophan, phosphate, and glucose was lower in the prototrophic strain than in a non-acetic acid-treated control. These findings are consistent with two mechanisms by which nutrient uptake may be inhibited. Intracellular adenosine triphosphate (ATP) levels were severely decreased upon acetic acid treatment, which likely slowed ATP-dependent proton symport, the major form of transport in yeast for nutrients other than glucose. In addition, the expression of genes encoding some nutrient transporters was repressed by acetic acid, including HXT1 and HXT3 that encode glucose transporters that operate by facilitated diffusion. These results illustrate how commonly used genetic markers in yeast deletion libraries complicate the effort to isolate strains with increased acetic acid resistance.

  1. High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae.

    OpenAIRE

    Sakai, Y.; Goh, T K; Tani, Y

    1993-01-01

    We have developed a transformation system which uses autonomous replicating plasmids for a methylotrophic yeast, Candida boidinii. Two autonomous replication sequences, CARS1 and CARS2, were newly cloned from the genome of C. boidinii. Plasmids having both a CARS fragment and the C. boidinii URA3 gene transformed C. boidinii ura3 cells to Ura+ phenotype at frequencies of up to 10(4) CFU/micrograms of DNA. From Southern blot analysis, CARS plasmids seemed to exist in polymeric forms as well as...

  2. 一种用于筛选启动子元件库的酵母检测载体的构建及初步应用%Construction and preliminary applications of a Saccharomyces cerevisiae detection plasmid using for screening promoter elements

    Institute of Scientific and Technical Information of China (English)

    王志芳; 王志标; 李丽娜; 安建梅; 王伟; 程克棣; 孔建强

    2013-01-01

    Synthetic biology of natural products is the design and construction of new biological systems by transferring a metabolic pathway of interest products into a chassis. Large-scale production of natural products is achieved by coordinate expression of multiple genes involved in genetic pathway of desired products. Promoters are cis-elements and play important roles in the balance of the metabolic pathways controlled by multiple genes by regulating gene expression. A detection plasmid of Saccharomyces cerevisiae was constructed based on DsRed-Monomer gene encoding for a red fluorescent protein. This plasmid was used for screening the efficient promoters applying for multiple gene-controlled pathways. First of all, eight pairs of primers specific to DsRed-Monomer gene were synthesized. The rapid cloning of DsRed-Monomer gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. AH cloned sequences were confirmed by DNA sequencing. A vector named pEASYDs-M containing full-length DsRed-Monomer gene was constructed and was used as the template for the construction of S.cerevisiae expression vector named for pYeDP60-Ds-M. pYeDP60-Ds-M was then transformed into S.cerevisiae for heterologous expression of DsRed-Monomer gene. SDS-PAGE, Western blot and fluorescence microscopy results showed that the recombinant DsRed-Monomer protein was expressed successfully in S.cerevisiae. The well-characterized, DsRed-Monomer gene was then cloned into a yeast expression vector pGBT9 to obtain a promoter detection plasmid pGBT9Red. For determination efficacy of pGBT9Red, six promoters (including four inducible promoters and two constitutive promoters) were cloned by PCR from the S.cerevisiae genome, and cloned into pGBT9Red by placing upstream of DsRed-Monomer gene, separately. The fluorescence microscopy results indicated that the six promoters (GAL1, GAL2, GAL7, GAL10, TEF2 and PGK1) can regulate the expression of Ds

  3. Improved production of fatty acids by Saccharomyces cerevisiae through screening a cDNA library from the oleaginous yeast Yarrowia lipolytica.

    Science.gov (United States)

    Shi, Shuobo; Ji, Haichuan; Siewers, Verena; Nielsen, Jens

    2016-02-01

    Biological production of fatty acid (FA)-derived products has gained increasing attention to replace petroleum-based fuels and chemicals. FA biosynthesis is highly regulated, and usually it is challenging to design rational engineering strategies. In addition, the conventional 'one sample at a time' method for lipid determination is time consuming and laborious, and it is difficult to screen large numbers of samples. Here, a method for detecting free FAs in viable cells using Nile red staining was developed for use in large-scale screening. Following optimization of the method, it was used for screening a cDNA library from the oleaginous yeast Yarrowia lipolytica for identification of genes/enzymes that were able to enhance free FA accumulation in Saccharomyces cerevisiae. Several novel enzymes resulting in increasing FA accumulation were discovered. These targets include a GPI anchor protein, malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, FA hydroxylase, farnesyltransferase, anoctamin, dihydrolipoamide dehydrogenase and phosphatidylethanolamine-binding protein. The best enzyme resulted in a 2.5-fold improvement in production of free FAs. Our findings not only provide a novel method for high-throughput evaluation of the content of free FAs, but also give new insight into how enzymes from Y. lipolytica may increase the production of fatty acids in S. cerevisiae.

  4. Co-segregation of yeast plasmid sisters under monopolin-directed mitosis suggests association of plasmid sisters with sister chromatids

    OpenAIRE

    Liu, Yen-Ting; Ma, Chien-Hui; Jayaram, Makkuni

    2013-01-01

    The 2-micron plasmid, a high copy extrachromosomal element in Saccharomyces cerevisiae, propagates itself with nearly the same stability as the chromosomes of its host. Plasmid stability is conferred by a partitioning system consisting of the plasmid-coded proteins Rep1 and Rep2 and a cis-acting locus STB. Circumstantial evidence suggests that the partitioning system couples plasmid segregation to chromosome segregation during mitosis. However, the coupling mechanism has not been elucidated. ...

  5. Plasmid Biopharmaceuticals.

    Science.gov (United States)

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A

    2014-12-01

    Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

  6. An improved method of xylose utilization by recombinant Saccharomyces cerevisiae.

    Science.gov (United States)

    Ma, Tien-Yang; Lin, Ting-Hsiang; Hsu, Teng-Chieh; Huang, Chiung-Fang; Guo, Gia-Luen; Hwang, Wen-Song

    2012-10-01

    The aim of this study was to develop a method to optimize expression levels of xylose-metabolizing enzymes to improve xylose utilization capacity of Saccharomyces cerevisiae. A xylose-utilizing recombinant S. cerevisiae strain YY2KL, able to express nicotinamide adenine dinucleotide phosphate, reduced (NADPH)-dependent xylose reductase (XR), nicotinamide adenine dinucleotide (NAD(+))-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK), showed a low ethanol yield and sugar consumption rate. To optimize xylose utilization by YY2KL, a recombinant expression plasmid containing the XR gene was transformed and integrated into the aur1 site of YY2KL. Two recombinant expression plasmids containing an nicotinamide adenine dinucleotide phosphate (NADP(+))-dependent XDH mutant and XK genes were dually transformed and integrated into the 5S ribosomal DNA (rDNA) sites of YY2KL. This procedure allowed systematic construction of an S. cerevisiae library with different ratios of genes for xylose-metabolizing enzymes, and well-grown colonies with different xylose fermentation capacities could be further selected in yeast protein extract (YPX) medium (1 % yeast extract, 2 % peptone, and 2 % xylose). We successfully isolated a recombinant strain with a superior xylose fermentation capacity and designated it as strain YY5A. The xylose consumption rate for strain YY5A was estimated to be 2.32 g/gDCW/h (g xylose/g dry cell weight/h), which was 2.34 times higher than that for the parent strain YY2KL (0.99 g/gDCW/h). The ethanol yield was also enhanced 1.83 times by this novel method. Optimal ratio and expression levels of xylose-metabolizing enzymes are important for efficient conversion of xylose to ethanol. This study provides a novel method that allows rapid and effective selection of ratio-optimized xylose-utilizing yeast strains. This method may be applicable to other multienzyme systems in yeast.

  7. Genome-Wide Transposon Mutagenesis in Saccharomyces cerevisiae and Candida albicans

    Science.gov (United States)

    Xu, Tao; Bharucha, Nikë; Kumar, Anuj

    2016-01-01

    Transposon mutagenesis is an effective method for generating large sets of random mutations in target DNA, with applicability toward numerous types of genetic screens in prokaryotes, single-celled eukaryotes, and metazoans alike. Relative to methods of random mutagenesis by chemical/UV treatment, transposon insertions can be easily identified in mutants with phenotypes of interest. The construction of transposon insertion mutants is also less labor-intensive on a genome-wide scale than methods for targeted gene replacement, although transposon insertions are not precisely targeted to a specific residue, and thus coverage of the target DNA can be problematic. The collective advantages of transposon mutagenesis have been well demonstrated in studies of the budding yeast Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans, as transposon mutagenesis has been used extensively for phenotypic screens in both yeasts. Consequently, we present here protocols for the generation and utilization of transposon-insertion DNA libraries in S. cerevisiae and C. albicans. Specifically, we present methods for the large-scale introduction of transposon insertion alleles in a desired strain of S. cerevisiae. Methods are also presented for transposon mutagenesis of C. albicans, encompassing both the construction of the plasmid-based transposon-mutagenized DNA library and its introduction into a desired strain of Candida. In total, these methods provide the necessary information to implement transposon mutagenesis in yeast, enabling the construction of large sets of identifiable gene disruption mutations, with particular utility for phenotypic screening in nonstandard genetic backgrounds. PMID:21815095

  8. Expression and secretion of Aspergillus niger glucoamylase in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    李文清; 何鸣; 罗进贤

    1995-01-01

    Aspergillus niger glucoamylase GA 1 cDNA was inserted in between the yeast PGK promoter and terminator on plasmid pMA91. The resultant plasmid pMAG69 was introduced into Saccharomyces cerevisiae GRF18 by protoplast transformation. The A niger GA I cDNA was expressed efficiently under the contiol of PGK promoter and 99% of the gene products were secreted into the culture medium using its own signal sequence The recombmant yeast can digest 87% of starch in 2 d in the medium containing 10% starch. The recombinant plasmid pMAG69 can exist stably in 5. cerevisiae.

  9. Library

    OpenAIRE

    Dulaney, Ronald E. Jr.

    1997-01-01

    This study began with the desire to design a public town library of the future and became a search for an inkling of what is essential to Architecture. It is murky and full of contradictions. It asks more than it proposes, and the traces of its windings are better ordered through collage than logical synthesis. This study is neither a thesis nor a synthesis. When drawing out the measure of this study it may be beneficial to state what it attempts to place at the ...

  10. Improved production of fatty acids by Saccharomyces cerevisiae through screening a cDNA library from the oleaginous yeast Yarrowia lipolytica

    DEFF Research Database (Denmark)

    Shi, Shuobo; Ji, Haichuan; Siewers, Verena;

    2016-01-01

    for screening a cDNA library from the oleaginous yeast Yarrowia lipolytica for identification of genes/enzymes that were able to enhance free FA accumulation in Saccharomyces cerevisiae. Several novel enzymes resulting in increasing FA accumulation were discovered. These targets include a GPI anchor protein......, malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, FA hydroxylase, farnesyltransferase, anoctamin, dihydrolipoamide dehydrogenase and phosphatidylethanolamine-binding protein. The best enzyme resulted in a 2.5-fold improvement in production of free FAs. Our findings not only provide a novel......' method for lipid determination is time consuming and laborious, and it is difficult to screen large numbers of samples. Here, a method for detecting free FAs in viable cells using Nile red staining was developed for use in large-scale screening. Following optimization of the method, it was used...

  11. Stable persistence of the yeast plasmid by hitchhiking on chromosomes during vegetative and germ-line divisions of host cells

    OpenAIRE

    Sau, Soumitra; Liu, Yen-Ting; Ma, Chien-Hui; Jayaram, Makkuni

    2015-01-01

    The chromosome-like stability of the Saccharomyces cerevisiae plasmid 2 micron circle likely stems from its ability to tether to chromosomes and segregate by a hitchhiking mechanism. The plasmid partitioning system, responsible for chromosome-coupled segregation, is comprised of 2 plasmid coded proteins Rep1 and Rep2 and a partitioning locus STB. The evidence for the hitchhiking model for mitotic plasmid segregation, although compelling, is almost entirely circumstantial. Direct tests for pla...

  12. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation

    OpenAIRE

    Timothy Hoggard; Ivan Liachko; Cassaundra Burt; Troy Meikle; Katherine Jiang; Gheorghe Craciun; Dunham, Maitreya J.; Fox, Catherine A.

    2016-01-01

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chrom...

  13. Deficient sumoylation of yeast 2-micron plasmid proteins Rep1 and Rep2 associated with their loss from the plasmid-partitioning locus and impaired plasmid inheritance.

    Directory of Open Access Journals (Sweden)

    Jordan B Pinder

    Full Text Available The 2-micron plasmid of the budding yeast Saccharomyces cerevisiae encodes copy-number amplification and partitioning systems that enable the plasmid to persist despite conferring no advantage to its host. Plasmid partitioning requires interaction of the plasmid Rep1 and Rep2 proteins with each other and with the plasmid-partitioning locus STB. Here we demonstrate that Rep1 stability is reduced in the absence of Rep2, and that both Rep proteins are sumoylated. Lysine-to-arginine substitutions in Rep1 and Rep2 that inhibited their sumoylation perturbed plasmid inheritance without affecting Rep protein stability or two-hybrid interaction between Rep1 and Rep2. One-hybrid and chromatin immunoprecipitation assays revealed that Rep1 was required for efficient retention of Rep2 at STB and that sumoylation-deficient mutants of Rep1 and Rep2 were impaired for association with STB. The normal co-localization of both Rep proteins with the punctate nuclear plasmid foci was also lost when Rep1 was sumoylation-deficient. The correlation of Rep protein sumoylation status with plasmid-partitioning locus association suggests a theme common to eukaryotic chromosome segregation proteins, sumoylated forms of which are found enriched at centromeres, and between the yeast 2-micron plasmid and viral episomes that depend on sumoylation of their maintenance proteins for persistence in their hosts.

  14. Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bojsen, Rasmus K; Andersen, Kaj Scherz; Regenberg, Birgitte

    2012-01-01

    to produce an ECM and respond to quorum sensing, and multi-cellular aggregates have lowered susceptibility to antifungals. Adhesion is mediated by a family of cell surface proteins of which Flo11 has been shown to be essential for biofilm development. FLO11 expression is regulated via a number of regulatory...... pathways including the protein kinase A and a mitogen-activated protein kinase pathway. Advanced genetic tools and resources have been developed for S. cerevisiae including a deletion mutant-strain collection in a biofilm-forming strain background and GFP-fusion protein collections. Furthermore, S...

  15. Plasmid cloning vehicle for Haemophilus influenzae and Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Clayton, N.L.; Setlow, J.K.

    1982-09-01

    A new plasmid cloning vehicle (pDM2) was used to introduce a library of Haemophilus influenzae chromosomal fragments into H. influenzae. Transformants of the higly recombination-defective rec-1 mutant were more likely to contain exclusively recombinant plasmids after exposure to ligated DNA mixtures than was the wild type. pDM2 could replicate in Escherichia coli K-12.

  16. Improvement of oxidative stress tolerance in Saccharomyces cerevisiae through global transcription machinery engineering.

    Science.gov (United States)

    Zhao, Hongwei; Li, Jingyuan; Han, Beizhong; Li, Xuan; Chen, Jingyu

    2014-05-01

    Excessive oxidative stress poses significant damage to yeast cells during fermentation process, and finally affects fermentation efficiency and the quality of products. In this paper, global transcription machinery engineering was employed to elicit Saccharomyces cerevisiae phenotypes of higher tolerance against oxidative stress caused by H2O2. Two strains from two plasmid-based mutagenesis libraries (Spt15 and Taf25), which exhibited significant increases in oxidative stress tolerance, were successfully isolated. At moderate H2O2 shock (≤3.5 mM), a positive correlation was found between the outperformance in cell growth of the oxidation-tolerate strains and H2O2 concentration. Several mutations were observed in the native transcription factors, which resulted in a different transcriptional profile compared with the control. Catalase and superoxide dismutase activities of the two mutants increased under H2O2 stress conditions. Fermentation experiments revealed that the mutant strain taf25-3 has a shorter lag phase compared to the control one, indicating that taf25-3 had improved adaptation ability to H2O2-induced oxidative stress and higher fermentation efficiency. Our study demonstrated that several amino acid substitutions in general transcription factors (Spt15 and Taf25) could modify the cellular oxidation defense systems and improve the anti-oxidation ability of S. cerevisiae. It could make the industrial ethanol fermentation more efficient and cost-effective by using the strain of higher stress tolerance. PMID:24633583

  17. Plasmid segregation mechanisms

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Gerdes, Kenn; Charbon, Gitte Ebersbach

    2005-01-01

    Bacterial plasmids encode partitioning (par) loci that ensure ordered plasmid segregation prior to cell division. par loci come in two types: those that encode actin-like ATPases and those that encode deviant Walker-type ATPases. ParM, the actin-like ATPase of plasmid R1, forms dynamic filaments...... that segregate plasmids paired at mid-cell to daughter cells. Like microtubules, ParM filaments exhibit dynamic instability (i.e., catastrophic decay) whose regulation is an important component of the DNA segregation process. The Walker box ParA ATPases are related to MinD and form highly dynamic, oscillating...... filaments that are required for the subcellular movement and positioning of plasmids. The role of the observed ATPase oscillation is not yet understood. However, we propose a simple model that couples plasmid segregation to ParA oscillation. The model is consistent with the observed movement...

  18. SPP1-mediated plasmid transduction.

    OpenAIRE

    Canosi, U; Lüder, G; Trautner, T A

    1982-01-01

    The virulent Bacillus subtilis phage SPP1 transduces plasmid DNA. Plasmid-transducing phages contain only plasmid DNA. Such DNA represents a concatemer of monomeric plasmid molecules with the molecular weight of mature SPP1 DNA. Biological parameters of plasmid transduction are described.

  19. Plasmid addiction systems: perspectives and applications in biotechnology

    OpenAIRE

    Kroll, Jens; Klinter, Stefan; Schneider,Cornelia; Voß, Isabella; Steinbüchel, Alexander

    2010-01-01

    Summary Biotechnical production processes often operate with plasmid‐based expression systems in well‐established prokaryotic and eukaryotic hosts such as Escherichia coli or Saccharomyces cerevisiae, respectively. Genetically engineered organisms produce important chemicals, biopolymers, biofuels and high‐value proteins like insulin. In those bioprocesses plasmids in recombinant hosts have an essential impact on productivity. Plasmid‐free cells lead to losses in the entire product recovery a...

  20. A yeast mutation that stabilizes a plasmid bearing a mutated ARS1 element.

    OpenAIRE

    Thrash-Bingham, C; Fangman, W L

    1989-01-01

    To identify the trans-acting factors involved in autonomously replicating sequence (ARS) function, we initiated a screen for Saccharomyces cerevisiae mutants capable of stabilizing a plasmid that contains a defective ARS element. The amm (altered minichromosome maintenance) mutations recovered in this screen defined at least four complementation groups. amm1, a mutation that has been studied in detail, gave rise to a 17-fold stabilization of one defective ARS1 plasmid over the level seen in w...

  1. Site-specific recombination of the circular 2 microns-like plasmid pKD1 requires integrity of the recombinase gene A and of the partitioning genes B and C.

    OpenAIRE

    Bianchi, M. M.

    1992-01-01

    In the circular plasmid pKD1, which stably replicates in Kluyveromyces lactis, the three open reading frames encode a site-specific recombinase (gene A) and two proteins involved in mitotic stability (genes B and C). A recombination analysis of plasmids in which gene B or C is inactivated reveals that unlike the 2 microns plasmid of Saccharomyces cerevisiae, these genes are also required for the site specificity of plasmid recombination.

  2. Chlamydial plasmids and bacteriophages.

    Science.gov (United States)

    Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław

    2015-01-01

    Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

  3. Cloning and Analysis of a Large Plasmid pBMB165 from Bacillus thuringiensis Revealed a Novel Plasmid Organization

    OpenAIRE

    Yueying Wang; Donghai Peng; Zhaoxia Dong; Lei Zhu; Suxia Guo; Ming Sun

    2013-01-01

    In this study, we report a rapid cloning strategy for large native plasmids via a contig linkage map by BAC libraries. Using this method, we cloned a large plasmid pBMB165 from Bacillus thuringiensis serovar tenebrionis strain YBT-1765. Complete sequencing showed that pBMB165 is 77,627 bp long with a GC-content of 35.36%, and contains 103 open reading frames (ORFs). Sequence analysis and comparison reveals that pBMB165 represents a novel plasmid organization: it mainly consists of a pXO2-like...

  4. Adaption of Saccharomyces cerevisiae expressing a heterologous protein

    DEFF Research Database (Denmark)

    Krogh, Astrid Mørkeberg; Beck, Vibe; Højlund Christensen, Lars;

    2008-01-01

    Production of the heterologous protein, bovine aprotinin, in Saccharomyces cerevisiae was shown to affect the metabolism of the host cell to various extent depending on the strain genotype. Strains with different genotypes, industrial and laboroatory, respectively, were investigated. The maximal ...... result of the adaptation. Determination of the level of mRNA encoding aprotinin and the plasmid copy number pointed to different mechanisms responsible for the decline in aprotinin yield in the different strains. (C) 2008 Elsevier B.V. All rights reserved....

  5. The partitioning and copy number control systems of the selfish yeast plasmid: an optimized molecular design for stable persistence in host cells

    OpenAIRE

    Yen-Ting-Liu,; Sau, Saumitra; Ma, Chien-Hui; Kachroo, Aashiq H.; Rowley, Paul A.; Chang, Keng-Ming; Fan, Hsiu-Fang; Jayaram, Makkuni

    2014-01-01

    The multi-copy 2 micron plasmid of Saccharomyces cerevisiae, a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely towards efficient replication, equal segregation and copy number maintenance. A pa...

  6. Syrinx 2A: an improved lambda phage vector designed for screening DNA libraries by recombination in vivo.

    OpenAIRE

    Lutz, C T; Hollifield, W C; Seed, B.; Davie, J M; Huang, H V

    1987-01-01

    The Syrinx 2A phage and pi AN13 plasmid were designed for screening of DNA libraries by homologous recombination in vivo. Syrinx 2A carries multiple cloning sites and a recently identified lambda gene, rap (recombination adept with plasmid), required for efficient phage-plasmid recombination. We describe a rapid, reliable, and technically easy method to screen Syrinx 2A libraries, expand the resulting phage-plasmid cointegrates, and subclone plasmid in as little as 2 days. Recombination scree...

  7. Plasmids encoding therapeutic agents

    Science.gov (United States)

    Keener, William K.

    2007-08-07

    Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

  8. The 2-micron plasmid as a nonselectable, stable, high copy number yeast vector

    Science.gov (United States)

    Ludwig, D. L.; Bruschi, C. V.

    1991-01-01

    The endogenous 2-microns plasmid of Saccharomyces cerevisiae has been used extensively for the construction of yeast cloning and expression plasmids because it is a native yeast plasmid that is able to be maintained stably in cells at high copy number. Almost invariably, these plasmid constructs, containing some or all 2-microns sequences, exhibit copy number levels lower than 2-microns and are maintained stably only under selective conditions. We were interested in determining if there was a means by which 2-microns could be utilized for vector construction, without forfeiting either copy number or nonselective stability. We identified sites in the 2-microns plasmid that could be used for the insertion of genetic sequences without disrupting 2-microns coding elements and then assessed subsequent plasmid constructs for stability and copy number in vivo. We demonstrate the utility of a previously described 2-microns recombination chimera, pBH-2L, for the manipulation and transformation of 2-microns as a pure yeast plasmid vector. We show that the HpaI site near the STB element in the 2-microns plasmid can be utilized to clone yeast DNA of at least 3.9 kb with no loss of plasmid stability. Additionally, the copy number of these constructs is as high as levels reported for the endogenous 2-microns.

  9. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked?

    Science.gov (United States)

    Million-Weaver, Samuel; Camps, Manel

    2014-09-01

    Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

  10. Plasmid-to-plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1986-01-01

    No recombination between plasmids was observed after conjugal transfer of a plasmid into a cell carrying another plasmid. Two types of such recombination took place after transformation, one type being Rec/sup +/ dependent and suggesting a preferred site of recombination. The other much rarer type was at least partially Rec/sup +/ independent.

  11. [The cloning and expression of the gene for beta-galactosidase from Candida pseudotropicalis yeasts in Saccharomyces cerevisiae cells].

    Science.gov (United States)

    Tretiak, K A; Zakal'skiĭ, A E; Gudz', S P

    1998-01-01

    The gene of beta-galactosidase of lactose-assimilating yeast Candida pseudotropicalis was cloned in pG2 and pBG2-3 hybrid shuttle vectors and expressed in Saccharomyces cerevisiae laboratory strains under the control of own promoter. The plasmids were able to replicate autonomously with relative stability in transformants of baker's yeasts. The availability of glucose or lactose in the medium influenced the recombinant plasmid stability and the expression of the cloned gene. A number of experiments have shown that the LAC+ phenotype in pG2-transformed Saccharomyces cerevisiae was due to the expression of the Candida pseudotropicalis lactose permease gene that is probably located in SaIG1/XhoI DNA fragment about 4.3 kb long. Southern hybridization experiments showed that LAC(+)-transformants of Saccharomyces cerevisiae contained both autonomously-replicative, and integrative pG2 plasmid.

  12. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method.

    OpenAIRE

    J Field; Nikawa, J; Broek, D; MacDonald, B.; Rodgers, L; Wilson, I A; Lerner, R A; Wigler, M

    1988-01-01

    We developed a method for immunoaffinity purification of Saccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cerevisiae alcohol dehydrogenase promoter ADH1. A monoclonal antibody previously raised against the peptide was used to purify adenylyl cyclase by affinity chromatography. The purified enzyme appeared to be ...

  13. Directed Evolution towards Increased Isoprenoid Production in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Carlsen, Simon; Nielsen, Michael Lynge; Kielland-Brandt, Morten;

    diversity. The most common way of producing these compounds is by organic synthesis. Organic synthesis does however have several disadvantages for production of secondary metabolites such as low yields due to the complex structures, which makes this way of production economically unfeasible. Microbial...... for discovering new genetic perturbations, which would results in and increased production of isoprenoids by S. cerevisiae has been very limited. This project is focus on creating diversity within a lycopene producing S. cerevisiae strain by construction of gDNA-, cDNA-, and transposon-libraries. The diversified...

  14. A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52.

    OpenAIRE

    Firmenich, A A; Elias-Arnanz, M; Berg, P

    1995-01-01

    To understand the mechanisms involved in homologous recombination, we have performed a search for Saccharomyces cerevisiae mutants unable to carry out plasmid-to-chromosome gene conversion. For this purpose, we have developed a colony color assay in which recombination is induced by the controlled delivery of double-strand breaks (DSBs). Recombination occurs between a chromosomal mutant ade2 allele and a second plasmid-borne ade2 allele where DSBs are introduced via the site-specific HO endon...

  15. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...... gene product had no PRPP synthase activity. In contrast, expression of five pairwise combinations of PRS genes resulted in the formation of active PRPP synthase. These combinations were PRS1 PRS2, PRS1 PRS3, and PRS1 PRS4, as well as PRS5 PRS2 and PRS5 PRS4. None of the remaining five possible pairwise...... combinations of PRS genes appeared to produce active enzyme. Extract of an E. coli strain containing a plasmid-borne PRS1 gene and a chromosome-borne PRS3 gene contained detectable PRPP synthase activity, whereas extracts of strains containing PRS1 PRS2, PRS1 PRS4, PRS5 PRS2, or PRS5 PRS4 contained...

  16. Persistence Mechanisms of Conjugative Plasmids

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes

    2009-01-01

    maintenance in the host cell. These importantly include the ability to self-mobilize in a process termed conjugative transfer, which may occur across species barriers. Other plasmid stabilizing mechanisms include the multimer resolution system, active partitioning, and post-segregational-killing of plasmid......Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting the...... successful propagation and long-term continued existence of these extra-chromosomal elements is extensive. Apart from the accessory genetic elements that may provide plasmid-harboring cells a selective advantage, special focus is placed on the mechanisms conjugative plasmids employ to ensure their stable...

  17. Conjugative plasmids of Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Emilia Pachulec

    Full Text Available Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones and with and without different tetM determinants (Dutch and American type tetM determinants have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233 or containing Dutch (pEP5289 or American (pEP5050 type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1alpha, beta, gamma, delta and epsilon subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids

  18. [Regulation of isoprenoid pathway for enhanced production of linalool in Saccharomyces cerevisiae].

    Science.gov (United States)

    Sun, Mingxue; Liu, Jidong; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2013-06-01

    Linalool is an important monoterpene, and widely used in food, pharmaceutical and cosmetic industry. The low concentration in plants and the difficulties in extraction restrict its large scale production. Saccharomyces cerevisiae can provide the monoterpene precursor, geranyl diphosphate (GPP) through its endogenous isoprenoid pathway. Therefore, it could be used as the host for monoterpene production. However, the weak metabolic flux through the isoprenoid pathway leads to the insufficient supply of GPP, and results in low monoterpene productivity. In order to increase the metabolic flux, we constructed the integrated expression plasmid pRS305-tHMG1 and free expression plasmid pYLIS-IDI1 to enhance the expression levels of isopentenyl diphosphate isomerase (IDI1) and a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase gene (tHMG1). The two plasmids were separately transformed into S. cerevisiae CEN.PK2-1C, resulting in strains LS01 and LS02. The plasmid pYLIS-IDI1 was further transformed into strain LS01, resulting in strain LS03. GC-MS analysis showed that the linalool concentration was increased by 1.3 times and reached (127.71 +/- 7.68) microg/L. In conclusion, enhancement of the supply of GPP precursors through the regulation of isoprenoid pathway could increase the linalool production in S. cerevisiae.

  19. Phenotypic plasticity in bacterial plasmids.

    Science.gov (United States)

    Turner, Paul E

    2004-01-01

    Plasmid pB15 was previously shown to evolve increased horizontal (infectious) transfer at the expense of reduced vertical (intergenerational) transfer and vice versa, a key trade-off assumed in theories of parasite virulence. Whereas the models predict that susceptible host abundance should determine which mode of transfer is selectively favored, host density failed to mediate the trade-off in pB15. One possibility is that the plasmid's transfer deviates from the assumption that horizontal spread (conjugation) occurs in direct proportion to cell density. I tested this hypothesis using Escherichia coli/pB15 associations in laboratory serial culture. Contrary to most models of plasmid transfer kinetics, my data show that pB15 invades static (nonshaking) bacterial cultures only at intermediate densities. The results can be explained by phenotypic plasticity in traits governing plasmid transfer. As cells become more numerous, the plasmid's conjugative transfer unexpectedly declines, while the trade-off between transmission routes causes vertical transfer to increase. Thus, at intermediate densities the plasmid's horizontal transfer can offset selection against plasmid-bearing cells, but at high densities pB15 conjugates so poorly that it cannot invade. I discuss adaptive vs. nonadaptive causes for the phenotypic plasticity, as well as potential mechanisms that may lead to complex transfer dynamics of plasmids in liquid environments. PMID:15166133

  20. Plasmid acquisition in microgravity

    Science.gov (United States)

    Juergensmeyer, Margaret A.; Juergensmeyer, Elizabeth A.; Guikema, James A.

    1995-01-01

    In microgravity, bacteria often show an increased resistance to antibiotics. Bacteria can develop resistance to an antibiotic after transformation, the acquisition of DNA, usually in the form of a plasmid containing a gene for resistance to one or more antibiotics. In order to study the capacity of bacteria to become resistant to antibiotics in microgravity, we have modified the standard protocol for transformation of Escherichia coli for use in the NASA-flight-certified hardware package, The Fluid Processing Apparatus (FPA). Here we report on the ability of E. coli to remain competent for long periods of time at temperatures that are readily available on the Space Shuttle, and present some preliminary flight results.

  1. Engineering the oxygen sensing regulation results in an enhanced recombinant human hemoglobin production by Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Martínez, José L.; Liu, Lifang; Petranovic, Dina;

    2015-01-01

    engineering also allowed the generation of different genetically modified organisms for the production of recombinant human hemoglobin. Several studies have showed very promising results using the bacterium Escherichia coli as a production platform, reporting hemoglobin titers above 5% of the total cell...... the generation of a set of plasmids to produce functional human hemoglobin in Saccharomyces cerevisiae, with final titers of active hemoglobin exceeding 4% of the total cell protein. In this study, we propose a strategy for further engineering S. cerevisiae by altering the oxygen sensing pathway by deleting...

  2. Plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.

    1982-01-01

    DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.

  3. Evolved plasmid-host interactions reduce plasmid interference cost.

    Science.gov (United States)

    Yano, Hirokazu; Wegrzyn, Katarznya; Loftie-Eaton, Wesley; Johnson, Jenny; Deckert, Gail E; Rogers, Linda M; Konieczny, Igor; Top, Eva M

    2016-09-01

    Antibiotic selection drives adaptation of antibiotic resistance plasmids to new bacterial hosts, but the molecular mechanisms are still poorly understood. We previously showed that a broad-host-range plasmid was poorly maintained in Shewanella oneidensis, but rapidly adapted through mutations in the replication initiation gene trfA1. Here we examined if these mutations reduced the fitness cost of TrfA1, and whether this was due to changes in interaction with the host's DNA helicase DnaB. The strains expressing evolved TrfA1 variants showed a higher growth rate than those expressing ancestral TrfA1. The evolved TrfA1 variants showed a lower affinity to the helicase than ancestral TrfA1 and were no longer able to activate the helicase at the oriV without host DnaA. Moreover, persistence of the ancestral plasmid was increased upon overexpression of DnaB. Finally, the evolved TrfA1 variants generated higher plasmid copy numbers than ancestral TrfA1. The findings suggest that ancestral plasmid instability can at least partly be explained by titration of DnaB by TrfA1. Thus under antibiotic selection resistance plasmids can adapt to a novel bacterial host through partial loss of function mutations that simultaneously increase plasmid copy number and decrease unfavorably high affinity to one of the hosts' essential proteins. PMID:27121483

  4. Featured Library: Parrish Library

    OpenAIRE

    Kirkwood, Hal P, Jr

    2015-01-01

    The Roland G. Parrish Library of Management & Economics is located within the Krannert School of Management at Purdue University. Between 2005 - 2007 work was completed on a white paper that focused on a student-centered vision for the Management & Economics Library. The next step was a massive collection reduction and a re-envisioning of both the services and space of the library. Thus began a 3 phase renovation from a 2 floor standard, collection-focused library into a single floor, 18,000s...

  5. Cypriot libraries

    OpenAIRE

    John F. Harvey

    1982-01-01

    Describes the current state of librarianship and bibliography in Cyprus, with separate sections for the Greek and Turkish sectors. Although there is no national library in the Greek sector there are 5 types of public library: Nicosia public library; Limassol, Larnaca and Paphos public libraries; community libraries; mobile libraries; and foreign cultural centre libraries. Schools and colleges in the Greek centre are well provided with libraries and most government departments sponsor special ...

  6. CRISPR-Cas9 Genome Engineering in Saccharomyces cerevisiae Cells.

    Science.gov (United States)

    Ryan, Owen W; Poddar, Snigdha; Cate, Jamie H D

    2016-01-01

    This protocol describes a method for CRISPR-Cas9-mediated genome editing that results in scarless and marker-free integrations of DNA into Saccharomyces cerevisiae genomes. DNA integration results from cotransforming (1) a single plasmid (pCAS) that coexpresses the Cas9 endonuclease and a uniquely engineered single guide RNA (sgRNA) expression cassette and (2) a linear DNA molecule that is used to repair the chromosomal DNA damage by homology-directed repair. For target specificity, the pCAS plasmid requires only a single cloning modification: replacing the 20-bp guide RNA sequence within the sgRNA cassette. This CRISPR-Cas9 protocol includes methods for (1) cloning the unique target sequence into pCAS, (2) assembly of the double-stranded DNA repair oligonucleotides, and (3) cotransformation of pCAS and linear repair DNA into yeast cells. The protocol is technically facile and requires no special equipment. It can be used in any S. cerevisiae strain, including industrial polyploid isolates. Therefore, this CRISPR-Cas9-based DNA integration protocol is achievable by virtually any yeast genetics and molecular biology laboratory. PMID:27250940

  7. Yeast 2-microns plasmid DNA replication in vitro: purification of the CDC8 gene product by complementation assay.

    OpenAIRE

    Arendes, J; Kim, K. C.; Sugino, A

    1983-01-01

    Extracts of the yeast Saccharomyces cerevisiae support DNA replication on exogenous yeast 2-microns plasmid DNA templates. A crude extract from a S. cerevisiae cell division cycle mutant, cdc8-1, expressed the temperature-sensitive phenotype since it could be inactivated at 42 degrees C in vitro. This heat-inactivated extract was fully complemented by the addition of either wild-type or cdc8-1 single-stranded DNA binding protein (SSB). restoration by SSB of the activity of the mutant cell ext...

  8. 1株中度嗜盐菌质粒基因组文库的构建及2个膜蛋白基因的克隆和序列分析%Construction of Plasmid Genomic Library of One Moderately Halophilic Bacteria & Cloning, Sequence Analysis of Two Membrane Protein Genes

    Institute of Scientific and Technical Information of China (English)

    朱文华; 董冠群; 滕长财; 侯娟; 吕彦; 孙业盈

    2012-01-01

    中度嗜盐菌是生活在高盐环境中的微生物,以1株从蜢子虾酱中分离的中度嗜盐菌盐脱氮枝芽胞杆菌(Virgibacillus halodenitrificans)为研究对象,通过酶切,筛选DNA片段然后连接pUC19载体并转化大肠埃希菌,构建了该菌株的质粒基因组文库,推测可获得5×103个阳性克隆,覆盖约15 Mb的遗传信息.随机选取部分克隆进行测序,在测序结果中筛选到了2个具有完整ORF的膜蛋白基因分别为编码Na+H+逆向转运蛋白的NhaC基因,编码一离子通道蛋白的MscS基因,GenBank登陆号分别为JX849200、JX849202.其中NhaC基因ORF全长为1 446 bp,编码481个氨基酸,包括10个跨膜结构域;MscS基因的全长ORF为822 bp,编码273个氨基酸的蛋白质,包括3个跨膜区,与其他菌种的序列比对表明第3个跨膜区保守性最强.%Moderately halophilic bacteria are microorganism living in high salt environment. A moderately halophilic bacteria Virgibacillus halodenitrijicans was separated from a shrimp paste as research object. Plasmid genomic library of the strain was constructed. DNA fragments were selected after genomic DNA digestion by restriction enzyme, and then were linked with pUC19 vector and transformed into E. coli to construct the plasmid genomic library of the strain. It was speculated that about 5 × 103 positive clones were obtained, covering about 15 Mb of genetic information. Part of the clones was selected to carry out sequencing. From the sequencing results two membrane protein genes possessing complete ORF respectively encoding NhaC gene of Na+ H + reverse transporter protein, MscS genes of encoding an ion channel protein were screened. The CenBank accession numbers of the two genes were JX849200, JX849202 respectively. Among them the NhaC gene was 1 446 bp length ORF, encoded 481 amino acids, including 10 transmem-brane domains. MscS gene had 822 bp length ORF, encoded a protein of 273 amino acids, including 3 transmembrane domains. The

  9. Plasmid construction using recombination activity in the fission yeast Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Ayako Chino

    Full Text Available BACKGROUND: Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC, is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date. METHODOLOGY/PRINCIPAL FINDINGS: We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp. No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe. CONCLUSIONS/SIGNIFICANCE: We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different

  10. Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    M. Pádula

    1999-09-01

    Full Text Available In the present study, we analyzed DNA damage induced by phycocyanin (PHY in the presence of visible light (VL using a set of repair endonucleases purified from Escherichia coli. We demonstrated that the profile of DNA damage induced by PHY is clearly different from that induced by molecules that exert deleterious effects on DNA involving solely singlet oxygen as reactive species. Most of PHY-induced lesions are single strand breaks and, to a lesser extent, base oxidized sites, which are recognized by Nth, Nfo and Fpg enzymes. High pressure liquid chromatography coupled to electrochemical detection revealed that PHY photosensitization did not induce 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo at detectable levels. DNA repair after PHY photosensitization was also investigated. Plasmid DNA damaged by PHY photosensitization was used to transform a series of Saccharomyces cerevisiae DNA repair mutants. The results revealed that plasmid survival was greatly reduced in rad14 mutants, while the ogg1 mutation did not modify the plasmid survival when compared to that in the wild type. Furthermore, plasmid survival in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant. The results reported here indicate that lethal lesions induced by PHY plus VL are repaired differently by prokaryotic and eukaryotic cells. Morever, nucleotide excision repair seems to play a major role in the recognition and repair of these lesions in Saccharomyces cerevisiae.

  11. One-hybrid screens at the Saccharomyces cerevisiae HMR locus identify novel transcriptional silencing factors.

    Science.gov (United States)

    Andrulis, Erik D; Zappulla, David C; Alexieva-Botcheva, Krassimira; Evangelista, Carlos; Sternglanz, Rolf

    2004-01-01

    In Saccharomyces cerevisiae, genes located at the telomeres and the HM loci are subject to transcriptional silencing. Here, we report results of screening a Gal4 DNA-binding domain hybrid library for proteins that cause silencing when targeted to a silencer-defective HMR locus. PMID:15020450

  12. Effects of genes exerting growth inhibition and plasmid stability on plasmid maintenance.

    OpenAIRE

    Boe, L; Gerdes, K; Molin, S

    1987-01-01

    Plasmid stabilization mediated by the parA+ and parB+ genes of the R1 plasmid and the ccd+ and sop+ genes of the F plasmid was tested on a mini-R1 plasmid and a pBR322 plasmid derivative. The mini-R1 plasmid is thought to be unstably inherited owing to a low copy number and to random segregation of the plasmid at cell division, whereas cells harboring the pBR322 derivative used in this work are lost through competition with plasmid-free cells, mainly as a result of the shorter generation time...

  13. Progressive Rearrangement of Telomeric Sequences Added to Both the ITR Ends of the Yeast Linear pGKL Plasmid

    Directory of Open Access Journals (Sweden)

    Gunge Norio

    2003-01-01

    Full Text Available Relocation into the nucleus of the yeast cytoplasmic linear plasmids was studied using a monitor plasmid pCLU1. In Saccharomyces cerevisiae, the nuclearly-relocated pCLU1 replicated in a linear form (termed pTLU-type plasmid which carried the host telomeric repeats TG1-3 of 300-350 bp at both ends. The telomere sequences mainly consisted of a major motif TGTGTGGGTGTGG which was complementary to part of the RNA template of yeast telomerase and were directly added to the very end of the pCLU1-terminal element ITR (inverted terminal repeat, suggesting that the ITR end played a role as a substrate of telomerase. The telomere sequences varied among isolated pTLU-type plasmids, but the TG1-3 organization was symmetrically identical on both ends of any one plasmid. During cell growth under non-selective condition, the telomeric repeat sequences were progressively rearranged on one side, but not on the opposite side of pTLU plasmid ends. This indicates that the mode of telomeric DNA replication or repair differed between both ends. Clonal analysis showed that the intense rearrangement of telomeric DNA was closely associated with extreme instability of pTLU plasmids.

  14. [Constructing recombinant plasmid pSH-CUP and knockout of acid trehalase gene in baker's yeast].

    Science.gov (United States)

    He, Dongqin; Xiao, Dongguang; Lv, Ye

    2008-02-01

    The ATH1 gene encoded acid trehalase in Saccharomyces cerevisiae. The gene disruption cassette combined the heterologous dominant kan(r) resistance marker with a Cre/loxP-mediated marker removal procedure. The gene disruption cassette was produced by PCR using the same long oligonucleotides comprising 50 nucleotides that annealed to sites upstream or downstream of the genomic target sequence to be deleted. After transformation of the linear disruption cassettes with a Cre/loxP-mediated marker into the cells of Saccharomyces cerevisiae BY-6, selected transformants were checked by PCR for correct the integration of the cassette and concurrent deletion of the chromosomal target sequence. The copper-resistance gene (CUP1-MT1) was cloned into pSH47, which yielded pSH-CUP. The recombinant plasmid pSH-CUP was transformed into the cells of Saccharomyces cerevisiae BY-6(delta ATH1, G418(r)), and transformants were selected for copper resistance. Upon expression of the Cre recombinase results in removal of the kan(r) gene, leaving behind a single loxP site at the chromosomal locus. Construction of the recombinant plasmid pSH-CUP avoided inserting non-yeast gene and made the loxP - kanMX - loxP gene disruption cassette more conventional for eukaryotic organism gene disruption.

  15. Fungal genomics beyond Saccharomyces cerevisiae?

    DEFF Research Database (Denmark)

    Hofmann, Gerald; Mcintyre, Mhairi; Nielsen, Jens

    2003-01-01

    Fungi are used extensively in both fundamental research and industrial applications. Saccharomyces cerevisiae has been the model organism for fungal research for many years, particularly in functional genomics. However, considering the diversity within the fungal kingdom, it is obvious that the a......Fungi are used extensively in both fundamental research and industrial applications. Saccharomyces cerevisiae has been the model organism for fungal research for many years, particularly in functional genomics. However, considering the diversity within the fungal kingdom, it is obvious...

  16. Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bomholt, Julie; Helix Nielsen, Claus; Scharff-Poulsen, Peter;

    2013-01-01

    In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tag......In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C...... and generated a monodisperse protein preparation. A single Ni-affinity chromatography step was used to obtain almost pure Aquaporin-1. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes....

  17. Library Automation

    OpenAIRE

    Dhakne, B. N.; Giri, V. V; Waghmode, S. S.

    2010-01-01

    New technologies library provides several new materials, media and mode of storing and communicating the information. Library Automation reduces the drudgery of repeated manual efforts in library routine. By use of library automation collection, Storage, Administration, Processing, Preservation and communication etc.

  18. Bacterial Plasmids in Antarctic Natural Microbial Assemblages

    OpenAIRE

    Kobori, Hiromi; Sullivan, Cornelius W.; Shizuya, Hiroaki

    1984-01-01

    Samples of psychrophilic and psychrotrophic bacteria were collected from sea ice, seawater, sediments, and benthic or ice-associated animals in McMurdo Sound, Antarctica. A total of 155 strains were isolated and tested for the presence of plasmids by DNA agarose gel electrophoresis. Thirty-one percent of the isolates carried at least one kind of plasmid. Bacterial isolates taken from sediments showed the highest plasmid incidence (42%), and isolates from seawater showed the lowest plasmid inc...

  19. Plasmid required for virulence of Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Watson, B.; Currier, T.C.; Gordon, M.P.; Chilton, M.D.; Nester, E.W.

    1975-07-01

    The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37/sup 0/C is shown to be due to loss of a large plasmid (1.2 x 10/sup 8/ daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6, is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 x A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.

  20. In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

    DEFF Research Database (Denmark)

    Carattoli, Alessandra; Zankari, Ea; García-Fernández, Aurora;

    2014-01-01

    In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft...

  1. Plasmid maintenance functions encoded on Dictyostelium discoideum nuclear plasmid Ddp1.

    OpenAIRE

    Hughes, J E; H. Kiyosawa; Welker, D L

    1994-01-01

    All of the plasmid-carried genes expressed during vegetative growth are essential for long-term maintenance of plasmid Ddp1 in the nucleus of Dictyostelium discoideum. Deletion of Ddp1 genes expressed only during development had no detectable effect on plasmid maintenance. Deletion of vegetatively expressed genes, either singly or in pairs, resulted in (i) a rapid loss of plasmid from cells grown in the absence of selection for plasmid retention, (ii) variation in the proportion of monomer to...

  2. RNAi-Assisted Genome Evolution (RAGE) in Saccharomyces cerevisiae.

    Science.gov (United States)

    Si, Tong; Zhao, Huimin

    2016-01-01

    RNA interference (RNAi)-assisted genome evolution (RAGE) applies directed evolution principles to engineer Saccharomyces cerevisiae genomes. Here, we use acetic acid tolerance as a target trait to describe the key steps of RAGE. Briefly, iterative cycles of RNAi screening are performed to accumulate multiplex knockdown modifications, enabling directed evolution of the yeast genome and continuous improvement of a target phenotype. Detailed protocols are provided on the reconstitution of RNAi machinery, creation of genome-wide RNAi libraries, identification and integration of beneficial knockdown cassettes, and repeated RAGE cycles. PMID:27581294

  3. One library to make them all: Streamlining yeast library creation by a SWAp-Tag (SWAT) strategy

    Science.gov (United States)

    Zalckvar, Einat; Goldman, Omer; Ben-Dor, Shifra; Schütze, Conny; Wiedemann, Nils; Knop, Michael; Khmelinskii, Anton; Schuldiner, Maya

    2016-01-01

    The yeast Saccharomyces cerevisiae is ideal for systematic studies relying on collections of modified strains (libraries). Despite the significance of yeast libraries and the immense variety of available tags and regulatory elements, only a few such libraries exist as their construction is extremely expensive and laborious. To overcome these limitations we developed a SWAp-Tag method (SWAT), in which one parental library can be modified easily and efficiently to give rise to an endless variety of libraries of choice. We showcase the versatility of the SWAT approach by constructing and investigating a library of ~1,800 strains carrying a SWAT-GFP module at the amino termini of endomembrane proteins and then using it to create two new libraries (mCherry or seamless GFP). Our work demonstrates how the SWAT method enables fast and effortless creation of yeast libraries, opening the door for endless new ways to systematically study cell biology. PMID:26928762

  4. One library to make them all: streamlining the creation of yeast libraries via a SWAp-Tag strategy.

    Science.gov (United States)

    Yofe, Ido; Weill, Uri; Meurer, Matthias; Chuartzman, Silvia; Zalckvar, Einat; Goldman, Omer; Ben-Dor, Shifra; Schütze, Conny; Wiedemann, Nils; Knop, Michael; Khmelinskii, Anton; Schuldiner, Maya

    2016-04-01

    The yeast Saccharomyces cerevisiae is ideal for systematic studies relying on collections of modified strains (libraries). Despite the significance of yeast libraries and the immense variety of available tags and regulatory elements, only a few such libraries exist, as their construction is extremely expensive and laborious. To overcome these limitations, we developed a SWAp-Tag (SWAT) method that enables one parental library to be modified easily and efficiently to give rise to an endless variety of libraries of choice. To showcase the versatility of the SWAT approach, we constructed and investigated a library of ∼1,800 strains carrying SWAT-GFP modules at the amino termini of endomembrane proteins and then used it to create two new libraries (mCherry and seamless GFP). Our work demonstrates how the SWAT method allows fast and effortless creation of yeast libraries, opening the door to new ways of systematically studying cell biology.

  5. Cloning and expression of the yeast galactokinase gene in an Escherichia coli plasmid

    International Nuclear Information System (INIS)

    The construction and isolation of a plasmid, derived from pBR322, which carries a BglII restriction fragment of DNA containing the galactokinase gene from Saccharomyces cerevisiae is described. This was accomplished by the following procedure: (1) Purified galactokinase mRNA, labelled with 125I, was hybridized to BglII digests of yeast DNA employing Southern's filter transfer technique to identify a restriction fragment containing the galactokinase gene. (2) This fragment was partially purified by agarose gel electrophoresis, ligated into the BamHI site of pBR322 and transrormed into Escherichia coli to generate a clone bank containing the galactokinase gene. (3) This bank was screened by in situ colony hybridization with galactokinase mRNA resulting in the identification of a plasmid carrying this gene. This plasmid DNA hybridized with the galactokinase mRNA to the same extent in the presence or absence of a large excess of unlabelled mRNA from cells that were not induced for galactokinase synthesis, while the same amount of unlabelled galactose-induced mRNA reduced the hybridization by 95%. When this plasmid was introduced into an E. coli strain deleted for the galactose operon it caused the synthesis of low levels of yeast galactokinase activity. (Auth.)

  6. Library statistics.

    OpenAIRE

    Melita Ambrožič

    1991-01-01

    The contribution deals with the purpose, beginnings and development of library statistics and the strivings for international standardization in this field. International recommendations are presented, as well as the ISO standard for library statistics. An overview is given of the theoretical contributions and statistical practice in Slovenian librarianship. Cautionary notice on the limitations of the applicability of library statistics in determining library performance is given and the inte...

  7. Digital Libraries

    CERN Document Server

    Papy, Fabrice

    2008-01-01

    Of vital interest to all librarians and information specialists, this book presents all aspects of the effects of digitization of today's and tomorrow's libraries. From social to technical issues, Digital Libraries includes chapters on the growth of the role of librarian, the reader experience, cataloging, search engines, OPAC, law, ergonomic studies, and the future of libraries.

  8. Development Of An Efficient Glycerol Utilizing Saccharomyces Cerevisiae Strain Via Adaptive Laboratory Evolution

    DEFF Research Database (Denmark)

    Strucko, Tomas; Zirngibl, Katharina; Tharwat Tolba Mohamed, Elsayed;

    2015-01-01

    that popular wild-type laboratory yeast strains, commonly applied in metabolic engineering studies, did not grow or grew very slowly in glycerol medium.In this work, an adaptive laboratory evolution approach to obtain S. cerevisiae strains with an improved ability to grow on glycerol was applied. A broad array...... catabolism in yeast. The knowledge acquired in this study may be further applied for rational S. cerevisiae strain improvement for using glycerol as a carbon source in industrial biotechnology processes. This work is a part of the DeYeastLibrary consortium financed by ERA-IB DeYeastLibrary - Designer yeast...... strain library optimized for metabolic engineering applications http://www.era-ib.net/deyeast-library...

  9. Homemade Site Directed Mutagenesis of Whole Plasmids

    Science.gov (United States)

    Laible, Mark; Boonrod, Kajohn

    2009-01-01

    Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers. PMID:19488024

  10. pLS010 plasmid vector

    Science.gov (United States)

    Lacks, Sanford A.; Balganesh, Tanjore S.

    1988-01-01

    Disclosed is recombinant plasmid pLS101, consisting essentially of a 2.0 Kb malM gene fragment ligated to a 4.4 Kb T.sub.c r DNA fragment, which is particularly useful for transforming Gram-positive bacteria. This plasmid contains at least four restriction sites suitable for inserting exogeneous gene sequences. Also disclosed is a method for plasmid isolation by penicillin selection, as well as processes for enrichment of recombinant plasmids in Gram-positive bacterial systems.

  11. ANALYSIS OF A MODEL OF PLASMID-BEARING, PLASMID-FREE COMPETITION IN A PULSED CHEMOSTAT

    OpenAIRE

    XIANGYUN SHI; XINYU SONG; XUEYONG ZHOU

    2006-01-01

    We introduce and study a chemostat model with plasmid-bearing, plasmid-free competition and impulsive effect. According to the stability analysis of the boundary periodic solution, we obtain the invasion threshold of the plasmid-free organism and plasmid-bearing organism. Furthermore, by using standard techniques of bifurcation theory, we prove the system has a positive τ-periodic solution, which shows that the impulsive effect destroys the equilibria of the unforced continuous system and ini...

  12. Effect of chromosome homology an plasmid transformation and plasmid conjugal transfer in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1984-05-14

    The pairing between plasmid and the homologous part of the chromosome associated with plasmid establishment may differ from the pairing which results from integration of a homologous region of the plasmid into the chromosome. Thus the rate of novobiocin transformation decreases with duplication of the chromosomal portion in pMB2, but the rate of establishment of the plasmid increases with this duplication. A model to explain these data is given. 17 references, 5 figures, 4 tables.

  13. The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis

    NARCIS (Netherlands)

    Cordes, C.; Meima, R; Twiest, B; Kazemier, B; Venema, G; vanDijl, JM; Bron, S

    1996-01-01

    The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the

  14. Cloning and expression in Saccharomyces cerevisiae of chit2 gene from Beauveria bassiana

    Institute of Scientific and Technical Information of China (English)

    SONG Jin-zhu; YANG Xiao-xue; WANG Yun; YANG Qian

    2009-01-01

    To study recycled trashes from shrimps and crabs in the sea through chitinase secreted by microor-ganisms, the chitinase gene chit2 was cloned and sequenced from Beauveria bassiana by the polymerase chain reaction (PCR), and was ligated into the yeast expression vector pYES2. The expression vector plasmid was transformed into Saccharomyces cerevisiae H158. Gene expression took place upon induction with 2% galac-tose. The measurement of enzyme activity shows that the expression production can be expressed in active forms and secreted to the medium. The enzyme activity approaches the peak of 0. 63 U/mL when the culture time is 36 h.

  15. Plasmid ColVBtrp maintenance in Erwinia carotovora.

    OpenAIRE

    Schukin, N N

    1981-01-01

    Plasmid ColVBtrp maintenance in Erwinia carotovora cells was followed by measuring kinetics of elimination of plasmid genetic markers and loss of plasmid deoxyribonucleic acid. An E. carotovora mutant stably carrying plasmid ColVBtrp was isolated. Besides stable plasmid maintenance, the mutant showed altered sensitivity to male-specific phage MS2, sensitivity to drugs, and colony morphology.

  16. Molecular cloning of eucaryotic genes required for excision repair of UV-irradiated DNA: isolation and partial characterization of the RAD3 gene of Saccharomyces cerevisiae.

    OpenAIRE

    Naumovski, L; Friedberg, E C

    1982-01-01

    We describe the molecular cloning of a 6-kilobase (kb) fragment of yeast chromosomal DNA containing the RAD3 gene of Saccharomyces cerevisiae. When present in the autonomously replicating yeast cloning vector YEp24, this fragment transformed two different UV-sensitive, excision repair-defective rad3 mutants of S. cerevisiae to UV resistance. The same result was obtained with a variety of other plasmids containing a 4.5-kb subclone of the 6-kb fragment. The UV sensitivity of mutants defective ...

  17. Library Locations

    Data.gov (United States)

    Allegheny County / City of Pittsburgh / Western PA Regional Data Center — Carnegie Library of Pittsburgh locations including address, coordinates, phone number, square footage, and standard operating hours.

  18. Cystathionine accumulation in Saccharomyces cerevisiae.

    OpenAIRE

    Ono, B; Suruga, T; Yamamoto, M.; Yamamoto, S.; Murata, K; Kimura, A; Shinoda, S; Ohmori, S.

    1984-01-01

    A cysteine-dependent strain of Saccharomyces cerevisiae and its prototrophic revertants accumulated cystathionine in cells. The cystathionine accumulation was caused by a single mutation having a high incidence of gene conversion. The mutation was designated cys3 and was shown to cause loss of gamma-cystathionase activity. Cysteine dependence of the initial strain was determined by two linked and interacting mutations, cys3 and cys1 . Since cys1 mutations cause a loss of serine acetyltransfer...

  19. Self-transmissible nif plasmid (pEA9) of Enterobacter agglomerans 339: molecular cloning and evidence for the existence of similar nif clusters on dissimilar plasmids in Enterobacter strains.

    Science.gov (United States)

    Steibl, H D; Siddavattam, D; Klingmüller, W

    1995-11-01

    A cosmid library was generated to the 200-kb self-transmissible nif plasmid pEA9 isolated from Enterobacter agglomerans 339. The cosmid clone identified to contain the complete nif cluster was used to determine the nif gene organization and the physical map. The restriction pattern and nif gene organization of this nif cluster showed remarkable similarities to the nif cluster identified on the 110-kb plasmid pEA3 of Enterobacter agglomerans 333. Nucleotide sequence of several randomly selected regions of the nif cluster of pEA9 showed 96% similarity when compared to the known sequences of the nif cluster of pEA3. However, the homology ended abruptly at the flanking regions of the nif clusters and no similarity could be detected with the rest of the DNA of these plasmids. This reveals the existence of similar nif clusters on dissimilar plasmids, implying the horizontal transfer of the entire nif gene cluster.

  20. America's Star Libraries: Top-Rated Libraries

    Science.gov (United States)

    Lance, Keith Curry; Lyons, Ray

    2009-01-01

    "Library Journal"'s national rating of public libraries, the "LJ" Index of Public Library Service 2009, Round 2, identifies 258 "star" libraries. Created by Keith Curry Lance and Ray Lyons and based on 2007 data from the IMLS, it rates 7,268 public libraries. The top libraries in each group get five, four, or three stars. All included libraries,…

  1. Libraries - Iowa DNR - NRGIS Library

    Data.gov (United States)

    NSGIC GIS Inventory (aka Ramona) — The Natural Resources Geographic Information System (NRGIS) Library is a Geographic Information System (GIS) repository developed and maintained by the GIS Section...

  2. Privatizing Libraries

    Science.gov (United States)

    Jerrard, Jane; Bolt, Nancy; Strege, Karen

    2012-01-01

    This timely special report from ALA Editions provides a succinct but comprehensive overview of the "privatization" of public libraries. It provides a history of the trend of local and state governments privatizing public services and assets, and then examines the history of public library privatization right up to the California legislation…

  3. Library Use

    DEFF Research Database (Denmark)

    Konzack, Lars

    2012-01-01

    A seminar paper about a survey of role-playing games in public libraries combined with three cases and a presentation of a model.......A seminar paper about a survey of role-playing games in public libraries combined with three cases and a presentation of a model....

  4. Translation and stability of an Escherichia coli beta-galactosidase mRNA expressed under the control of pyruvate kinase sequences in Saccharomyces cerevisiae.

    OpenAIRE

    Purvis, I J; Loughlin, L; Bettany, A J; Brown, A. J.

    1987-01-01

    Plasmids were assembled in which the coding region of the pyruvate kinase (PYK) gene of Saccharomyces cerevisiae was replaced by that of the B-galactosidase (LacZ) gene from Escherichia coli. Analysis of the resultant, chimaeric transcripts from low copy number, centromeric plasmids indicated that this substitution caused a dramatic reduction in the steady-state level of the messenger RNA (mRNA). This fluctuation cannot be wholly accounted for by the 2-fold decrease in mRNA stability observed...

  5. Bifurcation Analysis of a Chemostat Model of Plasmid-Bearing and Plasmid-Free Competition with Pulsed Input

    OpenAIRE

    Zhong Zhao; Baozhen Wang; Liuyong Pang; Ying Chen

    2014-01-01

    A chemostat model of plasmid-bearing and plasmid-free competition with pulsed input is proposed. The invasion threshold of the plasmid-bearing and plasmid-free organisms is obtained according to the stability of the boundary periodic solution. By use of standard techniques of bifurcation theory, the periodic oscillations in substrate, plasmid-bearing, and plasmid-free organisms are shown when some conditions are satisfied. Our results can be applied to control bioreactor aimed at producing co...

  6. Progress in Metabolic Engineering of Saccharomyces cerevisiae

    OpenAIRE

    Nevoigt, Elke

    2008-01-01

    Summary: The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic...

  7. A Candida albicans gene expressed in Saccharomyces cerevisiae results in a distinct pattern of mRNA processing.

    Science.gov (United States)

    Iborra, A; Sentandreu, R; Gozalbo, D

    1996-09-01

    Two plasmids (derived from YCplac22 and YEplac112) carrying a Candida albicans gene (including the 5' non-coding promoter sequences) coding for a 30 kDa membrane-bound protein, were used to transform Saccharomyces cerevisiae cells. A 30 kDa protein was immunodetected by Western blot in the membrane fraction of transformants. Northern analysis showed the presence of three mRNA species (of about 1.1, 0.7 and 0.5 kb) hybridizing with the C. albicans gene as a probe. The same result was obtained using the 5' and 3' regions of the gene as probes, whereas only a 1.1 kb mRNA was found in C. albicans and none was detected in S. cerevisiae control transformants. Thus, heterologous expression of this gene in S. cerevisiae results in a distinct pattern of mRNA processing, either due to the location on plasmid vectors and/or to differences in the mRNA processing systems in the two microorganisms.

  8. Surface display of malolactic enzyme from Oenococcus oeni on Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhang, Xiuyan; Hou, Xiaoyan; Liang, Fang; Chen, Fusheng; Wang, Xiaohong

    2013-04-01

    In order to display malolactic enzyme (MLE) on the cell surface of Saccharomyces cerevisiae, a yeast cell surface display plasmid pADH1-AGG was constructed by fusing the α-factor signal encoding sequence (267 bp) and the C-terminal half of α-agglutinin encoding sequence (1,645 bp) into the plasmid pADH1. The pADH1-AGG could successfully express and anchor the enhanced green fluorescent protein (EGFP) onto the yeast cell surface when the EGFP was used to verify its function. Then the pADH1-MLE was constructed by inserting the MLE encoding sequence (1,600 bp) into the pADH1-AGG and introduced into S. cerevisiae cells. The positive strain carrying pADH1-MLE was confirmed by use of the 6× His monoclonal antibody and fluorescein isothiocyanate-conjugated goat anti-mouse IgG. All results indicated that the MLE was displayed successfully on the cell surface of positive transformant. The MLE activity of genetically engineered yeast strain could turn 21.11 % L-malate into lactic acid after 12 h reaction with L-malate. The constructed yeast strain might be used to conduct malolactic fermentation (MLF) in wine to solve the important issues of sluggish MLF, microbial spoilage, and adverse metabolic substances produced by the lactic acid bacteria. PMID:23446978

  9. Plasmids as Tools for Containment.

    Science.gov (United States)

    García, José L; Díaz, Eduardo

    2014-10-01

    Active containment systems are a major tool for reducing the uncertainty associated with the introduction of monocultures, genetically engineered or not, into target habitats for a large number of biotechnological applications (e.g., bioremediation, bioleaching, biopesticides, biofuels, biotransformations, live vaccines, etc.). While biological containment reduces the survival of the introduced organism outside the target habitat and/or upon completion of the projected task, gene containment strategies reduce the lateral spread of the key genetic determinants to indigenous microorganisms. In fundamental research, suicide circuits become relevant tools to address the role of gene transfer, mainly plasmid transfer, in evolution and how this transfer contributes to genome plasticity and to the rapid adaptation of microbial communities to environmental changes. Many lethal functions and regulatory circuits have been used and combined to design efficient containment systems. As many new genomes are being sequenced, novel lethal genes and regulatory elements are available, e.g., new toxin-antitoxin modules, and they could be used to increase further the current containment efficiencies and to expand containment to other organisms. Although the current containment systems can increase the predictability of genetically modified organisms in the environment, containment will never be absolute, due to the existence of mutations that lead to the appearance of surviving subpopulations. In this sense, orthogonal systems (xenobiology) appear to be the solution for setting a functional genetic firewall that will allow absolute containment of recombinant organisms. PMID:26104372

  10. Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid integration

    OpenAIRE

    Bertoni, Carmen; Jarrahian, Sohail; Wheeler, Thurman M.; LI, YINING; Olivares, Eric C.; Michele P Calos; Rando, Thomas A.

    2005-01-01

    Plasmid-mediated gene therapy can restore dystrophin expression in skeletal muscle in the mdx mouse, a model of Duchenne muscular dystrophy. However, sufficient long-term expression and distribution of dystrophin remain a hurdle for translating this technology into a viable treatment for Duchenne muscular dystrophy. To improve plasmid-mediated gene therapy for muscle diseases, we studied the effects of targeted plasmid integration using a phage integrase (φC31) that can mediate the integratio...

  11. Characterization of a Haemophilus ducreyi mobilizing plasmid.

    OpenAIRE

    McNicol, P J; Albritton, W L; Ronald, A R

    1986-01-01

    The OriV site of Haemophilus ducreyi mobilizing plasmid pHD147, determined by replication in Escherichia coli polA, is located close to the OriT site. The OriT site, located by recombination-proficient and -deficient cells, and the OriV site map in a region of pHD147 homologous to the beta-lactamase-specifying plasmids of H. ducreyi and Neisseria gonorrhoeae.

  12. Protein diversity confers specificity in plasmid segregation.

    Science.gov (United States)

    Fothergill, Timothy J G; Barillà, Daniela; Hayes, Finbarr

    2005-04-01

    The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation. PMID:15805511

  13. Plasmid and chromosome segregation in prokaryotes

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Gerdes, Kenn

    2000-01-01

    Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic......-like apparatus in prokaryotes. The identification of chromosomal homologues of the well-characterized plasmid partitioning genes indicates that there could be a general mechanism of bacterial DNA partitioning. Udgivelsesdato: July 1...

  14. Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.

  15. Positive epistasis between co-infecting plasmids promotes plasmid survival in bacterial populations

    OpenAIRE

    San Millan, Alvaro; Heilbron, Karl; MacLean, R. Craig

    2014-01-01

    Plasmids have a key role in the horizontal transfer of genes among bacteria. Although plasmids are catalysts for bacterial evolution, it is challenging to understand how they can persist in bacterial populations over the long term because of the burden they impose on their hosts (the ‘plasmid paradox'). This paradox is especially perplexing in the case of ‘small' plasmids, which are unable to self-transfer by conjugation. Here, for the first time, we investigate how interactions between co-in...

  16. Mini-F plasmid genes that couple host cell division to plasmid proliferation.

    OpenAIRE

    Ogura, T; Hiraga, S

    1983-01-01

    A mechanism for stable maintenance of plasmids, besides the replication and partition mechanisms, has been found to be specified by genes of a mini-F plasmid. An oriC plasmid carrying both a mini-F segment necessary for partition [coordinates 46.4-49.4 kilobase pairs (kb) on the F map] and another segment (42.9-43.6 kb), designated ccd (coupled cell division), is more stably maintained than are oriC plasmids carrying only the partition segment; the stability is comparable to that of the paren...

  17. Adaptive Plasmid Evolution Results in Host-Range Expansion of a Broad-Host-Range Plasmid

    OpenAIRE

    De Gelder, Leen; Williams, Julia J.; Ponciano, José M; Sota, Masahiro; Eva M. Top

    2008-01-01

    Little is known about the range of hosts in which broad-host-range (BHR) plasmids can persist in the absence of selection for plasmid-encoded traits, and whether this “long-term host range” can evolve over time. Previously, the BHR multidrug resistance plasmid pB10 was shown to be highly unstable in Stenotrophomonas maltophilia P21 and Pseudomonas putida H2. To investigate whether this plasmid can adapt to such unfavorable hosts, we performed evolution experiments wherein pB10 was maintained ...

  18. Deletion of host histone acetyltransferases and deacetylases strongly affects Agrobacterium-mediated transformation of Saccharomyces cerevisiae.

    Science.gov (United States)

    Soltani, Jalal; van Heusden, Gerard Paul H; Hooykaas, Paul J J

    2009-09-01

    Agrobacterium tumefaciens is a plant pathogen that genetically transforms plant cells by transferring a part of its Ti-plasmid, the T-strand, to the host cell. Under laboratory conditions, it can also transform cells from many different nonplant organisms, including the yeast Saccharomyces cerevisiae. Collections of S. cerevisiae strains have been developed with systematic deletion of all coding sequences. Here, we used these collections to identify genes involved in the Agrobacterium-mediated transformation (AMT) of S. cerevisiae. We found that deletion of genes (GCN5, NGG1, YAF9 and EAF7) encoding subunits of the SAGA, SLIK, ADA and NuA4 histone acetyltransferase complexes highly increased the efficiency of AMT, while deletion of genes (HDA2, HDA3 and HST4) encoding subunits of histone deacetylase complexes decreased AMT. These effects are specific for AMT as the efficiency of chemical (lithium acetate) transformation was not or only slightly affected by these deletions. Our data are consistent with a positive role of host histone deacetylation in AMT.

  19. Elimination of multicopy plasmid R6K by bleomycin.

    OpenAIRE

    Attfield, P V; Pinney, R. J.

    1985-01-01

    Bleomycin eliminated multicopy plasmid R6K from growing cells of Escherichia coli AB1157 but failed to cure either of the low-copy plasmids R1 or R46. Measurements of R6K-encoded beta-lactamase and of covalently closed plasmid DNA indicated that the drug causes a progressive reduction in plasmid copy number.

  20. Behavior of IncQ Plasmids in Agrobacterium tumefaciens

    NARCIS (Netherlands)

    Hille, Jacques; Schilperoort, Rob

    1981-01-01

    Inc-Q plasmids were introduced into Agrobacterium tumefuciens, by mobilization from Escherichia coli with an Inc-P plasmid, or by transformation with purified plasmid DNA. It was found that they were stably maintained. The presence of an Inc-Q plasmid did not influence tumorigenicity. These results

  1. Plasmid Evolution and Interaction between the Plasmid Addiction Stability Systems of Two Related Broad-Host-Range IncQ-Like Plasmids

    OpenAIRE

    Deane, Shelly M.; Rawlings, Douglas E

    2004-01-01

    Plasmid pTC-F14 contains a plasmid stability system called pas (plasmid addiction system), which consists of two proteins, a PasA antitoxin and a PasB toxin. This system is closely related to the pas of plasmid pTF-FC2 (81 and 72% amino acid identity for PasA and PasB, respectively) except that the pas of pTF-FC2 contains a third protein, PasC. As both pTC-F14 and pTF-FC2 are highly promiscuous broad-host-range plasmids isolated from bacteria that share a similar ecological niche, the plasmid...

  2. Historical Events That Spawned the Field of Plasmid Biology.

    Science.gov (United States)

    Kado, Clarence I

    2014-10-01

    This chapter revisits the historical development and outcome of studies focused on the transmissible, extrachromosomal genetic elements called plasmids. Early work on plasmids involved structural and genetic mapping of these molecules, followed by the development of an understanding of how plasmids replicate and segregate during cell division. The intriguing property of plasmid transmission between bacteria and between bacteria and higher cells has received considerable attention. The utilitarian aspects of plasmids are described, including examples of various plasmid vector systems. This chapter also discusses the functional attributes of plasmids needed for their persistence and survival in nature and in man-made environments. The term plasmid biology was first conceived at the Fallen Leaf Lake Conference on Promiscuous Plasmids, 1990, Lake Tahoe, California. The International Society for Plasmid Biology was established in 2004 (www.ISPB.org).

  3. Relationship between plasmid content and auxotype in Neisseria gonorrhoeae isolates.

    OpenAIRE

    Dillon, J R; Pauzé, M

    1981-01-01

    One hundred and forty strains of Neisseria gonorrhoeae, representing 12 different auxotype groups, were examined for differences in plasmid content. Most auxotype groups harbored a phenotypically cryptic 2,6-megadalton plasmid; a few groups also carried a 24.5-megadalton plasmid which has been previously characterized as a transfer plasmid. However, isolates of the proline-, citrulline-, and uracil-requiring (PCU-) auxotype were consistently free of plasmids. The correlation between auxotype ...

  4. Plasmid stability and maintenance of copy number using natural marker

    OpenAIRE

    Hamzah Basil Mohammed; Sudhakar Malla

    2015-01-01

    Present study was conducted to study the plasmid stability with the help of natural plasmid isolated from the bacteria which lodges the ink gland of the sea squid and emits bioluminescence. Isolated bacterial strain was identified by using 16srRNA sequencing and its plasmid DNA was used for the experimental studies. The plasmid is found to be responsible for the bioluminescence. The stability of this plasmid was studied in shake flask method using the different sugar sources (Gluc...

  5. America's Star Libraries

    Science.gov (United States)

    Lyons, Ray; Lance, Keith Curry

    2009-01-01

    "Library Journal"'s new national rating of public libraries, the "LJ" Index of Public Library Service, identifies 256 "star" libraries. It rates 7,115 public libraries. The top libraries in each group get five, four, or three Michelin guide-like stars. All included libraries, stars or not, can use their scores to learn from their peers and improve…

  6. Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated, sorted and matured in the fission yeast Schizosaccharomyces pombe.

    Science.gov (United States)

    Simeon, A; Egner, R; Gascon, S; Suarez-Rendueles, P

    1995-03-01

    Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPYsc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 mu derived plasmid. Immunoblot analysis revealed that CPYsc produced in the fission yeast has a higher molecular mass than mature CPYsc produced by the budding yeast. CPYsc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPYsc produced by S. cerevisiae. CPYsc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPYsc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.

  7. COMBINATORIAL LIBRARIES

    DEFF Research Database (Denmark)

    1997-01-01

    The invention provides a method for the production of a combinatorial library of compound of general formula (I) using solid phase methodologies. The cleavage of the array of immobilised compounds of the phthalimido type from the solid support matrix is accomplished by using an array of dinucleop......The invention provides a method for the production of a combinatorial library of compound of general formula (I) using solid phase methodologies. The cleavage of the array of immobilised compounds of the phthalimido type from the solid support matrix is accomplished by using an array of...... dinucleophiles, e.g. hydrazines (hydrazinolysis) or N-hydroxylamines, whereby a combinatorial dimension is introduced in the cleavage step. The invention also provides a compound library....

  8. Glucose repression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kayikci, Omur; Nielsen, Jens

    2015-01-01

    Glucose is the primary source of energy for the budding yeast Saccharomyces cerevisiae. Although yeast cells can utilize a wide range of carbon sources, presence of glucose suppresses molecular activities involved in the use of alternate carbon sources as well as it represses respiration and...... gluconeogenesis. This dominant effect of glucose on yeast carbon metabolism is coordinated by several signaling and metabolic interactions that mainly regulate transcriptional activity but are also effective at post-transcriptional and post-translational levels. This review describes effects of glucose repression...... on yeast carbon metabolism with a focus on roles of the Snf3/Rgt2 glucose-sensing pathway and Snf1 signal transduction in establishment and relief of glucose repression....

  9. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Weinert, Brian Tate; Iesmantavicius, Vytautas; Moustafa, Tarek;

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation...

  10. Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli.

    Science.gov (United States)

    Godiska, Ronald; Mead, David; Dhodda, Vinay; Wu, Chengcang; Hochstein, Rebecca; Karsi, Attila; Usdin, Karen; Entezam, Ali; Ravin, Nikolai

    2010-04-01

    Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain numerous tandem or inverted repeats. Common vectors also induce transcription and translation of inserted fragments, which can select against recombinant clones containing open reading frames or repetitive DNA. Conversely, transcription from cloned promoters can interfere with plasmid stability. We have therefore developed a novel Escherichia coli cloning vector (termed 'pJAZZ' vector) that is maintained as a linear plasmid. Further, it contains transcriptional terminators on both sides of the cloning site to minimize transcriptional interference between vector and insert. We show that this vector stably maintains a variety of inserts that were unclonable in conventional plasmids. These targets include short nucleotide repeats, such as those of the expanded Fragile X locus, and large AT-rich inserts, such as 20-kb segments of genomic DNA from Pneumocystis, Plasmodium, Oxytricha or Tetrahymena. The pJAZZ vector shows decreased size bias in cloning, allowing more uniform representation of larger fragments in libraries. PMID:20040575

  11. TOTAL ANTIOXIDANT ACTIVITY OF YEAST SACCHAROMYCES CEREVISIAE

    OpenAIRE

    Blažena Lavová; Dana Urminská

    2013-01-01

    Antioxidants are health beneficial compounds that can protect cells and macromolecules (e.g. fats, lipids, proteins and DNA) from the damage of reactive oxygen species (ROS). Sacchamomyces cerevisiae are know as organisms with very important antioxidative enzyme systems such as superoxide dismutase or catalase. The total antioxidant activity (mmol Trolox equivalent – TE.g-1 d.w.) of Saccharomyces cerevisiae was measured by 2,2´-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) during the yeas...

  12. Expression of the denV gene of coliphage T4 in UV-sensitive rad mutants of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    A plasmid containing the denV gene from bacteriophage T4, under the control of the yeast alcohol dehydrogenase I (ADC1) promoter, conferred a substantial increase in UV resistance in the UV-sensitive Saccharomyces cerevisiae mutants rad1-2 and rad3-2. The UV resistance of the denV+ yeast cells was cell cycle dependent and correlated well with the level of the denV gene product as measured by immunoblotting and by a photoreversal assay for pyrimidine dimer-DNA glycosylase activity

  13. RSC1 and RSC2 Are Required for Expression of Mid-Late Sporulation-Specific Genes in Saccharomyces cerevisiae

    OpenAIRE

    Bungard, David; Reed, Michelle; Winter, Edward

    2004-01-01

    Rsc1 and Rsc2 are alternative bromodomain-containing subunits of the ATP-dependent RSC chromatin remodeling complex in Saccharomyces cerevisiae. Smk1 is a sporulation-specific mitogen-activated protein kinase homolog that is required for the postmeiotic events of spore formation. In this study we show that RSC1 and RSC2 are haploinsufficient for spore formation in a smk1 hypomorph. Moreover, diploids lacking Rsc1 or Rsc2 show a subset of smk1-like phenotypes. High-copy-number RSC1 plasmids do...

  14. BioShuttle-mediated Plasmid Transfer

    Directory of Open Access Journals (Sweden)

    Klaus Braun, Leonie von Brasch, Ruediger Pipkorn, Volker Ehemann, Juergen Jenne, Herbert Spring, Juergen Debus, Bernd Didinger, Werner Rittgen, Waldemar Waldeck

    2007-01-01

    Full Text Available An efficient gene transfer into target tissues and cells is needed for safe and effective treatment of genetic diseases like cancer. In this paper, we describe the development of a transport system and show its ability for transporting plasmids. This non-viral peptide-based BioShuttle-mediated transfer system consists of a nuclear localization address sequence realizing the delivery of the plasmid phNIS-IRES-EGFP coding for two independent reporter genes into nuclei of HeLa cells. The quantification of the transfer efficiency was achieved by measurements of the sodium iodide symporter activity. EGFP gene expression was measured with Confocal Laser Scanning Microscopy and quantified with biostatistical methods by analysis of the frequency of the amplitude distribution in the CLSM images. The results demonstrate that the “BioShuttle”-Technology is an appropriate tool for an effective transfer of genetic material carried by a plasmid.

  15. BioShuttle-mediated Plasmid Transfer

    Science.gov (United States)

    Braun, Klaus; von Brasch, Leonie; Pipkorn, Ruediger; Ehemann, Volker; Jenne, Juergen; Spring, Herbert; Debus, Juergen; Didinger, Bernd; Rittgen, Werner; Waldeck, Waldemar

    2007-01-01

    An efficient gene transfer into target tissues and cells is needed for safe and effective treatment of genetic diseases like cancer. In this paper, we describe the development of a transport system and show its ability for transporting plasmids. This non-viral peptide-based BioShuttle-mediated transfer system consists of a nuclear localization address sequence realizing the delivery of the plasmid phNIS-IRES-EGFP coding for two independent reporter genes into nuclei of HeLa cells. The quantification of the transfer efficiency was achieved by measurements of the sodium iodide symporter activity. EGFP gene expression was measured with Confocal Laser Scanning Microscopy and quantified with biostatistical methods by analysis of the frequency of the amplitude distribution in the CLSM images. The results demonstrate that the “BioShuttle”-Technology is an appropriate tool for an effective transfer of genetic material carried by a plasmid. PMID:18026568

  16. Plasmid and chromosome partitioning: surprises from phylogeny

    DEFF Research Database (Denmark)

    Gerdes, Kenn; Møller-Jensen, Jakob; Bugge Jensen, Rasmus

    2000-01-01

    and chromosomes from prokaryotic organisms. All known plasmid-encoded par loci specify three components: a cis-acting centromere-like site and two trans-acting proteins that form a nucleoprotein complex at the centromere (i.e. the partition complex). The proteins are encoded by two genes in an operon...... that is autoregulated by the par-encoded proteins. In all cases, the upstream gene encodes an ATPase that is essential for partitioning. Recent cytological analyses indicate that the ATPases function as adaptors between a host-encoded component and the partition complex and thereby tether plasmids and chromosomal...... origin regions to specific subcellular sites (i.e. the poles or quarter-cell positions). Two types of partitioning ATPases are known: the Walker-type ATPases encoded by the par/sop gene family (type I partitioning loci) and the actin-like ATPase encoded by the par locus of plasmid R1 (type II...

  17. The GDI1 genes from Kluyveromyces lactis and Pichia pastoris: cloning and functional expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Brummer, M H; Richard, P; Sundqvist, L; Väänänen, R; Keränen, S

    2001-07-01

    The nucleotide sequences of 2.8 kb and 2.9 kb fragments containing the Kluyveromyces lactis and Pichia pastoris GDI1 genes, respectively, were determined. K. lactis GDI1 was found during sequencing of a genomic library clone, whereas the P. pastoris GDI1 was obtained from a genomic library by complementing a Saccharomyces cerevisiae sec19-1 mutant strain. The sequenced DNA fragments contain open reading frames of 1338 bp (K.lactis) and 1344 bp (P. pastoris), coding for polypeptides of 445 and 447 residues, respectively. Both sequences fully complement the S. cerevisiae sec19-1 mutation. They have high degrees of homology with known GDP dissociation inhibitors from yeast species and other eukaryotes. PMID:11447595

  18. BioShuttle-mediated Plasmid Transfer

    OpenAIRE

    Braun, Klaus; von Brasch, Leonie; Pipkorn, Ruediger; Ehemann, Volker; Jenne, Juergen; Spring, Herbert; Debus, Juergen; Didinger, Bernd; Rittgen, Werner; Waldeck, Waldemar

    2007-01-01

    An efficient gene transfer into target tissues and cells is needed for safe and effective treatment of genetic diseases like cancer. In this paper, we describe the development of a transport system and show its ability for transporting plasmids. This non-viral peptide-based BioShuttle-mediated transfer system consists of a nuclear localization address sequence realizing the delivery of the plasmid phNIS-IRES-EGFP coding for two independent reporter genes into nuclei of HeLa cells. The quantif...

  19. Plasmid maintenance functions of the large virulence plasmid of Shigella flexneri.

    OpenAIRE

    Radnedge, L; Davis, M. A.; Youngren, B; Austin, S. J.

    1997-01-01

    The large virulence plasmid pMYSH6000 of Shigella flexneri contains a replicon and a plasmid maintenance stability determinant (Stb) on adjacent SalI fragments. The presence of a RepFIIA replicon on the SalI C fragment was confirmed, and the complete sequence of the adjacent SalI O fragment was determined. It shows homology to part of the transfer (tra) operon of the F plasmid. Stb stabilizes a partition-defective P1 miniplasmid in Escherichia coli. A 1.1-kb region containing a homolog of the...

  20. Plasmid transfer and plasmid-mediated genetic exchange in Brucella abortus.

    OpenAIRE

    Rigby, C E; Fraser, A.D.

    1989-01-01

    Naturally-occurring plasmids and gene transfer mechanisms have not yet been reported in brucellae. Here we show that Brucella abortus is capable of maintaining and transferring the broad-host-range plasmids pTH10 (IncP), pSa (IncW) and R751 (IncP), and describe pTH10-mediated transfer of B. abortus chromosomal genes to Escherichia coli. All three plasmids transferred by conjugation from E. coli to B. abortus S19, and from B. abortus S19 to B. abortus 292 (biovar 4). They were stably maintaine...

  1. A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid

    Directory of Open Access Journals (Sweden)

    Sinah Namita

    2012-08-01

    Full Text Available Abstract Background The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient quantities. We have developed a set of expression vectors that allows for ligation-independent cloning and rapid functional screening for protein expression in both E. coli and S. cerevisiae. Results A set of expression vectors was made that can express the same open reading frame in E. coli (via the T7 phage promoter and in S. cerevisiae (via the CUP1 or MET25 promoter. These plasmids also contain the essential elements for replication and selection in both cell types and have several advantages: they allow for cloning of genes by homologous recombination in yeast, protein expression can be determined before plasmid isolation and sequencing, and a GST-fusion tag is added to aid in soluble expression and purification. We have also included a TEV recognition site that allows for the specific cleavage of the fusion proteins to yield native proteins. Conclusions The dual promoter vectors can be used for rapid cloning, expression, and purification of target proteins from both prokaryotic and eukaryotic systems with the ability to study post-translation modifications.

  2. Competition between Plasmid-Bearing and Plasmid-Free Organisms in a Chemostat with Pulsed Input and Washout

    Directory of Open Access Journals (Sweden)

    Sanling Yuan

    2009-01-01

    Full Text Available We consider a model of competition between plasmid-bearing and plasmid-free organisms in the chemostat with pulsed input and washout. We investigate the subsystem with nutrient and plasmid-free organism and study the stability of the boundary periodic solutions, which are the boundary periodic solutions of the system. The stability analysis of the boundary periodic solution yields the invasion threshold of the plasmid-bearing organism. By using the standard techniques of bifurcation theory, we prove that above this threshold there are periodic oscillations in substrate, plasmid-free, and plasmid-bearing organisms. Numerical simulations are carried out to illustrate our results.

  3. Library news

    CERN Multimedia

    CERN Library

    2010-01-01

    The CERN Library has been providing electronic access to the "Techniques de l'Ingénieur" database for the past 8 months. As a reminder, this is a multidisciplinary database of over 4000 technical and scientific articles in French, covering a broad range of topics such as mechanical engineering, safety, electronics and the environment. In a few simple steps, you can create your own account, select the types of documents you are interested in and configure your settings so as to receive alerts when articles in your field of activity are published. You can now access this resource from outside CERN using the "remote access to electronic resources" service. Further information is available here. Direct access to the database. Remote access to electronic resources. If you have any questions or comments, don't hesitate to contact us at: library.desk@cern.ch.

  4. Yeast surface display for screening combinatorial polypeptide libraries.

    Science.gov (United States)

    Boder, E T; Wittrup, K D

    1997-06-01

    Display on the yeast cell wall is well suited for engineering mammalian cell-surface and secreted proteins (e.g., antibodies, receptors, cytokines) that require endoplasmic reticulum-specific post-translational processing for efficient folding and activity. C-terminal fusion to the Aga2p mating adhesion receptor of Saccharomyces cerevisiae has been used for the selection of scFv antibody fragments with threefold decreased antigen dissociation rate from a randomly mutated library. A eukaryotic host should alleviate expression biases present in bacterially propagated combinatorial libraries. Quantitative flow cytometric analysis enables fine discrimination of kinetic parameters for protein binding to soluble ligands.

  5. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and veter

  6. The fate of linear DNA in Saccharomyces cerevisiae and Candida glabrata: the role of homologous and non-homologous end joining.

    Directory of Open Access Journals (Sweden)

    Mary W Corrigan

    Full Text Available In vivo assembly of plasmids has become an increasingly used process, as high throughput studies in molecular biology seek to examine gene function. In this study, we investigated the plasmid construction technique called gap repair cloning (GRC in two closely related species of yeast - Saccharomyces cerevisiae and Candida glabrata. GRC utilizes homologous recombination (HR activity to join a linear vector and a linear piece of DNA that contains base pair homology. We demonstrate that a minimum of 20 bp of homology on each side of the linear DNA is required for GRC to occur with at least 10% efficiency. Between the two species, we determine that S. cerevisiae is slightly more efficient at performing GRC. GRC is less efficient in rad52 deletion mutants, which are defective in HR in both species. In dnl4 deletion mutants, which perform less non-homologous end joining (NHEJ, the frequency of GRC increases in C. glabrata, whereas GRC frequency only minimally increases in S. cerevisiae, suggesting that NHEJ is more prevalent in C. glabrata. Our studies allow for a model of the fate of linear DNA when transformed into yeast cells. This model is not the same for both species. Most significantly, during GRC, C. glabrata performs NHEJ activity at a detectable rate (>5%, while S. cerevisiae does not. Our model suggests that S. cerevisiae is more efficient at HR because NHEJ is less prevalent than in C. glabrata. This work demonstrates the determinants for GRC and that while C. glabrata has a lower efficiency of GRC, this species still provides a viable option for GRC.

  7. Plasmid maintenance and protein overproduction in selective recycle bioreactors.

    Science.gov (United States)

    Ogden, K L; Davis, R H

    1991-02-20

    A new plasmid construct has been used in conjunction with selective recycle to successfully maintain otherwise unstable plasmid-bearing E. coli cells in a continuous bioreactor and to produce significant amounts of the plasmid-encoded protein beta-lactamase. The plasmid is constructed so that pilin expression, which leads to bacterial flocculation, is under control of the tac operon. The plasmid-bearing cells are induced to flocculate in the separator, whereas cell growth and product synthesis occur in the main fermentation vessel without the inhibiting effects of pilin production. Selective recycle allows for the maintenance of the plasmid-bearing cells by separating flocculent, plasmid-bearing cells from nonflocculent, segregant cells in an inclined settler, and recycling only the plasmid-bearing cells to the reactor. As a result, product expression levels are maintained that are more than ten times the level achieved without selective recycle. All experimental data agree well with theoretical predictions. PMID:18597374

  8. SACCHAROMYCES CEREVISIAE AND ITS VALIDATION

    Directory of Open Access Journals (Sweden)

    Miroslav Ondrejovič

    2015-02-01

    Full Text Available The aim of this study was to optimize of independent variables as temperature, time and reaction ratio to output parameter of simultaneous enzyme saccharification and fermentation by Saccharomyces cerevisiae of pretreated wheat straw as model substrate via RSM (response surface methodology approach. As dependent variable, it was chosen ethanol yields characterizing effectivity of process. The optimal conditions were approximately temperature 100 °C, time 1 hour and reaction ratio 26 mL to 1 g of treated wheat straw with ethanol yields 141.9 mg.g-1. After calculating the optimal values, the validation analyze was carried out and it was found out that the predicted and experimentally verified dependent variable was in agreement with the optimal parameters (~ 95 %. Proposed model was tested for three lignocellulosic materials (winter wheat straw, alfalfa hay and maize straw as wheat straw used as model substrate and it was confirmed the possibility of its use for other agricultural residues with similar content of lignocellulose.

  9. Construction and characterization of a normalized whole-life-cycle cDNA library of rice

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A cDNA library with genomic complete coverage is a powerful tool for functional genomic studies. For studying the functions of rice genes on a large scale, a normalized whole-life-cycle cDNA library is constructed based on the strategy of saturation hybridization with genomic DNA using rice cultivar Minghui 63, an elite restorer line for a number of rice hybrids that are widely cultivated in China. This library consists of cDNA from 15 directionally cloned cDNA libraries constructed with different tissues from 9 developmental stages. For normalization, the denatured plasmids purified from the 15 directionally cloned libraries are mixed and hybridized with saturated genomic DNA labeled with magnetic beads in two complementary systems. Well-matched plasmids are captured from the hybridized genomic DNA and electroporated into competent DH10B E. coli for construction of the normalized whole-life-cycle cDNA library. This library consists of 62000 clones with an average insert length about 1.4 kb. Inverse Northern blotting shows that this cDNA library included many rarely expressed genes and tissue-specific genes. Sequencing of 10750 cDNA clones of this library reveals 6399 unique ESTs (expressed sequence tags), indicating that the non-redundancy of the library is about 59.5%. This library has been used to make cDNA microarrays for functional genomic studies.

  10. The influence of biofilms in the biology of plasmids

    OpenAIRE

    Cook, Laura C.C.; Dunny, Gary M.

    2014-01-01

    The field of plasmid biology has historically focused on bacteria growing in liquid culture. Surface attached communities of bacterial biofilms have recently been understood to be the normal environment of bacteria in the natural world. Thus, studies examining plasmid replication, maintenance, and transfer in biofilms are essential for a true understanding of bacterial plasmid biology. This chapter reviews the current knowledge of the interplay between bacterial biofilms and plasmids, focusin...

  11. The 2 micron plasmid purloins the yeast cohesin complex

    OpenAIRE

    Mehta, Shwetal; Yang, Xian Mei; Chan, Clarence S.; Dobson, Melanie J.; Jayaram, Makkuni; Velmurugan, Soundarapandian

    2002-01-01

    The yeast 2 micron plasmid achieves high fidelity segregation by coupling its partitioning pathway to that of the chromosomes. Mutations affecting distinct steps of chromosome segregation cause the plasmid to missegregate in tandem with the chromosomes. In the absence of the plasmid stability system, consisting of the Rep1 and Rep2 proteins and the STB DNA, plasmid and chromosome segregations are uncoupled. The Rep proteins, acting in concert, recruit the yeast cohesin complex to the STB locu...

  12. Postsegregational killing does not increase plasmid stability but acts to mediate the exclusion of competing plasmids

    OpenAIRE

    Cooper, Tim F; Heinemann, Jack A.

    2000-01-01

    Postsegregational killing (PSK) systems consist of a tightly linked toxin–antitoxin pair. Antitoxin must be continually produced to prevent the longer lived toxin from killing the cell. PSK systems on plasmids are widely believed to benefit the plasmid by ensuring its stable vertical inheritance. However, experimental tests of this “stability” hypothesis were not consistent with its predictions. We suggest an alternative hypothesis to explain the evolution of PSK: ...

  13. Characterization of Saccharomyces cerevisiae promoters for heterologous gene expression in Kluyveromyces marxianus.

    Science.gov (United States)

    Lee, Ki-Sung; Kim, Jun-Seob; Heo, Paul; Yang, Tae-Jun; Sung, Young-Je; Cheon, Yuna; Koo, Hyun Min; Yu, Byung Jo; Seo, Jin-Ho; Jin, Yong-Su; Park, Jae Chan; Kweon, Dae-Hyuk

    2013-03-01

    Kluyveromyces marxianus is now considered one of the best choices of option for industrial applications of yeast because the strain is able to grow at high temperature, utilizes various carbon sources, and grows fast. However, the use of K. marxianus as a host for industrial applications is still limited. This limitation is largely due to a lack of knowledge on the characteristics of the promoters since the time and amount of protein expression is strongly dependent on the promoter employed. In this study, four well-known constitutive promoters (P(CYC), P(TEF), P(GPD), and P(ADH)) of Saccharomyces cerevisiae were characterized in K. marxianus in terms of protein expression level and their stochastic behavior. After constructing five URA3-auxotrophic K. marxianus strains and a plasmid vector, four cassettes each comprising one of the promoters--the gene for the green fluorescence protein (GFP)--CYC1 terminator (T(CYC)) were inserted into the vector. GFP expression under the control of each one of the promoters was analyzed by reverse transcription PCR, fluorescence microscopy, and flow cytometer. Using these combined methods, the promoter strength was determined to be in the order of P(GPD) > P(ADH) ∼ P(TEF) > P(CYC). All promoters except for the P(CYC) exhibited three distinctive populations, including non-expressing cells, weakly expressing cells, and strongly expressing cells. The relative ratios between populations were strongly dependent on the promoter and culture time. Forward scattering was independent of GFP fluorescence intensity, indicating that the different fluorescence intensities were not just due to different cell sizes derived from budding. It also excluded the possibility that the non-expressing cells resulted from plasmid loss because plasmid stability was maintained at almost 100 % over the culture time. The same cassettes, cloned into a single copy plasmid pRS416 and transformed into S. cerevisiae, showed only one population. When the

  14. Compositional discordance between prokaryotic plasmids and host chromosomes

    NARCIS (Netherlands)

    M.W.J. van Passel; A. Bart; A.C.M. Luyf; A.H.C. van Kampen; A. van der Ende

    2006-01-01

    Background: Most plasmids depend on the host replication machinery and possess partitioning genes. These properties confine plasmids to a limited range of hosts, yielding a close and presumably stable relationship between plasmid and host. Hence, it is anticipated that due to amelioration the dinucl

  15. Plasmid Segregation: Spatial Awareness at the Molecular Level

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Gerdes, Kenn

    2007-01-01

    In bacteria, low-copy number plasmids ensure their stable inheritance by partition loci (par), which actively distribute plasmid replicates to each side of the cell division plane. Using time-lapse fluorescence microscopic tracking of segregating plasmid molecules, a new study provides novel...

  16. Cloning of Two Bacteriocin Genes from a Lactococcal Bacteriocin Plasmid

    NARCIS (Netherlands)

    Belkum, Marco J. van; Hayema, Bert Jan; Geis, Arnold; Kok, Jan; Venema, Gerard

    1989-01-01

    Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4

  17. Libraries for users services in academic libraries

    CERN Document Server

    Alvite, Luisa

    2010-01-01

    This book reviews the quality and evolution of academic library services. It revises service trends offered by academic libraries and the challenge of enhancing traditional ones such as: catalogues, repositories and digital collections, learning resources centres, virtual reference services, information literacy and 2.0 tools.studies the role of the university library in the new educational environment of higher educationrethinks libraries in academic contextredefines roles for academic libraries

  18. Competition between Plasmid-Bearing and Plasmid-Free Organisms in a Chemostat with Pulsed Input and Washout

    OpenAIRE

    Sanling Yuan; Yu Zhao; Anfeng Xiao

    2009-01-01

    We consider a model of competition between plasmid-bearing and plasmid-free organisms in the chemostat with pulsed input and washout. We investigate the subsystem with nutrient and plasmid-free organism and study the stability of the boundary periodic solutions, which are the boundary periodic solutions of the system. The stability analysis of the boundary periodic solution yields the invasion threshold of the plasmid-bearing organism. By using the standard techniques of bifurcation theory, w...

  19. Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae.

    Science.gov (United States)

    Samuelsson, T; Olsson, M

    1990-05-25

    A transfer RNA lacking modified nucleosides was produced by transcription in vitro of a cloned gene that encodes a Saccharomyces cerevisiae glycine tRNA. At least three different uridines (in nucleotide positions 13, 32, and 55) of this transcript tRNA are modified to pseudouridine by an extract of S. cerevisiae. Variants of the RNA substrate were also constructed that each had only one of these sites, thus allowing specific monitoring of pseudouridylation at different nucleotide positions. Using such RNAs to assay pseudouridine synthesis, enzymes producing this nucleoside were purified from an extract of S. cerevisiae. The activities corresponding to positions 13, 32, and 55 in the tRNA substrate could all be separated chromatographically, indicating that there is a separate enzyme for each of these sites. The enzyme specific for position 55 (denoted pseudouridine synthase 55) was purified approximately 4000-fold using a combination of DEAE-Sepharose, heparin-Sepharose, and hydroxylapatite.

  20. Sequence of closely related plasmids encoding bla(NDM-1 in two unrelated Klebsiella pneumoniae isolates in Singapore.

    Directory of Open Access Journals (Sweden)

    Ying-Tsong Chen

    Full Text Available BACKGROUND: Spread of the bla(NDM-1 gene that encodes the New Delhi metallo-β-lactamase (NDM-1 in Enterobacteriaceae is a major global health problem. Plasmids carrying bla(NDM-1 from two different multi-drug resistant Klebsiella pneumonia isolates collected in Singapore were completely sequenced and compared to known plasmids carrying bla(NDM-1. METHODOLOGY/PRINCIPAL FINDINGS: The two plasmids, pTR3 and pTR4, were transferred to Escherichia coli recipient strain J53 and completely sequenced by a shotgun approach using 3-kb paired-end libraries on 454. Although the K. pneumoniae strains were unrelated by molecular typing using PFGE and MLST, complete sequencing revealed that pTR3 and pTR4 are identical. The plasmid sequence is similar to the E. coli NDM-1-encoding plasmid p271A, which was isolated in Australia from a patient returning from Bangladesh. The immediate regions of the bla(NDM-1 gene in pTR3/4 are identical to that of p271A, but the backbone of our plasmid is much more similar to another IncN2 plasmid reported recently, pJIE137, which contained an additional 5.2-kb CUP (conserved upstream repeat regulon region in comparison to p271A. A 257-bp element bounded by imperfect 39-bp inverted repeats (IR and an incomplete version of this element flanking the 3.6-kb NDM-1-encoding region were identified in these plasmids and are likely to be the vestiges of an unknown IS. CONCLUSIONS: Although the hosts are not epidemiologically linked, we found that the plasmids bearing the bla(NDM-1 gene are identical. Comparative analyses of the conserved NDM-1-encoding region among different plasmids from K. pneumoniae and E. coli suggested that the transposable elements and the two unknown IR-associated elements flanking the NDM-1-encoding region might have aided the spreading of this worrisome resistance determinant.

  1. Cell Libraries

    Science.gov (United States)

    1994-01-01

    A NASA contract led to the development of faster and more energy efficient semiconductor materials for digital integrated circuits. Gallium arsenide (GaAs) conducts electrons 4-6 times faster than silicon and uses less power at frequencies above 100-150 megahertz. However, the material is expensive, brittle, fragile and has lacked computer automated engineering tools to solve this problem. Systems & Processes Engineering Corporation (SPEC) developed a series of GaAs cell libraries for cell layout, design rule checking, logic synthesis, placement and routing, simulation and chip assembly. The system is marketed by Compare Design Automation.

  2. Heterologous production of non-ribosomal peptide LLD-ACV in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Siewers, Verena; Chen, Xiao; Huang, Le;

    2009-01-01

    -(l-α-aminoadipyl)–l-cysteinyl–d-valine (ACV) as a model NRP. The Penicillium chrysogenum gene pcbAB encoding ACV synthetase was expressed in S. cerevisiae from a high-copy plasmid together with phosphopantetheinyl transferase (PPTase) encoding genes from Aspergillus nidulans, P. chrysogenum and Bacillus subtilis, and in all the three cases...... production of ACV was observed. To improve ACV synthesis, several factors were investigated. Codon optimization of the 5′ end of pcbAB did not significantly increase ACV production. However, a 30-fold enhancement was achieved by lowering the cultivation temperature from 30 to 20 °C. When ACVS and PPTase...... encoding genes were integrated into the yeast genome, a 6-fold decrease in ACV production was observed indicating that gene copy number was one of the rate-limiting factors for ACV production in yeast....

  3. THE INTEGRATED STATE OF THE ROLLING-CIRCLE PLASMID PTB913 IN THE COMPOSITE BACILLUS PLASMID PTB19

    NARCIS (Netherlands)

    OSKAM, L; HILLENGA, DJ; VENEMA, G; BRON, S

    1992-01-01

    pTB19, a 27 kb plasmid originating from a thermophilic Bacillus species, contains integrated copies of two rolling-circle type plasmids on a 10.6 kb DNA fragment. In the present study we analysed the part of pTB19 that contains the rolling-circle plasmid pTB913 and the region in between the two roll

  4. Characteristics of sterol uptake in Saccharomyces cerevisiae.

    OpenAIRE

    Lorenz, R T; Rodriguez, R J; Lewis, T A; Parks, L W

    1986-01-01

    A Saccharomyces cerevisiae sterol auxotroph, FY3 (alpha hem1 erg7 ura), was used to probe the characteristics of sterol uptake in S. cerevisiae. The steady-state cellular concentration of free sterol at the late exponential phase of growth could be adjusted within a 10-fold range by varying the concentration of exogenously supplied sterol. When cultured on 1 microgram of sterol ml-1, the cells contained a minimal cellular free-cholesterol concentration of 0.85 nmol/mg (dry weight) and were te...

  5. High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

    Directory of Open Access Journals (Sweden)

    Qureshi Nasib

    2006-05-01

    Full Text Available Abstract Background The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. Results We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved Cel

  6. Stable inheritance of plasmid R1 requires two different loci.

    OpenAIRE

    Gerdes, K; Larsen, J E; Molin, S

    1985-01-01

    The largest EcoRI fragment from plasmid R1 mediates a stability phenotype which is required to ensure the stable inheritance of this low-copy-number plasmid. When covalently linked to small, unstable R1 derivatives, this fragment makes the plasmids as stable as the wild-type R1 plasmid. A genetic analysis showed that two independently acting stabilization functions are encoded by this EcoRI fragment, both of which have the potential of partial stabilization of mini-R1 plasmids. The two loci a...

  7. DNA restriction-modification systems mediate plasmid maintenance.

    OpenAIRE

    Kulakauskas, S; Lubys, A; Ehrlich, S. D.

    1995-01-01

    Two plasmid-carried restriction-modification (R-M) systems, EcoRI (from pMB1 of Escherichia coli) and Bsp6I (from pXH13 of Bacillus sp. strain RFL6), enhance plasmid segregational stability in E. coli and Bacillus subtilis, respectively. Inactivation of the endonuclease or the presence of the methylase in trans abolish the stabilizing activity of the R-M systems. We propose that R-M systems mediate plasmid segregational stability by postsegregational killing of plasmid-free cells. Plasmid-enc...

  8. Library Services. Miscellaneous Papers.

    Science.gov (United States)

    International Federation of Library Associations, The Hague (Netherlands).

    Papers on library journal cooperation, interlibrary lending, library services to minorities, and school library media centers, which were presented at the 1983 International Federation of Library Associations (IFLA) conference, include: (1) "The Co-operation between Editors of Library Journals in Socialist Countries," in which Wolfgang Korluss…

  9. Library Research and Statistics.

    Science.gov (United States)

    Lynch, Mary Jo; Brier, David J.; Lebbin, Vickery K.; Halstead, Kent; Fox, Bette-Lee; Kremen, Maya L.; Miller, Marilyn L.; Shontz, Marilyn L.

    1998-01-01

    Provides nine articles: research on libraries and librarianship, 1997; changing faces of library education (ALA-accredited graduate program title changes); number of libraries in the U.S., Canada, and Mexico; highlights of NCES surveys; library acquisition expenditures; price indexes for public and academic libraries; state rankings of selected…

  10. Marketing the Virtual Library

    Science.gov (United States)

    Fagan, Jody Condit

    2009-01-01

    Far more people are familiar with their local public or college library facility than their library's website and online resources. In fact, according to a recent survey, 96% of Americans said they had visited a library in person, but less than one-third have visited their online library. Since everyone agrees that online library resources are…

  11. Role of Plasmid in Production of Acetobacter Xylinum Biofilms

    Directory of Open Access Journals (Sweden)

    Abbas Rezaee

    2005-01-01

    Full Text Available Acetobacter xylinum has the ability to produce cellulotic biofilms. Bacterial cellulose is expected to be used in many industrial or biomedical materials for its unique characteristics. A. xylinum contains a complex system of plasmid DNA molecules. A 44 kilobases (kb plasmid was isolated in wild type of A. xylinum. To improve the cellulose producing ability of A. xylinum, role of the plasmid in production of cellulose was studied. The comparisons between wild type and cured cells of A. xylinum showed that there is considerably difference in cellulose production. In order to study the relationship between plasmid and the rate of cellulose production, bacteria were screened for plasmid profile by a modified method for preparation of plasmid. This method yields high levels of pure plasmid DNA that can be used for common molecular techniques, such as digestion and transformation, with high efficiency.

  12. Modeling sRNA-regulated Plasmid Maintenance

    CERN Document Server

    Gong, Chen Chris

    2016-01-01

    We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin's mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, ...

  13. Cell Surface Display of Four Types of Solanum nigrum Metallothionein on Saccharomyces cerevisiae for Biosorption of Cadmium.

    Science.gov (United States)

    Wei, Qinguo; Zhang, Honghai; Guo, Dongge; Ma, Shisheng

    2016-05-28

    We displayed four types of Solanum nigrum metallothionein (SMT) for the first time on the surface of Saccharomyces cerevisiae using an α-agglutinin-based display system. The SMT genes were amplified by RT-PCR. The plasmid pYES2 was used to construct the expression vector. Transformed yeast strains were confirmed by PCR amplification and custom sequencing. Surface-expressed metallothioneins were indirectly indicated by the enhanced cadmium sorption capacity. Flame atomic absorption spectrophotometry was used to examine the concentration of Cd(2+) in this study. The transformed yeast strains showed much higher resistance ability to Cd(2+) compared with the control. Strikingly, their Cd(2+) accumulation was almost twice as much as that of the wild-type yeast cells. Furthermore, surface-engineered yeast strains could effectively adsorb ultra-trace cadmium and accumulate Cd(2+) under a wide range of pH levels, from 3 to 7, without disturbing the Cu(2+) and Hg(2+). Four types of surfaceengineered Saccharomyces cerevisiae strains were constructed and they could be used to purify Cd(2+)-contaminated water and adsorb ultra-trace cadmium effectively. The surface-engineered Saccharomyces cerevisiae strains would be useful tools for the bioremediation and biosorption of environmental cadmium contaminants. PMID:26838339

  14. Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Julie Bomholt

    Full Text Available In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tagged with yeast enhanced GFP for quantification of functional expression, determination of sub-cellular localization, estimation of in vivo folding efficiency and establishment of a purification protocol. Aquaporin-1 was found to constitute 8.5 percent of total membrane protein content after expression at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30°C was due to in vivo mal-folding. Reduction of the expression temperature to 15°C almost completely prevented Aquaporin-1 mal-folding. Bioimaging of live yeast cells revealed that recombinant Aquaporin-1 accumulated in the yeast plasma membrane. A detergent screen for solubilization revealed that CYMAL-5 was superior in solubilizing recombinant Aquaporin-1 and generated a monodisperse protein preparation. A single Ni-affinity chromatography step was used to obtain almost pure Aquaporin-1. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes.

  15. Primers-4-Yeast: a comprehensive web tool for planning primers for Saccharomyces cerevisiae.

    Science.gov (United States)

    Yofe, Ido; Schuldiner, Maya

    2014-02-01

    The budding yeast Saccharomyces cerevisiae is a key model organism of functional genomics, due to its ease and speed of genetic manipulations. In fact, in this yeast, the requirement for homologous sequences for recombination purposes is so small that 40 base pairs (bp) are sufficient. Hence, an enormous variety of genetic manipulations can be performed by simply planning primers with the correct homology, using a defined set of transformation plasmids. Although designing primers for yeast transformations and for the verification of their correct insertion is a common task in all yeast laboratories, primer planning is usually done manually and a tool that would enable easy, automated primer planning for the yeast research community is still lacking. Here we introduce Primers-4-Yeast, a web tool that allows primers to be designed in batches for S. cerevisiae gene-targeting transformations, and for the validation of correct insertions. This novel tool enables fast, automated, accurate primer planning for large sets of genes, introduces consistency in primer planning and is therefore suggested to serve as a standard in yeast research. Primers-4-Yeast is available at: http://www.weizmann.ac.il/Primers-4-Yeast

  16. Engineering the oxygen sensing regulation results in an enhanced recombinant human hemoglobin production by Saccharomyces cerevisiae.

    Science.gov (United States)

    Martínez, José L; Liu, Lifang; Petranovic, Dina; Nielsen, Jens

    2015-01-01

    Efficient production of appropriate oxygen carriers for transfusions (blood substitutes or artificial blood) has been pursued for many decades, and to date several strategies have been used, from synthetic polymers to cell-free hemoglobin carriers. The recent advances in the field of metabolic engineering also allowed the generation of different genetically modified organisms for the production of recombinant human hemoglobin. Several studies have showed very promising results using the bacterium Escherichia coli as a production platform, reporting hemoglobin titers above 5% of the total cell protein content. However, there are still certain limitations regarding the protein stability and functionality of the recombinant hemoglobin produced in bacterial systems. In order to overcome these limitations, yeast systems have been proposed as the eukaryal alternative. We recently reported the generation of a set of plasmids to produce functional human hemoglobin in Saccharomyces cerevisiae, with final titers of active hemoglobin exceeding 4% of the total cell protein. In this study, we propose a strategy for further engineering S. cerevisiae by altering the oxygen sensing pathway by deleting the transcription factor HAP1, which resulted in an increase of the final recombinant active hemoglobin titer exceeding 7% of the total cellular protein.

  17. Heterologous production of non-ribosomal peptide LLD-ACV in Saccharomyces cerevisiae.

    Science.gov (United States)

    Siewers, Verena; Chen, Xiao; Huang, Le; Zhang, Jie; Nielsen, Jens

    2009-11-01

    Non-ribosomal peptides (NRPs) are a diverse family of secondary metabolites with a broad range of biological activities. We started to develop an eukaryotic microbial platform based on the yeast Saccharomyces cerevisiae for heterologous production of NRPs using delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine (ACV) as a model NRP. The Penicillium chrysogenum gene pcbAB encoding ACV synthetase was expressed in S. cerevisiae from a high-copy plasmid together with phosphopantetheinyl transferase (PPTase) encoding genes from Aspergillus nidulans, P. chrysogenum and Bacillus subtilis, and in all the three cases production of ACV was observed. To improve ACV synthesis, several factors were investigated. Codon optimization of the 5' end of pcbAB did not significantly increase ACV production. However, a 30-fold enhancement was achieved by lowering the cultivation temperature from 30 to 20 degrees C. When ACVS and PPTase encoding genes were integrated into the yeast genome, a 6-fold decrease in ACV production was observed indicating that gene copy number was one of the rate-limiting factors for ACV production in yeast.

  18. One-step generation of error-prone PCR libraries using Gateway® technology

    Directory of Open Access Journals (Sweden)

    Gruet Antoine

    2012-01-01

    Full Text Available Abstract Background Error-prone PCR (epPCR libraries are one of the tools used in directed evolution. The Gateway® technology allows constructing epPCR libraries virtually devoid of any background (i.e., of insert-free plasmid, but requires two steps: the BP and the LR reactions and the associated E. coli cell transformations and plasmid purifications. Results We describe a method for making epPCR libraries in Gateway® plasmids using an LR reaction without intermediate BP reaction. We also describe a BP-free and LR-free sub-cloning method for in-frame transferring the coding sequence of selected clones from the plasmid used to screen the library to another one devoid of tag used for screening (such as the green fluorescent protein. We report preliminary results of a directed evolution program using this method. Conclusions The one-step method enables producing epPCR libraries of as high complexity and quality as does the regular, two-step, protocol for half the amount of work. In addition, it contributes to preserve the original complexity of the epPCR product.

  19. A tetO Toolkit To Alter Expression of Genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Cuperus, Josh T; Lo, Russell S; Shumaker, Lucia; Proctor, Julia; Fields, Stanley

    2015-07-17

    Strategies to optimize a metabolic pathway often involve building a large collection of strains, each containing different versions of sequences that regulate the expression of pathway genes. Here, we develop reagents and methods to carry out this process at high efficiency in the yeast Saccharomyces cerevisiae. We identify variants of the Escherichia coli tet operator (tetO) sequence that bind a TetR-VP16 activator with differential affinity and therefore result in different TetR-VP16 activator-driven expression. By recombining these variants upstream of the genes of a pathway, we generate unique combinations of expression levels. Here, we built a tetO toolkit, which includes the I-OnuI homing endonuclease to create double-strand breaks, which increases homologous recombination by 10(5); a plasmid carrying six variant tetO sequences flanked by I-OnuI sites, uncoupling transformation and recombination steps; an S. cerevisiae-optimized TetR-VP16 activator; and a vector to integrate constructs into the yeast genome. We introduce into the S. cerevisiae genome the three crt genes from Erwinia herbicola required for yeast to synthesize lycopene and carry out the recombination process to produce a population of cells with permutations of tetO variants regulating the three genes. We identify 0.7% of this population as making detectable lycopene, of which the vast majority have undergone recombination at all three crt genes. We estimate a rate of ∼20% recombination per targeted site, much higher than that obtained in other studies. Application of this toolkit to medically or industrially important end products could reduce the time and labor required to optimize the expression of a set of metabolic genes. PMID:25742460

  20. Agricultural Libraries and Information.

    Science.gov (United States)

    Russell, Keith W., Ed.; Pisa, Maria G., Ed.

    1990-01-01

    Eleven articles address issues relating to agricultural libraries and information, including background on agricultural libraries and information, trend management, document delivery, reference services, user needs and library services, collection development, technologies for international information management, information sources,…

  1. Italian library associations

    Directory of Open Access Journals (Sweden)

    Ksenija Petaros-Kmetec

    2004-01-01

    Full Text Available In Italy, five library associations of national significance function at present. There are special associations of ecclesiastic libraries, prison libraries, architecture libraries and libraries with artistic material. The role of the general national association, covering all types of libraries including documentation centres, is played by the Italian Library Association. It strive for the development of a contemporary Italian library system comparable to international standards, monitors library legislation, promotes education for librarians and keeps the librarians and the broader public informed about the importance of libraries and librarianship for society. The activity and efforts of the association are reflected through their website offering much information and links to similar sites. ILA presents and realises its activities for both, the librarians and the public users. A great deal of actions promoting libraries and the Library Association might be interesting for Slovenia and perhaps transferred to our environment.

  2. Tangential Ultrafiltration of Aqueous "Saccharomyces Cerevisiae" Suspensions

    Science.gov (United States)

    Silva, Carlos M.; Neves, Patricia S.; Da Silva, Francisco A.; Xavier, Ana M. R. B.; Eusebio, M. F. J.

    2008-01-01

    Experimental work on ultrafiltration is presented to illustrate the practical and theoretical principles of this separation technique. The laboratory exercise comprises experiments with pure water and with aqueous "Saccharomyces cerevisiae" (from commercial Baker's yeast) suspensions. With this work students detect the characteristic phenomena…

  3. Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering.

    Science.gov (United States)

    Chen, Yingying; Stabryla, Lisa; Wei, Na

    2016-01-29

    Development of acetic acid-resistant Saccharomyces cerevisiae is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target, WHI2 (encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance in S. cerevisiae. Overexpression of WHI2 significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. The WHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression of WHI2 gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 in S. cerevisiae. Meanwhile, the whi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response in S. cerevisiae. Additionally, overexpression of WHI2 and of a cognate phosphatase gene, PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production.

  4. Determination of Plasmid Segregational Stability in a Growing Bacterial Population.

    Science.gov (United States)

    Kramer, M Gabriela

    2016-01-01

    Bacterial plasmids are extensively used as cloning vectors for a number of genes for academic and commercial purposes. Moreover, attenuated bacteria carrying recombinant plasmids expressing genes with anti-tumor activity have shown promising therapeutic results in animal models of cancer. Equitable plasmid distribution between daughter cells during cell division, i.e., plasmid segregational stability, depends on many factors, including the plasmid copy number, its replication mechanism, the levels of recombinant gene expression, the type of bacterial host, and the metabolic burden associated with all these factors. Plasmid vectors usually code for antibiotic-resistant functions, and, in order to enrich the culture with bacteria containing plasmids, antibiotic selective pressure is commonly used to eliminate plasmid-free segregants from the growing population. However, administration of antibiotics can be inconvenient for many industrial and therapeutic applications. Extensive ongoing research is being carried out to develop stably-inherited plasmid vectors. Here, I present an easy and precise method for determining the kinetics of plasmid loss or maintenance for every ten generations of bacterial growth in culture. PMID:26846807

  5. Comparative analysis of plasmids in the genus Listeria.

    Directory of Open Access Journals (Sweden)

    Carsten Kuenne

    Full Text Available BACKGROUND: We sequenced four plasmids of the genus Listeria, including two novel plasmids from L. monocytogenes serotype 1/2c and 7 strains as well as one from the species L. grayi. A comparative analysis in conjunction with 10 published Listeria plasmids revealed a common evolutionary background. PRINCIPAL FINDINGS: All analysed plasmids share a common replicon-type related to theta-replicating plasmid pAMbeta1. Nonetheless plasmids could be broadly divided into two distinct groups based on replicon diversity and the genetic content of the respective plasmid groups. Listeria plasmids are characterized by the presence of a large number of diverse mobile genetic elements and a commonly occurring translesion DNA polymerase both of which have probably contributed to the evolution of these plasmids. We detected small non-coding RNAs on some plasmids that were homologous to those present on the chromosome of L. monocytogenes EGD-e. Multiple genes involved in heavy metal resistance (cadmium, copper, arsenite as well as multidrug efflux (MDR, SMR, MATE were detected on all listerial plasmids. These factors promote bacterial growth and survival in the environment and may have been acquired as a result of selective pressure due to the use of disinfectants in food processing environments. MDR efflux pumps have also recently been shown to promote transport of cyclic diadenosine monophosphate (c-di-AMP as a secreted molecule able to trigger a cytosolic host immune response following infection. CONCLUSIONS: The comparative analysis of 14 plasmids of genus Listeria implied the existence of a common ancestor. Ubiquitously-occurring MDR genes on plasmids and their role in listerial infection now deserve further attention.

  6. Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells.

    OpenAIRE

    Gerdes, K; Rasmussen, P. B.; Molin, S

    1986-01-01

    The stability locus parB+ of plasmid R1 has been found to specify a unique type of plasmid maintenance function. Two genes, hok (host killing) and sok (suppressor of killing), are required for the stabilizing activity. The hok gene encodes a highly toxic gene product, whose overexpression causes a rapid killing and a concomitant dramatic change in morphology of the host cell. The other gene, sok, was found to encode a product that counteracts the hok gene-mediated killing. The parB+ region wa...

  7. A comprehensive library of fluorescent transcriptional reporters for Escherichia coli.

    Science.gov (United States)

    Zaslaver, Alon; Bren, Anat; Ronen, Michal; Itzkovitz, Shalev; Kikoin, Ilya; Shavit, Seagull; Liebermeister, Wolfram; Surette, Michael G; Alon, Uri

    2006-08-01

    E. coli is widely used for systems biology research; there exists a need, however, for tools that can be used to accurately and comprehensively measure expression dynamics in individual living cells. To address this we present a library of transcriptional fusions of gfp to each of about 2,000 different promoters in E. coli K12, covering the great majority of the promoters in the organism. Each promoter fusion is expressed from a low-copy plasmid. We demonstrate that this library can be used to obtain highly accurate dynamic measurements of promoter activity on a genomic scale, in a glucose-lactose diauxic shift experiment. The library allowed detection of about 80 previously uncharacterized transcription units in E. coli, including putative internal promoters within previously known operons, such as the lac operon. This library can serve as a tool for accurate, high-resolution analysis of transcription networks in living E. coli cells.

  8. Molecular characterization of "plasmid-free" antibiotic-resistant Haemophilus influenzae.

    OpenAIRE

    Roberts, M C; Smith, A. L.

    1980-01-01

    We examined 14 multiresistant and 8 ampicillin- or tetracycline-resistant Haemophilus influenzae isolates and 4 ampicillin-resistant H. parainfluenzae isolates for plasmid deoxyribonucleic acid. Sixteen strains carried plasmids. Both "plasmid-free" and plasmid-carrying isolates transferred the antibiotic resistance by conjugation. All transconjugants carried plasmid deoxyribonucleic acid, suggesting that the apparent plasmid-free strains contained R plasmids encoding for antibiotic resistance.

  9. Permissiveness of soil microbial communities towards broad host range plasmids

    DEFF Research Database (Denmark)

    Klümper, Uli

    for plasmids carrying antibiotic resistance genes is increasingly suspected to majorly contribute to the emergence of multi-resistant pathogens. More specifically, I examined what fraction of a soil microbial community is permissive to plasmids, identified the phylogenetic identity of this fraction and studied......Horizontal transfer of mobile genetic elements facilitates adaptive and evolutionary processes in bacteria. Among the known mobile genetic elements, plasmids can confer their hosts with accessory adaptive traits, such as antibiotic or heavy metal resistances, or additional metabolic pathways....... Plasmids are implicated in the rapid spread of antibiotic resistance and the emergence of multi-resistant pathogenic bacteria, making it crucial to be able to quantify, understand, and, ideally, control plasmid transfer in mixed microbial communities. The fate of plasmids in microbial communities...

  10. Transformation of Haemophilus influenzae by plasmid RSF0885

    Energy Technology Data Exchange (ETDEWEB)

    Notani, N.K.; Setlow, J.K.; McCarthy, D.; Clayton, N.L.

    1981-12-01

    Plasmid RSF0885, which conferred ampicillin resistance, transformed competent Haemophilus influenzae cells with low efficiency (maximun, less than 0.01%). As judged by competition experiments and uptake of radioactivity, plasmid RSF0885 deoxyribonucleic acid was taken up into competent H. influenzae cells several orders of magnitude less efficiently than H. influenzae chromosomal deoxyribonucleic acid. Plasmid RSF0885 transformed cells with even lower efficiency than could be accounted for by the low uptake. Transformation was not affected by rec-1 and rec-2 mutations in the recipient, and strains cured of the plasmid did not show increased transformation. Plasmid molecules cut once with a restriction enzyme that made blunt ends did not transform. Transformation was favored by the closed circular form of the plasmid.

  11. Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

    OpenAIRE

    Pueschel, Laura; Li, Hongshan; Hymes, Matthew

    2011-01-01

    We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine ...

  12. Processes for the production of pharmaceutical grade plasmid DNA

    OpenAIRE

    Voß, Carsten

    2008-01-01

    Plasmid DNA is currently used in gene therapy and genetic vaccination as a vector system for the delivery of therapeutic genes. Clinical trials as well as future therapeutics demand large amounts of high quality plasmid DNA that fulfils the specifications set by regulatory authorities. This thesis describes the development, analysis, and evaluation of pharmaceutical plasmid DNA production processes comprising cultivation, product isolation, and purification as well as stability assessment dur...

  13. Identification of plasmid partition function in coryneform bacteria.

    OpenAIRE

    Kurusu, Y; Satoh, Y.; Inui, M.; Kohama, K; Kobayashi, M.; Terasawa, M.; Yukawa, H

    1991-01-01

    We have identified and characterized a partition function that is required for stable maintenance of plasmids in the coryneform bacteria Brevibacterium flavum MJ233 and Corynebacterium glutamicum ATCC 31831. This function is localized to a HindIII-NspV fragment (673 bp) adjacent to the replication region of the plasmid, named pBY503, from Brevibacterium stationis IFO 12144. The function was independent of copy number control and was not associated directly with plasmid replication functions. ...

  14. Pathogenomics of the Virulence Plasmids of Escherichia coli

    OpenAIRE

    Johnson, Timothy J.; Lisa K. Nolan

    2009-01-01

    Summary: Bacterial plasmids are self-replicating, extrachromosomal elements that are key agents of change in microbial populations. They promote the dissemination of a variety of traits, including virulence, enhanced fitness, resistance to antimicrobial agents, and metabolism of rare substances. Escherichia coli, perhaps the most studied of microorganisms, has been found to possess a variety of plasmid types. Included among these are plasmids associated with virulence. Several types of E. col...

  15. Safety and efficacy of DNA vaccines: Plasmids vs. minicircles

    OpenAIRE

    Stenler, Sofia; Blomberg, Pontus; Smith, Ci Edvard

    2014-01-01

    While DNA vaccination using plasmid vectors is highly attractive, there is a need for further vector optimization regarding safety, stability, and efficiency. In this commentary, we review the minicircle vector (MC), which is an entity devoid of plasmid bacterial sequences, as an alternative to the traditional plasmid construct. The commentary highlights the recent discovery by Stenler et al. (2014) that the small size of an MC enables improved resistance to the shearing forces associated wit...

  16. Comparative genetic organization of incompatibility group P degradative plasmids.

    OpenAIRE

    Burlage, R S; Bemis, L A; Layton, A C; Sayler, G. S.; Larimer, F

    1990-01-01

    Plasmids that encode genes for the degradation of recalcitrant compounds are often examined only for characteristics of the degradative pathways and ignore regions that are necessary for plasmid replication, incompatibility, and conjugation. If these characteristics were known, then the mobility of the catabolic genes between species could be predicted and different catabolic pathways might be combined to alter substrate range. Two catabolic plasmids, pSS50 and pSS60, isolated from chlorobiph...

  17. Effects of rpt1, rpt4 and rpt6 td mutants on GAL1/10 gene expression in Saccharomyces cerevisiae

    OpenAIRE

    Román, Lorena Casado 1991

    2012-01-01

    The aim of this project was to study the roles of Rpt1, Rpt4 and Rpt6 in transcriptional regulation of the GAL network. Therefore, three temperature sentitive degron (td) mutants were created by integrating recombinant plasmids into Saccharomyces cerevisiae chromosomes. Under galactose induction, degradation of Rpt4 caused a decrease in GAL1 and GAL10 mRNA levels, Rpt1 degradation did not cause any detectable effect and Rpt6 degradation caused an increase in GAL1 and GAL10 mRNA transcribed...

  18. The Library Building Tomorrow.

    Science.gov (United States)

    Waters, Richard L.

    1987-01-01

    Examination of the library of tomorrow speculates about the impact of changes in the functions of government, technology, demographics, lifestyle, and values on the role of the library. A facility for the contemporary public library is described that can both accommodate traditional services and respond to changes in the library's role. (17…

  19. Growing Competition for Libraries.

    Science.gov (United States)

    Gibbons, Susan

    2001-01-01

    Describes the Questia subscription-based online academic digital books library. Highlights include weaknesses of the collection; what college students want from a library; importance of marketing; competition for traditional academic libraries that may help improve library services; and the ability of Questia to overcome barriers and…

  20. The library marketing toolkit

    CERN Document Server

    Potter, Ned

    2012-01-01

    A guide that offers coverage of various elements of library marketing and branding for different sectors including archives and academic, public and special libraries. It is suitable for those who are involved in promoting their library or information service, whether at an academic, public or special library or in archives or records management.

  1. Economics of Academic Libraries.

    Science.gov (United States)

    Baumol, William J.; Marcus, Matityahu

    An analysis is conducted of economic issues pertinent to library planning in higher education in the face of rising costs and diminishing financial support. The individual chapters deal with: 1) growth rates in large university libraries; 2) library costs in colleges and universities; 3) cost trends and long-range plans; 4) library data; and 5) a…

  2. The New Library Professional

    Science.gov (United States)

    Wilder, Stanley

    2007-01-01

    This article discusses what the growing generation gap among library employees mean for academic research libraries and for the profession. Viewed collectively, the members of the under-35 cohort are a harbinger of a new kind of academic library professional, one whose traits bear directly on the ability of libraries to thrive amid the continuing…

  3. Teleporting the library?

    DEFF Research Database (Denmark)

    Heilesen, Simon

    2009-01-01

    In 2007, six Danish public libraries established a virtual library, Info Island DK, in Second Life. This article discusses the library project in terms of design. The design processes include the planning and implementation of the virtual library structure and its equipment, as well as the organi...

  4. Prison Libraries Inside Out.

    Science.gov (United States)

    Singer, Glen

    2000-01-01

    Discussion of prison libraries provides an inside look at the correctional institution environment, prison security concerns, inmate patrons and library use, library collections and services, librarians and staff, the day-to-day operation of a prison library, and future possibilities and needs. (Author/LRW)

  5. Inside Prison Libraries.

    Science.gov (United States)

    Vogel, Brenda; And Others

    1989-01-01

    Issues related to prison libraries are discussed in six articles. Topics covered include the history of American penitentiary ideology; standards for prison libraries; the controversy as to whether prison libraries should serve prisoners or be used as penological tools; and the lack of knowledge about prison libraries within the general library…

  6. Extended Library Hours

    OpenAIRE

    Devarai, Rajashekhar S.; Devarai, Kanyakumari S.

    1997-01-01

    Extension of library hours is useful both for public & LIS professional. Five laws, need for extended hours, problems of extended library hours, implication of extended library hours. ROCLOLIB are the related topics covered in the paper to highlight the importance of extended library hours.

  7. Degradative Plasmid and Heavy Metal Resistance Plasmid Naturally Coexist in Phenol and Cyanide Assimilating Bacteria

    OpenAIRE

    Bahig E.  Deeb; Abdullah D. Altalhi

    2009-01-01

    Problem statement: Heavy metals are known to be powerful inhibitors of xenobiotics biodegradation activities. Alleviation the inhibitory effect of these metals on the phenol biodegradation activities in presence of heavy metals resistant plasmid was investigated. Approach: Combination of genetic systems of degradation of xenobiotic compound and heavy metal resistance was one of the approaches to the creation of polyfunctional strains for bioremediation of s...

  8. Photonic plasmid stability of transformed Salmonella typhimurium: A comparison of three unique plasmids

    Science.gov (United States)

    Acquiring a highly stable photonic plasmid in transformed Salmonella typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella typhimurium (S. typh-lux) u...

  9. Photonic plasmid stability of transformed Salmonella Typhimurium: A comparison of three unique plasmids

    Directory of Open Access Journals (Sweden)

    Lay Donald

    2009-07-01

    Full Text Available Abstract Background Acquiring a highly stable photonic plasmid in transformed Salmonella Typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella Typhimurium (S. typh-lux using three different plasmids and characterize their respective photonic properties. Results In presence of ampicillin (AMP, S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 plasmids exhibited 100% photon-emitting colonies over a 10-d study period. Photon emitters of S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 without AMP selection decreased over time (P 7 to 1 × 109 CFU, P 0.05; although photonic emissions across a range of bacterial concentrations were not different (1 × 104 to 1 × 106 CFU, P > 0.05. For very low density bacterial concentrations imaged in 96 well plates photonic emissions were positively correlated with bacterial concentration (P 3 to 1 × 105 CFU low to high were different in the 96-well plate format (P Conclusion These data characterize photon stability properties for S. typh-lux transformed with three different photon generating plasmids that may facilitate real-time Salmonella tracking using in vivo or in situ biophotonic paradigms.

  10. Photonic Plasmid Stability of Transformed Salmonella Typhimurium: A Comparison of Three Unique Plasmids

    Science.gov (United States)

    Background: Acquiring a highly stable photonic plasmid in transformed Salmonella Typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella Typhimurium (S....

  11. Bacteriophages Limit the Existence Conditions for Conjugative Plasmids

    Science.gov (United States)

    Wood, A. Jamie; Dytham, Calvin; Pitchford, Jonathan W.; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A.

    2015-01-01

    ABSTRACT Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. PMID:26037122

  12. Plasmid genes required for microcin B17 production.

    Science.gov (United States)

    San Millán, J L; Kolter, R; Moreno, F

    1985-09-01

    The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production.

  13. Identification of plasmid partition function in coryneform bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Kurusu, Yasurou; Satoh, Yukie; Inui, Masayuki; Kohama, Keiko; Kobayashi, Miki; Terasawa, Masato; Yukawa, Hideaki (Mitsubishi Petrochemical Co., Ltd., Ibaraki (Japan))

    1991-03-01

    The authors have identified and characterized a partition function that is required for stable maintenance of plasmids in the coryneform bacteria Brevibacterium flavum MJ233 and Corynebacterium glutamicum ATCC 31831. This function is localized to a HindIII-NspV fragment (673 bp) adjacent to the replication region of the plasmid, named pBY503, from Brevibacterium stationis IFO 12144. The function was independent of copy number control and was not associated directly with plasmid replication functions. This fragment was able to stabilize the unstable plasmids in cis but not in trans.

  14. Plasmids foster diversification and adaptation of bacterial populations in soil.

    Science.gov (United States)

    Heuer, Holger; Smalla, Kornelia

    2012-11-01

    It is increasingly being recognized that the transfer of conjugative plasmids across species boundaries plays a vital role in the adaptability of bacterial populations in soil. There are specific driving forces and constraints of plasmid transfer within bacterial communities in soils. Plasmid-mediated genetic variation allows bacteria to respond rapidly with adaptive responses to challenges such as irregular antibiotic or metal concentrations, or opportunities such as the utilization of xenobiotic compounds. Cultivation-independent detection and capture of plasmids from soil bacteria, and complete sequencing have provided new insights into the role and ecology of plasmids. Broad host range plasmids such as those belonging to IncP-1 transfer a wealth of accessory functions which are carried by similar plasmid backbones. Plasmids with a narrower host range can be more specifically adapted to particular species and often transfer genes which complement chromosomally encoded functions. Plasmids seem to be an ancient and successful strategy to ensure survival of a soil population in spatial and temporal heterogeneous conditions with various environmental stresses or opportunities that occur irregularly or as a novel challenge in soil.

  15. Characterization of a plasmid from moderately halophilic eubacteria

    OpenAIRE

    Fernández Castillo, Rosario; Vargas, C.; Nieto Gutiérrez, Joaquín José; Ventosa Ucero, Antonio; Ruiz Berraquero, Francisco

    1992-01-01

    A plasmid has been isolated for the first time from moderately halophilic eubacteria. Halomonas elongata, Halomonrrs halmphila, Deleya halophila and Vibrio costkola were found to harbour an 11.5 kbp plasmid (pMH1). The plasmid was isolated and characterized after transformation into Escherichia coh'JM101 cells. A restriction map was constructed, and unique restriction sites for EcoRI, EcoRV and ClaI were detected. The occurrence of such a plasmid in the original halophilic strains was confirm...

  16. Degradative Plasmid and Heavy Metal Resistance Plasmid Naturally Coexist in Phenol and Cyanide Assimilating Bacteria

    Directory of Open Access Journals (Sweden)

    Bahig E.  Deeb

    2009-01-01

    Full Text Available Problem statement: Heavy metals are known to be powerful inhibitors of xenobiotics biodegradation activities. Alleviation the inhibitory effect of these metals on the phenol biodegradation activities in presence of heavy metals resistant plasmid was investigated. Approach: Combination of genetic systems of degradation of xenobiotic compound and heavy metal resistance was one of the approaches to the creation of polyfunctional strains for bioremediation of soil after co-contamination with organic pollutants and heavy metals. Results: A bacterial strain Pseudomonas putida PhCN (pPhCN1, pPhCN2 had been obtained. This bacterium contained two plasmids, a 120 Kb catabolic plasmid that encode for breakdown of phenol (pPhCN1 and pPhCN2 plasmid (100 Kb that code for cadmium and copper resistant. Cyanide assimilation by this bacterium was encoded by chromosomal genes. The inhibitory effect of cadmium (Cd2+ or copper (Cu2+ on the degradation of phenol and cyanide by P. putida strains PhCN and PhCN1 (contained pPhCN1 were investigated. The resistant strain PhCN showed high ability to degrade phenol and cyanide in presence of Cd2+ or Cu2+ comparing with the sensitive strain PhCN1. In addition, Cd2+ or Cu2+ was also found to exert a strong inhibitory effect on the C23O dioxygenase enzyme activity in the presence of cyanide as a nitrogen source. Conclusion: The presence of heavy metal resistance plasmid alleviated the inhibitory effect of metals on the phenol and cyanide assimilation by resistant strain.

  17. TOTAL ANTIOXIDANT ACTIVITY OF YEAST SACCHAROMYCES CEREVISIAE

    Directory of Open Access Journals (Sweden)

    Blažena Lavová

    2013-02-01

    Full Text Available Antioxidants are health beneficial compounds that can protect cells and macromolecules (e.g. fats, lipids, proteins and DNA from the damage of reactive oxygen species (ROS. Sacchamomyces cerevisiae are know as organisms with very important antioxidative enzyme systems such as superoxide dismutase or catalase. The total antioxidant activity (mmol Trolox equivalent – TE.g-1 d.w. of Saccharomyces cerevisiae was measured by 2,2´-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid during the yeast cultivation. It was found that the total antioxidant activity was the highest (1.08 mmol TE.g-1 d.w. in the strain Kolín after 32 hours of cultivation and the lowest (0.26 mmol TE.g-1 d.w. in the strain Gyöng after 12 hours of cultivation.

  18. Cell Wall Assembly in Saccharomyces cerevisiae

    OpenAIRE

    Lesage, Guillaume; Bussey, Howard

    2006-01-01

    An extracellular matrix composed of a layered meshwork of β-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and it...

  19. Phosphate transport and sensing in Saccharomyces cerevisiae.

    OpenAIRE

    Wykoff, D D; O'Shea, E K

    2001-01-01

    Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate; however, little is known about how phosphate concentrations are sensed. The similarity of Pho84p, a high-affinity phosphate transporter in Saccharomyces cerevisiae, to the glucose sensors Snf3p and Rgt2p has led to the hypothesis that Pho84p is an inorganic phosphate sensor. Furthermore, pho84Delta strains have defects in phosphate signaling; they constitutively express PHO5, a phosphate starvat...

  20. Viruses and prions of Saccharomyces cerevisiae

    OpenAIRE

    Wickner, Reed B.; Fujimura, Tsutomu; Esteban, Rosa

    2013-01-01

    Saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including dsRNA viruses, ssRNA viruses, and prions. Studies of the mechanisms of virus and prion replication, virus structure, and structure of the amyloid filaments that are the basis of yeast prions have been at the forefront of such studies in these classes of infectious entities. Yeast has been particularly useful in defining the interactions of the infectious elements with cellular compone...

  1. Stationary phase in the yeast Saccharomyces cerevisiae.

    OpenAIRE

    Werner-Washburne, M; Braun, E.; Johnston, G C; Singer, R A

    1993-01-01

    Growth and proliferation of microorganisms such as the yeast Saccharomyces cerevisiae are controlled in part by the availability of nutrients. When proliferating yeast cells exhaust available nutrients, they enter a stationary phase characterized by cell cycle arrest and specific physiological, biochemical, and morphological changes. These changes include thickening of the cell wall, accumulation of reserve carbohydrates, and acquisition of thermotolerance. Recent characterization of mutant c...

  2. Identification of coated vesicles in Saccharomyces cerevisiae

    OpenAIRE

    1984-01-01

    Clathrin-coated vesicles were found in yeast, Saccharomyces cerevisiae, and enriched from spheroplasts by a rapid procedure utilizing gel filtration on Sephacryl S-1000. The coated vesicles (62-nm diam) were visualized by negative stain electron microscopy and clathrin triskelions were observed by rotary shadowing. The contour length of a triskelion leg was 490 nm. Coated vesicle fractions contain a prominent band with molecular weight of approximately 185,000 when analyzed by SDS PAGE. The p...

  3. America's Star Libraries, 2010: Top-Rated Libraries

    Science.gov (United States)

    Lyons, Ray; Lance, Keith Curry

    2010-01-01

    The "LJ" Index of Public Library Service 2010, "Library Journal"'s national rating of public libraries, identifies 258 "star" libraries. Created by Ray Lyons and Keith Curry Lance, and based on 2008 data from the IMLS, it rates 7,407 public libraries. The top libraries in each group get five, four, or three stars. All included libraries, stars or…

  4. Saccharomyces cerevisiae metabolism in ecological context

    Science.gov (United States)

    Jouhten, Paula; Ponomarova, Olga; Gonzalez, Ramon; Patil, Kiran R.

    2016-01-01

    The architecture and regulation of Saccharomyces cerevisiae metabolic network are among the best studied owing to its widespread use in both basic research and industry. Yet, several recent studies have revealed notable limitations in explaining genotype–metabolic phenotype relations in this yeast, especially when concerning multiple genetic/environmental perturbations. Apparently unexpected genotype–phenotype relations may originate in the evolutionarily shaped cellular operating principles being hidden in common laboratory conditions. Predecessors of laboratory S. cerevisiae strains, the wild and the domesticated yeasts, have been evolutionarily shaped by highly variable environments, very distinct from laboratory conditions, and most interestingly by social life within microbial communities. Here we present a brief review of the genotypic and phenotypic peculiarities of S. cerevisiae in the context of its social lifestyle beyond laboratory environments. Accounting for this ecological context and the origin of the laboratory strains in experimental design and data analysis would be essential in improving the understanding of genotype–environment–phenotype relationships. PMID:27634775

  5. Homology of plasmids in strains of unicellular cyanobacteria

    NARCIS (Netherlands)

    Hondel, C.A.M.J.J. van den; Keegstra, W.; Borrias, W.E.; Arkel, G.A. van

    1979-01-01

    Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron micr

  6. Purification of large plasmids with methacrylate monolithic columns.

    Science.gov (United States)

    Krajnc, Nika Lendero; Smrekar, Franci; Cerne, Jasmina; Raspor, Peter; Modic, Martina; Krgovic, Danijela; Strancar, Ales; Podgornik, Ales

    2009-08-01

    The rapid evolution of gene therapy and DNA vaccines results in an increasing interest in producing large quantities of pharmaceutical grade plasmid DNA. Most current clinical trials involve plasmids of 10 kb or smaller in size, however, future requirements for multigene vectors including extensive control regions may require the production of larger plasmids, e. g., 20 kb and bigger. The objective of this study was to examine certain process conditions for purification of large plasmids with the size of up to 93 kb. Since there is a lack of knowledge about production and purification of bigger plasmid DNA, cell lysis and storage conditions were investigated. The impact of chromatographic system and methacrylate monolithic column on the degradation of plasmid molecules under nonbinding conditions at different flow rates was studied. Furthermore, capacity measurements varying salt concentration in loading buffer were performed and the capacities up to 13 mg of plasmid per mL of the monolithic column were obtained. The capacity flow independence in the range from 130 to 370 cm/h was observed. Using high resolution monolithic column the separation of linear and supercoiled isoforms of large plasmids was obtained. Last but not least, since the baseline separation of RNA and pDNA was achieved, the one step purification on larger CIM DEAE 8 mL tube monolithic column was performed and the fractions were analyzed by CIM analytical monolithic columns. PMID:19598166

  7. Two novel conjugative plasmids from a single strain of Sulfolobus

    NARCIS (Netherlands)

    Erauso, G.; Stedman, K.M.; Werken, van de H.J.G.; Zillig, W.; Oost, van der J.

    2006-01-01

    Two conjugative plasmids (CPs) were isolated and characterized from the same 'Sulfolobus islandicus' strain, SOG2/4, The plasmids were separated from each other and transferred into Sulfolobus soltataricus. One has a high copy number and is not stable (pSOG1) whereas the other has a low copy number

  8. Functional analysis of three plasmids from Lactobacillus plantarum

    NARCIS (Netherlands)

    Kranenburg, R. van; Golic, N.; Bongers, R.; Leer, R.J.; Vos, W.M. de; Siezen, R.J.; Kleerebezem, M.

    2005-01-01

    Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. Th

  9. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.

    2013-01-01

    Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papil

  10. Examination of uropathogenic Escherichia coli strains conferring large plasmids

    Directory of Open Access Journals (Sweden)

    SUHARTONO

    2010-04-01

    Full Text Available Suhartono (2010 Examination of uropathogenic Escherichia coli strains conferring large plasmids. Biodiversitas 11: 59-64. Of major uropathogens, Escherichia coli has been widely known as a main pathogen of UTIs globally and has considerable medical and financial consequences. A strain of UPEC, namely E. coli ST131, confers a large plasmid encoding cephalosporinases (class C β-lactamase or AmpC that may be disseminated through horizontal transfer among bacterial populations. Therefore, it is worth examining such large plasmids by isolating, purifying, and digesting the plasmid with restriction enzymes. The examination of the large plasmids was conducted by isolating plasmid DNA visualized by agarose gel electrophoresis as well as by PFGE. The relationship of plasmids among isolates was carried out by HpaI restriction enzyme digestion. Of 36 isolates of E. coli ST 131, eight isolates possessed large plasmids, namely isolates 3, 9, 10, 12, 17, 18, 26 and 30 with the largest molecular size confirmed by agarose gel electrophoresis and PFGE was ~42kb and ~118kb respectively. Restriction enzyme analysis revealed that isolates 9, 10, 12, 17 and 18 have the common restriction patterns and those isolates might be closely related.

  11. Construction and Application of R Prime Plasmids, Carrying Different Segments of an Octopine Ti Plasmid from Agrobacterium tumefaciens, for Complementation of vir Genes

    NARCIS (Netherlands)

    Hille, Jacques; Klasen, Ina; Schilperoort, Rob

    1982-01-01

    Several R prime plasmids have been obtained with high efficiency, by enclosing the R plasmid replicator, in an R::Ti cointegrate plasmid, between two copies of the transposon Tn1831, in the same orientation. These R primes carry different segments of an octopine Ti plasmid, and are compatible with T

  12. Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters.

    Science.gov (United States)

    Shi, Shuobo; Valle-Rodríguez, Juan Octavio; Siewers, Verena; Nielsen, Jens

    2014-09-01

    In recent years, significant advances have been made to engineer robust microbes for overproducing biochemical products from renewable resources. These accomplishments have to a large extend been based on plasmid based methods. However, plasmid maintenance may cause a metabolic burden on the host cell and plasmid-based overexpression of genes can result in genetically unstable strains, which contributes to loss in productivity. Here, a chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum-based diesel. An integration construct was designed and integrated into chromosomal delta sequences by repetitive transformation, which resulted in 1-6 copies of the integration construct per genome. The corresponding FAEE production increased up to 34 mg/L, which is an about sixfold increase compared to the equivalent plasmid-based producer. The integrated cassette in the yeast genome was stably maintained in nonselective medium after deletion of RAD52 which is essential for efficient homologous recombination. To obtain a further increase of FAEE production, genes encoding endogenous acyl-CoA binding protein (ACB1) and a bacterial NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN) were overexpressed in the final integration strain, which resulted in another 40% percent increase in FAEE production. Our integration strategy enables easy engineering of strains with adjustable gene copy numbers integrated into the genome and this allows for an easy evaluation of the effect of the gene copy number on pathway flux. It therefore represents a valuable tool for introducing and expressing a heterologous pathway in yeast. PMID:24752598

  13. A novel, highly regulated, rapidly inducible system for the expression of chicken progesterone receptor, cPRA, in Saccharomyces cerevisiae.

    Science.gov (United States)

    Poletti, A; Weigel, N L; McDonnell, D P; Schrader, W T; O'Malley, B W; Conneely, O M

    1992-05-01

    A rapidly inducible and tightly regulated system for the expression of protein in yeast is based on a chimeric promoter constructed of two copies of a vitellogenin-estrogen-response element (ERE) which are inserted upstream from the promoter of the yeast gene encoding iso-1-cytochrome c. The chimeric promoter was inserted in a yeast expression plasmid upstream from the coding sequence of ubiquitin fused in frame to a cDNA encoding the full-length chicken progesterone receptor A (cPRA). The resultant plasmid (YEpA2) was co-transformed in Saccharomyces cerevisiae with a plasmid which encodes the human estrogen receptor. Estradiol (E2)-induced transactivation of the chimeric promoter results in transcription of the cPRA gene from YEpA2, and synthesis of cPRA. The fusion protein, ubiquitin-cPRA, is rapidly cleaved in vivo to produce cPRA. Analysis of samples by Western immunoblot shows that cPRA is almost undetectable in the absence of E2, and that treatment with 50 nM E2 results in a 500-1000-fold induction of cPRA (0.06-0.3% of the total protein) after 1 h. The plasmid-expressed soluble receptor is stable and demonstrates the correct affinity for its ligand. We have prepared yeast extracts using enzymatic digestion of the cell wall with oxalyticase followed by hypotonic shock. This has resulted in a dramatic increase in the % of receptor which binds hormone compared to previous studies which used mechanical disruption techniques. The cPRA is biologically active since it activates transcription of a co-transformed reporter gene containing its response element.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1316867

  14. High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe.

    OpenAIRE

    Okazaki, K.; Okazaki, N.; Kume, K.; Jinno, S; Tanaka, K; Okayama, H

    1990-01-01

    We describe a highly efficient alkali cation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of fission yeast Schizosaccharomyces pombe mutants. cDNA libraries constructed with the pcD or pcD2 vector are transduced into yeast by cotransfection with a linearized vector, which allows an enhanced homologous recombination between the yeast vector and the library plasmid leading to the efficient formation of concatemers containing pcD molecules. The tran...

  15. Construction of a eukaryotic expression plasmid of Humanin

    Institute of Scientific and Technical Information of China (English)

    LUO Ben-yan; CHEN Xiang-ming; TANG Min; CHEN Feng; CHEN Zhi

    2005-01-01

    Objective: To construct a eukaryotic expression plasmid pcDNA3.1 (-)-Humanin. Methods: The recombinant plasm pGEMEX- 1-Humanin was digested with restriction endonucleases BamH I and Hind Ⅲ and the Humanin gene fragments, abo 100 bp length, were obtained. Then the Humanin gene fragments were inserted into eukaryotic expression vector pcDNA3.1 (-) and the recombinant plasmids pcDNA3. l(-)-Humanin were identified by sequencing. Results: Recombinant plasmid DNA succesfully produced a band which had the same size as that of the Humanin positive control. The sequence of recombinant plasmids accorded with the Humnain gene sequence. Conclusions: A eukaryotic expression plasmid of Humanin was successfully constructed.

  16. Emergence of Plasmid-Borne dfrA14 Trimethoprim Resistance Gene in Shigella sonnei.

    Science.gov (United States)

    Miranda, Alfonso; Ávila, Bárbara; Díaz, Patricia; Rivas, Lina; Bravo, Karen; Astudillo, Javier; Bueno, Constanza; Ulloa, María T; Hermosilla, Germán; Del Canto, Felipe; Salazar, Juan C; Toro, Cecilia S

    2016-01-01

    The most common mechanism of trimethoprim (TMP)-resistance is the acquisition of dihydrofolate reductase enzyme resistant to this drug. Previous molecular characterization of TMP-genes resistance in Chilean isolates of Shigella sonnei searching for dfrA1 and dfrA8, showed solely the presence of dfrA8 (formerly dhfrIIIc). However, these genetic markers were absent in S. sonnei strains further isolated during an outbreak in 2009. To identify the TMP-resistance gene in these strains, a genomic DNA library from a TMP-resistant (TMP(R)) S. sonnei representative strain for the outbreak was used to clone, select and identify a TMP-resistance marker. The TMP(R) clone was sequenced by primer walking, identifying the presence of the dfrA14 gene in the sul2-strA'-dfrA14-'strA-strB gene arrangement, harbored in a native 6779-bp plasmid. The same plasmid was isolated by transforming with a ~4.2 MDa plasmid extracted from several TMP(R) S. sonnei strains into Escherichia coli. This plasmid, named pABC-3, was present only in dfrA14-positive strains and was homologous to a previously described pCERC-1, but different due to the absence of an 11-bp repetitive unit. The distribution of dfrA1, dfrA8, and dfrA14 TMP-resistance genes was determined in 126 TMP(R) S. sonnei isolates. Most of the strains (96%) carried only one of the three TMP-resistance genes assessed. Thus, all strains obtained during the 2009-outbreak harbored only dfrA14, whereas, dfrA8 was the most abundant gene marker before outbreak and, after the outbreak dfrA1 seems have appeared in circulating strains. According to PFGE, dfrA14-positive strains were clustered in a genetically related group including some dfrA1- and dfrA8-positive strains; meanwhile other genetic group included most of the dfrA8-positive strains. This distribution also correlated with the isolation period, showing a dynamics of trimethoprim genetic markers prevalent in Chilean S. sonnei strains. To our knowledge, dfrA14 gene associated to a small

  17. Antibiotic resistance of vibrio cholerae: special considerations of R-plasmids.

    Science.gov (United States)

    Kuwahara, S

    1978-09-01

    Studies on the transmission of R plasmid by conjugation between enterobacteria and vibrio or related bacteria were reviewed. The majority of the reports confirmed successful transmission from enterobacteria to Vibrio cholerae and related species, although the transmission frequencies were extremely low and the transmitted R plasmid was very unstable except for thermosensitive kanamycin plasmid and usual R plasmid coexisting with P plasmid. Strains of V. cholerae and Aeromonas liquefaciens as well as A. salmonicida bearing R plasmid were detected in nature. R plasmid was relatively unstable in V. cholerae strains with which transmission of R plasmid to enterobacteria was confirmed. At present, only 3 R plasmids have been obtained from naturally occurring strains of V. cholerae. Although the 2 European plasmids belong to the C incompatibility group with 98 megadalton closed covalent circular DNA molecule, one plasmid belongs to the J group with more than 25 megadalton molecular weight, and no CCC of satelite DNA was detected in bacteria harboring this plasmid.

  18. Plasmid DNA induces dodecyl triethyl ammonium bromide to aggregate into vesicle

    Institute of Scientific and Technical Information of China (English)

    Xiang Mei Ran; Xia Guo; Jia Tong Ding

    2012-01-01

    Single-chained cationic surfactant dodecyl triethyl ammonium bromide and plasmid DNA together can form vesicles once the concentration of plasmid DNA reaches a critical value (Ccvc).Bigger the size of plasmid DNA,higher the value of Ccvc.

  19. Bacterial Mitosis: ParM of Plasmid R1 Moves Plasmid DNA by an Actin-like Insertional Polymerization Mechanism

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette;

    2003-01-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...

  20. Bifurcation Analysis of a Chemostat Model of Plasmid-Bearing and Plasmid-Free Competition with Pulsed Input

    Directory of Open Access Journals (Sweden)

    Zhong Zhao

    2014-01-01

    to the stability of the boundary periodic solution. By use of standard techniques of bifurcation theory, the periodic oscillations in substrate, plasmid-bearing, and plasmid-free organisms are shown when some conditions are satisfied. Our results can be applied to control bioreactor aimed at producing commercial producers through genetically altered organisms.

  1. Construction of small-insert and large-insert metagenomic libraries.

    Science.gov (United States)

    Simon, Carola; Daniel, Rolf

    2010-01-01

    The vast majority of the Earth's biological diversity is hidden in uncultured and yet uncharacterized microbial genomes. The construction of metagenomic libraries is a cultivation-independent molecular approach to assess this unexplored genetic reservoir. In the last few years, a high number of novel biocatalysts have been identified by function-based or sequence-based screening of metagenomic libraries. Here, we describe detailed protocols for the construction of metagenomic small-insert and large-insert libraries in plasmids and fosmids, respectively, from environmental DNA. PMID:20830554

  2. Correctional Libraries, Library Standards, and Diversity.

    Science.gov (United States)

    Shirley, Glennor L.

    2003-01-01

    Respondents to a survey of prison librarians (n=35) regarding how they cope with demands for materials and services related to diversity issues felt that adherence to Library Standards for Adult Correctional Institutions was often impeded by security concerns. Racial differences between library providers and prisoners was not considered relevant.…

  3. 'Digital Library: A New Concept of Library

    OpenAIRE

    Deshmukh A. Hadi A. Bari

    2012-01-01

    This article throws light on introduction of Digital Library, Definition of Digital Library, Need, Objective, Function, Concept; Advantages & Disadvantages of Digital Library and it also describe components of Digital Library.

  4. Plasmid stability in immobilized and free recombinant Escherichia coli JM105(pKK223-200): importance of oxygen diffusion, growth rate, and plasmid copy number.

    OpenAIRE

    de Taxis du Poët, P; Arcand, Y; Bernier, R.; Barbotin, J N; Thomas, D.

    1987-01-01

    Stability of the plasmid pKK223-200 in Escherichia coli JM105 was studied for both free and immobilized cells during continuous culture. The relationship between plasmid copy number, xylanase activity, which was coded for by the plasmid, and growth rate and culture conditions involved complex interactions which determined the plasmid stability. Generally, the plasmid stability was enhanced in cultured immobilized cells compared with free-cell cultures. This stability was associated with modif...

  5. Positive selection and compensatory adaptation interact to stabilize non-transmissible plasmids

    OpenAIRE

    Millan, A. San; Peña-Miller, R.; Toll-Riera, M.; Halbert, Z. V.; McLean, A R; Cooper, B. S.; Maclean, R. C.

    2014-01-01

    Plasmids are important drivers of bacterial evolution, but it is challenging to understand how plasmids persist over the long term because plasmid carriage is costly. Classical models predict that horizontal transfer is necessary for plasmid persistence, but recent work shows that almost half of plasmids are non-transmissible. Here we use a combination of mathematical modelling and experimental evolution to investigate how a costly, non-transmissible plasmid, pNUK73, can be maintained in popu...

  6. Kluyveromyces lactis maintains Saccharomyces cerevisiae intron-encoded splicing signals.

    OpenAIRE

    Deshler, J O; Larson, G P; Rossi, J J

    1989-01-01

    The actin (ACT) gene from the budding yeast Kluyveromyces lactis was cloned, and the nucleotide sequence was determined. The gene had a single intron 778 nucleotides in length which possessed the highly conserved splicing signals found in Saccharomyces cerevisiae introns. We demonstrated splicing of heterologous ACT transcripts in both K. lactis and S. cerevisiae.

  7. NIAID Biodefense Image Library

    Science.gov (United States)

    ... Content Marketing Share this: Main Content Area Image Library These high-resolution (300 dpi) images may be ... for Disease Control and Prevention public health image library . Smallpox Vaccination Study-1 Smallpox vaccination dilution study: ...

  8. National Library of Medicine

    Science.gov (United States)

    ... ideas about NLM's third century, the future of big data, and the role of libraries in supporting research ... ideas about NLM's third century, the future of big data, and the role of libraries in supporting research ...

  9. Libraries - LIBRARIES_ISL_IN: Libraries in Indiana (Indiana State Library, Point Shapefile)

    Data.gov (United States)

    NSGIC GIS Inventory (aka Ramona) — LIBRARIES_ISL_IN is a point shapefile showing the locations of 528 libraries included in Microsoft Excel spreadsheets that are downloadable from the Web page named...

  10. Reutilizing Existing Library Space.

    Science.gov (United States)

    Davis, Marlys Cresap

    1987-01-01

    This discussion of the reutilization of existing library space reviews the decision process and considerations for implementation. Two case studies of small public libraries which reassigned space to better use are provided, including floor plans. (1 reference) (MES)

  11. Facility Focus: Libraries.

    Science.gov (United States)

    College Planning & Management, 2003

    2003-01-01

    Describes the designs of the Ferris State University Library for Information, Technology and Education (FLITE), and the Meyer Library and Information Technology Center at Southwest Missouri State University. Includes photographs. (EV)

  12. Libraries serving dialogue

    CERN Document Server

    Dupont, Odile

    2014-01-01

    This book based on experiences of libraries serving interreligious dialogue, presents themes like library tools serving dialogue between cultures, collections dialoguing, children and young adults dialoguing beyond borders, story telling as dialog, librarians serving interreligious dialogue.

  13. Library staff development course.

    OpenAIRE

    Eaton, E K

    1981-01-01

    The Moody Medical Library at the University of Texas Medical Branch plans, presents, and evaluates regularly a staff development program for its employees, including librarians and clerical and technical staff. The program's purpose is to provide continuing education for the library staff while concurrently: (1) providing information concerning specific library services and programs; (2) illustrating the interrelationship of the departments and divisions within the library; (3) developing a s...

  14. Effects of spaceflight on polysaccharides of Saccharomyces cerevisiae cell wall.

    Science.gov (United States)

    Liu, Hong-Zhi; Wang, Qiang; Liu, Xiao-Yong; Tan, Sze-Sze

    2008-12-01

    Freeze-dried samples of four Saccharomyces cerevisiae strains, namely, FL01, FL03, 2.0016, and 2.1424, were subjected to spaceflight. After the satellite's landing on Earth, the samples were recovered and changes in yeast cell wall were analyzed. Spaceflight strains of all S. cerevisiae strains showed significant changes in cell wall thickness (P growth curve analysis showed spaceflight S. cerevisiae 2.0016 had a faster growth rate, shorter lag phase periods, higher final biomass, and higher content of beta-glucan. Genetic stability analysis showed that prolonged subculturing of spaceflight strain S. cerevisiae 2.0016 did not lead to the appearance of variants, indicating that the genetic stability of S. cerevisiae 2.0016 mutant could be sufficient for its exploitation of beta-glucan production. PMID:18797865

  15. Nuclear and mitochondrial genome instability induced by senna (Cassia angustifolia Vahl.) aqueous extract in Saccharomyces cerevisiae strains.

    Science.gov (United States)

    Silva, C R; Caldeira-de-Araújo, A; Leitão, A C; Pádula, M

    2014-11-27

    Cassia angustifolia Vahl. (senna) is commonly used in self-medication and is frequently used to treat intestine constipation. A previous study involving bacteria and plasmid DNA suggested the possible toxicity of the aqueous extract of senna (SAE). The aim of this study was to extend the knowledge concerning SAE genotoxicity mechanisms because of its widespread use and its risks to human health. We investigated the impact of SAE on nuclear DNA and on the stability of mitochondrial DNA in Saccharomyces cerevisiae (wt, ogg1, msh6, and ogg1msh6) strains, monitoring the formation of petite mutants. Our results demonstrated that SAE specifically increased Can(R) mutagenesis only in the msh6 mutant, supporting the view that SAE can induce misincorporation errors in DNA. We observed a significant increase in the frequency of petite colonies in all studied strains. Our data indicate that SAE has genotoxic activity towards both mitochondrial and nuclear DNA.

  16. The Library Lobby

    Science.gov (United States)

    Ralston, Anthony

    1971-01-01

    The library is examined as a university-wide resource and as a research facility. The use of the library relative to computing facilities is also considered as is the cost and value of library usage in relation to computer usage. (Author)

  17. PAL: Positional Astronomy Library

    Science.gov (United States)

    Jenness, T.; Berry, D. S.

    2016-06-01

    The PAL library is a partial re-implementation of Pat Wallace's popular SLALIB library written in C using a Gnu GPL license and layered on top of the IAU's SOFA library (or the BSD-licensed ERFA) where appropriate. PAL attempts to stick to the SLA C API where possible.

  18. Body Basics Library

    Science.gov (United States)

    ... a Friend Who Cuts? About the Body Basics Library KidsHealth > For Teens > About the Body Basics Library Print A A A Text Size Did you ... system, part, and process works. Use this medical library to find out about basic human anatomy, how ...

  19. Public Library Finance.

    Science.gov (United States)

    Mason, Marilyn Gell

    This study reviews trends in public library finance; examines recent political, economic, and technological changes; and assesses the impact of these changes on public library services. A history of the public library in America is presented, as well as an analysis of the principles of economics and public finance which reveals that current…

  20. Supervision in Libraries.

    Science.gov (United States)

    Bailey, Martha J.

    Although the literature of library administration draws extensively on that of business management, it is difficult to compare library supervision to business or industrial supervision. Library supervisors often do not have managerial training and may consider their management role as secondary. The educational level of the staff they supervise…

  1. A Truly Bookless Library

    Science.gov (United States)

    Kolowich, Steve

    2011-01-01

    The difference between the University of Texas at San Antonio's Applied Engineering and Technology Library and other science-focused libraries is not that its on-site collection is also available electronically. It is that its on-site collection is only available electronically. The idea of libraries with no bound books has been a recurring theme…

  2. Changing State Digital Libraries

    Science.gov (United States)

    Pappas, Marjorie L.

    2006-01-01

    Research has shown that state virtual or digital libraries are evolving into websites that are loaded with free resources, subscription databases, and instructional tools. In this article, the author explores these evolving libraries based on the following questions: (1) How user-friendly are the state digital libraries?; (2) How do state digital…

  3. Learning Boost C++ libraries

    CERN Document Server

    Mukherjee, Arindam

    2015-01-01

    If you are a C++ programmer who has never used Boost libraries before, this book will get you up-to-speed with using them. Whether you are developing new C++ software or maintaining existing code written using Boost libraries, this hands-on introduction will help you decide on the right library and techniques to solve your practical programming problems.

  4. The Electronic, Eclectic Library.

    Science.gov (United States)

    Dowlin, Kenneth E.

    1980-01-01

    Utilization of telecommunications and computer technology to increase access to information is identified as a primary goal for public libraries. To further service in the future, libraries should consider database services, online access to library resources, online community conferencing, community databases, network resource sharing,…

  5. School Libraries and Innovation

    Science.gov (United States)

    McGrath, Kevin G.

    2015-01-01

    School library programs have measured success by improved test scores. But how do next-generation school libraries demonstrate success as they strive to be centers of innovation and creativity? These libraries offer solutions for school leaders who struggle to restructure existing systems built around traditional silos of learning (subjects and…

  6. Reforming Prison Libraries.

    Science.gov (United States)

    Coyle, William J.

    1989-01-01

    Discusses the current widespread acceptance of the public library model for prison libraries, in which preferences of the inmates are the chief consideration in programing and collection development. It is argued that this model results in recreational programs and collections that fail to fulfill the prison library's role in education and…

  7. Spanish Museum Libraries Network.

    Science.gov (United States)

    Lopez de Prado, Rosario

    This paper describes the creation of an automated network of museum libraries in Spain. The only way in which the specialized libraries in the world today can continue to be active and to offer valid information is to automate the service they offer, and create network libraries with cooperative plans. The network can be configured with different…

  8. Marketing Academic Libraries

    Science.gov (United States)

    Mallon, Melissa, Ed.

    2013-01-01

    Ask any academic librarian if marketing their library and its services is an important task, and the answer will most likely be a resounding "yes!" Particularly in economically troubled times, librarians are increasingly called upon to promote their services and defend their library's worth. Since few academic libraries have in-house marketing…

  9. 紫杉二烯生物合成模块与不同底盘的适配%Fitness of Taxadiene Biosynthetic Modules with Different S. cerevisiae Chassis

    Institute of Scientific and Technical Information of China (English)

    张正伟; 丁明珠; 元英进

    2014-01-01

    以酿酒酵母BY4742及其单敲菌株作为底盘细胞,优化底盘细胞甲羟戊酸途径,上调并融合表达牻牛儿基牻牛儿基焦磷酸( GGPP)合成的相关基因,引入人工合成的外源GGPP合成酶基因与紫杉二烯合成酶基因,构建了多载体紫杉二烯生物合成模块;还利用酵母组装技术,通过对紫杉二烯合成路径相关基因进行模块化设计组装,构建了依托单一着丝粒( CEN)质粒的紫杉二烯生物合成模块.将构建的2个模块与不同底盘细胞进行适配,使紫杉二烯产量获得了数倍提升,最高产量可达74.84 mg/L.%In the research works of constructing taxadiene aritficaial synthetic cell, S. cerevisiae is a common-ly used chassis. The highest taxadiene yield in S. cerevisiae that has been reported was 8. 7 mg/L. In this work, the fitness of different taxadiene biosynthetic modules with different S. cerevisiae chassis was studied to elevate the taxadiene yield to a higher level. We chose the S. cerevisiae strains BY4742 and the single knoc-kout strains of BY4742 as chassis, constructed the multi-plasmids taxadiene biosynthetic module by optimizing the mevalonic acid( MVA) pathway with tHMGR, importing the synthetic genes GGPPSsa and tTS, up-regula-ting and co-expressing the genes BTS1 and ERG20 which are correlated with the synthesis of geranylgeranyl diphosphate(GGPP). Then we redesigned and reconstructed the module inserted into a single CEN plasmid by the method of DNA assembler. Through fitting the different modules with different chassis, we acquired strains with diffe-rent taxadiene yields, the highest yield was 74. 84 mg/L in the strain with the single-plasmid module and the chassis of YNL280C. The results indicated that the single-plasmid taxadiene biosynthetic module was more stable in the chassis than the multi-plasmids one, and the regulation of the pathways correlated with the MVA pathway will also influent the taxadiene yield.

  10. Isocitrate lyase localisation in Saccharomyces cerevisiae cells.

    Science.gov (United States)

    Chaves, R S; Herrero, P; Ordiz, I; Angeles del Brio, M; Moreno, F

    1997-10-01

    The isocitrate lyase from Saccharomyces cerevisiae was only located in the cell cytoplasm. This protein was found not to be associated with cell organelles, even under growth conditions that induce peroxisome proliferation. This conclusion is supported by experiments carried out by damaging the protoplast plasma membrane with DEAE-dextran, by differential centrifugation of osmotically lysed protoplast and by using the green fluorescent protein (GFP) of Aequorea victoria as a reporter fusion tag to localise the subcellular compartment to which isocitrate lyase is targeted.

  11. Plasmid Copy Number Determination by Quantitative Polymerase Chain Reaction.

    Science.gov (United States)

    Anindyajati; Artarini, A Anita; Riani, Catur; Retnoningrum, Debbie S

    2016-01-01

    Recombinant therapeutic proteins are biopharmaceutical products that develop rapidly for years. Recombinant protein production in certain hosts requires vector expression harboring the gene encoding the corresponding protein. Escherichia coli is the prokaryote organism mostly used in recombinant protein production, commonly using a plasmid as the expression vector. Recombinant protein production is affected by plasmid copy number harboring the encoded gene, hence the determination of plasmid copy number also plays an important role in establishing a recombinant protein production system. On the industrial scale, a low copy number of plasmids are more suitable due to their better stability. In the previous study we constructed pCAD, a plasmid derived from the low copy number pBR322 plasmid. This study was aimed to confirm pCAD's copy number by quantitative polymerase chain reaction (qPCR). Plasmid copy number was determined by comparing the quantification signal from the plasmid to those from the chromosome. Copy number was then calculated by using a known copy number plasmid as a standard. Two pairs of primers, called tdk and ori, were designed for targeting a single gene tdk in the chromosome and a conserved domain in the plasmid's ori, respectively. Primer quality was analyzed in silico using PrimerSelect DNASTAR and PraTo software prior to in vitro evaluation on primer specificity and efficiency as well as optimization of qPCR conditions. Plasmid copy number determination was conducted on E. coli lysates harboring each plasmid, with the number of cells ranging from 10(2)-10(5) cells/μL. Cells were lysed by incubation at 95ºC for 10 minutes, followed by immediate freezing at -4°C. pBR322 plasmid with the copy number of ~19 copies/cell was used as the standard, while pJExpress414-sod plasmid possessing the high copy number pUC ori was also determined to test the method being used. In silico analysis based on primer-primer and primer-template interactions showed

  12. An updated view of plasmid conjugation and mobilization in Staphylococcus.

    Science.gov (United States)

    Ramsay, Joshua P; Kwong, Stephen M; Murphy, Riley J T; Yui Eto, Karina; Price, Karina J; Nguyen, Quang T; O'Brien, Frances G; Grubb, Warren B; Coombs, Geoffrey W; Firth, Neville

    2016-01-01

    The horizontal gene transfer facilitated by mobile genetic elements impacts almost all areas of bacterial evolution, including the accretion and dissemination of antimicrobial-resistance genes in the human and animal pathogen Staphylococcus aureus. Genome surveys of staphylococcal plasmids have revealed an unexpected paucity of conjugation and mobilization loci, perhaps suggesting that conjugation plays only a minor role in the evolution of this genus. In this letter we present the DNA sequences of historically documented staphylococcal conjugative plasmids and highlight that at least 3 distinct and widely distributed families of conjugative plasmids currently contribute to the dissemination of antimicrobial resistance in Staphylococcus. We also review the recently documented "relaxase-in trans" mechanism of conjugative mobilization facilitated by conjugative plasmids pWBG749 and pSK41, and discuss how this may facilitate the horizontal transmission of around 90% of plasmids that were previously considered non-mobilizable. Finally, we enumerate unique sequenced S. aureus plasmids with a potential mechanism of mobilization and predict that at least 80% of all non-conjugative S. aureus plasmids are mobilizable by at least one mechanism. We suggest that a greater research focus on the molecular biology of conjugation is essential if we are to recognize gene-transfer mechanisms from our increasingly in silico analyses. PMID:27583185

  13. Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

    International Nuclear Information System (INIS)

    Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores

  14. Investigation of plasmid-induced growth defect in Pseudomonas putida.

    Science.gov (United States)

    Mi, Jia; Sydow, Anne; Schempp, Florence; Becher, Daniela; Schewe, Hendrik; Schrader, Jens; Buchhaupt, Markus

    2016-08-10

    Genetic engineering in bacteria mainly relies on the use of plasmids. But despite their pervasive use for physiological studies as well as for the design and optimization of industrially used production strains, only limited information about plasmid induced growth defects is available for different replicons and organisms. Here, we present the identification and characterization of such a phenomenon for Pseudomonas putida transformants carrying the pBBR1-derived plasmid pMiS1. We identified the kanamycin resistance gene and the transcription factor encoding rhaR gene to be causal for the growth defect in P. putida. In contrast, this effect was not observed in Escherichia coli. The plasmid-induced growth defect was eliminated after introduction of a mutation in the plasmid-encoded rep gene, thus enabling construction of the non-toxic variant pMiS4. GFP reporters construct analyses and qPCR experiments revealed a distinctly lowered plasmid copy number for pMiS4, which is probably the reason for alleviation of the growth defect by this mutation. Our work expands the knowledge about plasmid-induced growth defects and provides a useful low-copy pBBR1 replicon variant. PMID:27287537

  15. Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

    Energy Technology Data Exchange (ETDEWEB)

    Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I. (Department of Agriculture, College Station, TX (USA))

    1990-08-01

    Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.

  16. Multilocus sequence typing of IncN plasmids

    DEFF Research Database (Denmark)

    García-Fernández, Aurora; Villa, Laura; Moodley, Arshnee;

    2011-01-01

    OBJECTIVES: Incompatibility group N (IncN) plasmids have been associated with the dissemination of antimicrobial resistance and are a major vehicle for the spread of blaVIM-1 in humans and blaCTX-M-1 in animals. A plasmid multilocus sequence typing (pMLST) scheme was developed for rapid categoriz......OBJECTIVES: Incompatibility group N (IncN) plasmids have been associated with the dissemination of antimicrobial resistance and are a major vehicle for the spread of blaVIM-1 in humans and blaCTX-M-1 in animals. A plasmid multilocus sequence typing (pMLST) scheme was developed for rapid...... in different countries from both animals and humans belonged to ST1, suggesting dissemination of an epidemic plasmid through the food chain. Fifteen of 17 plasmids carrying blaVIM-1 from Klebsiella pneumoniae and Escherichia coli, isolated during a 5year period in Greece were assigned to ST10, suggesting...... that spread and persistence of this particular IncN-carrying blaVIM-1 lineage in Greece. CONCLUSIONS: This study proposes the use of pMLST as a suitable and rapid method for identification of IncN epidemic plasmid lineages. The recent spread of blaCTX-M-1 among humans and animals seems to be associated...

  17. Manipulations in the Peripheral Stalk of the Saccharomyces cerevisiae F1F0-ATP Synthase*

    Science.gov (United States)

    Welch, Amanda K.; Bostwick, Caleb J.; Cain, Brian D.

    2011-01-01

    The Saccharomyces cerevisiae F1F0-ATP synthase peripheral stalk is composed of the OSCP, h, d, and b subunits. The b subunit has two membrane-spanning domains and a large hydrophilic domain that extends along one side of the enzyme to the top of F1. In contrast, the Escherichia coli peripheral stalk has two identical b subunits, and subunits with substantially altered lengths can be incorporated into a functional F1F0-ATP synthase. The differences in subunit structure between the eukaryotic and prokaryotic peripheral stalks raised a question about whether the two stalks have similar physical and functional properties. In the present work, the length of the S. cerevisiae b subunit has been manipulated to determine whether the F1F0-ATP synthase exhibited the same tolerances as in the bacterial enzyme. Plasmid shuffling was used for ectopic expression of altered b subunits in a strain carrying a chromosomal disruption of the ATP4 gene. Wild type growth phenotypes were observed for insertions of up to 11 and a deletion of four amino acids on a nonfermentable carbon source. In mitochondria-enriched fractions, abundant ATP hydrolysis activity was seen for the insertion mutants. ATPase activity was largely oligomycin-insensitive in these mitochondrial fractions. In addition, very poor complementation was seen in a mutant with an insertion of 14 amino acids. Lengthier deletions yielded a defective enzyme. The results suggest that although the eukaryotic peripheral stalk is near its minimum length, the b subunit can be extended a considerable distance. PMID:21257750

  18. Sequence analysis of plasmid in Klebsiella pneumoniae KF3%一株多重耐药肺炎克雷伯菌KF3的质粒研究

    Institute of Scientific and Technical Information of China (English)

    陆红云; 张洪勤; 姚晓鼎; 王军荣; 席亚丽; 周明明; 周铁丽; 包其郁; 李劲松

    2010-01-01

    Objective To study the structures of the plasmids of Klebsiella pneumoniae KF3 at the genome metagenome level througth with whole plasmid DNA sequencing, to analyze the functional genes carried by plasmid and to identify the correlation of resistance and pathogenicity between the plasmids and the host strains. Methods The alkaline lysis method was used to extract plasmids. We constructed the small insert pUC18 library and the large insert Forsmid library, sequenced and used the Phred / Phrap / Consed package to assemble these sequences and gained a complete sequence. The open reading frame(ORFs) were predicted by the Glimmer software and annotated, analyzed the functions of these genes. Results We successfully constructed the pUC18 library and the Fosmid libraries for the plasmid DNA and obtained three circular double-stranded DNA plasmids: pKF3-70 (69 477 bp), pKF3-90 (91 327 bp) and pKF3-147 ( 147 416 bp). There were drug resistant genes, conjugative transfer genes and mobile DNA elements identified on three plasmids. Conclusion The three plasmids of KF3 could be transferred among different strains. It would lead to the dissemination of the resistant genes.%目的 通过对肺炎克雷伯菌KF3质粒DNA全序列测定,从基因组水平研究质粒DNA的结构、功能基因和与宿主菌耐药相关性.方法 碱裂解法提取质粒DNA,构建质粒DNA文库并测序.采用Phred/Phrap/Consed软件包进行序列拼接,Glimmer软件预测开放阅读框架(ORF)及功能分析.结果 构建包含3个质粒DNA的pUC18文库和Fosmid文库,测序获得3个质粒全序列.功能注释分析发现3个质粒均为可接合转移质粒,编码大量耐药相关基因.结论 肺炎克雷伯菌KF3的3个质粒都是可接合转移质粒,将耐药基因在细菌间进行水平转移,造成了耐药菌的播散.

  19. Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Shuhei Noda

    Full Text Available To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC, which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

  20. Separation of plasmid DNA topoisomers by multimodal chromatography.

    Science.gov (United States)

    Silva-Santos, A Rita; Alves, Cláudia P A; Prazeres, Duarte Miguel F; Azevedo, Ana M

    2016-06-15

    The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample. PMID:27033004

  1. Library/vendor relationships

    CERN Document Server

    Brooks, Sam

    2014-01-01

    A view of the mutual dependence between libraries and vendorsAs technology advances, libraries are forced to reach beyond their own resources to find effective ways to maintain accuracy and superior service levels. Vendors provide databases and integrated library systems that perform those functions for profit. Library/Vendor Relationships examines the increasing cooperation in which libraries find they must participate in, and vice versa, with the vendors that provide system infrastructure and software. Expert contributors provide insights from all sides of this unique collaboration, offering

  2. Molecular and process design for rotavirus-like particle production in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Palomares Laura A

    2011-05-01

    Full Text Available Abstract Background Virus-like particles (VLP have an increasing range of applications including vaccination, drug delivery, diagnostics, gene therapy and nanotechnology. These developments require large quantities of particles that need to be obtained in efficient and economic processes. Production of VLP in yeast is attractive, as it is a low-cost protein producer able to assemble viral structural proteins into VLP. However, to date only single-layered VLP with simple architecture have been produced in this system. In this work, the first steps required for the production of rotavirus-like particles (RLP in S. cerevisiae were implemented and improved, in order to obtain the recombinant protein concentrations required for VLP assembly. Results The genes of the rotavirus structural proteins VP2, VP6 and VP7 were cloned in four Saccharomyces cerevisiae strains using different plasmid and promoter combinations to express one or three proteins in the same cell. Performance of the best constructs was evaluated in batch and fed-batch cultures using a complete synthetic media supplemented with leucine, glutamate and succinate. The strain used had an important effect on recombinant protein concentration, while the type of plasmid, centromeric (YCp or episomal (YEp, did not affect protein yields. Fed-batch culture of the PD.U-267 strain resulted in the highest concentration of rotavirus proteins. Volumetric and specific productivities increased 28.5- and 11-fold, respectively, in comparison with batch cultures. Expression of the three rotavirus proteins was confirmed by immunoblotting and RLP were detected using transmission electron microscopy. Conclusions We present for the first time the use of yeast as a platform to express multilayered rotavirus-like particles. The present study shows that the combined use of molecular and bioprocess tools allowed the production of triple-layered rotavirus RLP. Production of VLP with complex architecture in yeasts

  3. Library system of Italy

    Directory of Open Access Journals (Sweden)

    Nataša Gerbec

    2003-01-01

    Full Text Available In the European extent, Italy is the cradle of libraries and library sciences. In the past, Italian national public libraries played an important role through their vast book treasury. But only during the last thirty years have public libraries been developed following the Anglo-American public library model. Italy does not have any uniform or general legislation concerning libraries. On the state level, this area is regulated by some separate acts, while on the regional level there is a collection of various acts and regulations. Libraries are not strictly divided into general categories. It is required that the professionals engaged in Italian libraries should have secondary or university education. The level of their professional tasks depends on the type of library and its capacity. The competency for the development in the field of librarianship is assigned to The Ministry of Cultural and Environment Heritage as well as to its subordinate institutions (Central Institute for the Union catalogue of Italian Libraries and for Bibliographic Information, Central Institute for Book Pathology, Observatory for International Libraries Programmes.

  4. The participatory public library

    DEFF Research Database (Denmark)

    Rasmussen, Casper Hvenegaard

    2016-01-01

    Purpose From collection to connection has been a buzzword in the library world for more than a decade. This catchy phrase indicates that users are seen not only as borrowers, but as active participants. The aim of this paper is to investigate and analyse three questions in relation to user...... participation in public libraries in a Nordic perspective. How can participation in public libraries be characterised? Why should libraries deal with user participation? What kinds of different user participation can be identified in public libraries? Design/methodology/approach The paper uses a selection...... of theoretical approaches and practical examples to obtain a varied understanding of user participation in public libraries. Research fields outside library and information science have developed a wide range of theoretical approaches on user participation. Examples from cultural policy, museum studies...

  5. Libraries in Society

    DEFF Research Database (Denmark)

    Kristiansson, Michael; Skouvig, Laura

    brought about by the boom of the internet and the advent of the post-modern globalised knowledge-based and network-organised society. Finally, the paper outlines how theoretical and strategic library development can benefit from academic considerations on the dialectics between openness and restrictedness......The purpose of the paper is to investigate the phenomenon of openness in relation to library development. The term openness is presented and related to library development from historical and theoretical perspectives. The paper elaborates on the differences over time on to how openness has been...... understood in a library setting. Historically, openness in form of the open shelves played a crucial role in developing the modern public library. The paper examines this openness-centred library policy as adopted by Danish public libraries in the beginning of the 20th century by applying the theories...

  6. Biosorption of cesium by saccharomyces cerevisia

    International Nuclear Information System (INIS)

    The characteristics of Cs+ biosorption by Saccharornyces cerevisia was investigated, including the biosorption kinetics, biosorption equilibrium, isotherm as well as the IR spectrum of biomass pre- and post-biosorption. The experimental results show that the process of Cs+ biosorption onto the biomass of Saccharornyces cerevisia can be devided into two stages, the first stage is physical sorption and the sorption equilibrium is very quickly reached (within 20 min). The biosorption kinetics can be described by the pseudo second-order equation quite well (R2=0.989), the kinetic parameters k2 and qe are 3.56 x 10-3 g/(mg·min) and 7.18 mg/g, respectively. The equilibrium isotherm data can be fitted with Langmuir and Freundlich models, with the maximum biosorptive capacity of 10.13 mg/g. Both the IR spectra of the biomass pre- and post-biosorption almost are same, and it indicates that the biosorption of Cs+ does not change the structure of the biomass, however, some adsorptive peaks shift. (authors)

  7. Response of Saccharomyces cerevisiae to cadmium stress

    Energy Technology Data Exchange (ETDEWEB)

    Moreira, Luciana Mara Costa; Ribeiro, Frederico Haddad; Neves, Maria Jose [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia], e-mail: luamatu@uol.com.br; Porto, Barbara Abranches Araujo; Amaral, Angela M.; Menezes, Maria Angela B.C. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Lab. de Ativacao Neutronica], e-mail: menezes@cdtn.br; Rosa, Carlos Augusto [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Microbiologia], e-mail: carlrosa@icb.ufmg

    2009-07-01

    The intensification of industrial activity has been greatly contributing with the increase of heavy metals in the environment. Among these heavy metals, cadmium becomes a serious pervasive environmental pollutant. The cadmium is a heavy metal with no biological function, very toxic and carcinogenic at low concentrations. The toxicity of cadmium and several other metals can be mainly attributed to the multiplicity of coordination complexes and clusters that they can form. Some aspects of the cellular response to cadmium were extensively investigated in the yeast Saccharomyces cerevisiae. The primary site of interaction between many toxic metals and microbial cells is the plasma membrane. Plasma-membrane permeabilisation has been reported in a variety of microorganisms following cadmium exposure, and is considered one mechanism of cadmium toxicity in the yeast. In this work, using the yeast strain S. cerevisiae W303-WT, we have investigated the relationships between Cd uptake and release of cellular metal ions (K{sup +} and Na{sup +}) using neutron activation technique. The neutron activation was an easy, rapid and suitable technique for doing these metal determinations on yeast cells; was observed the change in morphology of the strains during the process of Cd accumulation, these alterations were observed by Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) during incorporation of cadmium. (author)

  8. Generation of recombinant porcine parvovirus virus-like particles in Saccharomyces cerevisiae and development of virus-specific monoclonal antibodies.

    Science.gov (United States)

    Tamošiūnas, Paulius Lukas; Petraitytė-Burneikienė, Rasa; Lasickienė, Rita; Akatov, Artiomas; Kundrotas, Gabrielis; Sereika, Vilimas; Lelešius, Raimundas; Žvirblienė, Aurelija; Sasnauskas, Kęstutis

    2014-01-01

    Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.

  9. Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae.

    Science.gov (United States)

    Bao, Zehua; Xiao, Han; Liang, Jing; Zhang, Lu; Xiong, Xiong; Sun, Ning; Si, Tong; Zhao, Huimin

    2015-05-15

    One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.

  10. Expression and secretion of the Candida wickerhamii extracellular beta-glucosidase gene, bglB, in Saccharomyces cerevisiae.

    Science.gov (United States)

    Skory, C D; Freer, S N; Bothast, R J

    1996-11-01

    The yeast Candida wickerhamii exports a cell-associated beta-glucosidase that is active against cellobiose and all soluble cellodextrins. Because of its unique ability to tolerate end-product inhibition by glucose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory. Using several different promoters and constructs, bglB was expressed in the hosts Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. Expression was initially performed in E. coli using either the lacZ or tac promoter. This resulted in intracellular expression of the BglB protein with the protein being rapidly fragmented. Secretion and glycosylation of active beta-glucosidase was achieved with several different S. cerevisiae constructs utilizing either the adh1 or the gal1 promoter on 2-micro replicating plasmids. When either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to be hyperglycosylated. Expression in P. pastoris was also examined to determine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate. Using the alcohol oxidase promoter in conjunction with either the pho1 or the alpha-factor secretion signal, the recombinant enzyme was successfully secreted and glycosylated in P. pastoris. However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activity. PMID:8929394

  11. Influenza Plasmid DNA Vaccines: Progress and Prospects.

    Science.gov (United States)

    Bicho, Diana; Queiroz, João António; Tomaz, Cândida Teixeira

    2015-01-01

    Current influenza vaccines have long been used to fight flu infectious; however, recent advances highlight the importance of produce new alternatives. Even though traditional influenza vaccines are safe and usually effective, they need to be uploaded every year to anticipate circulating flu viruses. This limitation together with the use of embryonated chicken eggs as the substrate for vaccine production, is time-consuming and could involve potential biohazards in growth of new virus strains. Plasmid DNA produced by prokaryote microorganisms and encoding foreign proteins had emerged as a promising therapeutic tool. This technology allows the expression of a gene of interest by eukaryotic cells in order to induce protective immune responses against the pathogen of interest. In this review, we discuss the strategies to choose the best DNA vaccine to be applied in the treatment and prevention of influenza. Specifically, we give an update of influenza DNA vaccines developments, all involved techniques, their main characteristics, applicability and technical features to obtain the best option against influenza infections.

  12. Flight Software Math Library

    Science.gov (United States)

    McComas, David

    2013-01-01

    The flight software (FSW) math library is a collection of reusable math components that provides typical math utilities required by spacecraft flight software. These utilities are intended to increase flight software quality reusability and maintainability by providing a set of consistent, well-documented, and tested math utilities. This library only has dependencies on ANSI C, so it is easily ported. Prior to this library, each mission typically created its own math utilities using ideas/code from previous missions. Part of the reason for this is that math libraries can be written with different strategies in areas like error handling, parameters orders, naming conventions, etc. Changing the utilities for each mission introduces risks and costs. The obvious risks and costs are that the utilities must be coded and revalidated. The hidden risks and costs arise in miscommunication between engineers. These utilities must be understood by both the flight software engineers and other subsystem engineers (primarily guidance navigation and control). The FSW math library is part of a larger goal to produce a library of reusable Guidance Navigation and Control (GN&C) FSW components. A GN&C FSW library cannot be created unless a standardized math basis is created. This library solves the standardization problem by defining a common feature set and establishing policies for the library s design. This allows the libraries to be maintained with the same strategy used in its initial development, which supports a library of reusable GN&C FSW components. The FSW math library is written for an embedded software environment in C. This places restrictions on the language features that can be used by the library. Another advantage of the FSW math library is that it can be used in the FSW as well as other environments like the GN&C analyst s simulators. This helps communication between the teams because they can use the same utilities with the same feature set and syntax.

  13. COMPARISON OF THE EXPRESSION IN Saccharomyces cerevisiae OF ENDOGLUCANASE II FROM Trichoderma reesei AND ENDOGLUCANASE I FROM Aspergillus aculeatus

    Directory of Open Access Journals (Sweden)

    Weina Zhang,

    2012-07-01

    Full Text Available Two distinct expression cassettes were synthesized by overlapping PCR for expressing the endoglucanase I gene (egl1 from Aspergillus aculeatus and the endoglucanase II gene (egl2 from Trichoderma reesei in a Saccharomyces cerevisiae host. One contained the anchored sequence from the S. cerevisiae cwp2 gene, while the other did not. The low and high copy number plasmids YCplac33 and YEplac195 were used. The enzymatic activities and viscosity changes in the YP-CMC medium varied between the eight recombinant yeast strains produced, and the greatest values were obtained with the YE-TrEII’ strain, which had an activity of 347.7 U/g dry cell weight (DCW and viscosity at 12 h of 4.7% of the initial control value, respectively; YE-TrEII’ was YEplac195-based and contained T. reesei egl2 and no Cwp2 sequence. Strains YC-AaEI and YC-TrEII showed the lowest enzyme activitiy (80.5 and 30.4 U/g DCW, respectively and viscosity changes at 12 h (20.5 and 26.2% of the initial control viscosity, respectively, which were YCplac33-based and contained the Cwp2 sequence. The results showed that gene copy number was the most significant factor to influence the expression of endoglucanases in S. cerevisiae, and the existence of Cwp2 sequence led to decreased enzymatic level and viscosity-reducing performance, while it was shown not to realize efficient surface display of these two endoglucanases.Keywords

  14. Genome Sequence and Analysis of a Stress-Tolerant, Wild-Derived Strain of Saccharomyces cerevisiae Used in Biofuels Research.

    Science.gov (United States)

    McIlwain, Sean J; Peris, David; Sardi, Maria; Moskvin, Oleg V; Zhan, Fujie; Myers, Kevin S; Riley, Nicholas M; Buzzell, Alyssa; Parreiras, Lucas S; Ong, Irene M; Landick, Robert; Coon, Joshua J; Gasch, Audrey P; Sato, Trey K; Hittinger, Chris Todd

    2016-01-01

    The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. The Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics. PMID:27172212

  15. Assessing Library Automation and Virtual Library Development in Four Academic Libraries in Oyo, Oyo State, Nigeria

    Science.gov (United States)

    Gbadamosi, Belau Olatunde

    2011-01-01

    The paper examines the level of library automation and virtual library development in four academic libraries. A validated questionnaire was used to capture the responses from academic librarians of the libraries under study. The paper discovers that none of the four academic libraries is fully automated. The libraries make use of librarians with…

  16. Plasmid pGA1 from Corynebacterium glutamicum codes for a gene product that positively influences plasmid copy number.

    OpenAIRE

    Nesvera, J; Pátek, M; Hochmannová, J; Abrhámová, Z; Becvárová, V; Jelínkova, M; Vohradský, J

    1997-01-01

    The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheria...

  17. Characterization of the Double-Partitioning Modules of R27: Correlating Plasmid Stability with Plasmid Localization

    OpenAIRE

    Trevor D Lawley; Taylor, Diane E.

    2003-01-01

    Plasmid R27 contains two independent partitioning modules, designated Par1 and Par2, within transfer region 2. Par1 is member of the type I partitioning family (Walker-type ATPase), and Par2 is a member of the type II partitioning family (actin-type ATPase). Stability tests of cloned Par1 and Par2 and insertional disruptions of Par1 and Par2 within R27 demonstrated that Par1 is the major stability determinant whereas Par2 is the minor stability determinant. Creation of double-partitioning mut...

  18. Partial Rescue of pos5 Mutants by YEF1 and UTR1 Genes in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    Yong-Fu LI; Feng SHI

    2006-01-01

    Three NAD kinase homologs, encoded by UTR1, POS5 and YEF1 genes, are found in the yeast Saccharomyces cerevisiae and proven to be important sources of NADPH for the cell. Pos5p, existing in the mitochondrial matrix, is critical for higher temperature endurance and mitochondrial functions, such as glycerol usability and arginine biosynthesis. Through constructing the high-copy expression plasmids of YEF1 and UTR1, which contained the green fluorescent protein reporter tag at their 3' terminus, and introducing them into POS5 gene deletion mutants (i.e. pos5, utr1pos5, yef1pos5 and utr1yef1pos5), the high-copy YEF1 and UTR1 plasmids carrying transformants for pos5 mutants were obtained. Their temperature sensitivity and growth phenotype on media with glycerol as the sole carbon source, or on media without arginine, were checked. Results showed the partial rescue of mitochondrial dysfunctions and temperature sensitivity of pos5 mutants by the high-copy YEF1 gene, and of glycerol growth defect and temperature sensitivity by the high-copy UTR1 gene, which confirmed the potential supplying ability of Yef1p and Utr1p for mitochondrial NADP(H) and implied the weak transport of NADP from cytosol to mitochondria. However, even through the green fluorescent protein reporter label, the subcellular localization of Yef1p and Utr1p in yeast cells could not be observed, which indicated the low expression level of these two NAD kinase homologs.

  19. A series of template plasmids for Escherichia coli genome engineering.

    Science.gov (United States)

    Deb, Shalini S; Reshamwala, Shamlan M S; Lali, Arvind M

    2016-06-01

    Metabolic engineering strategies often employ multi-copy episomal vectors to overexpress genes. However, chromosome-based overexpression is preferred as it avoids the use of selective pressure and reduces metabolic burden on the cell. We have constructed a series of template plasmids for λ Red-mediated Escherichia coli genome engineering. The template plasmids allow construction of genome integrating cassettes that can be used to integrate single copies of DNA sequences at predetermined sites or replace promoter regions. The constructed cassettes provide flexibility in terms of expression levels achieved and antibiotics used for selection, as well as allowing construction of marker-free strains. The modular design of the template plasmids allows replacement of genetic parts to construct new templates. Gene integration and promoter replacement using the template plasmids are illustrated. PMID:27071533

  20. Construction and Identification of Plasmid pTA-TUB2

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    An about 1.40 Kb target gene fragment was yielded by PCR amplification with the plasmid pRB 129,which was identified by restriction enzyme digestion that the PCR product was TU B2 gene.The gene was digested by the restriction enzyme and was linked with pTA plasmid to construct pTA-TU B2 plasmid.The plasmid was transformed into Chaetomium spp.by PEG method and the transformation rate was 27/(2×105) and it is nine times higher than that of pRB 129.The transformants can grow on the PDA containing 1 000 μg*mL-1 carbendazim,which is 1 000 times higher than the original Chaetomium spp.The resistance was stable after 10 times transfer on non-selective medium.

  1. Characterization of Nocardia Plasmid pXT107

    Institute of Scientific and Technical Information of China (English)

    Hai-Yang XIA; Yong-Qiang TIAN; Ran ZHANG; Kai-Chun LIN; Zhong-Jun QIN

    2006-01-01

    Nocardia, Rhodococcus and Streptornyces, all members of the actinomycetes family, are Gram-positive eubacteria with high G+C content and able to form mycelium. We report here a newly identified plasmid pXT107 of Nocardia sp. 107, one of the smallest circular plasmids found in Nocardia.The complete nucleotide sequence of pXT107 consisted of 4335 bp with 65% G+C content, and encoded one replication extragenic palindromic (Rep) and six hypothetical proteins. The Rep, double-strand origin and single-strand origin of pXT107 resembled those of typical rolling-circle-replication plasmids, such as pNI100 of Nocardia, pRE8424 of Rhodococcus and plJ101 of Streptomyces. The Escherichia coli-Nocardia shuttle plasmid pHAQ22, containing thc rep gene of pXT107, is able to propagate in Nocardia but not in Streptomyces.

  2. Single Guide RNA Library Design and Construction.

    Science.gov (United States)

    Wang, Tim; Lander, Eric S; Sabatini, David M

    2016-03-01

    This protocol describes how to generate a single guide RNA (sgRNA) library for use in genetic screens. There are many online tools available for predicting sgRNA sequences with high target specificity and/or cleavage activity. Here, we refer the user to genome-wide sgRNA sequence predictions that we have developed for both the human and mouse and that are available from the Broad Institute website. Once a set of target genes and corresponding sgRNA sequences has been identified, customized oligonucleotide pools can be rapidly synthesized by a number of commercial vendors. Thereafter, as described here, the oligonucleotides can be efficiently cloned into an appropriate lentiviral expression vector backbone. The resulting plasmid pool can then be packaged into lentiviral particles and used to generate knockouts in any cell line of choice. PMID:26933249

  3. Pharmaceutical development of the plasmid DNA vaccine pDERMATT

    OpenAIRE

    Quaak, S.G.L.

    2009-01-01

    The discovery of tumor specific antigens and self tolerance mechanisms against these antigens led to the assumption that antigens circulating at sufficient concentration levels could break this self tolerance mechanism and evoke immunological antitumor effects. pDERMATT (plasmid DNA encoding recombinant MART-1 and tetanus toxin fragment-c) is a plasmid that encodes for MART-1, a melanoma associated antigen that is expressed in a large fraction of melanomas. In animal models administration of ...

  4. Isolation and properties of plasmids from Deinococcus radiodurans Sark

    International Nuclear Information System (INIS)

    Radioresistant bacterium, Deinococcus radiodurans, can repair completely almost all of DNA damages including double strand breaks induced by gamma-rays up to about 5 kGy. In order to reveal the repair mechanism, it is necessary to develop a cloning vector available for the genetic analysis. We tried to isolate plasmids from D.radiodurans Sark strain. In the present paper the isolation and properties of plasmids were described. (author)

  5. Plasmid transfer and genetic recombination by protoplast fusion in staphylococci.

    OpenAIRE

    Götz, F; Ahrné, S.; Lindberg, M

    1981-01-01

    The experimental conditions for plasmid transfer and genetic recombination in Staphylococcus aureus and some coagulase-negative staphylococci by protoplast fusion are described. Protoplasts were prepared by treatment with lysostaphin and lysozyme in a buffered medium with 0.7 to 0.8 M sucrose. Regeneration of cell walls was accomplished on a hypertonic agar medium containing succinate and bovine serum albumin. Transfer of plasmids occurred after treatment of the protoplast mixtures with polye...

  6. Transfer of conjugative plasmids among bacteria under environmentally relevant conditions

    DEFF Research Database (Denmark)

    Musovic, Sanin

    at spredningskapacitet af en konjugerbare plasmid, der koder for kviksølv resistens via merA genet, finder sted under substrat begrænsede forhold til syntetisk bakterielt samfund. Plasmid overførsel var meget forhøjet ved kontinuert udsættelse af mikrokosms for en høj koncentration af kviksølv. De forskellige vækstrater...

  7. Construction of a bioluminescence reporter plasmid for Francisella tularensis

    OpenAIRE

    Bina, Xiaowen R.; Miller, Mark A.; James E Bina

    2010-01-01

    A Francisella tularensis shuttle vector that constitutively expresses the Photorhabdus luminescens lux operon in type A and type B strains of F. tularensis was constructed. The bioluminescence reporter plasmid was introduced into the live vaccine strain of F. tularensis and used to follow F. tularensis growth in a murine intranasal challenge model in real time by bioluminescence imaging. The results show that the new bioluminescence reporter plasmid represents a useful tool for tularemia rese...

  8. LIBRARY SURVEY 2001

    CERN Multimedia

    2001-01-01

    The primary role of the library is to make sure that you can do YOUR work in the most efficient way possible. To ensure that we continue to match our services to your information needs, the library regularly gathers the views and opinions of its readers in a variety of ways, [link for e-version: http://library/library_general/statistics/library_statistics_ surveys.html], including user surveys. The last survey was carried out in 1996. One of the most visible results of that survey was the extension of the library desk service until seven o'clock in the evening, to meet the demand for greater access to library materials. Now the 'electronic library' is becoming more important than the physical one, we feel it is once again time to ensure that we are providing the services and information you need, in the most effective way possible. We also want to make sure you are aware of the full range of services that the library provides. Please spare just a few minutes to fill out our survey at http://library.cern.ch/su...

  9. News from the Library

    CERN Multimedia

    CERN Library

    2010-01-01

    The LHC Library to be merged with the Central Library. Not everyone knows that CERN Scientific Information Service currently counts three physical libraries on site. The Central Library is located in Building 52 and there are two satellite libraries located respectively in building 30 (the LHC Library) and in building 864 on Prévessin site (the SPS Library). Moreover, the Legal Service Library is located in Building 60. In the past, there have been at CERN up to 6 satellite libraries; they were essential at a time when information was only in paper form and having multiple copies of documents located in several places at CERN was useful to facilitate scientific research. Today, this need is less critical as most of our resources are online. That is why, following a SIPB (Scientific Information Policy Board) decision, the collections of the LHC Library will be merged this summer with the Central collection. This reorganization and centralization of resources will improve loan services. The SP...

  10. Plasmid vector with temperature-controlled gene expression

    International Nuclear Information System (INIS)

    In plasmid pBR327, a fragment 169 b.p. long including promotor p3 of the bla gene has been deleted. The deletional derivative so obtained (pSP2) has been used to construct a recombinant plasmid bearing a fragment of phage λ DNA with the p/sub R/ promotor and the gene of the temperature-sensitive repressor cI. It has been shown that the plasmid vector so constructed (pCE119) with promotor cR performs repressor-cI-controlled transcription of the bla gene, as a result of which induction for an hour at 420C leads to an almost 100-fold increase in the amount of product of the bla gene as compared with that at 320C. The possibility of the use of plasmid cPE119 for the expression of other genes has been demonstrated for the case of the semisynthetic β-galactosidase gene of E. coli. In this case, on induction of the cells with recombinant plasmid pCEZ12 for 3 hours at 420C, a 300-fold increase in the amount of active β-galactosidase, as compared with that at 320C, was observed. It is important to point out that under these conditions (at 420C), at least 99% of the cells containing the plasmid retain the phenotype lacZ+, which indicates the stability of the proposed vector system

  11. Plasmid copy number noise in monoclonal populations of bacteria

    Science.gov (United States)

    Wong Ng, Jérôme; Chatenay, Didier; Robert, Jérôme; Poirier, Michael Guy

    2010-01-01

    Plasmids are extra chromosomal DNA that can confer to their hosts’ supplementary characteristics such as antibiotic resistance. Plasmids code for their copy number through their own replication frequency. Even though the biochemical networks underlying the plasmid copy number (PCN) regulation processes have been studied and modeled, no measurement of the heterogeneity in PCN within a whole population has been done. We have developed a fluorescent-based measurement system, which enables determination of the mean and noise in PCN within a monoclonal population of bacteria. Two different fluorescent protein reporters were inserted: one on the chromosome and the other on the plasmid. The fluorescence of these bacteria was measured with a microfluidic flow cytometry device. We show that our measurements are consistent with known plasmid characteristics. We find that the partitioning system lowers the PCN mean and standard deviation. Finally, bacterial populations were allowed to grow without selective pressure. In this case, we were able to determine the plasmid loss rate and growth inhibition effect.

  12. Plasmid-free T7-based Escherichia coli expression systems.

    Science.gov (United States)

    Striedner, Gerald; Pfaffenzeller, Irene; Markus, Luchner; Nemecek, Sabine; Grabherr, Reingard; Bayer, Karl

    2010-03-01

    In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid-free system was proven in chemostat cultivation for 40 generations in a non-induced and for 10 generations in a fully induced state. For this reason plasmid-free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid-free systems in upstream-processing. PMID:19891007

  13. Construction and Use of Flow Cytometry Optimized Plasmid-Sensor Strains

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Oregaard, Gunnar; Sørensen, Søren Johannes;

    2009-01-01

    stability of the plasmid is high. The method presented here relies on a phenotypic (green fluorescence protein) marker, which is switched on if the host bacteria loses the residing plasmid. The incorporation of flow cytometry for single-cell detection and discrimination between plasmid-free and plasmid...

  14. Comparative analysis of conjugative plasmids mediating gentamicin resistance in Staphylococcus aureus.

    OpenAIRE

    Goering, R. V.; Ruff, E A

    1983-01-01

    Five gentamicin-resistant clinical isolates of Staphylococcus aureus were found to contain self-transmissible plasmids of 32 to 37 megadaltons in size. Restriction endonuclease digests of the plasmids were markedly similar to those of reference plasmids of unrelated geographical origin, thus suggesting the significant contribution of common conjugal plasmids to the emergence of gentamicin resistance in S. aureus populations.

  15. Poly purine.pyrimidine sequences upstream of the beta-galactosidase gene affect gene expression in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brahmachari Samir K

    2001-10-01

    Full Text Available Abstract Background Poly purine.pyrimidine sequences have the potential to adopt intramolecular triplex structures and are overrepresented upstream of genes in eukaryotes. These sequences may regulate gene expression by modulating the interaction of transcription factors with DNA sequences upstream of genes. Results A poly purine.pyrimidine sequence with the potential to adopt an intramolecular triplex DNA structure was designed. The sequence was inserted within a nucleosome positioned upstream of the β-galactosidase gene in yeast, Saccharomyces cerevisiae, between the cycl promoter and gal 10Upstream Activating Sequences (UASg. Upon derepression with galactose, β-galactosidase gene expression is reduced 12-fold in cells carrying single copy poly purine.pyrimidine sequences. This reduction in expression is correlated with reduced transcription. Furthermore, we show that plasmids carrying a poly purine.pyrimidine sequence are not specifically lost from yeast cells. Conclusion We propose that a poly purine.pyrimidine sequence upstream of a gene affects transcription. Plasmids carrying this sequence are not specifically lost from cells and thus no additional effort is needed for the replication of these sequences in eukaryotic cells.

  16. The Library International Partnerweek 2011

    DEFF Research Database (Denmark)

    Presentation at the Library International Partnerweek, held at Copenhagen Technical Library at the Copenhagen University College of Engineering. Participant: Ms. Carmen Priesto Estravid from Madrid Technical University, E.U.I.T. Obras Públicas, Library. Spain Ms.Tuulikki Hattunen from TUAS Library....... Finland Ms. Anitta Ôrm from Kemi-Tornio UAS Library. Finland Mr. Manfred Walter from HTW-Berlin. Germany Mr. Peter Hald from Copenhagen Technical Library. Denmark Mr. Ole Micahelsen from Copenhagen Technical Library. Denmark...

  17. Libraries in Alabama: MedlinePlus

    Science.gov (United States)

    ... LibraryLibraries in Alabama URL of this page: https://medlineplus.gov/libraries/alabama.html Libraries in Alabama ... Suite 205 Birmingham, AL 35205 205-918-2130 http://www.asmi.org/library.php?page=library Birmingham ...

  18. Pleiotropic effects of heterozygosity at the mating-type locus of the yeast Saccharomyces cerevisiae on repair, recombination and transformation.

    Science.gov (United States)

    Durand, J; Birdsell, J; Wills, C

    1993-12-01

    Sexual (MAT a/alpha) and asexual (MAT a/a) strains of the yeast Saccharomyces cerevisiae, which are completely isogenic except at the MAT locus, were compared in their response to ultraviolet radiation. The effects of UV on survival, mitotic intragenic recombination, photoreactivation, and transformation efficiency with UV-irradiated plasmid DNA were examined. The sexual strain had enhanced survival and higher rates of mitotic intragenic recombination compared with the asexual strain. Exposure to visible light subsequent to irradiation increased the survival of both sexual and asexual strains, and decreased their rates of mitotic intragenic recombination. Similar results were obtained by Haladus and Zuk (1980) in their examination of sexual strains homozygous for rad6-1, and wild-type sexuals. Our sexual strain was also consistently more proficient at transforming plasmid DNA, whether that DNA had been irradiated or not. When pre-irradiated with 25 J/m2 of UV, MAT a/alpha cells transformed more efficiently than MAT a/a cells. When subsequently exposed to light, the ability of these pre-irradiated cells to transform decreased for both strains with increasing irradiation of the plasmid. A smaller decrease in transformation efficiency occurred when cells of both strains were kept in the dark. When pre-irradiated with 100 J/m2, the MAT a/alpha cells showed a 2-fold increase in their transformation efficiency of both irradiated and unirradiated plasmids by up to 2-fold, a phenomenon not seen in the MAT a/a cells even when pre-irradiated with much higher doses of UV. This increase in transformation efficiency was not, however, seen in the MAT a/alpha cells when they were exposed to visible light after UV irradiation. These results suggest that cells with the MAT a/alpha genotype have a UV-inducible system that increases the efficiency of transformation in the absence of visible light. This increase in transformation is not an induced increase in the repair of plasmid DNA

  19. The CERN Library

    CERN Multimedia

    Hester, Alec G

    1968-01-01

    Any advanced research centre needs a good Library. It can be regarded as a piece of equipment as vital as any machine. At the present time, the CERN Library is undergoing a number of modifications to adjust it to the changing scale of CERN's activities and to the ever increasing flood of information. This article, by A.G. Hester, former Editor of CERN COURIER who now works in the Scientific Information Service, describes the purposes, methods and future of the CERN Library.

  20. Gaming in the library

    OpenAIRE

    Walsh, Andrew

    2014-01-01

    This workshop will cover the uses of play, games and gamification in a library setting. Using examples such as Lemontree at the University of Huddersfield, non-digital games, particularly card games such as SEEK!, and play materials such as Lego and Playdough, we will show how these ideas can work in a practical library setting. Attendees will work hands on to consider how they can create and use play and games within their own library settings.

  1. Digital Libraries: Contents & Services

    OpenAIRE

    Nazim, Mohammad; Saraf, Sanjiv

    2005-01-01

    Explains what a digital library is and how it is designed to support access to digital contents and services. In a broad sense a digital library is simply an on-line system providing access to a wide variety of contents and services. Contents include virtually any kind of electronic material such as various kinds of electronic media (text, images, video, etc.), licensed databases of journals, articles and abstracts and description of physical collections. Digital Libraries offer different typ...

  2. Synchronization of the Budding Yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Foltman, Magdalena; Molist, Iago; Sanchez-Diaz, Alberto

    2016-01-01

    A number of model organisms have provided the basis for our understanding of the eukaryotic cell cycle. These model organisms are generally much easier to manipulate than mammalian cells and as such provide amenable tools for extensive genetic and biochemical analysis. One of the most common model organisms used to study the cell cycle is the budding yeast Saccharomyces cerevisiae. This model provides the ability to synchronise cells efficiently at different stages of the cell cycle, which in turn opens up the possibility for extensive and detailed study of mechanisms regulating the eukaryotic cell cycle. Here, we describe methods in which budding yeast cells are arrested at a particular phase of the cell cycle and then released from the block, permitting the study of molecular mechanisms that drive the progression through the cell cycle.

  3. Oscillations in glycolysis in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kloster, Antonina; Olsen, Lars Folke

    2012-01-01

    . The amplitude dependence on cell density shows the same behavior as that observed in cells in a CSTR. Furthermore, the amplitude decreases with increasing inhibition of the three ATPases (i) F0F1 ATPase, (ii) plasma membrane ATPase (Pma1p) and (iii) vacuolar ATPase (V-ATPase). The amplitude of the oscillations...... of membrane-bound ATPases . In addition we also studied a recent detailed model of glycolysis and found that, although thismodel faithfully reproduces the oscillations of glycolytic intermediates observed experimentally, it is not able to explain the role of ATPase activity on the oscillations.......Wehave investigated the glycolytic oscillations, measured as NADH autofluorescence, in the yeast Saccharomyces cerevisiae in a batch reactor. Specifically, we have tested the effect of cell density and a number of inhibitors or activators of ATPase activity on the amplitude of the oscillations...

  4. Molecular Basis for Saccharomyces cerevisiae Biofilm Development

    DEFF Research Database (Denmark)

    Andersen, Kaj Scherz

    In this study, I sought to identify genes regulating the global molecular program for development of sessile multicellular communities, also known as biofilm, of the eukaryotic microorganism, Saccharomyces cerevisiae (yeast). Yeast biofilm has a clinical interest, as biofilms can cause chronic...... infections in humans. Biofilm is also interesting from an evolutionary standpoint, as an example of primitive multicellularity. By using a genome-wide screen of yeast deletion mutants, I show that 71 genes are essential for biofilm formation. Two-thirds of these genes are required for transcription of FLO11......, but only a small subset is previously described as regulators of FLO11. These results reveal that the regulation of biofilm formation and FLO11 is even more complex than what has previously been described. I find that the molecular program for biofilm formation shares many essential components with two...

  5. Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction.

    OpenAIRE

    Orbach, M J; Jackson, E N

    1982-01-01

    Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls int...

  6. Tyrosine Partners Coordinate DNA Nicking by the Salmonella typhimurium Plasmid pCU1 Relaxase Enzyme

    OpenAIRE

    Nash, Rebekah P.; Niblock, Franklin C.; Redinbo, Matthew R.

    2011-01-01

    Conjugative plasmid transfer results in the spread of antibiotic resistance genes and virulence factors between bacterial cells. Plasmid transfer is dependent upon the DNA nicking activity of a plasmid-encoded relaxase enzyme. Tyrosine residues within the relaxase cleave the DNA plasmid nic site in a highly sequence-specific manner. The conjugative resistance plasmid pCU1 encodes a relaxase with four tyrosine residues surrounding its active site (Y18,19,26,27). We use activity assays to demon...

  7. Linear Plasmid SLP2 Is Maintained by Partitioning, Intrahyphal Spread, and Conjugal Transfer in Streptomyces▿

    OpenAIRE

    Hsu, Chin-Chen; Chen, Carton W.

    2009-01-01

    Low-copy-number plasmids generally encode a partitioning system to ensure proper segregation after replication. Little is known about partitioning of linear plasmids in Streptomyces. SLP2 is a 50-kb low-copy-number linear plasmid in Streptomyces lividans, which contains a typical parAB partitioning operon. In S. lividans and Streptomyces coelicolor, a parAB deletion resulted in moderate plasmid loss and growth retardation of colonies. The latter was caused by conjugal transfer from plasmid-co...

  8. Probing glycolytic and membrane potential oscillations in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Poulsen, Allan K.; Andersen, Ann Zahle; Brasen, Jens Christian;

    2008-01-01

    We have investigated glycolytic oscillations under semi-anaerobic conditions in Saccharomyces cerevisiae by means of NADH fluorescence, measurements of intracellular glucose concentration, and mitochondrial membrane potential. The glucose concentration was measured using an optical nanosensor, wh...

  9. Large-Scale Overexpression and Purification of ADARs from Saccharomyces cerevisiae for Biophysical and Biochemical Studies

    Science.gov (United States)

    Macbeth, Mark R.; Bass, Brenda L.

    2008-01-01

    Many biochemical and biophysical analyses of enzymes require quantities of protein that are difficult to obtain from expression in an endogenous system. To further complicate matters, native adenosine deaminases that act on RNA (ADARs) are expressed at very low levels, and overexpression of active protein has been unsuccessful in common bacterial systems. Here we describe the plasmid construction, expression, and purification procedures for ADARs overexpressed in the yeast Saccharomyces cerevisiae. ADAR expression is controlled by the Gal promoter, which allows for rapid induction of transcription when the yeast are grown in media containing galactose. The ADAR is translated with an N-terminal histidine tag that is cleaved by the tobacco etch virus protease, generating one nonnative glycine residue at the N-terminus of the ADAR protein. ADARs expressed using this system can be purified to homogeneity, are highly active in deaminating RNA, and are produced in quantities (from 3 to 10 mg of pure protein per liter of yeast culture) that are sufficient for most biophysical studies. PMID:17662848

  10. A Synthetic Interaction between CDC20 and RAD4 in Saccharomyces cerevisiae upon UV Irradiation

    Directory of Open Access Journals (Sweden)

    Bernadette Connors

    2014-01-01

    Full Text Available Regulation of DNA repair can be achieved through ubiquitin-mediated degradation of transiently induced proteins. In Saccharomyces cerevisiae, Rad4 is involved in damage recognition during nucleotide excision repair (NER and, in conjunction with Rad23, recruits other proteins to the site of damage. We identified a synthetic interaction upon UV exposure between Rad4 and Cdc20, a protein that modulates the activity of the anaphase promoting complex (APC/C, a multisubunit E3 ubiquitin ligase complex. The moderately UV sensitive Δrad4 strain became highly sensitive when cdc20-1 was present, and was rescued by overexpression of CDC20. The double mutant is also deficient in elicting RNR3-lacZ transcription upon exposure to UV irradiation or 4-NQO compared with the Δrad4 single mutant. We demonstrate that the Δrad4/cdc20-1 double mutant is defective in double strand break repair by way of a plasmid end-joining assay, indicating that Rad4 acts to ensure that damaged DNA is repaired via a Cdc20-mediated mechanism. This study is the first to present evidence that Cdc20 may play a role in the degradation of proteins involved in nucleotide excision repair.

  11. Large-scale overexpression and purification of ADARs from Saccharomyces cerevisiae for biophysical and biochemical studies.

    Science.gov (United States)

    Macbeth, Mark R; Bass, Brenda L

    2007-01-01

    Many biochemical and biophysical analyses of enzymes require quantities of protein that are difficult to obtain from expression in an endogenous system. To further complicate matters, native adenosine deaminases that act on RNA (ADARs) are expressed at very low levels, and overexpression of active protein has been unsuccessful in common bacterial systems. Here we describe the plasmid construction, expression, and purification procedures for ADARs overexpressed in the yeast Saccharomyces cerevisiae. ADAR expression is controlled by the Gal promoter, which allows for rapid induction of transcription when the yeast are grown in media containing galactose. The ADAR is translated with an N-terminal histidine tag that is cleaved by the tobacco etch virus protease, generating one nonnative glycine residue at the N-terminus of the ADAR protein. ADARs expressed using this system can be purified to homogeneity, are highly active in deaminating RNA, and are produced in quantities (from 3 to 10mg of pure protein per liter of yeast culture) that are sufficient for most biophysical studies.

  12. Heterologous Expression of Amylase Gene from Saccharomycopsis fibuligera in an Industrial Strain of Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    LIU Zeng-ran; ZHANG Guang-yi; LONG Zhang-fu; LIU Shi-gui

    2005-01-01

    An α-amylase encoding gene was amplified by polymerase chain reaction from Saccharomycopsis fibuligera and inserted into a shuttle vector YEp352,together with the yeast phosphoglycerate kinase 1 promoter and α-factor signal gene. The recombinant expression plasmid pLA8α was transformed into an industrial strain of Saccharomyces cerevisiae Sc-11. The activity of the α-amylase produced by the transformant Sc-11-pLA8α was 6.3 U/mL and the starch utilization rate in YPS medium was 42 %. The purified amylase was analyzed by SDS-PAGE,showing a molecular weight of 55×103 protein band. Furthermore, the residual sugar, ethanol and some volatile compounds in the fermented worts under simulating brewing conditions were determined by chromatographic analyses. The fermentation characteristics of Sc-11-pLA8α were similar to that of Sc-11 and only minor changes in the concentration of flavor compounds could be observed.

  13. Biosorption of 241Am by immobilized Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Americium-241 is one of the most serious radioactive contaminating nuclides due to its high toxicity and long half-life. The encouraging biosorption of 241Am from aqueous solutions by free Saccharomyces cerevisiae (S. cerevisiae) has been observed in our previous experiments. 241Am biosorption by immobilized S. cerevisiae and the effect of the various experimental conditions on the adsorption were investigated. The results indicated that the 241Am biosorption by immobilized S. cerevisiae is still very efficient, and immobilized S. cerevisiae can be used repeatedly or continuously. The biosorption equilibrium was achieved within 2 hours, and more than 92% of 241Am was removed by immobilized S. cerevisiae in the pH 1-4 range. No significant differences in 241Am biosorption were observed at 15-45 deg C. The immobilized S. cerevisiae, even after used repeatedly for 6 times, still could adsorb more than 90% of 241Am in solutions of 1.08 MBq/l (8.5 μg/l). At this moment, the total adsorption capacity for 241Am was more than 63.3 KBq/g globe (0.5 μg/g), but has not reached saturation yet. The 241Am left in solutions with initial concentration of 1.08 MBq/l (8.5 μg/l) was noted as low as ∼10 Bq/l (∼8.0 x 10-5 μg/l) after adsorption by the immobilized S. cerevisiae for 3 times. (author)

  14. Research on biosorption of uranium by saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The effects of pH and the granularity of S. cerevisiae on the biosorption capacity were examined in order to study the properties of the biosorption of uranium from effluent by Saccharomyces cerevisiae. The isotherm was drawn. From the isotherm, the equations of Langmuir and Freundlich were achieved. The results showed the highest biosorption capacity was obtained when the pH value was about 6 and the granularity was 0.15-0.13 mm

  15. FCLib: The Feature Characterization Library.

    Energy Technology Data Exchange (ETDEWEB)

    Gentile, Ann C.; Doyle, Wendy S. K.; Kegelmeyer, W. Philip [Sandia National Laboratories, Livermore, CA; Ulmer, Craig D. [Sandia National Laboratories, Livermore, CA

    2008-11-01

    The Feature Characterization Library (FCLib) is a software library that simplifies the process of interrogating, analyzing, and understanding complex data sets generated by finite element applications. This document provides an overview of the library, a description of both the design philosophy and implementation of the library, and examples of how the library can be utilized to extract understanding from raw datasets.

  16. An Leabharlann : The Irish Library

    OpenAIRE

    Sliney, Marjory

    2010-01-01

    Academic libraries in Challenging Times / John Cox; Public Libraries and the Recession / Liam Ronayne; Social Networks / Jane Burns; Poetry Promotion in Public libraries / Brian Hegarty & Clare Thornley; Conference Reports: Conference on Conceptions of Library and Information Sciencw / Eva Hornung; LILAC 2010 / Aoife Geraghty; Books: Acquisitions; Information Policy; Academic Libraries; Marketing; Student Research Support; News from the Stacks; Obituary – Bob McKee

  17. Plasmids and rickettsial evolution: insight from Rickettsia felis.

    Directory of Open Access Journals (Sweden)

    Joseph J Gillespie

    Full Text Available BACKGROUND: The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG or spotted fever group (SFG rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives also occur in AG (but not SFG rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFdelta, is an artifact of the original genome assembly. CONCLUSION/SIGNIFICANCE: Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in

  18. Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains

    Directory of Open Access Journals (Sweden)

    Hana Šuranská

    2016-03-01

    Full Text Available Abstract In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were employed to differentiate among indigenous S. cerevisiae strains. This combination of the methods provides a rapid and relatively simple approach for identification of yeast of S. cerevisiae at strain level. In total, 120 isolates were identified and grouped by molecular approaches and 45 of the representative strains were tested for selected important oenological properties including ethanol, sulfur dioxide and osmotic stress tolerance, intensity of flocculation and desirable enzymatic activities. Their ability to produce and utilize acetic/malic acid was examined as well; in addition, H2S production as an undesirable property was screened. The oenological characteristics of indigenous isolates were compared to a commercially available S. cerevisiae BS6 strain, which is commonly used as the starter culture. Finally, some indigenous strains coming from organically treated grape berries were chosen for their promising oenological properties and these strains will be used as the starter culture, because application of a selected indigenous S. cerevisiae strain can enhance the regional character of the wines.

  19. In vitro screening of probiotic properties of Saccharomyces cerevisiae var. boulardii and food-borne Saccharomyces cerevisiae strains

    DEFF Research Database (Denmark)

    van der Aa Kuhle, Alis; Skovgaard, Kerstin; Jespersen, Lene

    2005-01-01

    .6-16.8%) recorded for two isolates from blue veined cheeses. Merely 25% of the S. cerevisiae var. boulardii strains displayed good adhesive properties (16.2-28.0%). The expression of the proinflammatory cytokine IL-1α decreased strikingly in IPEC-J2 cells exposed to a Shiga-like toxin 2e producing Escherichia coli...... strain when the cells were pre- and coincubated with S. cerevisiae var. boulardii even though this yeast strain was low adhesive (5.4%), suggesting that adhesion is not a mandatory prerequisite for such a probiotic effect. A strain of S. cerevisiae isolated from West African sorghum beer exerted similar...... effects hence indicating that food-borne strains of S. cerevisiae may possess probiotic properties in spite of low adhesiveness. © 2004 Elsevier B.V. All rights reserved....

  20. Libraries at the Ready

    Science.gov (United States)

    Celano, Donna C.; Neuman, Susan B.

    2016-01-01

    Because English language learners enter kindergarten at a distinct disadvantage, Celano and Neuman examine the role public libraries can play in rallying around these young children to better prepare them for school. The authors document a new program called Every Child Ready to Read, which recently launched in 4,000 public libraries across the…

  1. Small Public Library Management

    Science.gov (United States)

    Pearlmutter, Jane; Nelson, Paul

    2012-01-01

    Anyone at the helm of a small public library knows that every little detail counts. But juggling the responsibilities that are part and parcel of the job is far from easy. Finally, here's a handbook that includes everything administrators need to keep a handle on library operations, freeing them up to streamline and improve how the organization…

  2. Library Classification 2020

    Science.gov (United States)

    Harris, Christopher

    2013-01-01

    In this article the author explores how a new library classification system might be designed using some aspects of the Dewey Decimal Classification (DDC) and ideas from other systems to create something that works for school libraries in the year 2020. By examining what works well with the Dewey Decimal System, what features should be carried…

  3. Library Censorship after Webster.

    Science.gov (United States)

    Lee, Earl

    1989-01-01

    Discusses the potential of the Supreme Court's recent decision in Webster v. Reproductive Health Services, which upheld the Missouri anti-abortion statutes, to encourage state sponsored fiscal censorship in libraries. The types of library services and materials that might be affected are identified, and the First Amendment implications are…

  4. Dumping the "Library."

    Science.gov (United States)

    Crowley, Bill

    1998-01-01

    Discusses an alternative to abandoning the word "library" for "information" in graduate education. Recommends patient, consistent effort by library- and information-science educators to convince academic librarians that if they accept the standards of the teaching/research faculty, including the need to earn a Ph.D., it will raise the prestige of…

  5. Libraries on the Way

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    An NGO campaigns to help rural students find the joy of reading Tan Li joined the Gan Quan Library,a Trural library in Chengde,north China’s Hebei Province,after graduating from the Shanxi University of Finance and Economics in July.

  6. Attribution of Library Costs

    Science.gov (United States)

    Drake, Miriam A.

    1977-01-01

    Universities conduct a variety of cost-allocation studies that require the collection and analysis of the library cost-data. Cost accounting methods are used in most studies; however, costs are attributed to library user groups in a variety of ways. Cost accounting studies are reviewed and allocation methods are discussed. (Author)

  7. University Libraries in Transition.

    Science.gov (United States)

    Hyatt, James A.

    1986-01-01

    College and university libraries are experiencing change in the ways they provide services and in their responses to rising costs and reduced financial support. These conditions result from three major phenomena: the information explosion, the technology revolution, and escalating library costs. (MLW)

  8. Facility Focus: Libraries.

    Science.gov (United States)

    College Planning & Management, 1997

    1997-01-01

    Describes four operating libraries located at Union Theological Seminary in Virginia, Emory University in Georgia, Kean College in New Jersey, and Community College of Philadelphia. Their size, cost, date completed, architect, and various features are provided. Despite technological advances, the college library is a feasible part of higher…

  9. Libraries for Small Museums.

    Science.gov (United States)

    Anderson, Linda M.

    Presented are the very basic requirements for establishing a small special library operating under a limited budget. Physical plant organization, cataloging, book processing, circulation procedures, book selection and ordering and instructions for typists are covered. Although the practices discussed were established for a museum library, what is…

  10. Homelessness in Public Libraries

    Science.gov (United States)

    Wong, Yi Ling

    2009-01-01

    This paper takes a theoretical and practical approach in defining the "problem" of homelessness in libraries. The author examines three fundamental problems on homelessness. The three fundamental questions are: (a) Who are the homeless? (b) Why are they homeless? (c) What are their information needs in libraries? These questions are important in…

  11. Financing Public Library Construction.

    Science.gov (United States)

    Rohlf, Robert H.; Stoffel, Lester L.

    1987-01-01

    Reviews financing options available to Illinois public libraries for construction or expansion, including general obligation bonds, mortgage funds, building reserve funds, building fund levies, lease back arrangements, sale of air or ground development rights, interest on special funds, gift funds and grants, Library Service and Construction Act…

  12. The Undergraduate Library Collection.

    Science.gov (United States)

    Stewart, Rolland C.

    The development of undergraduate library collections is shown under two aspects: (1) the formation of the basic collection of the Undergraduate Library of the University of Michigan, and (2) the problems, practical and theoretial, encountered in the day-to-day effort to maintain the collection. The budget is the sire of all selection criteria.…

  13. Maintaining Precious Library Resources.

    Science.gov (United States)

    Wiens, Janet

    2002-01-01

    Using the examples of libraries at Southeast Missouri State University and the University of North Texas, discusses the big-picture approach and extensive communication among all users necessary for library maintenance efforts. Addresses establishing a master plan, prioritizing projects, having the right staff, and communicating. (EV)

  14. The Memory Library

    DEFF Research Database (Denmark)

    Olesen-Bagneux, Ole

    2014-01-01

    of classification and retrieval processes is presented. The key element is to understand the library both as a physical structure and as a structure in the memory of the Alexandrian scholars. In this article, these structures are put together so to propose a new interpretation of the library....

  15. Library Reference Services.

    Science.gov (United States)

    Miller, Constance; And Others

    1985-01-01

    Seven articles on library reference services highlight reference obsolescence in academic libraries, major studies of unobtrusive reference tests, methods for evaluating reference desk performance, reference interview evaluation, problems of reference desk control, online searching by end users, and reference collection development in…

  16. Global Warming's Library Challenge

    Science.gov (United States)

    Meyer, Jennifer

    2008-01-01

    Like every institution that uses energy, consumes resources, and engages in construction or renovation, libraries have an impact on the environment and on the critical problem of climate change. Taking action to protect library collections is not only an idealistic professional goal but also a very practical one. Disaster preparation measures and…

  17. Academic Libraries in Japan

    Science.gov (United States)

    Cullen, Rowena; Nagata, Haruki

    2008-01-01

    Academic libraries in Japan are well resourced by international standards, and support Japan's internationally recognized research capability well, but there are also ways in which they reflect Japan's strong bureaucratic culture. Recent changes to the status of national university libraries have seen a new interest in customer service, and…

  18. Prison Libraries: Bibliography.

    Science.gov (United States)

    Gillespie, David M.

    An Alphabetically arranged bibliography, listing 485 entries representing 518 citations taken from "Poole's Index to Periodical Literature" 1802-1906, "Cannon's Bibliography of Library Economy," and "Library Literature" 1921 to mid-1970. Other sources used include: The Index to the Journal of Correctional Education; ten bibliographies taken from…

  19. Library Consortia in Hungary

    Science.gov (United States)

    Csajbok, Edit; Szluka, Peter; Vasas, Livia

    2012-01-01

    During the last two decades many Hungarian libraries have developed considerably, beyond what was considered possible prior to 1989 and the beginning of events signaling the end of Communism in the country. Some of the modernization of library services has been realized through participation in cooperative agreements. Many smaller and larger…

  20. Library Automation Style Guide.

    Science.gov (United States)

    Gaylord Bros., Liverpool, NY.

    This library automation style guide lists specific terms and names often used in the library automation industry. The terms and/or acronyms are listed alphabetically and each is followed by a brief definition. The guide refers to the "Chicago Manual of Style" for general rules, and a notes section is included for the convenience of individual…

  1. Plasma-activated air mediates plasmid DNA delivery in vivo.

    Science.gov (United States)

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Bulysheva, Anna; Heller, Richard

    2016-01-01

    Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets. PMID:27110584

  2. Improvement of acetic acid tolerance of Saccharomyces cerevisiae using a zinc-finger-based artificial transcription factor and identification of novel genes involved in acetic acid tolerance.

    Science.gov (United States)

    Ma, Cui; Wei, Xiaowen; Sun, Cuihuan; Zhang, Fei; Xu, Jianren; Zhao, Xinqing; Bai, Fengwu

    2015-03-01

    Acetic acid is present in cellulosic hydrolysate as a potent inhibitor, and the superior acetic acid tolerance of Saccharomyces cerevisiae ensures good cell viability and efficient ethanol production when cellulosic raw materials are used as substrates. In this study, a mutant strain of S. cerevisiae ATCC4126 (Sc4126-M01) with improved acetic acid tolerance was obtained through screening strains transformed with an artificial zinc finger protein transcription factor (ZFP-TF) library. Further analysis indicated that improved acetic acid tolerance was associated with improved catalase (CAT) activity. The ZFP coding sequence associated with the improved phenotype was identified, and real-time RT-PCR analysis revealed that three of the possible genes involved in the enhanced acetic acid tolerance regulated by this ZFP-TF, namely YFL040W, QDR3, and IKS1, showed decreased transcription levels in Sc4126-M01 in the presence of acetic acid, compared to those in the control strain. Sc4126-M01 mutants having QDR3 and IKS1 deletion (ΔQDR3 and ΔIKS1) exhibited higher acetic acid tolerance than the wild-type strain under acetic acid treatment. Glucose consumption rate and ethanol productivity in the presence of 5 g/L acetic acid were improved in the ΔQDR3 mutant compared to the wild-type strain. Our studies demonstrated that the synthetic ZFP-TF library can be used to improve acetic acid tolerance of S. cerevisiae and that the employment of an artificial transcription factor can facilitate the exploration of novel functional genes involved in stress tolerance of S. cerevisiae. PMID:25698512

  3. Free Library Data?

    Directory of Open Access Journals (Sweden)

    Raymond Bérard

    2011-02-01

    Full Text Available As library materials are catalogued by public organisations and librarians are active promoters of the principles of open access, one would expect library data to be freely available to all. Yet this is not the case. Why then do so few libraries make their data available free of charge? This article reviews the diverging, often restrictive policies and the interests (commercial and strategic at stake. It presents a panorama of the current situation, the actors and interests involved. It addresses the legal aspects and the obstacles and it shows how data produced by libraries can be made freely available to other knowledge organisations while retaining and developing the collective organisations and services built by library networks over the years. The aim of the ‘free the data movement’ is to share and reuse bibliographic data in a new ecosystem where all the actors are involved, both users and providers, not just librarians.

  4. Las Campanas Stellar Library

    Science.gov (United States)

    Chilingarian, Igor; Zolotukhin, Ivan; Beletsky, Yuri; Worthey, Guy

    2015-08-01

    Stellar libraries are fundamental tools required to understand stellar populations in star clusters and galaxies as well as properties of individual stars. Comprehensive libraries exist in the optical domain, but the near-infrared (NIR) domain stays a couple of decades behind. Here we present the Las Campanas Stellar Library project aiming at obtaining high signal-to-noise intermediate-resolution (R=8000) NIR spectra (0.83library the largest homogeneous collection of stellar spectra covering the entire NIR domain. We also re-calibrated in flux and wavelength the two existing optical stellar libraries, INDO-US and UVES-POP and followed up about 400 non-variable stars in the NIR in order to get complete optical-NIR coverage. Worth mentioning that our current sample includes about 80 AGB stars and a few dozens of bulge/LMC/SMC stars.

  5. Motivation and library management

    Directory of Open Access Journals (Sweden)

    Tatjana Likar

    2000-01-01

    Full Text Available The present article deals with motivation, its relation to management and its role and use in librarianship in our country and abroad. The countries where librarianship is well developed started to deal with library management and questions of motivation of library workers decades ago, whereas elsewhere the subject is at its start. The prerequisite for modern policy making is attention to the elements of modern library management. Librarians, library managers and directors of libraries should create a work environment providing long term satisfaction with work by means of certain knowledge and tools. The level of motivation of the staff is influenced by the so called higher factors deriving from the work process itself and related to work contents: achieve¬ment, recognition, trust and work itself. Extrinsic factors (income, interpersonal relations, technology of administration, company policy, working conditions, work con¬trol, personal security, job security and position... should exercise lesser impact on the level of motivation.

  6. Library Legislation: Some General Considerations

    Science.gov (United States)

    Ladenson, Alex

    1970-01-01

    Library service has become a concern of government at all levels with each having its specific role to play. This introductory statement to this issue of Library Trends" indicates the major substantive areas of library legislation. (Author/NH)

  7. Lest We Forget: Prison Libraries.

    Science.gov (United States)

    Koons, Phil

    1988-01-01

    This discussion of prison library programs in Ohio covers: (1) financial support; (2) the nature of prison library work; (3) a typical day in the library; and (4) rewards of prison librarianship. (1 reference) (MES)

  8. Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae

    DEFF Research Database (Denmark)

    Johnson, Timothy J.; Bielak, Eliza Maria; Fortini, Daniela;

    2012-01-01

    and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid......, based upon these sequences and subtypes, was then developed. Use of this revised typing procedure revealed that IncX plasmid occurrence among bacterial populations is much more common than had previously been acknowledged. Thus, this revised procedure can be used to better discern the occurrence of Inc...

  9. Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

    DEFF Research Database (Denmark)

    Reisner, A.; Molin, Søren; Zechner, E.L.

    2002-01-01

    An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage lambda recombination...... functions using very short regions of homology. Initial manipulation of the IncFII target plasmids R1 and R1drd19 indicated that the linear targeting DNA should be devoid of all extraneous homologies to. the target molecule for optimal insertion specificity. Indeed, a simple recombination assay proved...... resistance genes and fluorescent markers. The choice of 5' non-homologous extensions in primer pairs used for amplifying the marker cassettes determines the site specificity of the targeting DNA. This methodology is applicable to the modification of all plasmids that replicate in E coli and is not restricted...

  10. A novel plasmid pEA68 of Erwinia amylovora and the description of a new family of plasmids.

    Science.gov (United States)

    Ismail, Emadeldeen; Blom, Jochen; Bultreys, Alain; Ivanović, Milan; Obradović, Aleksa; van Doorn, Joop; Bergsma-Vlami, Maria; Maes, Martine; Willems, Anne; Duffy, Brion; Stockwell, Virginia O; Smits, Theo H M; Puławska, Joanna

    2014-12-01

    Recent genome analysis of Erwinia amylovora, the causal agent of fire blight disease on Rosaceae, has shown that the chromosome is highly conserved among strains and that plasmids are the principal source of genomic diversity. A new circular plasmid, pEA68, was found in E. amylovora strain 692 (LMG 28361), isolated in Poland from Sorbus (mountain ash) with fire blight symptoms. Annotation of the 68,763-bp IncFIIa-type plasmid revealed that it contains 79 predicted CDS, among which two operons (tra, pil) are associated with mobility. The plasmid is maintained stably in E. amylovora and does not possess genes associated with antibiotic resistance or known virulence genes. Curing E. amylovora strain 692 of pEA68 did not influence its virulence in apple shoots nor amylovoran synthesis. Of 488 strains of E. amylovora from seventeen countries, pEA68 was only found in two additional strains from Belgium. Although the spread of pEA68 is currently limited to Europe, pEA68 comprises, together with pEA72 and pEA78 both found in North America, a new plasmid family that spans two continents.

  11. Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence

    NARCIS (Netherlands)

    van Diepeningen, Anne D; Debets, Alfons J M; Slakhorst, S Marijke; Hoekstra, Rolf F

    2008-01-01

    Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with t

  12. Anion exchange purification of plasmid DNA using expanded bed adsorption.

    Science.gov (United States)

    Ferreira, G N; Cabral, J M; Prazeres, D M

    2000-01-01

    Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH = 8.0) buffer at an upward flow of 300 cmh-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh-1. Purification factors of 36 +/- 1 fold, 26 +/- 0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed-values of 35 +/- 2 and 5 +/- 0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.

  13. The development of plasmid-free strains of Agrobacterium tumefaciens by using incompatibility with a Rhizobium meliloti plasmid to eliminate pAtC58.

    Science.gov (United States)

    Hynes, M F; Simon, R; Pühler, A

    1985-03-01

    Agrobacterium tumefaciens strains LBA275 and LBA290 were cured of their cryptic plasmid pAtC58 by the introduction of the Rhizobium meliloti plasmid pRme41a, which is incompatible with pAtC58. pRme41a and pTiC58, the resident Ti plasmid of LBA275, were subsequently eliminated by growth at supraoptimal temperature (40 degrees C). The resulting plasmid-free Agrobacterium strains, UBAPF1 and UBAPF2, have proved extremely useful for the study of Rhizobium plasmids. The loss of the cryptic plasmid pAtC58 has no effect on the tumor-forming ability of the Agrobacterium strains; when the Ti plasmid is present, normal tumors are formed on Kalanchoe daigremontiana. PMID:4001194

  14. Libraries Today, Libraries Tomorrow: Contemporary Library Practices and the Role of Library Space in the L

    Directory of Open Access Journals (Sweden)

    Ana Vogrinčič Čepič

    2013-09-01

    Full Text Available ABSTRACTPurpose: The article uses sociological concepts in order to rethink the changes in library practices. Contemporary trends are discussed with regard to the changing nature of working habits, referring mostly to the new technology, and the (emergence of the third space phenomenon. The author does not regard libraries only as concrete public service institutions, but rather as complex cultural forms, taking in consideration wider social context with a stress on users’ practices in relation to space.Methodology/approach: The article is based on the (self- observation of the public library use, and on the (discourse analysis of internal library documents (i.e. annual reports and plans and secondary sociological literature. As such, the cultural form approach represents a classic method of sociology of culture.Results: The study of relevant material in combination with direct personal experiences reveals socio-structural causes for the change of users’ needs and habits, and points at the difficulty of spatial redefinition of libraries as well as at the power of the discourse.Research limitations: The article is limited to an observation of users’ practices in some of the public libraries in Ljubljana and examines only a small number of annual reports – the discoveries are then further debated from the sociological perspective.Originality/practical implications: The article offers sociological insight in the current issues of the library science and tries to suggest a wider explanation that could answer some of the challenges of the contemporary librarianship.

  15. Cloning vectors for expression of cDNA libraries in mammalian cells.

    OpenAIRE

    Murphy, A.J.; Efstratiadis, A

    1987-01-01

    We have constructed a series of compound cloning vectors (lambda ZD vectors), each consisting of phage lambda arms carrying a modified version of the retroviral expression vector pZIP-neoSV (x)1. cDNA, inserted into a cloning site present in the retroviral vector component, is cloned with high efficiency using the lambda system. A cDNA library in plasmids is then released by homologous recombination between the retroviral long terminal repeats. Retroviral transduction is achieved by transient...

  16. Characterization of atypical Aeromonas salmonicida isolates by ribotyping and plasmid profiling

    DEFF Research Database (Denmark)

    Pedersen, Karl; Dalsgaard, Inger; Larsen, J.L.

    1996-01-01

    ) and plasmid profiles. Most epidemiologically unrelated strains had different ribotypes, whereas isolates from the same outbreak were identical. All strains, except one, carried one or more large plasmids (>55 kbp) and all strains, except two, additionally carried one or more smaller plasmids. Many strains...... isolated from the same outbreak showed different plasmid profiles although some plasmids were identical. The results suggest the existence of several atypical Aer, salmonicida. It also seems that ribotypes are stable properties for these bacteria while the plasmids are more labile....

  17. Restriction Fragment Length Polymorphisms of Virulence Plasmids in Rhodococcus equi

    OpenAIRE

    Takai, Shinji; Shoda, Masato; Sasaki, Yukako; Tsubaki, Shiro; Fortier, Guillaume; Pronost, Stephane; Rahal, Karim; Becu, Teotimo; Begg, Angela; Browning, Glenn; Nicholson, Vivian M.; Prescott, John F.

    1999-01-01

    Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries—Argentina, Australia, Canada, France, and Japan—were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested...

  18. Sustained plasmid DNA release from dissolving mineral coatings

    OpenAIRE

    Choi, Siyoung; Murphy, William L.

    2010-01-01

    Calcium phosphate (Ca-P) minerals such as hydroxyapatite are able to bind a diverse range of biological molecules due to the presence of anions and cations in their crystal structure. The well-characterized ability of Ca-P minerals to bind and release plasmid DNA, coupled with the ability of biodegradable Ca-P coatings to form on the surface of common biomaterials, provides a potential mechanism for controlled release of plasmid DNA from various biomaterials. In this study we hypothesized tha...

  19. Marketing the hospital library.

    Science.gov (United States)

    Bridges, Jane

    2005-01-01

    Many librarians do not see themselves as marketers, but marketing is an essential role for hospital librarians. Library work involves education, and there are parallels between marketing and education as described in this article. It is incumbent upon hospital librarians actively to pursue ways of reminding their customers about library services. This article reinforces the idea that marketing is an element in many of the things that librarians already do, and includes a list of suggested marketing strategies intended to remind administrators, physicians, and other customers that they have libraries in their organizations. PMID:15982957

  20. Gender, Technology, and Libraries

    OpenAIRE

    Lamont, Melissa

    2009-01-01

    Information technology (IT) is vitally important to many organizations, including libraries. Yet a review of employment statistics and a citation analysis show that men make up the majority of the IT workforce, in libraries and in the broader workforce. Research from sociology, psychology, and women’s studies highlights the organizational and social issues that inhibit women. Understanding why women are less evident in library IT positions will help inform measures to remedy the gender dispar...

  1. CRNL library serials list

    International Nuclear Information System (INIS)

    A list of 1900 serial publications (periodicals, society transactions and proceedings, annuals and directories, indexes, newspapers, etc.) is presented with volumes and years held by the Main Library. This library is the largest in AECL as well as one of the largest scientific and technical libraries in North America, and functions as a Canadian resource for nuclear information. A main alphabetical list is followed by broad subject field lists representing research interests, and lists of abstract and index serials, general bibliographic serials, conference indexes, press releases, English translations, and original language journals

  2. Co-expression of a Saccharomyces diastaticus glucoamylase-encoding gene and a Bacillus amyloliquefaciens alpha-amylase-encoding gene in Saccharomyces cerevisiae.

    Science.gov (United States)

    Steyn, A J; Pretorius, I S

    1991-04-01

    A glucoamylase-encoding gene (STA2) from Saccharomyces diastaticus and an alpha-amylase-encoding gene (AMY) from Bacillus amyloliquefaciens were cloned separately into a yeast-integrating shuttle vector (YIp5), generating recombinant plasmids pSP1 and pSP2, respectively. The STA2 and AMY genes were jointly cloned into YIp5, generating plasmid pSP3. Subsequently, the dominant selectable marker APH1, encoding resistance to Geneticin G418 (GtR), was cloned into pSP3, resulting in pSP4. For enhanced expression of GtR, the APH1 gene was fused to the GAL10 promoter and terminated by the URA3 terminator, resulting in pSP5. Plasmid pSP5 was converted to a circular minichromosome (pSP6) by the addition of the ARS1 and CEN4 sequences. Laboratory strains of Saccharomyces cerevisiae transformed with plasmids pSP1 through pSP6, stably produced and secreted glucoamylase and/or alpha-amylase. Brewers' and distillers' yeast transformed with pSP6 were also capable of secreting amylolytic enzymes. Yeast transformants containing pSP1, pSP2 and pSP3 assimilated soluble starch with an efficiency of 69%, 84% and 93%, respectively. The major starch hydrolysis products produced by crude amylolytic enzymes found in the culture broths of the pSP1-, pSP2- and pSP3-containing transformants, were glucose, glucose and maltose (1:1), and glucose and maltose (3:1), respectively. These results confirmed that co-expression of the STA2 and AMY genes synergistically enhanced starch degradation.

  3. Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyes cerevisiae improves ethanol production

    DEFF Research Database (Denmark)

    Roca, Christophe Francois Aime; Nielsen, Jens; Olsson, Lisbeth

    2003-01-01

    Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase...

  4. Arsenate and phosphate interaction in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    GENG Chun-nu; ZHU Yong-guan

    2006-01-01

    In the present study, arsenate(As(Ⅴ)) and phosphate(P(Ⅴ)) interactions were investigated in growth, uptake and RNA content in yeast(Saccharomyces cerevisiae). Yeast grew slowly with As(Ⅴ) concentrations increasing in the medium. However, the maximal population density was almost the same among different As(Ⅴ) treatments. It was in the late log phase that yeast growth was augmented by low As(Ⅴ), which was maybe due to the fact that methionine metabolism was stressed by vitamin B6 deprivation, so As(Ⅴ)treatments did not affect maximal population density. However, with P (Ⅴ) concentrations increasing, the maximal population density increased. Therefore, the maximal population density was determined by P (Ⅴ) concentrations in the medium but not by As (Ⅴ)concentrations in the medium. Ycf1p(a tonoplast transpor) transports As(GS)3 into the vacuole, but arsenic(As) remaining in the thalli was 1.27% with As(Ⅴ) exposure for 60 h, from which it can be speculated that the percentage of As transported into vacuole should be lower than 1.27%. However, the percentage of As pumped out of cell was 71.49% with As (Ⅴ) exposure for 68 h. Although two pathways (extrusion and sequestration) were involved in As detoxification in yeast, the extrusion pathway played a major role in As detoxification. RNA content was the highest in the early-log phase and was reduced by As(Ⅴ).

  5. Functional profiling of the Saccharomyces cerevisiae genome.

    Science.gov (United States)

    Giaever, Guri; Chu, Angela M; Ni, Li; Connelly, Carla; Riles, Linda; Véronneau, Steeve; Dow, Sally; Lucau-Danila, Ankuta; Anderson, Keith; André, Bruno; Arkin, Adam P; Astromoff, Anna; El-Bakkoury, Mohamed; Bangham, Rhonda; Benito, Rocio; Brachat, Sophie; Campanaro, Stefano; Curtiss, Matt; Davis, Karen; Deutschbauer, Adam; Entian, Karl-Dieter; Flaherty, Patrick; Foury, Francoise; Garfinkel, David J; Gerstein, Mark; Gotte, Deanna; Güldener, Ulrich; Hegemann, Johannes H; Hempel, Svenja; Herman, Zelek; Jaramillo, Daniel F; Kelly, Diane E; Kelly, Steven L; Kötter, Peter; LaBonte, Darlene; Lamb, David C; Lan, Ning; Liang, Hong; Liao, Hong; Liu, Lucy; Luo, Chuanyun; Lussier, Marc; Mao, Rong; Menard, Patrice; Ooi, Siew Loon; Revuelta, Jose L; Roberts, Christopher J; Rose, Matthias; Ross-Macdonald, Petra; Scherens, Bart; Schimmack, Greg; Shafer, Brenda; Shoemaker, Daniel D; Sookhai-Mahadeo, Sharon; Storms, Reginald K; Strathern, Jeffrey N; Valle, Giorgio; Voet, Marleen; Volckaert, Guido; Wang, Ching-yun; Ward, Teresa R; Wilhelmy, Julie; Winzeler, Elizabeth A; Yang, Yonghong; Yen, Grace; Youngman, Elaine; Yu, Kexin; Bussey, Howard; Boeke, Jef D; Snyder, Michael; Philippsen, Peter; Davis, Ronald W; Johnston, Mark

    2002-07-25

    Determining the effect of gene deletion is a fundamental approach to understanding gene function. Conventional genetic screens exhibit biases, and genes contributing to a phenotype are often missed. We systematically constructed a nearly complete collection of gene-deletion mutants (96% of annotated open reading frames, or ORFs) of the yeast Saccharomyces cerevisiae. DNA sequences dubbed 'molecular bar codes' uniquely identify each strain, enabling their growth to be analysed in parallel and the fitness contribution of each gene to be quantitatively assessed by hybridization to high-density oligonucleotide arrays. We show that previously known and new genes are necessary for optimal growth under six well-studied conditions: high salt, sorbitol, galactose, pH 8, minimal medium and nystatin treatment. Less than 7% of genes that exhibit a significant increase in messenger RNA expression are also required for optimal growth in four of the tested conditions. Our results validate the yeast gene-deletion collection as a valuable resource for functional genomics.

  6. A highly selectable and highly transferable Ti plasmid to study conjugal host range and Ti plasmid dissemination in complex ecosystems.

    Science.gov (United States)

    Teyssier-Cuvelle, S; Oger, P; Mougel, C; Groud, K; Farrand, S K; Nesme, X

    2004-07-01

    A conjugal donor system, ST2, was constructed to study the conjugal dissemination of a Ti plasmid to wild-type recipient bacteria in vitro and in situ. The system consisted of a polyauxotrophic derivative of C58 harboring a hyperconjugative and highly selectable Ti plasmid, pSTiEGK, which was constructed by inserting a multiple antibiotic resistance cassette in the traM- mcpA region of pTiC58Delta accR. ST2 transfers pSTiEGK constitutively at frequencies up to 10(-1) to plasmidless Agrobacterium recipients. The host range of pSTiEGK includes all the known genomic species of Agrobacterium, indigenous soil agrobacteria and some Rhizobium and Phyllobacterium spp. All transconjugants became pathogenic upon acquisition of the Ti plasmid and were also able to transfer pSTiEGK by conjugation. This host range was indistinguishable from that of its wild-type parent pTiC58, and therefore pSTiEGK constitute a valid proxy to study the dissemination of Ti plasmids directly in the environment. Transconjugants can be selected on a combination of four antibiotics, which efficiently prevents the growth of the indigenous microbiota present in complex environments. The transfer of pSTiEGK to members of the genus Agrobacterium was affected primarily by the plasmid content of the recipient strain (10(3)- to 10(5)-fold reduction), e.g., the presence of incompatible plasmids. As a consequence, a species should be considered permissive to Ti transfer whenever one permissive isolate is found. PMID:15164241

  7. DNA sequence analysis of plasmids from multidrug resistant Salmonella enterica serotype Heidelberg isolates.

    Directory of Open Access Journals (Sweden)

    Jing Han

    Full Text Available Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.

  8. Genetic Characterization of ExPEC-Like Virulence Plasmids among a Subset of NMEC.

    Science.gov (United States)

    Nicholson, Bryon A; West, Aaron C; Mangiamele, Paul; Barbieri, Nicolle; Wannemuehler, Yvonne; Nolan, Lisa K; Logue, Catherine M; Li, Ganwu

    2016-01-01

    Neonatal Meningitis Escherichia coli (NMEC) is one of the most common causes of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or neurologic deficits in survivors. Large plasmids have been shown experimentally to increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9 ExPEC-like plasmids were isolated from NMEC and sequenced to identify the core and accessory plasmid genes of ExPEC-like virulence plasmids in NMEC and create an expanded plasmid phylogeny. Results showed sequenced virulence plasmids carry a strongly conserved core of genes with predicted functions in five distinct categories including: virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The major functions of virulence-associated and plasmid core genes serve to increase in vivo fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate NMEC's survival in the host's low iron environment, and systems to enhance bacterial resistance to host innate immunity. Phylogenetic analysis based on these core plasmid genes showed that at least two lineages of ExPEC-like plasmids could be discerned. Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could not be differentiated based solely on the genes of the core plasmid genome. PMID:26800268

  9. Genetic Characterization of ExPEC-Like Virulence Plasmids among a Subset of NMEC.

    Directory of Open Access Journals (Sweden)

    Bryon A Nicholson

    Full Text Available Neonatal Meningitis Escherichia coli (NMEC is one of the most common causes of neonatal bacterial meningitis in the US and elsewhere resulting in mortality or neurologic deficits in survivors. Large plasmids have been shown experimentally to increase the virulence of NMEC in the rat model of neonatal meningitis. Here, 9 ExPEC-like plasmids were isolated from NMEC and sequenced to identify the core and accessory plasmid genes of ExPEC-like virulence plasmids in NMEC and create an expanded plasmid phylogeny. Results showed sequenced virulence plasmids carry a strongly conserved core of genes with predicted functions in five distinct categories including: virulence, metabolism, plasmid stability, mobile elements, and unknown genes. The major functions of virulence-associated and plasmid core genes serve to increase in vivo fitness by adding multiple iron uptake systems to the genetic repertoire to facilitate NMEC's survival in the host's low iron environment, and systems to enhance bacterial resistance to host innate immunity. Phylogenetic analysis based on these core plasmid genes showed that at least two lineages of ExPEC-like plasmids could be discerned. Further, virulence plasmids from Avian Pathogenic E. coli and NMEC plasmids could not be differentiated based solely on the genes of the core plasmid genome.

  10. Ultrastructural changes of Saccharomyces cerevisiae in response to ethanol stress.

    Science.gov (United States)

    Ma, Manli; Han, Pei; Zhang, Ruimin; Li, Hao

    2013-09-01

    In the fermentative process using Saccharomyces cerevisiae to produce bioethanol, the performance of cells is often compromised by the accumulation of ethanol. However, the mechanism of how S. cerevisiae responds against ethanol stress remains elusive. In the current study, S. cerevisiae cells were cultured in YPD (yeast extract - peptone - dextrose) medium containing various concentrations of ethanol (0%, 2.5%, 5%, 7.5%, 10%, and 15% (v/v)). Compared with the control group without ethanol, the mean cell volume of S. cerevisiae decreased significantly in the presence of 7.5% and 10% ethanol after incubation for 16 h (P < 0.05), and in the presence of 15% ethanol at all 3 sampling time points (1, 8, and 16 h) (P < 0.05). The exposure of S. cerevisiae cells to ethanol also led to an increase in malonyldialdehyde content (P < 0.05) and a decrease in sulfhydryl group content (P < 0.05). Moreover, the observations through transmission electron microscopy enabled us to relate ultrastructural changes elicited by ethanol with the cellular stress physiology. Under ethanol stress, the integrity of the cell membrane was compromised. The swelling or distortion of mitochondria together with the occurrence of a single and large vacuole was correlated with the addition of ethanol. These results suggested that the cell membrane is one of the targets of ethanol, and the degeneration of mitochondria promoted the accumulation of intracellular reactive oxygen species.

  11. Removing cadmium from electroplating wastewater by waste saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    DAI Shu-juan; WEI De-zhou; ZHOU Dong-qin; JIA Chun-yun; WANG Yu-juan; LIU Wen-gang

    2008-01-01

    The appropriate condition and scheme of removing cadmium from electroplating wastewater were investigated by adsorption-precipitation method using waste saccharomyces cerevisiae(WSC) as sorbent. Effect factors on biosorption of cadmium in cadmium-containing electroplating wastewater by waste saccharomyces cerevisiae and precipitation process of waste saccharomyces cerevisiae after adsorbing cadmium were studied. The results show that removal rate of cadmium is over 88% after 30 min adsorbing under the condition of cadmium concentration 26 mg/L, the dosage of waste saccharomyces cerevisiae 16.25 g/L, temperature 18 ℃, pH 6.0 and precipitation time 4 h. Biosorption-precipitation method is effective to remove cadmium in cadmium-containing electroplating wastewater by waste saccharomyces cerevisiae. The SEM, infrared spectroscopy and Zeta-potential of the cells show that chemical chelating is the main adsorption form; electrostatic attraction, hydrogen bonding and van der Waals force all function in adsorption process; and ―NH2―,―C=O―,―C=O―NH―,―CH3, ―OH are the main adsorption groups.

  12. Divergence in wine characteristics produced by wild and domesticated strains of Saccharomyces cerevisiae

    OpenAIRE

    Katie E Hyma; Saerens, Sofie M; Verstrepen, Kevin J.; Justin C Fay

    2011-01-01

    The budding yeast Saccharomyces cerevisiae is the primary species used by wine makers to convert sugar into alcohol during wine fermentation. Saccharomyces cerevisiae is found in vineyards, but is also found in association with oak trees and other natural sources. Although wild strains of S. cerevisiae as well as other Saccharomyces species are also capable of wine fermentation, a genetically distinct group of S. cerevisiae strains is primarily used to produce wine, consistent with the idea t...

  13. Web design for libraries

    CERN Document Server

    Rubenstein, Charles

    2014-01-01

    Having a clear, attractive, and easy-to-navigate website that allows users to quickly find what they want is essential for any organization-including a library. This workbook makes website creation easy-no HTML required.

  14. Library Architecture: Some Observations

    Directory of Open Access Journals (Sweden)

    Bernhard Fabian

    2002-07-01

    Full Text Available There are plenty of libraries (among them many university and research libraries which do not provide adequate work-places. Chairs may have been selected for their stylish look rather than for their physical comfort. Desk lamps may have been deemed unnecessary (they might have distorted the overall impression which the reading room was expected to make And so on. I keep wondering how many librarians have spent some time in their libraries as readers, and have assessed their reading rooms from the user’s point of view. Have they been in a cubicle? Or have they read a book under glaring neon lights? Do they know how well their air-conditioning works? I know a library in which the only window that can be opened is in the librarian’s office.

  15. European Digital Mathematics Library

    OpenAIRE

    Rakosnik, Jiri; Pavlov, Radoslav

    2013-01-01

    The aim of this paper is to survey the European Digital Mathematics Library project goals and achievements as well as an outlook for sustainable development. “Making mathematics literature published in Europe available online” www.eudml.org

  16. Presidential Electronic Records Library

    Data.gov (United States)

    National Archives and Records Administration — PERL (Presidential Electronic Records Library) used to ingest and provide internal access to the Presidential electronic Records of the Reagan, Bush, and Clinton...

  17. Working in the Library

    CERN Multimedia

    Maximilien Brice

    2009-01-01

    Head Librarian Jens Vigen seeking information on the first discussions concerning the construction of the Large Hadron Collider in the LEP Tunnel (1984), here assisted by two of the library apprentices, Barbara Veyre and Dina-Elisabeth Bimbu (seated).

  18. Iowa DNR - NRGIS Library

    Data.gov (United States)

    Iowa State University GIS Support and Research Facility — The Natural Resources Geographic Information System (NRGIS) Library is a Geographic Information System (GIS) repository developed and maintained by the GIS Section...

  19. USGS Photographic Library

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — The U.S. Geological Survey Denver Library maintains a collection of over 400,000 photographs taken during geologic studies of the United States and its territories...

  20. Archives Library Information Center

    Data.gov (United States)

    National Archives and Records Administration — ALIC is an online library catalog of books, periodicals, and other materials contained in Archives I and II and book collections located in other facilities.

  1. Increasing Public Library Productivity.

    Science.gov (United States)

    Samuelson, Howard

    1981-01-01

    Suggests ways of improving productivity for public libraries faced with increased accountability, dwindling revenues, and continuing inflation. Techniques described include work simplification, work analysis, improved management, and employee motivation. (RAA)

  2. Display of wasp venom allergens on the cell surface of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Poulsen Lars K

    2010-09-01

    Full Text Available Abstract Background Yeast surface display is a technique, where the proteins of interest are expressed as fusions with yeast surface proteins and thus remain attached to the yeast cell wall after expression. Our purpose was to study whether allergens expressed on the cell surface of baker's yeast Saccharomyces cerevisiae preserve their native allergenic properties and whether the yeast native surface glycoproteins interfere with IgE binding. We chose to use the major allergens from the common wasp Vespula vulgaris venom: phospholipase A1, hyaluronidase and antigen 5 as the model. Results The proteins were expressed on the surface as fusions with a-agglutinin complex protein AGA2. The expression was confirmed by fluorescent cytometry (FACS after staining the cells with antibody against a C-tag attached to the C-terminal end of the allergens. Phospholipase A1 and hyaluronidase retained their enzymatic activities. Phospholipase A1 severely inhibited the growth of the yeast cells. Antigen 5 - expressing yeast cells bound IgE antibodies from wasp venom allergic patient sera but not from control sera as demonstrated by FACS. Moreover, antigen 5 - expressing yeast cells were capable of mediating allergen-specific histamine release from human basophils. Conclusions All the three major wasp venom allergens were expressed on the yeast surface. A high-level expression, which was observed only for antigen 5, was needed for detection of IgE binding by FACS and for induction of histamine release. The non-modified S. cerevisiae cells did not cause any unspecific reaction in FACS or histamine release assay despite the expression of high-mannose oligosaccharides. In perspective the yeast surface display may be used for allergen discovery from cDNA libraries and possibly for sublingual immunotherapy as the cells can serve as good adjuvant and can be produced in large amounts at a low price.

  3. Library Publishing is Special: Selection and Eligibility in Library Publishing

    OpenAIRE

    Royster, Paul

    2014-01-01

    Traditional publishing is based on ownership, commerce, paid exchanges, and scholarship as a commodity, while library activities are based on a service model of sharing resources and free exchange. I believe library publishing should be based on those values and should not duplicate or emulate traditional publishing. University presses have mixed views of library publishing, and libraries should not adopt those attitudes. Library publishers are not gatekeepers; their mission is dissemination....

  4. Library Widget for Moodle

    OpenAIRE

    Mariela Hristova

    2013-01-01

    Any course within a course management system is generally considered the intellectual space of the professor teaching it. Research tools and guides, such as search boxes for discovery services or links to course-specific and subject-specific guides, are created and maintained by librarians. In trying to get our tools and services closer to where students spend their time devoted to coursework, Oakland University libraries have developed a library widget – a self-serve code generator that allo...

  5. Digital library usability studies

    CERN Document Server

    Eden, Bradford Lee

    2005-01-01

    Each summer, circulation staff in my library inventories a section of the stacks andbrings collection issues to the attention of appropriate bibliographers. Since I amresponsible for the economics collection, I see an array of government documents thathave managed to elude the cataloging process. Many of these titles are decades old,having squatted in the library undisturbed and uncirculated since our online catalogwas implemented in 1990.

  6. Digital libraries : an overview

    OpenAIRE

    Jange, Suresh; Angadi, Mallikarjun

    2001-01-01

    During the past recent years, there has been tremendous development reaming the concept of digital libraries-a knowledge base that can be stored and retrieved through on-line networks. Digital libraries are the most complex form of information systems that support digital document preservation, distributed database management, hypertext, filtering, information retrieval and selective dissemination of information. This has really overcome geographical barrier offering wide range of academic,...

  7. At the Library

    Institute of Scientific and Technical Information of China (English)

    任孝伟

    2000-01-01

    Jim wants to borrow a book from a new library(图书馆).He comes to the library with Jack.They can't see any assistants(图书管理员)there,but only some robots(机器人)standing there.Then Jim says to the robot,""Hey,give me the book."" But the robot doesn't work.

  8. Python library reference

    OpenAIRE

    Rossum, van, M.A.J.

    1995-01-01

    Python is an extensible, interpreted, object-oriented programming language. It supports a wide range of applications, from simple text processing scripts to interactive WWW browsers. While the Python Reference Manual describes the exact syntax and semantics of the language, it does not describe the standard library that is distributed with the language, and which greatly enhances its immediate usability. This library contains built-in modules (written in C) that provide access to system funct...

  9. The Global Library

    OpenAIRE

    Schöpfel, Joachim

    2012-01-01

    The Agenda 21 is the United Nations' action plan for the 21st century in favour of sustainable development. Launched at the Rio Earth Summit in 1992, its 40 chapters include social, economic and ecological actions to be implemented by local authorities, governments, but also by corporate companies and services. Twenty years after the Earth Summit, our proposal is to apply the Agenda 21 to library management and development and to move on to the global or sustainable library. Based on the conc...

  10. Marketingstrategies for academic libraries

    OpenAIRE

    Jung, Claudia

    2008-01-01

    This assignment is about the development of a general strategic marketing plan for academic libraries in Germany and can be used as a guideline for libraries that want to develop concrete marketing strategies for several products and services. Two examples of marketing projects are at its end presented for linking theoretical approaches to practice. Finally the development of an own marketing strategy for “information literacy” builds the last part of the assignment.

  11. Hispanic College Students Library Experience

    Science.gov (United States)

    Lumley, Risa; Newman, Eric; Brown, Haakon T.

    2015-01-01

    This study looks at undergraduate Hispanic students' interpretations and current perceptions of the academic library's purpose, usefulness and value. What are the reasons to use the library? What are the barriers to use? This study will examine academic libraries' move toward electronic library materials and what it means for Hispanic students.…

  12. Trends in State Library Cooperation.

    Science.gov (United States)

    Kittel, Dorothy A.

    This nine page pamphlet describes the development of such federal library legislation as the Library Services Act (1956), the Library Services and Construction Act (1964), the Elementary and Secondary Education Act, the Higher Education Act, and the Medical Library Assistance Act (1964). The effect of this legislation on new forms of intertype…

  13. Survey of Ohio's Prison Libraries.

    Science.gov (United States)

    Liggett, Joanna M.

    1996-01-01

    Prison libraries present a growing employment opportunity for librarians, but there is little statistical or descriptive information about prison libraries. A survey was conducted of Ohio prison libraries to create a profile and increase appreciation and understanding of them. Information was collected about library users, types of materials,…

  14. Sports Education Library Information Services

    Institute of Scientific and Technical Information of China (English)

    许晓峰

    2014-01-01

    Library website, is the first window for readers to understand library information services. Sports Education academy library fully take advantage of homepage, combine open access resource searched with the library collections, after targeted collection, selection, sorting, processing, clustering or reorganization, establish a navigation system of open access resource of physical Sports Education.

  15. Interior Design Trends in Libraries.

    Science.gov (United States)

    Sager, Don, Ed.

    2000-01-01

    Four contributing authors discuss perspectives on current trends in library interior design. Articles include: "Trends in Library Furnishings: A Manufacturer's Perspective" (Andrea Johnson); "Libraries, Architecture, and Light: The Architect's Perspective" (Rick McCarthy); "The Library Administrator's Perspective" (Chadwick Raymond); and "The…

  16. Trends in Philippine Library History.

    Science.gov (United States)

    Hernandez, Vicente S.

    This paper divides Philippine library history into three periods, establishing a relationship between historical events and library trends. During the Spanish period, modern library trends were introduced through the establishment of the Sociedad Economica in 1780, but did not influence Philippine library culture until the later part of the 19th…

  17. Plasmid-determined copper resistance in Pseudomonas syringae from impatiens

    Energy Technology Data Exchange (ETDEWEB)

    Cooksey, D.A. (Univ. of California, Riverside (USA))

    1990-01-01

    A strain of Pseudomonas syringae was recently identified as the cause of a new foliar blight of impatiens. The bacterium was resistant to copper compounds, which are used on a variety of crops for bacterial and fungal disease control. The bacterium contained a single 47-kilobase plasmid (pPSI1) that showed homology to a copper resistance operon previously cloned and characterized from P. syringae pv. tomato plasmid pPT23D (D. Cooksey, Appl. Environ. Microbiol. 53:454-456, 1987). pPSI1 was transformed by electroporation into a copper-sensitive P. syringae strain, and the resulting transformants were copper resistant. A physical map of pPSI1 was constructed, and the extent of homology to pPT23D outside the copper resistance operon was determined in Southern hybridizations. The two plasmids shared approximately 20 kilobases of homologous DNA, with the remainder of each plasmid showing no detectable homology. The homologous regions hybridized strongly, but there was little or no conservation of restriction enzyme recognition sites.

  18. Studying plasmid horizontal transfer in situ: a critical review

    DEFF Research Database (Denmark)

    Sørensen, Søren Johannes; Bailey, Mark; Hansen, Lars Hestbjerg;

    2005-01-01

    This review deals with the prospective, experimental documentation of horizontal gene transfer (HGT) and its role in real-time, local adaptation. We have focused on plasmids and their function as an accessory and/or adaptive gene pool. Studies of the extent of HGT in natural environments have...

  19. New tetracycline resistance determinant on R plasmids from Vibrio anguillarum.

    OpenAIRE

    Aoki, T.; Satoh, T.; Kitao, T.

    1987-01-01

    Two classes of tetracycline resistance determinants on R plasmids were detected in Vibrio anguillarum strains isolated from ayu (sweat fish; Plecoglossus altivelis) farms in Japan. Tetracycline resistance genes categorized as class B were prevalent from 1973 to 1977; however, a new tetracycline resistance gene, which was not classified into tetracycline resistance determinant class A, B, C, or D, has been prevalent since 1981.

  20. Use of plasmid DNA for induction of protective immunity

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    2004-01-01

    Vaccines based on plasmid DNA have been tested for a number of fish pathogens but so far it is only in case of the rhabdoviruses, where the technology has been a real break through in vaccine research. Aspects of dose, time-course and mechanisms of protection, as well as practical use are discussed....

  1. Pharmaceutical development of the plasmid DNA vaccine pDERMATT

    NARCIS (Netherlands)

    Quaak, S.G.L.

    2009-01-01

    The discovery of tumor specific antigens and self tolerance mechanisms against these antigens led to the assumption that antigens circulating at sufficient concentration levels could break this self tolerance mechanism and evoke immunological antitumor effects. pDERMATT (plasmid DNA encoding recombi

  2. Stability of Integrated Plasmids in the Chromosome of Lactococcus lactis

    NARCIS (Netherlands)

    Leenhouts, Kees J.; Kok, Jan; Venema, Gerhardus

    1990-01-01

    Derivatives of plasmids pBR322, pUB110, pSC101, and pTB19, all containing an identical fragment of lactococcal chromosomal DNA, were integrated via a Campbell-like mechanism into the same chromosomal site of Lactococcus lactis MG1363, and the transformants were analyzed for the stability of the inte

  3. Plasmid containing a DNA ligase gene from Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Griffin, K.; Setlow, J.K.

    1984-05-01

    A ligase gene from Haemophilus influenzae was cloned into the shuttle vector pDM2. Although the plasmid did not affect X-ray sensitivity, it caused an increase in UV sensitivity of the wild-type but not excision-defective H. influenzae and a decrease in UV sensitivity of the rec-1 mutant. 14 references, 2 figures.

  4. NSUF Irradiated Materials Library

    Energy Technology Data Exchange (ETDEWEB)

    Cole, James Irvin [Idaho National Lab. (INL), Idaho Falls, ID (United States)

    2015-09-01

    The Nuclear Science User Facilities has been in the process of establishing an innovative Irradiated Materials Library concept for maximizing the value of previous and on-going materials and nuclear fuels irradiation test campaigns, including utilization of real-world components retrieved from current and decommissioned reactors. When the ATR national scientific user facility was established in 2007 one of the goals of the program was to establish a library of irradiated samples for users to access and conduct research through competitively reviewed proposal process. As part of the initial effort, staff at the user facility identified legacy materials from previous programs that are still being stored in laboratories and hot-cell facilities at the INL. In addition other materials of interest were identified that are being stored outside the INL that the current owners have volunteered to enter into the library. Finally, over the course of the last several years, the ATR NSUF has irradiated more than 3500 specimens as part of NSUF competitively awarded research projects. The Logistics of managing this large inventory of highly radioactive poses unique challenges. This document will describe materials in the library, outline the policy for accessing these materials and put forth a strategy for making new additions to the library as well as establishing guidelines for minimum pedigree needed to be included in the library to limit the amount of material stored indefinitely without identified value.

  5. Characterization of the Lactobacillus plantarum plasmid pCD033 and generation of the plasmid free strain L. plantarum 3NSH.

    Science.gov (United States)

    Heiss, Silvia; Grabherr, Reingard; Heinl, Stefan

    2015-09-01

    Lactobacillus plantarum CD033, a strain isolated from grass silage in Austria, harbors a 7.9 kb plasmid designated pCD033. Sequence analysis identified 14 open reading frames and 8 of these were supposed to be putative coding sequences. Gene annotation revealed no putative essential genes being plasmid encoded, but a plasmid addiction system based on a PemI/PemK-like toxin-antitoxin system, able to stabilize plasmid maintenance. Absence of a replication initiation protein, a double strand origin as well as a single strand origin on plasmid pCD033 suggests replication via a new type of theta mechanism, whereby plasmid replication is potentially initiated and regulated by non-coding RNA. Detailed examination of segregational stability of plasmid vectors consisting of pCD033-fragments, combined with a selection marker, resulted in definition of a stably maintained minimal replicon. A gene encoding a RepB/OrfX-like protein was found to be not essential for plasmid replication. Alignment of the amino acid sequence of this protein with related proteins unveiled a highly conserved amino acid motif (LLDQQQ). L. plantarum CD033 was cured of pCD033 resulting in the novel plasmid free strain L. plantarum 3NSH. Plasmid curing demonstrated that no essential features are provided by pCD033 under laboratory conditions.

  6. Service Innovation In Academic Libraries

    DEFF Research Database (Denmark)

    Scupola, Ada; Nicolajsen, Hanne Westh

    2010-01-01

    Purpose – The purpose of this article is to investigate whether management and employees in academic libraries involve users in library service innovations and what these user roles are. Design/methodology/approach – The article first reviews the literature focusing on innovation, new product...... development, new service development and library science with specific focus on users and management. Subsequently the research uses a case study approach to investigate management and customer involvement in a Danish academic library. Findings – Results from the case study show that academic libraries...... in academic library service innovations on the basis of an in-depth case study of a Danish academic library....

  7. Survival and evolution of a large multidrug resistance plasmid in new clinical bacterial hosts

    DEFF Research Database (Denmark)

    Porse, Andreas; Schønning, Kristian; Munck, Christian;

    2016-01-01

    and population sequencing to show that the long-term persistence and molecular integrity of the plasmid is highly influenced by multiple factors within a 25 kb plasmid region constituting a host-dependent burden. In the E. coli hosts investigated here, improved plasmid stability readily evolves via IS......Large conjugative plasmids are important drivers of bacterial evolution and contribute significantly to the dissemination of antibiotic resistance. Although plasmid borne multidrug resistance is recognized as one of the main challenges in modern medicine, the adaptive forces shaping the evolution...... of these plasmids within pathogenic hosts are poorly understood. Here we study plasmid-host adaptations following transfer of a 73 kb conjugative multidrug resistance plasmid to naïve clinical isolates of Klebsiella pneumoniae and Escherichia coli We use experimental evolution, mathematical modelling...

  8. Design of expanded bed supports for the recovery of plasmid DNA by anion exchange adsorption

    DEFF Research Database (Denmark)

    Theodossiou, Irini; Søndergaard, M.; Thomas, Owen R. T.

    2001-01-01

    In this study we detail the rational design of new chromatographic adsorbents tailored for the capture of plasmid DNA. Features present on current chromatographic supports that can significantly enhance plasmid binding capacity have been identified in packed bed chromatography experiments...

  9. Conservation of plasmids among plant-pathogenic Pseudomonas syringae isolates of diverse origins.

    Science.gov (United States)

    von Bodman, S B; Shaw, P D

    1987-05-01

    Thirty isolates of Pseudomonas syringae pv. tabaci, pv. angulata (pathogens on tobacco), pv. coronafaciens, and pv. striafaciens (pathogens on oats) were examined for plasmid DNAs. The strains were obtained from plants throughout the world, some over 50 years ago. Of the 22 tobacco pathogens, 16 contain predominantly one type of plasmid, the pJP27.00 type. The remaining six tobacco-specific strains do not harbor detectable plasmids. The oat pathogens contain one, two, or three plasmids. DNA homology studies indicate that the plasmid DNAs are highly conserved. More importantly, the plasmids harbored by strains isolated from one host plant are conserved most stringently; e.g., the plasmids from the tobacco pathogens are, with one exception, indistinguishable by restriction endonuclease digestion and Southern hybridization. There is also extensive homology among plasmids indigenous to the oat-specific P. syringae pv. coronafaciens and pv. striafaciens strains. PMID:3628554

  10. Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption

    DEFF Research Database (Denmark)

    Scalcinati, Gionata; Otero, José Manuel; Van Vleet, Jennifer R. H.;

    2012-01-01

    Industrial biotechnology aims to develop robust microbial cell factories, such as Saccharomyces cerevisiae, to produce an array of added value chemicals presently dominated by petrochemical processes. Xylose is the second most abundant monosaccharide after glucose and the most prevalent pentose...... sugar found in lignocelluloses. Significant research efforts have focused on the metabolic engineering of S. cerevisiae for fast and efficient xylose utilization. This study aims to metabolically engineer S. cerevisiae, such that it can consume xylose as the exclusive substrate while maximizing carbon...... of this strain was employed to further elucidate the observed physiology confirms a strongly up-regulated glyoxylate pathway enabling respiratory metabolism. The resulting strain is a desirable platform for the industrial production of biomass-related products using xylose as a sole carbon source....

  11. Cadmium-induced oxidative stress in Saccharomyces cerevisiae.

    Science.gov (United States)

    Muthukumar, Kannan; Nachiappan, Vasanthi

    2010-12-01

    The present study was undertaken to determine the effect of cadmium (Cd) on the antioxidant status of the yeast Saccharomyces cerevisiae. S. cerevisiae serves as a good eukaryotic model system for the study of the molecular mechanisms of oxidative stress. We investigated the adaptative response of S. cerevisiae exposed to Cd. Yeast cells could tolerate up to 100 microM Cd and an inhibition in the growth and viability was observed. Exposure of yeast cells to Cd showed an increase in malondialdehyde and glutathione. The activities of catalase, superoxide dismutase and glutathione peroxidase were also high in Cd-exposed cells. The incorporation of Cd led to significant increase in iron, zinc and inversely the calcium, copper levels were reduced. The results suggest that antioxidants were increased and are involved in the protection against macromolecular damage during oxidative stress; presumably, these enzymes are essential for counteracting the pro-oxidant effects of Cd. PMID:21355423

  12. Transcription of ColE1Ap mbeC induced by conjugative plasmids from twelve different incompatibility groups.

    OpenAIRE

    Selvaratnam, S; Gealt, M A

    1993-01-01

    Although nonconjugative mobilizable plasmids require helping functions of conjugative plasmids in order to be mobilized into recipients, at least some genes from the nonconjugative plasmids may be induced to assist in the DNA transfer process. Conjugative plasmids from 12 different incompatibility groups mobilized the nonconjugative plasmid ColE1Ap between Escherichia coli strains. Introduction of any of the conjugative plasmids into the ColE1Ap-containing strain resulted in an induction of m...

  13. Plasmid profiling of bacterial isolates from confined environments

    Science.gov (United States)

    van Houdt, Rob; Provoost, Ann; Coninx, Ilse; Leys, Natalie; Mergeay, Max

    Plasmid profiling of bacterial isolates from confined environments R. Van Houdt, I. Coninx, A. Provoost, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. Human exploration of extreme and isolated hostile environments such as space requires special confined small volume habitats to protect and house the crew. However, human confinement in such small volume habitats has restrictions on waste disposal and personal hygiene and inevitably generates a particular community of microorganisms within the habitat. These microorganisms are mainly originating from the crew (skin, mucous membranes, upper respiratory tract, mouth, and gastrointestinal tract) but also include the residing environmental microorganisms. Earth-based confined habitats such as the Antarctic Research Station Concordia are used as test beds for long-duration spaceflights to study the physiologic and psychological adaptation to isolated environments. The dynamics of the environmental microbial population in such a test bed could render additional insights in assessing the potential health risks in long-duration space missions. Not only total bacterial contamination levels are important, but it is essential to identify also the predominant microbial taxa and their mobile genetic elements (MGE). These MGEs could be exchanged between bacteria by horizontal gene transfer and may alter the pathogenic potential since they often carry antibiotic resistance or more in general adaptation-enhancing traits. In this study several bacterial strains isolated in the Concordia research station were examined for their plasmid content. An optimized protocol for extraction of large plasmids showed the present of at least one plasmid in 50% of the strains. For all strains the minimal inhibitory concentration of a range of antibiotics was determined indicating resistance to

  14. Vectorial acylation in Saccharomyces cerevisiae. Fat1p and fatty acyl-CoA synthetase are interacting components of a fatty acid import complex

    DEFF Research Database (Denmark)

    Zou, Zhiying; Tong, Fumin; Færgeman, Nils J.;

    2003-01-01

    In Saccharomyces cerevisiae Fat1p and fatty acyl-CoA synthetase (FACS) are hypothesized to couple import and activation of exogenous fatty acids by a process called vectorial acylation. Molecular genetic and biochemical studies were used to define further the functional and physical interactions...... and Fat1p play distinct roles in the fatty acid import process. When expressed from a 2-mu plasmid, Fat1p contributes significant oleoyl-CoA synthetase activity, which indicates vectorial esterification and metabolic trapping are the driving forces behind import. Evidence of a physical interaction between...... as trap were active when tested using the yeast two-hybrid system. Third, co-expressed, differentially tagged Fat1p and Faa1p or Faa4p were co-immunoprecipitated. Collectively, these data support the hypothesis that fatty acid import by vectorial acylation in yeast requires a multiprotein complex, which...

  15. Comparison of electrically mediated and liposome-complexed plasmid DNA delivery to the skin

    OpenAIRE

    Heller, Loree C.; Jaroszeski, Mark J; Coppola, Domenico; Heller, Richard

    2008-01-01

    Background Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo, including the skin. We have previously demonstrated efficient delivery of plasmid DNA to the skin utilizing a custom-built four-plate electrode. The experiments described here further evaluate cutaneous plasmid delivery using in vivo electroporation. Plasmid expression levels are compared to those after liposome mediated delivery. Methods Enhanced electrically-mediated delivery, and ...

  16. Identification of tetracycline-resistant R-plasmids in Streptococcus agalactiae (group B).

    OpenAIRE

    Burdett, V

    1980-01-01

    In this report, 30 tetracycline-resistant clinical isolates of group B Streptococcus were examined to assess the extent to which tetracycline resistance is plasmid mediated. Of these, 27 showed no physical or genetic evidence of plasmid-mediated resistance; however, one conjugative and two small (3.5 X 10(6)-dalton) multicopy non-self-transmissible tetracycline resistance plasmids were identified. The conjugative plasmid was transmissible to Streptococcus faecalis as well as to Streptococcus ...

  17. Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

    DEFF Research Database (Denmark)

    Klümper, Uli; Riber, Leise; Dechesne, Arnaud;

    2014-01-01

    Conjugal plasmids can provide microbes with full complements of new genes and constitute potent vehicles for horizontal gene transfer. Conjugal plasmid transfer is deemed responsible for the rapid spread of antibiotic resistance among microbes. While broad host range plasmids are known to transfer...... bacteria and can, therefore, directly connect large proportions of the soil bacterial gene pool. This finding reinforces the evolutionary and medical significances of these plasmids....

  18. Incidence of Plasmids in Thermus spp. Isolated in Yellowstone National Park

    OpenAIRE

    Munster, Michael J.; Munster, Ann P.; Sharp, Richard J.

    1985-01-01

    Forty-eight strains of Thermus spp. were isolated from thermal sites in Yellowstone National Park, Wyo., and 62.5% showed evidence of plasmid DNA. Attempts to assign function to the plasmid DNA were unsuccessful, and the presence of plasmid DNA could not be correlated with antibiotic or heavy metal resistance. A number of these cryptic plasmids are now being investigated for their potential as vectors for molecular cloning in Thermus spp.

  19. Conservation of Plasmid-Encoded Traits among Bean-Nodulating Rhizobium Species

    OpenAIRE

    Brom, Susana; Girard, Lourdes; García-de los Santos, Alejandro; Sanjuan-Pinilla, Julio M.; Olivares, José; Sanjuan, Juan

    2002-01-01

    Rhizobium etli type strain CFN42 contains six plasmids. We analyzed the distribution of genetic markers from some of these plasmids in bean-nodulating strains belonging to different species (Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum, and Sinorhizobium fredii). Our results indicate that independent of geographic origin, R. etli strains usually share not only the pSym plasmid but also other plasmids containing symbiosis-related genes, with a similar organi...

  20. Modulation of ColE1-like Plasmid Replication for Recombinant Gene Expression

    OpenAIRE

    Camps, Manel

    2010-01-01

    ColE1-like plasmids constitute the most popular vectors for recombinant protein expression. ColE1 plasmid replication is tightly controlled by an antisense RNA mechanism that is highly dynamic, tuning plasmid metabolic burden to the physiological state of the host. Plasmid homeostasis is upset upon induction of recombinant protein expression because of non-physiological levels of expression and because of the frequently biased amino acid composition of recombinant proteins. Disregulation of p...

  1. Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

    OpenAIRE

    Yarmolinsky, M B; Hansen, E B; Jafri, S; Chattoraj, D K

    1989-01-01

    P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomp...

  2. Stability in Escherichia coli of an antibiotic resistance plasmid from Bacteroides fragilis.

    OpenAIRE

    Rashtchian, A; Booth, S J

    1981-01-01

    A Bacteroides fragilis strain resistant to penicillin G, tetracycline, and clindamycin was screened for the presence of plasmid deoxyribonucleic acid (DNA). Agarose gel electrophoresis of ethanol-precipitated DNA from cleared lysates of this strain revealed two plasmid DNA bands. The molecular weights of the plasmids were estimated by their relative mobility in agarose gel and compared with standard plasmids with known molecular weights. The molecular weights were 3.40 +/- 0.20 x 10(6) and 1....

  3. Properties of R1162, a broad-host-range, high-copy-number plasmid.

    OpenAIRE

    R. MEYER; Hinds, M; Brasch, M.

    1982-01-01

    Regions of plasmid DNA encoding characteristic properties of the IncQ (P-4) group plasmid R1162 were identified by mutagenesis and in vitro cloning. Coding sequences sufficient for expression of incompatibility and efficient conjugal mobilization by plasmid R751 were found to be linked to the origin of DNA replication. In contrast, there was a region remote from the origin, and active in trans, that was required for plasmid maintenance. A derivative that was temperature sensitive for stabilit...

  4. Characterization of different plasmid-borne dihydropteroate synthases mediating bacterial resistance to sulfonamides.

    OpenAIRE

    Swedberg, G; Sköld, O

    1980-01-01

    Plasmid-borne resistance to sulfonamides was studied in both newly isolated and earlier characterized R plasmids. Two different classes of drug-resistant dihydropteroate synthases were found to be responsible for most cases of plasmid-mediated sulfonamide resistance. The plasmid-coded enzymes could be completely separated from their chromosomal counterpart and also showed differences in heat stability and molecular size. The resistant and chromosomal enzymes could bind the normal substrate, p...

  5. The critical role of the linear plasmid lp36 in the infectious cycle of Borrelia burgdorferi

    OpenAIRE

    Mollie W Jewett; Lawrence, Kevin; Bestor, Aaron C; Tilly, Kit; Grimm, Dorothee; Shaw, Pamela; VanRaden, Mark; Gherardini, Frank; Rosa, Patricia A.

    2007-01-01

    Borrelia burgdorferi, the aetiological agent of Lyme disease, follows a life cycle that involves passage between the tick vector and the mammalian host. To investigate the role of the 36 kb linear plasmid, lp36 (also designated the B. burgdorferi K plasmid), in the infectious cycle of B. burgdorferi, we examined a clone lacking this plasmid, but containing all other plasmids known to be required for infectivity. Our results indicated that lp36 was not required for spirochete survival in the t...

  6. Spectral library searching in proteomics.

    Science.gov (United States)

    Griss, Johannes

    2016-03-01

    Spectral library searching has become a mature method to identify tandem mass spectra in proteomics data analysis. This review provides a comprehensive overview of available spectral library search engines and highlights their distinct features. Additionally, resources providing spectral libraries are summarized and tools presented that extend experimental spectral libraries by simulating spectra. Finally, spectrum clustering algorithms are discussed that utilize the same spectrum-to-spectrum matching algorithms as spectral library search engines and allow novel methods to analyse proteomics data.

  7. Library service to dental practitioners.

    OpenAIRE

    Ashin, E R

    1983-01-01

    Dental school libraries offer resources of value to dental practitioners, but do not always consider practitioners to be primary clientele. A survey was conducted among the sixty U.S. dental school libraries to examine policies and attitudes toward service to practitioners. Although library use by dentists is estimated to be low, most libraries are willing to serve them as long as it does not reduce the libraries' ability to assist students and faculty. Of the respondents, 57% replied that th...

  8. Influence of dough freezing on Saccharomyces cerevisiae metabolism

    Directory of Open Access Journals (Sweden)

    Pejin Dušanka J.

    2007-01-01

    Full Text Available The need to freeze dough is increasing in bakery production. Frozen dough can be stored for a long time without quality change. The capacity of bakery production can be increased in this way, and in the same time, the night shifts can be decreased. Yeast cells can be damaged by freezing process resulting in poor technological quality of dough after defrostation (longer fermentation of dough. The influence of frozen storage time of dough on survival percentage of Saccharomyces cerevisiae was investigated. Dough samples were taken after 1, 7, 14 and 28 days of frozen storage at -20°C. After defrosting, at room temperature, samples were taken from the surface and the middle part of dough (under aseptic conditions, and the percentage of living S. cerevisiae cells was determined. During frozen storage of dough, the number of living S. cerevisiae decreased. After 28 days of frozen storage, the percentage of live cells on the surface and inside the dough was 53,1% and 54,95%, respectively. The addition of k-carragenan to dough increased the percentage of living cells in the middle part of dough up to 64,63%. Pure cultures, isolated from survived S. cerevisia cells in frozen dough by agar plates method (Koch's method, were multiplied in optimal liquid medium for yeasts. The content of cytochromes in S. cerevisiae cells was determined by spectrophotometric method. The obtained results showed that the content of cytochromes in survived S. cerevisiae cells was not affected by dough freezing process. Growth rate and fermentative activity (Einchor's method were determined in multiplied cells.

  9. Plasmid vectors for Xylella fastidiosa utilizing a toxin-antitoxin system for plasmid stability in the absence of antibiotic selection

    Science.gov (United States)

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacte...

  10. Accumulation of gold using Baker's yeast, Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Authors have reported preconcentration of 152Eu, a long-lived fission product, by yeast cells, Saccharomyces cerevisiae. Gold being a precious metal is used in electroplating, hydrogenation catalyst, etc. Heterogeneous composition of samples and low concentration offers renewed interest in its selective extraction of gold using various extractants. Gold can be recovered from different solutions using various chemical reagents like amines, organophosphorus compounds, and extractants containing sulphur as donor atom, etc. In the present work, two different strains of baker's yeast, Saccharomyces cerevisiae have been used to study the preconcentration of gold at various experimental conditions

  11. Library 3.0 intelligent libraries and apomediation

    CERN Document Server

    Kwanya, Tom; Underwood, Peter

    2015-01-01

    The emerging generation of research and academic library users expect the delivery of user-centered information services. 'Apomediation' refers to the supporting role librarians can give users by stepping in when users need help. Library 3.0 explores the ongoing debates on the "point oh” phenomenon and its impact on service delivery in libraries. This title analyses Library 3.0 and its potential in creating intelligent libraries capable of meeting contemporary needs, and the growing role of librarians as apomediators. Library 3.0 is divided into four chapters. The first chapter introduces and places the topic in context. The second chapter considers "point oh” libraries. The third chapter covers library 3.0 librarianship, while the final chapter explores ways libraries can move towards '3.0'.

  12. Studies on the expression of plasmid-borne genes in the endosymbiotic state of Rhizobium leguminosarum

    NARCIS (Netherlands)

    Krol, A.J.M.

    1982-01-01

    The subject matter of the research reported in this thesis is the role of plasmid-borne genes of Rhizobium in symbiosis and nitrogen fixation. Plasmid DNA was isolated from Rhizobium leguminosarum strain PRE and the expression of plasmid DNA in nitrogen fixing nodules was investigated by hybridizati

  13. Presence and analysis of plasmids in human and animal associated Arcobacter species

    DEFF Research Database (Denmark)

    Douidah, Laid; De Zutter, Lieven; Van Nieuwerburgh, Filip;

    2014-01-01

    In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three sma...

  14. Novel plasmid conferring kanamycin and tetracycline resistance in turkey-derived Campylobacter jejuni strain 11601MD

    Science.gov (United States)

    In Campylobacter spp., resistance to the antibiotics kanamycin and tetracycline is frequently associated with plasmid-borne genes. However, relatively few plasmids of Campylobacter jejuni have been fully characterized to date. A novel plasmid (p11601MD; 44,095 bp.) harboring tet(O) was identified in...

  15. The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai.].

    Science.gov (United States)

    Ellul, P; Ríos, G; Atarés, A; Roig, L A; Serrano, R; Moreno, V

    2003-08-01

    An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species. PMID:12783167

  16. Xylose and xylose/glucose co-fermentation by recombinant Saccharomyces cerevisiae strains expressing individual hexose transporters.

    Science.gov (United States)

    Gonçalves, Davi L; Matsushika, Akinori; de Sales, Belisa B; Goshima, Tetsuya; Bon, Elba P S; Stambuk, Boris U

    2014-09-01

    Since the uptake of xylose is believed to be one of the rate-limiting steps for xylose ethanol fermentation by recombinant Saccharomyces cerevisiae strains, we transformed a hxt-null strain lacking the major hexose transporters (hxt1Δ-hxt7Δ and gal2Δ) with an integrative plasmid to overexpress the genes for xylose reductase (XYL1), xylitol dehydrogenase (XYL2) and xylulokinase (XKS1), and analyzed the impact that overexpression of the HXT1, HXT2, HXT5 or HXT7 permeases have in anaerobic batch fermentations using xylose, glucose, or xylose plus glucose as carbon sources. Our results revealed that the low-affinity HXT1 permease allowed the maximal consumption of sugars and ethanol production rates during xylose/glucose co-fermentations, but was incapable to allow xylose uptake when this sugar was the only carbon source. The moderately high-affinity HXT5 permease was a poor glucose transporter, and it also did not allow significant xylose uptake by the cells. The moderately high-affinity HXT2 permease allowed xylose uptake with the same rates as those observed during glucose consumption, even under co-fermentation conditions, but had the drawback of producing incomplete fermentations. Finally, the high-affinity HXT7 permease allowed efficient xylose fermentation, but during xylose/glucose co-fermentations this permease showed a clear preference for glucose. Thus, our results indicate that approaches to engineer S. cerevisiae HXT transporters to improve second generation bioethanol production need to consider the composition of the biomass sugar syrup, whereby the HXT1 transporter seems more suitable for hydrolysates containing xylose/glucose blends, whereas the HXT7 permease would be a better choice for xylose-enriched sugar streams.

  17. Nucleotide sequence analysis of enteropathogenic Escherichia coli (EPEC) adherence factor probe and development of PCR for rapid detection of EPEC harboring virulence plasmids.

    OpenAIRE

    Franke, J.; Franke, S.; SCHMIDT, H.; Schwarzkopf, A.; Wieler, L H; Baljer, G.; Beutin, L.; Karch, H

    1994-01-01

    The 1-kb BamHI-SalI fragment from plasmid pMAR2 termed the enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) probe was cloned in pUC19 and pK18. The nucleotide sequence of this fragment was determined, and a set of primers was designed to amplify a 397-bp region associated with pMAR2 by PCR. An analysis of the whole EAF sequence with database libraries indicated no significant homology to any known genes. However, between bases 701 and 787 of the fragment, an 82.8% homology betw...

  18. Increased Diversity of Libraries from Libraries: Chemoinformatic Analysis of Bis-Diazacyclic Libraries

    OpenAIRE

    López-Vallejo, Fabian; Nefzi, Adel; Bender, Andreas; Owen, John R.; Nabney, Ian T.; Houghten, Richard A.; Medina-Franco, Jose L.

    2011-01-01

    Combinatorial libraries continue to play a key role in drug discovery. To increase structural diversity, several experimental methods have been developed. However, limited efforts have been performed so far to quantify the diversity of the broadly used diversity-oriented synthetic (DOS) libraries. Herein we report a comprehensive characterization of 15 bis-diazacyclic combinatorial libraries obtained through libraries from libraries, which is a DOS approach. Using MACCS keys, radial and diffe...

  19. KTI11 and KTI13, Saccharomyces cerevisiae genes controlling sensitivity to G1 arrest induced by Kluyveromyces lactis zymocin.

    Science.gov (United States)

    Fichtner, Lars; Schaffrath, Raffael

    2002-05-01

    The Kluyveromyces lactis zymocin and its gamma-toxin subunit inhibit cell cycle progression of Saccharomyces cerevisiae. To identify S. cerevisiae genes conferring zymocin sensitivity, we complemented the unclassified zymocin-resistant kti11 and kti13 mutations using a single-copy yeast library. Thus, we identified yeast open reading frames (ORFs) YBL071w-A and YAL020c/ATS1 as KTI11 and KTI13 respectively. Disruption of KTI11 and KTI13 results in the complex tot phenotype observed for the gamma-toxin target site mutants, tot1-7, and includes zymocin resistance, thermosensitivity, hypersensitivity to drugs and slow growth. Both loci, KTI11 and KTI13, are actively transcribed protein-encoding genes as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and in vivo HA epitope tagging. Kti11p is highly conserved from yeast to man, and Kti13p/Ats1p is related to yeast Prp20p and mammalian RCC1, components of the Ran-GTP/GDP cycle. Combining disruptions in KTI11 or KTI13 with a deletion in TOT3/ELP3 coding for the RNA polymerase II (RNAPII) Elongator histone acetyltransferase (HAT) yielded synthetic effects on slow growth phenotype expression. This suggests genetic interaction and possibly links KTI11 and KTI13 to Elongator function. PMID:11994165

  20. Library Widget for Moodle

    Directory of Open Access Journals (Sweden)

    Mariela Hristova

    2013-01-01

    Full Text Available Any course within a course management system is generally considered the intellectual space of the professor teaching it. Research tools and guides, such as search boxes for discovery services or links to course-specific and subject-specific guides, are created and maintained by librarians. In trying to get our tools and services closer to where students spend their time devoted to coursework, Oakland University libraries have developed a library widget – a self-serve code generator that allows professors to select what tools and services they want to bring into their course space. This approach has proven to be flexible, because it does not depend on a library presence within the course management system. It also offers persistent presence within courses since professors can archive courses, including the library widget, at the end of a semester and restore them in the system in future semesters. We are using the library widget as a pilot to inform decisions on future full integration of such functionality into Moodle.

  1. Fission product data library

    International Nuclear Information System (INIS)

    A library is described of data for 584 isotopes of fission products, including decay constants, branching ratios (both burn-up and decay), the type of emitted radiation, relative and absolute yields, capture cross sections for thermal neutrons, and resonance integrals. When a detailed decay scheme is not known, the mean energies of beta particles and neutrino and gamma radiations are given. In the ZVJE SKODA system the library is named BIBFP and is stored on film No 49 of the NE 803 B computer. It is used in calculating the inventory of fission products in fuel elements (and also determining absorption cross sections for burn-up calculations, gamma ray sources, heat generation) and in solving radioactivity transport problems in the primary circuit. It may also be used in the spectrometric method for burn-up determination of fuel elements. The library comprises the latest literary data available. It serves as the basis for library BIBGRFP storing group constants of fission products with independent yields of isotopes from fission. This, in turn, forms the basis for the BIBDN library collecting data on the precursors of delayed neutron emitters. (author)

  2. A borderless Library

    CERN Multimedia

    CERN Library

    2010-01-01

    The CERN Library has a large collection of documents in online or printed format in all disciplines needed by physicists, engineers and technicians. However,  users sometimes need to read documents not available at CERN. But don’t worry! Thanks to its Interlibrary loan and document delivery service, the CERN Library can still help you. Just fill in the online form or email us. We will then locate the document in other institutions and order it for you free of charge. The CERN Library cooperates with the largest libraries in Europe, such as ETH (Eidgenössische Technische Hochschule) in Zurich, TIB (Technische Informationsbibliothek) in Hanover and the British Library in London. Thanks to our network and our expertise in document search, most requests are satisfied in record time: articles are usually served in .pdf version a few hours after the order, and books or other printed materials are delivered within a few days. It is possible to ask for all types of documents suc...

  3. Expansion of a Plasmid Classification System for Gram-Positive Bacteria and Determination of the Diversity of Plasmids in Staphylococcus aureus Strains of Human, Animal, and Food Origins

    OpenAIRE

    Lozano, Carmen; García-Migura, Lourdes; Aspiroz, Carmen; Zarazaga, Myriam; Torres, Carmen; Aarestrup, Frank Møller

    2012-01-01

    An expansion of a previously described plasmid classification was performed and used to reveal the plasmid content of a collection of 92 Staphylococcus aureus strains of different origins. rep genes of other genera were detected in Staphylococcus. S1 pulsed-field gel electrophoresis (PFGE) hybridizations were performed with 18 representative S. aureus strains, and a high number of plasmids of different sizes and organizations were detected.

  4. Determination of plasmid copy number reveals the total plasmid DNA amount is greater than the chromosomal DNA amount in Bacillus thuringiensis YBT-1520.

    Directory of Open Access Journals (Sweden)

    Chunying Zhong

    Full Text Available Bacillus thuringiensis is the most widely used bacterial bio-insecticide, and most insecticidal crystal protein-coding genes are located on plasmids. Most strains of B. thuringiensis harbor numerous diverse plasmids, although the plasmid copy numbers (PCNs of all native plasmids in this host and the corresponding total plasmid DNA amount remains unknown. In this study, we determined the PCNs of 11 plasmids (ranging from 2 kb to 416 kb in a sequenced B. thuringiensis subsp. kurstaki strain YBT-1520 using real-time qPCR. PCNs were found to range from 1.38 to 172, and were negatively correlated to plasmid size. The amount of total plasmid DNA (∼8.7 Mbp was 1.62-fold greater than the amount of chromosomal DNA (∼5.4 Mbp at the mid-exponential growth stage (OD(600 = 2.0 of the organism. Furthermore, we selected three plasmids with different sizes and replication mechanisms to determine the PCNs over the entire life cycle. We found that the PCNs dynamically shifted at different stages, reaching their maximum during the mid-exponential growth or stationary phases and remaining stable and close to their minimum after the prespore formation stage. The PCN of pBMB2062, which is the smallest plasmid (2062 bp and has the highest PCN of those tested, varied in strain YBT-1520, HD-1, and HD-136 (172, 115, and 94, respectively. These findings provide insight into both the total plasmid DNA amount of B. thuringiensis and the strong ability of the species to harbor plasmids.

  5. Role of the 85-Kilobase Plasmid and Plasmid-Encoded Virulence-Associated Protein A in Intracellular Survival and Virulence of Rhodococcus equi

    OpenAIRE

    Giguère, Steeve; Hondalus, Mary K.; Yager, Julie A.; Darrah, Patricia; Mosser, David M.; Prescott, John F.

    1999-01-01

    Rhodococcus equi is a facultative intracellular pathogen of macrophages and a cause of pneumonia in young horses (foals) and immunocompromised people. Isolates of R. equi from pneumonic foals typically contain large, 85- or 90-kb plasmids encoding a highly immunogenic virulence-associated protein (VapA). The objective of this study was to determine the role of the 85-kb plasmid and VapA in the intracellular survival and virulence of R. equi. Clinical isolates containing the plasmid and expres...

  6. Increased diversity of libraries from libraries: chemoinformatic analysis of bis-diazacyclic libraries.

    Science.gov (United States)

    López-Vallejo, Fabian; Nefzi, Adel; Bender, Andreas; Owen, John R; Nabney, Ian T; Houghten, Richard A; Medina-Franco, José L

    2011-05-01

    Combinatorial libraries continue to play a key role in drug discovery. To increase structural diversity, several experimental methods have been developed. However, limited efforts have been performed so far to quantify the diversity of the broadly used diversity-oriented synthetic libraries. Herein, we report a comprehensive characterization of 15 bis-diazacyclic combinatorial libraries obtained through libraries from libraries, which is a diversity-oriented synthetic approach. Using MACCS keys, radial and different pharmacophoric fingerprints as well as six molecular properties, it was demonstrated the increased structural and property diversity of the libraries from libraries over the individual libraries. Comparison of the libraries to existing drugs, NCI diversity, and the Molecular Libraries Small Molecule Repository revealed the structural uniqueness of the combinatorial libraries (mean similarity <0.5 for any fingerprint representation). In particular, bis-cyclic thiourea libraries were the most structurally dissimilar to drugs retaining drug-like character in property space. This study represents the first comprehensive quantification of the diversity of libraries from libraries providing a solid quantitative approach to compare and contrast the diversity of diversity-oriented synthetic libraries with existing drugs or any other compound collection.

  7. cDNA suppression subtraction library for screening down-regulated genes in gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jian-Jun Du; Ke-Feng Dou; Shu-You Peng; Hua-Sheng Xiao; Wei-Zhong Wang; Wen-Xian Guan; Zhong-Hua Wang; Zhi-Qing Gao; Ying-Bin Liu

    2003-01-01

    AIM: To establish cDNA suppression subtraction library with a high subtraction efficiency by counterpart normal gastric mucosa mixture mRNA subtracting gastric cancer cells mixture mRNA for screening down-regulated genes in gastric carcinoma.METHODS: RNA of gastric cancer tissues and counterpart normal gastric mucosa were respectively isolated in five patients with gastric cancer, and their mRNA was purified.cDNA suppression subtraction library was established by counterpart normal gastric mucosa mixture mRNA (tester)subtracting gastric cancer tissues mixture mRNA (driver) of five patients with gastric carcinoma. The library plasmids were transformed into competent bacteria DH5a after ligation of the library cDNA fragments with T vectors. Library plasmids were extracted after picking colonies and shaking bacteria overnight. Its subtraction efficiency was confirmed by PCR and reverse hybridization of a nylon filter onto which the colonies of bacteria were transfered with probes of reverse transcription products cDNA of gastric cancer tissues mRNA and counterpart normal gastric mucosa mRNA labeled with α-32P dCTP.RESULTS: mRNA purified from total RNA of gastric cancer tissues and counterpart normal gastric mucosa in five patients with gastric carcinoma revealed a good quality. cDNA suppression subtraction library constructed for screening down-regulated genes in gastric carcinoma represented a high subtraction efficiency. 86 % of differential expression in down-regulated genes between counterpart normal gastric mucosa and gastric carcinoma was confirmed.CONCLUSION: cDNA suppression subtraction library with a high subtraction efficiency for screening down-regulated genes in gastric carcinoma is successfully established.

  8. Plasmid-Chromosome Recombination of Irradiated Shuttle Vector DNA in African Green Monkey Kidney Cells.

    Science.gov (United States)

    Mudgett, John Stuart

    1987-09-01

    An autonomously replicating shuttle vector was used to investigate the enhancement of plasmid-chromosome recombination in mammalian host cells by ultraviolet light and gamma radiation. Sequences homologous to the shuttle vector were stably inserted into the genome of African Green Monkey kidney cells to act as the target substrate for these recombination events. The SV40- and pBR322-derived plasmid DNA was irradiated with various doses of radiation before transfection into the transformed mammalian host cells. The successful homologous transfer of the bacterial ampicillin resistance (amp^{rm r}) gene from the inserted sequences to replace a mutant amp^->=ne on the shuttle vector was identified by plasmid extraction and transformation into E. coli host cells. Ultraviolet light (UV) was found not to induce homologous plasmid-chromosome recombination, while gamma radiation increased the frequency of recombinant plasmids detected. The introduction of specific double -strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was found to be increased by plasmid UV irradiation, but not to change with time. Plasmid survival, recombination, and mutagenesis were not affected by treating the mammalian host cells with UV light prior to plasmid transfection. The amp^{rm r} recombinant plasmid molecules analyzed were found to be mostly the result of nonconservative exchanges which appeared to involve both homologous and possibly nonhomologous interactions with the host chromosome. The observation that these recombinant structures were obtained from all of the plasmid alterations investigated suggests a common mechanistic origin for plasmid -chromosome recombination in these mammalian cells.

  9. The stb operon balances the requirements for vegetative stability and conjugative transfer of plasmid R388.

    Directory of Open Access Journals (Sweden)

    Catherine Guynet

    2011-05-01

    Full Text Available The conjugative plasmid R388 and a number of other plasmids carry an operon, stbABC, adjacent to the origin of conjugative transfer. We investigated the role of the stbA, stbB, and stbC genes. Deletion of stbA affected both conjugation and stability. It led to a 50-fold increase in R388 transfer frequency, as well as to high plasmid loss. In contrast, deletion of stbB abolished conjugation but provoked no change in plasmid stability. Deletion of stbC showed no effect, neither in conjugation nor in stability. Deletion of the entire stb operon had no effect on conjugation, which remained as in the wild-type plasmid, but led to a plasmid loss phenotype similar to that of the R388ΔstbA mutant. We concluded that StbA is required for plasmid stability and that StbA and StbB control conjugation. We next observed the intracellular positioning of R388 DNA molecules and showed that they localize as discrete foci evenly distributed in live Escherichia coli cells. Plasmid instability of the R388ΔΔstbA mutant correlated with aberrant localization of the plasmid DNA molecules as clusters, either at one cell pole, at both poles, or at the cell center. In contrast, plasmid molecules in the R388ΔΔstbB mutant were mostly excluded from the cell poles. Thus, results indicate that defects in both plasmid maintenance and transfer are a consequence of variations in the intracellular positioning of plasmid DNA. We propose that StbA and StbB constitute an atypical plasmid stabilization system that reconciles two modes of plasmid R388 physiology: a maintenance mode (replication and segregation and a propagation mode (conjugation. The consequences of this novel concept in plasmid physiology will be discussed.

  10. Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Sánchez-López, Juan Francisco; González-Ibarra, Joaquín; Álvarez-Vargas, Aurelio; Milewski, Slawomir; Villagómez-Castro, Julio César; Cano-Canchola, Carmen; López-Romero, Everardo

    2015-06-01

    Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.

  11. Coupling between the Basic Replicon and the Kis-Kid Maintenance System of Plasmid R1: Modulation by Kis Antitoxin Levels and Involvement in Control of Plasmid Replication

    OpenAIRE

    Juan López-Villarejo; Damián Lobato-Márquez; Ramón Díaz-Orejas

    2015-01-01

    kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now repo...

  12. Coupling between the Basic Replicon and the Kis-Kid Maintenance System of Plasmid R1: Modulation by Kis Antitoxin Levels and Involvement in Control of Plasmid Replication

    Directory of Open Access Journals (Sweden)

    Juan López-Villarejo

    2015-02-01

    Full Text Available kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication.

  13. Stellar libraries for Gaia

    International Nuclear Information System (INIS)

    Gaia will observe up to a billion stellar sources. Automated algorithms are under development to derive the atmospheric parameters of all observed spectra, from low resolution optical spectra alone or in synergy with high resolution spectra in the near-IR Ca II triplet region. To do so, a large database of state-of-the-art stellar libraries has been produced for the Gaia community, computed using different codes optimized for specific purposes. The choice to use different spectral codes in different regions of the H-R diagram raises the problem of the coherence of the different spectra, specifically in the transition zones. We present a comparison between the libraries from the point of view of spectra simulations for training the Gaia algorithms. We also present the implementation of these libraries into a Simple Stellar Population code.

  14. Secure medical digital libraries.

    Science.gov (United States)

    Papadakis, I; Chrissikopoulos, V; Polemi, D

    2001-12-01

    In this paper, a secure medical digital library is presented. It is based on the CORBA specifications for distributed systems. The described approach relies on a three-tier architecture. Interaction between the medical digital library and its users is achieved through a Web server. The choice of employing Web technology for the dissemination of medical data has many advantages compared to older approaches, but also poses extra requirements that need to be fulfilled. Thus, special attention is paid to the distinguished nature of such medical data, whose integrity and confidentiality should be preserved at all costs. This is achieved through the employment of Trusted Third Parties (TTP) technology for the support of the required security services. Additionally, the proposed digital library employs smartcards for the management of the various security tokens that are used from the above services.

  15. DOLIB: Distributed Object Library

    Energy Technology Data Exchange (ETDEWEB)

    D`Azevedo, E.F.; Romine, C.H.

    1994-10-01

    This report describes the use and implementation of DOLIB (Distributed Object Library), a library of routines that emulates global or virtual shared memory on Intel multiprocessor systems. Access to a distributed global array is through explicit calls to gather and scatter. Advantages of using DOLIB include: dynamic allocation and freeing of huge (gigabyte) distributed arrays, both C and FORTRAN callable interfaces, and the ability to mix shared-memory and message-passing programming models for ease of use and optimal performance. DOLIB is independent of language and compiler extensions and requires no special operating system support. DOLIB also supports automatic caching of read-only data for high performance. The virtual shared memory support provided in DOLIB is well suited for implementing Lagrangian particle tracking techniques. We have also used DOLIB to create DONIO (Distributed Object Network I/O Library), which obtains over a 10-fold improvement in disk I/O performance on the Intel Paragon.

  16. Presence of Glycopeptide-Encoding Plasmids in Enterococcal Isolates from Food and Humans in Denmark

    DEFF Research Database (Denmark)

    Migura, Lourdes Garcia; Valenzuela, Antonio Jesus Sanchez; Jensen, Lars Bogø

    2011-01-01

    developed techniques for classification of plasmids. Replicons associated with sex pheromone-inducible plasmids were detected in all GR E. faecalis, whereas GR Enterococcus faecium contained plasmids known to be widely distributed among enterococci. vanA resistance is common in E. faecium isolates from meat...... elements (MGE) such as plasmids and transposons. Presence of MGE was tested in all GRE isolated from food in Denmark in 2005–2007 including the first vanA mediated Enterococcus faecalis isolated from food. The ability of these plasmids to transfer and persist among enterococci was investigated using newly...

  17. Enterococcus faecalis hemolysin-bacteriocin plasmids belong to the same incompatibility group.

    OpenAIRE

    Colmar, I; Horaud, T

    1987-01-01

    Plasmid pair coexistence was studied both among nine Enterococcus faecalis hemolysin-bacteriocin (Hly-Bcn) plasmids, including pJH2, pAD1, pAM gamma 1, and pIP964, and between pIP964 and five R plasmids. Some of the Hly-Bcn plasmids used were derivatives encoding resistance to erythromycin or tetracycline. The Hly-Bcn plasmids were incompatible with each other; 40 to 100% displacement was observed bilaterally for eight pairs and unilaterally for one pair. In contrast, pIP964 stably coexisted ...

  18. [Plasmids of streptomycetes strains isolated from soils of Ukraine with different anthropogenic loading].

    Science.gov (United States)

    Luk'ianchuk, V V; Polishchuk, L V; Matseliukh, B P

    2010-01-01

    Screening of plasmid DNA was carried out among 94 streptomycetes cultures which were isolated from the samples of Ukrainian soils with different anthropogenic contamination. Seventeen streptomycetes strains containing plasmid DNA were found. It is established that some cultures contain more than one plasmid (Streptomyces sp.M15, S.sp.T8, S.sp.T19). Depending on a molecular sizes the found plasmids were divided in 2 groups: 3 kb-15 kb, and 30 kb-70 kb. Research of a few morphological and physiological properties of plasmid strains of streptomycetes was carried out. The paper is presented in Ukrainian. PMID:21117293

  19. Investigation of diversity of plasmids carrying the blaTEM-52 gene

    DEFF Research Database (Denmark)

    Bielak, Eliza Maria; Bergenholtz, Rikke D.; Jørgensen, Mikael Skaanning;

    2011-01-01

    OBJECTIVES: To investigate the diversity of plasmids that carry blaTEM-52 genes among Escherichia coli and Salmonella enterica originating from animals, meat products and humans. METHODS: A collection of 22 blaTEM-52-encoding plasmids was characterized by restriction fragment length polymorphism...... of self-transfer to a plasmid-free E. coli recipient. CONCLUSIONS: The blaTEM-52 gene found in humans could have been transmitted on transferable plasmids originating from animal sources. Some of the blaTEM-52 plasmids carry replicons that differ from the classical ones. Two novel replicons were detected...

  20. Structural and genetic analyses of a par locus that regulates plasmid partition in Bacillus subtilis.

    OpenAIRE

    Chang, S.; Chang, S Y; Gray, O

    1987-01-01

    The Bacillus plasmid pLS11 partitions faithfully during cell division. Using a partition-deficient plasmid vector, we randomly cloned DNA fragments of plasmid pLS11 and identified the locus that regulates plasmid partition (par) by cis complementation in Bacillus subtilis. The cloned par gene conferred upon the vector plasmid a high degree of segregational stability. The par locus was mapped to a 167-base-pair segment on pLS11, and its nucleotide sequence was determined. The cloned par fragme...

  1. cmp, a cis-acting plasmid locus that increases interaction between replication origin and initiator protein.

    OpenAIRE

    Gennaro, M L; Novick, R P

    1986-01-01

    pT181, a 4.4-kilobase multicopy plasmid of Staphylococcus aureus, encodes a trans-acting initiator protein, RepC, which was rate limiting for replication. Deletions in a 500-base-pair region of the plasmid external to the minimal replicon decreased the ability of the plasmid to compete with a coexisting incompatible plasmid. These deletions, which define a region called cmp (for competition), appeared to affect the interaction of RepC and the plasmid origin of replication. However, in the hom...

  2. Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3

    OpenAIRE

    Al-Allaf, Faisal A.; Tolmachov, Oleg E.; Zambetti, Lia Paola; Tchetchelnitski, Viktoria; Mehmet, Huseyin

    2012-01-01

    Large-scale production of plasmid DNA to prepare therapeutic gene vectors or DNA-based vaccines requires a suitable bacterial host, which can stably maintain the plasmid DNA during industrial cultivation. Plasmid loss during bacterial cell divisions and structural changes in the plasmid DNA can dramatically reduce the yield of the desired recombinant plasmid DNA. While generating an HIV-based gene vector containing a bicistronic expression cassette 5′-Olig2cDNA-IRES-dsRed2-3′, we encountered ...

  3. The incC Sequence Is Required for R27 Plasmid Stability

    OpenAIRE

    Tassinari, Eleonora; Aznar, Sonia; Urcola, Imanol; Prieto, Alejandro; Hüttener, Mário; Juárez, Antonio

    2016-01-01

    IncHI plasmids account for multiple antimicrobial resistance in Salmonella and other enterobacterial genera. These plasmids are generally very stable in their bacterial hosts. R27 is the archetype of IncHI1 plasmids. A high percentage of the R27-encoded open reading frames (ORFs) (66.7%) do not show similarity to any known ORFs. We performed a deletion analysis of all non-essential R27 DNA sequences to search for hitherto non-identified plasmid functions that might be required for plasmid sta...

  4. Library resources on the Internet

    Science.gov (United States)

    Buchanan, Nancy L.

    1995-07-01

    Library resources are prevalent on the Internet. Library catalogs, electronic books, electronic periodicals, periodical indexes, reference sources, and U.S. Government documents are available by telnet, Gopher, World Wide Web, and FTP. Comparatively few copyrighted library resources are available freely on the Internet. Internet implementations of library resources can add useful features, such as full-text searching. There are discussion lists, Gophers, and World Wide Web pages to help users keep up with new resources and changes to existing ones. The future will bring more library resources, more types of library resources, and more integrated implementations of such resources to the Internet.

  5. Controlling hospital library theft

    OpenAIRE

    Cuddy, Theresa M.; Marchok, Catherine

    2003-01-01

    At Capital Health System/Fuld Campus (formerly Helene Fuld Medical Center), the Health Sciences Library lost many books and videocassettes. These materials were listed in the catalog but were missing when staff went to the shelves. The hospital had experienced a downsizing of staff, a reorganization, and a merger. When the library staff did an inventory, $10,000 worth of materials were found to be missing. We corrected the situation through a series of steps that we believe will help other li...

  6. Enterprise Reference Library

    Science.gov (United States)

    Bickham, Grandin; Saile, Lynn; Havelka, Jacque; Fitts, Mary

    2011-01-01

    Introduction: Johnson Space Center (JSC) offers two extensive libraries that contain journals, research literature and electronic resources. Searching capabilities are available to those individuals residing onsite or through a librarian s search. Many individuals have rich collections of references, but no mechanisms to share reference libraries across researchers, projects, or directorates exist. Likewise, information regarding which references are provided to which individuals is not available, resulting in duplicate requests, redundant labor costs and associated copying fees. In addition, this tends to limit collaboration between colleagues and promotes the establishment of individual, unshared silos of information The Integrated Medical Model (IMM) team has utilized a centralized reference management tool during the development, test, and operational phases of this project. The Enterprise Reference Library project expands the capabilities developed for IMM to address the above issues and enhance collaboration across JSC. Method: After significant market analysis for a multi-user reference management tool, no available commercial tool was found to meet this need, so a software program was built around a commercial tool, Reference Manager 12 by The Thomson Corporation. A use case approach guided the requirements development phase. The premise of the design is that individuals use their own reference management software and export to SharePoint when their library is incorporated into the Enterprise Reference Library. This results in a searchable user-specific library application. An accompanying share folder will warehouse the electronic full-text articles, which allows the global user community to access full -text articles. Discussion: An enterprise reference library solution can provide a multidisciplinary collection of full text articles. This approach improves efficiency in obtaining and storing reference material while greatly reducing labor, purchasing and

  7. Reference neutron activation library

    International Nuclear Information System (INIS)

    Many scientific endeavors require accurate nuclear data. Examples include studies of environmental protection connected with the running of a nuclear installation, the conceptual designs of fusion energy producing devices, astrophysics and the production of medical isotopes. In response to this need, many national and international data libraries have evolved over the years. Initially nuclear data work concentrated on materials relevant to the commercial power industry which is based on the fission of actinides, but recently the topic of activation has become of increasing importance. Activation of materials occurs in fission devices, but is generally overshadowed by the primary fission process. In fusion devices, high energy (14 MeV) neutrons produced in the D-T fusion reaction cause activation of the structure, and (with the exception of the tritium fuel) is the dominant source of activity. Astrophysics requires cross-sections (generally describing neutron capture) or its studies of nucleosynthesis. Many analytical techniques require activation analysis. For example, borehole logging uses the detection of gamma rays from irradiated materials to determine the various components of rocks. To provide data for these applications, various specialized data libraries have been produced. The most comprehensive of these have been developed for fusion studies, since it has been appreciated that impurities are of the greatest importance in determining the overall activity, and thus data on all elements are required. These libraries contain information on a wide range of reactions: (n,γ), (n,2n), (n,α), (n,p), (n,d), (n,t), (n,3He)and (n,n')over the energy range from 10-5 eV to 15 or 20 MeV. It should be noted that the production of various isomeric states have to be treated in detail in these libraries,and that the range of targets must include long-lived radioactive nuclides in addition to stable nuclides. These comprehensive libraries thus contain almost all the

  8. Knowledge management for libraries

    CERN Document Server

    Forrestal, Valerie

    2015-01-01

    Libraries are creating dynamic knowledge bases to capture both tacit and explicit knowledge and subject expertise for use within and beyond their organizations. In this book, readers will learn to move policies and procedures manuals online using a wiki, get the most out of Microsoft SharePoint with custom portals and Web Parts, and build an FAQ knowledge base from reference management applications such as LibAnswers. Knowledge Management for Libraries guides readers through the process of planning, developing, and launching th

  9. future of research libraries

    CERN Document Server

    Naryandas, Narakesari; Kindström, Daniel

    2014-01-01

    Research libraries have been an integral part of the scholarly communication system since that system emerged in its present form. They now face a period of unprecedentedly drastic and rapid change. This is caused, first and foremost, by the migration of much scholarly material to digital formats, raising the question of the future purpose of the 'library space'. Together with this come transfigurational changes to the communication change of recorded information, with the roles of authors , publishers, database producers and librarians and archivists all in a state of flux. Finally, new forms

  10. Wordpress for libraries

    CERN Document Server

    Haefele, Chad

    2015-01-01

    WordPress is not only the most popular blogging software in the world, but it is also a powerful content management system that runs more than 23 percent of all websites. The current version alone has been downloaded almost 20 million times, and the WordPress community has built more than 38,000 plugins to extend and enhance the system. Libraries are using this technology to create community-oriented websites, blogs, subject guides, digital archives, and more. This hands-on, practical book walks readers through the entire process of setting up a WordPress website for their library,

  11. Genetiese manipulering van die gis Saccharomyces cerevisiae betreffende polisakkariedbenutting

    Directory of Open Access Journals (Sweden)

    I. S. Pretoruis

    1992-07-01

    Full Text Available Die gis Saccharomyces cerevisiae word wêreldwyd as die belangrikste kommersiële mikro-organisme bestempel en geniet sogenaamde ABAV-status (Algemeen Beskou As Veilig weens dié gis se eeue lange verbintenis met voedselproduksie (bv. brood, wyn, bier, proteienaanvulling en geurstowwe.

  12. Analysis of the RNA Content of the Yeast "Saccharomyces Cerevisiae"

    Science.gov (United States)

    Deutch, Charles E.; Marshall, Pamela A.

    2008-01-01

    In this article, the authors describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. This set of experiments focuses on RNAs from the yeast "Saccharomyces cerevisiae", a…

  13. Sucrose and Saccharomyces cerevisiae: a relationship most sweet.

    Science.gov (United States)

    Marques, Wesley Leoricy; Raghavendran, Vijayendran; Stambuk, Boris Ugarte; Gombert, Andreas Karoly

    2016-02-01

    Sucrose is an abundant, readily available and inexpensive substrate for industrial biotechnology processes and its use is demonstrated with much success in the production of fuel ethanol in Brazil. Saccharomyces cerevisiae, which naturally evolved to efficiently consume sugars such as sucrose, is one of the most important cell factories due to its robustness, stress tolerance, genetic accessibility, simple nutrient requirements and long history as an industrial workhorse. This minireview is focused on sucrose metabolism in S. cerevisiae, a rather unexplored subject in the scientific literature. An analysis of sucrose availability in nature and yeast sugar metabolism was performed, in order to understand the molecular background that makes S. cerevisiae consume this sugar efficiently. A historical overview on the use of sucrose and S. cerevisiae by humans is also presented considering sugarcane and sugarbeet as the main sources of this carbohydrate. Physiological aspects of sucrose consumption are compared with those concerning other economically relevant sugars. Also, metabolic engineering efforts to alter sucrose catabolism are presented in a chronological manner. In spite of its extensive use in yeast-based industries, a lot of basic and applied research on sucrose metabolism is imperative, mainly in fields such as genetics, physiology and metabolic engineering.

  14. Recycling carbon dioxide during xylose fermentation by engineered Saccharomyces cerevisiae

    Science.gov (United States)

    In this study, we introduced the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase (PRK) into an engineered S. cerevisiae (SR8) harboring the XR/XDH pathway and up-regulated PPP 10, to enable CO2 recycling through a synthetic rPPP during xylose fermentation (Fig. 1). ...

  15. The Plasma Membrane of Saccharomyces cerevisiae : Structure, Function, and Biogenesis

    NARCIS (Netherlands)

    VANDERREST, ME; KAMMINGA, AH; NAKANO, A; ANRAKU, Y; POOLMAN, B; KONINGS, WN

    1995-01-01

    The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an extens

  16. Improving biomass sugar utilization by engineered Saccharomyces cerevisiae

    Science.gov (United States)

    The efficient utilization of all available sugars in lignocellulosic biomass, which is more abundant than available commodity crops and starch, represents one of the most difficult technological challenges for the production of bioethanol. The well-studied yeast Saccharomyces cerevisiae has played a...

  17. Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism.

    Science.gov (United States)

    Kim, Soo Rin; Park, Yong-Cheol; Jin, Yong-Su; Seo, Jin-Ho

    2013-11-01

    Efficient and rapid fermentation of all sugars present in cellulosic hydrolysates is essential for economic conversion of renewable biomass into fuels and chemicals. Xylose is one of the most abundant sugars in cellulosic biomass but it cannot be utilized by wild type Saccharomyces cerevisiae, which has been used for industrial ethanol production. Therefore, numerous technologies for strain development have been employed to engineer S. cerevisiae capable of fermenting xylose rapidly and efficiently. These include i) optimization of xylose-assimilating pathways, ii) perturbation of gene targets for reconfiguring yeast metabolism, and iii) simultaneous co-fermentation of xylose and cellobiose. In addition, the genetic and physiological background of host strains is an important determinant to construct efficient and rapid xylose-fermenting S. cerevisiae. Vibrant and persistent researches in this field for the last two decades not only led to the development of engineered S. cerevisiae strains ready for industrial fermentation of cellulosic hydrolysates, but also deepened our understanding of operational principles underlying yeast metabolism. PMID:23524005

  18. Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Solis Escalante, D.

    2015-01-01

    Glycolysis, a biochemical pathway that oxidizes glucose to pyruvate, is at the core of sugar metabolism in Saccharomyces cerevisiae (bakers’ yeast). Glycolysis is not only a catabolic route involved in energy conservation, but also provides building blocks for anabolism. From an applied perspective,

  19. Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Erdmann, Ralf; Veenhuis, Marten; Mertens, Daphne; Kunau, Wolf-H.

    1989-01-01

    Two mutants of Saccharomyces cerevisiae affected in peroxisomal assembly (pas mutants) have been isolated and characterized. Each strain contains a single mutation that results in (i) the inability to grow on oleic acid, (ii) accumulation of peroxisomal matrix enzymes in the cytosol, and (iii) absen

  20. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.

    Science.gov (United States)

    Bierman, M; Logan, R; O'Brien, K; Seno, E T; Rao, R N; Schoner, B E

    1992-07-01

    We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. All vectors contain the 760-bp oriT fragment from the IncP plasmid, RK2. Transfer functions need to be supplied in trans by the E. coli donor strain. We have incorporated into these vectors selectable antibiotic-resistance markers (AmR, ThR, SpR) that function in Streptomyces spp. and other features that should allow for: (i) integration via homologous recombination between cloned DNA and the Streptomyces spp. chromosome, (ii) autonomous replication, or (iii) site-specific integration at the bacteriophage phi C31 attachment site. Shuttle cosmids for constructing genomic libraries and bacteriophage P1 cloning vector capable of accepting approx. 100-kb fragments are also described. A simple mating procedure has been developed for the conjugal transfer of these vectors from E. coli to Streptomyces spp. that involves plating of the donor strain and either germinated spores or mycelial fragments of the recipient strain. We have shown that several of these vectors can be introduced into Streptomyces fradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.