Using "Pseudomonas Putida xylE" Gene to Teach Molecular Cloning Techniques for Undergraduates

Science.gov (United States)

Dong, Xu; Xin, Yi; Ye, Li; Ma, Yufang

2009-01-01

We have developed and implemented a serial experiment in molecular cloning laboratory course for undergraduate students majored in biotechnology. "Pseudomonas putida xylE" gene, encoding catechol 2, 3-dioxygenase, was manipulated to learn molecular biology techniques. The integration of cloning, expression, and enzyme assay gave students…

Molecular cloning and characterization of pathogenesis-related ...

African Journals Online (AJOL)

We described the cloning and characterization of pathogenesis-related protein 5 gene in maize, named ZmPR5 (GenBank Accession Number: HM230665). Molecular and bioinformatic analyses of ZmPR5 revealed an open reading frame (ORF) of 525 bp, encoding a protein of 175 amino acids (aa) and a deduced ...

Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

Science.gov (United States)

Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

2016-10-01

This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

Molecular cloning of cellulase genes from indigenous bacterial isolates

International Nuclear Information System (INIS)

Jong Bor Chyan; Pauline Liew Woan Ying; Mat Rasol Awang

2006-01-01

Indigenous cellulolytic bacterial isolates having high activities in degrading carboxymethyl cellulose (CMC) were isolated from local environments. Identification of these isolates were performed by molecular techniques. By using polymerase chain reaction (PCR) techniques, PCR products encoding cellulase gene were amplified from the total genomic DNAs. Purified PCR product was successfully cloned and expressed in Escherichia coli host system. The complete nucleotide sequences of the cellulase genes determined. The analysis of amino acid sequences deduced from the genes indicated that the cloned DNA fragments show high homology to those of endoglucanase genes of family GH5. All cloned genes consist of an N-terminal signal peptide, a catalytic domain of family 5 glycosyl hydrolase and a cellulose-binding domain of family III. (Author)

Molecular cloning and characterization of a novel human testis ...

Indian Academy of Sciences (India)

Molecular cloning and characterization of a novel human testis-specific gene by use of ... pared against 70 other libraries, and the hits showing >10- fold differences .... proteins or testis-development-related proteins such as TSP-. NY, TPX1 ...

Molecular cloning and characterization of a putative OGG_N domain ...

African Journals Online (AJOL)

Molecular cloning and characterization of a putative OGG_N domain from the camel, Camelus dromedarius. Farid Shokry Ataya, Mohammad Saud Alanazi, Dalia Fouad, Hehsam Mahmoud Saeed, Mohammad Bazzi ...

Hybridization improved bacteria resistance in abalone: Evidence from physiological and molecular responses.

Science.gov (United States)

Liang, Shuang; Luo, Xuan; You, Weiwei; Ke, Caihuan

2018-01-01

Hybridization is an effective way of improving germplasm in abalone, as it often generates benign traits in the hybrids. The hybrids of Haliotis discus hannai and H. gigantea have shown heterosis in terms of disease resistance than one or both parental species. In the present study, to elucidate the physiological and molecular mechanism of this heterosis, we analyzed the dynamic changes of several immune indexes including survival rate, total circulating haemocyte count (THC), phagocytic activity, reactive oxygen species level (ROS) and phenoloxidase activity (PO) in two parental species, H. discus hannai (DD) and H. gigantea (GG), and their reciprocal hybrids H. discus hannai ♀ × H. gigantea ♂ (DG), H. gigantea ♀ × H. discus hannai ♂ (GD) challenged with a mixture of Vibrio harveyi, V. alginolyticus and V. parahaemolyticus (which have been demonstrated to be pathogenic to abalone). Besides, we cloned and analyzed three important immune genes: heat shock protein 70 (hsp70), ferritin and cold shock domain protein (csdp) in H. discus hannai and H. gigantea, then further investigated their mRNA level changes in the four abalone genotypes after bacterial challenge. Results showed that these physiological and molecular parameters were significantly induced by bacterial exposure, and their changing patterns were obviously different between the four genotypes: (1) Survival rates of the two hybrids were higher than both parental species after bacterial exposure; (2) DG had higher THC than the other three genotypes; (3) Phagocytosis responded slower in the hybrids than in the parental species; (4) DD's ROS level was lower than the other three genotypes at 48 h post infection; (5) Phenoloxidase activity was lower in DD during the infection compared to the other genotypes; (6) mRNA levels of hsp70 and csdp, were always lower in at least one parental species (DD) than in the hybrids after the bacterial exposure. Results from this study indicate that the hybrids

Structural and functional characterization of a novel molluskan ortholog of TRAF and TNF receptor-associated protein from disk abalone (Haliotis discus discus).

Science.gov (United States)

Lee, Youngdeuk; Elvitigala, Don Anushka Sandaruwan; Whang, Ilson; Lee, Sukkyoung; Kim, Hyowon; Zoysa, Mahanama De; Oh, Chulhong; Kang, Do-Hyung; Lee, Jehee

2014-09-01

Immune signaling cascades have an indispensable role in the host defense of almost all the organisms. Tumor necrosis factor (TNF) signaling is considered as a prominent signaling pathway in vertebrate as well as invertebrate species. Within the signaling cascade, TNF receptor-associated factor (TRAF) and TNF receptor-associated protein (TTRAP) has been shown to have a crucial role in the modulation of immune signaling in animals. Here, we attempted to characterize a novel molluskan ortholog of TTRAP (AbTTRAP) from disk abalone (Haliotis discus discus) and analyzed its expression levels under pathogenic stress. The complete coding sequence of AbTTRAP consisted of 1071 nucleotides, coding for a 357 amino acid peptide, with a predicted molecular mass of 40 kDa. According to our in-silico analysis, AbTTRAP resembled the typical TTRAP domain architecture, including a 5'-tyrosyl DNA phosphodiesterase domain. Moreover, phylogenetic analysis revealed its common ancestral invertebrate origin, where AbTTRAP was clustered with molluskan counterparts. Quantitative real time PCR showed universally distributed expression of AbTTRAP in selected tissues of abalone, from which more prominent expression was detected in hemocytes. Upon stimulation with two pathogen-derived mitogens, lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), transcript levels of AbTTRAP in hemocytes and gill tissues were differentially modulated with time. In addition, the recombinant protein of AbTTRAP exhibited prominent endonuclease activity against abalone genomic DNA, which was enhanced by the presence of Mg(2+) in the medium. Collectively, these results reinforce the existence of the TNF signaling cascade in mollusks like disk abalone, further implicating the putative regulatory behavior of TTRAP in invertebrate host pathology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular Cloning Designer Simulator (MCDS): All-in-one molecular cloning and genetic engineering design, simulation and management software for complex synthetic biology and metabolic engineering projects.

Science.gov (United States)

Shi, Zhenyu; Vickers, Claudia E

2016-12-01

Molecular Cloning Designer Simulator (MCDS) is a powerful new all-in-one cloning and genetic engineering design, simulation and management software platform developed for complex synthetic biology and metabolic engineering projects. In addition to standard functions, it has a number of features that are either unique, or are not found in combination in any one software package: (1) it has a novel interactive flow-chart user interface for complex multi-step processes, allowing an integrated overview of the whole project; (2) it can perform a user-defined workflow of cloning steps in a single execution of the software; (3) it can handle multiple types of genetic recombineering, a technique that is rapidly replacing classical cloning for many applications; (4) it includes experimental information to conveniently guide wet lab work; and (5) it can store results and comments to allow the tracking and management of the whole project in one platform. MCDS is freely available from https://mcds.codeplex.com.

Anti-Inflammatory Effect of By-Products from Haliotis discus hannai in RAW 264.7 Cells

Directory of Open Access Journals (Sweden)

Ho-Seok Rho

2015-01-01

Full Text Available Several reports promoted the potential of shellfish due to their ability to act as antioxidant, anti-inflammatory, and antimicrobial agents. Pacific abalone, Haliotis discus hannai viscera is, reported to possess bioactivities such as antioxidative stress and anti-inflammatory. In this study, anti-inflammatory potential of mucus-secreting glands from shell-shucking waste of H. discus hannai was evaluated using RAW 264.7 mouse macrophage cell model. Results indicated that presence of H. discus hannai mucosubstance by-products (AM significantly lowered the nitric oxide (NO production along the expressional suppression of inflammatory mediators such as cytokines TNF-α, IL-1β, and IL-6 and enzymes iNOS and COX-2. Also, AM was shown to increase expression of anti-inflammatory response mediator HO-1. Presence of AM also scavenged the free radicals in vitro. In conclusion, by-products of H. discus hannai are suggested to possess notable anti-inflammatory potential which promotes the possibility of utilization as functional food ingredient.

Molecular cloning of osteoma-inducing replication-competent murine leukemia viruses from the RFB osteoma virus stock

DEFF Research Database (Denmark)

Pedersen, Lene; Behnisch, Werner; Schmidt, Jörg

1992-01-01

We report the molecular cloning of two replication-competent osteoma-inducing murine leukemia viruses from the RFB osteoma virus stock (M. P. Finkel, C. A. Reilly, Jr., B. O. Biskis, and I. L. Greco, p. 353-366, in C. H. G. Price and F. G. M. Ross, ed., Bone--Certain Aspects of Neoplasia, 1973......). Like the original RFB osteoma virus stock, viruses derived from the molecular RFB clones induced multiple osteomas in mice of the CBA/Ca strain. The cloned RFB viruses were indistinguishable by restriction enzyme analysis and by nucleotide sequence analysis of their long-terminal-repeat regions...

A universal mini-vector and an annealing of PCR products (APP)-based cloning strategy for convenient molecular biological manipulations.

Science.gov (United States)

Liu, Xia; Li, Tuoping; Hart, Darren J; Gao, Song; Wang, Hongling; Gao, Herui; Xu, Shumin; Zhang, Yifeng; Liu, Yifei; An, Yingfeng

2018-03-18

Currently, the most widely used strategies for molecular cloning are sticky-end ligation-based cloning, TA cloning, blunt-end ligation-based cloning and ligase-independent cloning. In this study we have developed a novel mini-vector pANY1 which can simultaneously meet the requirements of all these cloning strategies. In addition, the selection of appropriate restriction digestion sites is difficult in some cases because of the presence of internal sites. In this study, an annealing of PCR products (APP)-based sticky-end cloning strategy was introduced to avoid this issue. Additionally, false positives occur during molecular cloning, which increases the workload of isolating positive clones. The plasmid pANY1 contains a ccdB cassette between multiple cloning sites, which efficiently avoids these false positives. Therefore, this mini-vector should serve as a useful tool with wide applications in biosciences, agriculture, food technologies, etc. Copyright © 2018 Elsevier Inc. All rights reserved.

Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

Science.gov (United States)

Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

2001-01-01

This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

Experiences in using DISCUS for visualizing human communication

Science.gov (United States)

Groehn, Matti; Nieminen, Marko; Haho, Paeivi; Smeds, Riitta

2000-02-01

In this paper, we present further improvement to the DISCUS software that can be used to record and analyze the flow and constants of business process simulation session discussion. The tool was initially introduced in 'visual data exploration and analysis IV' conference. The initial features of the tool enabled the visualization of discussion flow in business process simulation sessions and the creation of SOM analyses. The improvements of the tool consists of additional visualization possibilities that enable quick on-line analyses and improved graphical statistics. We have also created the very first interface to audio data and implemented two ways to visualize it. We also outline additional possibilities to use the tool in other application areas: these include usability testing and the possibility to use the tool for capturing design rationale in a product development process. The data gathered with DISCUS may be used in other applications, and further work may be done with data ming techniques.

Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

Energy Technology Data Exchange (ETDEWEB)

Deymier, Martin J., E-mail: mdeymie@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Claiborne, Daniel T., E-mail: dclaibo@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Ende, Zachary, E-mail: zende@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Ratner, Hannah K., E-mail: hannah.ratner@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Kilembe, William, E-mail: wkilembe@rzhrg-mail.org [Zambia-Emory HIV Research Project (ZEHRP), B22/737 Mwembelelo, Emmasdale Post Net 412, P/BagE891, Lusaka (Zambia); Allen, Susan, E-mail: sallen5@emory.edu [Zambia-Emory HIV Research Project (ZEHRP), B22/737 Mwembelelo, Emmasdale Post Net 412, P/BagE891, Lusaka (Zambia); Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA (United States); Hunter, Eric, E-mail: eric.hunter2@emory.edu [Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329 (United States); Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA (United States)

2014-11-15

The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor.

Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning

International Nuclear Information System (INIS)

Deymier, Martin J.; Claiborne, Daniel T.; Ende, Zachary; Ratner, Hannah K.; Kilembe, William; Allen, Susan; Hunter, Eric

2014-01-01

The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual's diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens. - Highlights: • Our novel methodology demonstrates accurate amplification and cloning of full-length HIV-1 genomes. • A majority of plasma derived HIV variants from a chronically infected individual are infectious. • The transmitted/founder was more infectious than the majority of the variants from the chronically infected donor

Gene Transfer and Molecular Cloning of the Human NGF Receptor

Science.gov (United States)

Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

1986-04-01

Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

Molecular cloning of a peptidylglycine alpha-hydroxylating monooxygenase from sea anemones

DEFF Research Database (Denmark)

Hauser, F; Williamson, M; Grimmelikhuijzen, C J

1997-01-01

conserved regions of PHM, we have now cloned a PHM from the sea anemone Calliactis parasitica showing 42% amino acid sequence identity with rat PHM. Among the conserved (identical) amino acid residues are five histidine and one methionine residue, which bind two Cu2+ atoms that are essential for PHM...... activity. No cDNA coding for PAL could be identified, suggesting that sea anemone PAL is coded for by a gene that is different from the sea anemone PHM gene, a situation similar to the one found in insects. This is the first report on the molecular cloning of a cnidarian PHM. Udgivelsesdato: 1997-Dec-18...

Ferritin from the Pacific abalone Haliotis discus hannai: Analysis of cDNA sequence, expression, and activity.

Science.gov (United States)

Qiu, Reng; Kan, Yunchao; Li, Dandan

2016-02-01

Ferritin plays an important role in iron homeostasis due to its ability to bind and sequester large amounts of iron. In this study, the gene encoding a ferritin (HdhFer2) was cloned from Pacific abalone (Haliotis discus hannai). The full-length cDNA of HdhFer2 contains a 5'-UTR of 121 bp, an ORF of 516 bp, and a 3'-UTR of 252 bp with a polyadenylation signal sequence of AATAAA and a poly(A) tail. It also contains a 31 bp iron-responsive element (IRE) in the 5'-UTR position, which is conserved in many ferritins. HdhFer2 consists of 171 amino acid residues with a predicted molecular weight (MW) ∼19.8 kDa and a theoretical isoelectric point (PI) of 4.84. The deduced amino acid sequence of HdhFer2 contains two ferritin iron-binding region signatures (IBRSs). HdhFer2 mRNA was detected in a wide range of tissues and was dominantly expressed in the gill. Infection with the bacterial pathogen Vibrio anguillarum significantly upregulated HdhFer2 expression in a time-dependent manner. Recombinant HdhFer2 (rHdhFer2) purified from Escherichia coli was able to bind ferrous iron in a concentration-dependent manner. In summary, these results suggest that HdhFer2 is a crucial protein in the iron-withholding defense system, and plays an important role in the innate immune response of abalone. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enzymatic properties and primary structures of two α-amylase isozymes from the Pacific abalone Haliotis discus hannai

OpenAIRE

Kumagai, Yuya; Satoh, Takuya; Inoue, Akira; Ojima, Takao

2013-01-01

Two α-amylase (EC 3.2.1.1) isozymes, HdAmy58 and HdAmy82, with approximate molecular masses of 58 kDa and 82 kDa, respectively, were isolated from the digestive fluid of the Pacific abalone Haliotis discus hannai. Optimal temperatures and pHs for HdAmy58 and HdAmy82 were at 30℃ and 6.7, and 30℃ and 6.1, respectively. Both enzymes similarly degraded starch, glycogen, and maltooligosaccharides larger than maltotriose producing maltose and maltotriose as the major degradation products. However, ...

The 5' untranslated region of a novel infectious molecular clone of the dicistrovirus cricket paralysis virus modulates infection.

Science.gov (United States)

Kerr, Craig H; Wang, Qing S; Keatings, Kathleen; Khong, Anthony; Allan, Douglas; Yip, Calvin K; Foster, Leonard J; Jan, Eric

2015-06-01

Dicistroviridae are a family of RNA viruses that possesses a single-stranded positive-sense RNA genome containing two distinct open reading frames (ORFs), each preceded by an internal ribosome entry site that drives translation of the viral structural and nonstructural proteins, respectively. The type species, Cricket paralysis virus (CrPV), has served as a model for studying host-virus interactions; however, investigations into the molecular mechanisms of CrPV and other dicistroviruses have been limited as an established infectious clone was elusive. Here, we report the construction of an infectious molecular clone of CrPV. Transfection of in vitro-transcribed RNA from the CrPV clone into Drosophila Schneider line 2 (S2) cells resulted in cytopathic effects, viral RNA accumulation, detection of negative-sense viral RNA, and expression of viral proteins. Transmission electron microscopy, viral titers, and immunofluorescence-coupled transwell assays demonstrated that infectious viral particles are released from transfected cells. In contrast, mutant clones containing stop codons in either ORF decreased virus infectivity. Injection of adult Drosophila flies with virus derived from CrPV clones but not UV-inactivated clones resulted in mortality. Molecular analysis of the CrPV clone revealed a 196-nucleotide duplication within its 5' untranslated region (UTR) that stimulated translation of reporter constructs. In cells infected with the CrPV clone, the duplication inhibited viral infectivity yet did not affect viral translation or RNA accumulation, suggesting an effect on viral packaging or entry. The generation of the CrPV infectious clone provides a powerful tool for investigating the viral life cycle and pathogenesis of dicistroviruses and may further understanding of fundamental host-virus interactions in insect cells. Dicistroviridae, which are RNA viruses that infect arthropods, have served as a model to gain insights into fundamental host-virus interactions in

Identification of molecular performance from oil palm clones based on SSR markers

Science.gov (United States)

Putri, Lollie Agustina P.; Basyuni, M.; Bayu, Eva S.; Arvita, D.; Arifiyanto, D.; Syahputra, I.

2018-03-01

In Indonesia, the oil palms are an important economic crop, producing food and raw materials for the food, confectionary, cosmetics and oleo-chemical industrial demands of oil palm products. Clonal oil palm offers the potential for greater productivity because it is possible to establish uniform tree stands comprising identical copies (clones) of a limited number of highly productive oil palms. Unfortunately, tissue culture sometimes accentuates the expression of detects in oil palm, particularly when embryogenesis is induced in particullar callus for prolonged periods. This research is conducted by taking individual tree sample of clone germplasm two years old. The purpose of this research is to molecular performance analysis of some oil palm clones based on SSR markers. A total of 30 trees oil palm clones were used for analysis. In this experiment, the DNA profile diversity was assessed using five loci of oil palm’s specific SSR markers. The results of the experiment indicated out of 3 SSR markers (FR-0779, FR-3663 and FR-0782) showed monomorphic of PCR product and 2 SSR markers (FR-0783 and FR- 3745) showed polymorphic of PCR product. There are 10 total number of PCR product. These preliminary results demonstrated SSR marker can be used to evaluate genetic relatedness among trees of oil palm clones.

Molecular cloning and characterization of arginine kinase gene of Toxocara canis.

Science.gov (United States)

Sahu, Shivani; Samanta, S; Harish, D R; Sudhakar, N R; Raina, O K; Shantaveer, S B; Madhu, D N; Kumar, Ashok

2015-06-01

Toxocara canis is an important gastrointestinal nematode of dogs and also a causative agent of visceral larva migrans in humans. Arginine kinase (AK) gene is one of the important biomolecule of phosphagen kinase of T. canis which is emerging as an exciting novel diagnostic target in toxocarosis. The present study was carried out to clone and characterize AK gene of T. canis for future utilization as a diagnostic molecule. Total RNA was extracted from intact adult worms and reverse transcription was done with oligo dT primers to obtain complementary DNA (cDNA). Polymerase chain reaction (PCR) was carried out using cDNA as template with specific primers which amplified a product of 1,202 bp. The amplicon was cloned into pDrive cloning vector and clone was confirmed by colony PCR and restriction endonuclease analysis. Sequence analysis of the gene showed 99.8 and 77.9 % homology with the published AK gene of T. canis (EF015466.1) and Ascaris suum respectively. Structural analysis shown that the mature AK protein consist of 400 amino acids with a molecular wt of 45360.73 Da. Further expression studies are required for producing the recombinant protein for its evaluation in the diagnosis of T. canis infection in humans as well as in adult dogs.

Immersing Undergraduate Students in the Research Experience: A Practical Laboratory Module on Molecular Cloning of Microbial Genes

Science.gov (United States)

Wang, Jack T. H.; Schembri, Mark A.; Ramakrishna, Mathitha; Sagulenko, Evgeny; Fuerst, John A.

2012-01-01

Molecular cloning skills are an essential component of biological research, yet students often do not receive this training during their undergraduate studies. This can be attributed to the complexities of the cloning process, which may require many weeks of progressive design and experimentation. To address this issue, we incorporated an…

Molecular cloning and characterization of l-methionine γ-lyase from Streptomyces avermitilis.

Science.gov (United States)

Kudou, Daizou; Yasuda, Eri; Hirai, Yoshiyuki; Tamura, Takashi; Inagaki, Kenji

2015-10-01

A pyridoxal 5'-phosphate-dependent methionine γ-lyase (MGL) was cloned from Streptomyces avermitilis catalyzed the degradation of methionine to α-ketobutyrate, methanethiol, and ammonia. The sav7062 gene (1,242 bp) was corresponded to 413 amino acid residues with a molecular mass of 42,994 Da. The deduced amino acid sequence showed a high degree of similarity to those of other MGL enzymes. The sav7062 gene was overexpressed in Escherichia coli. The enzyme was purified to homogeneity and exhibited the MGL catalytic activities. We cloned the enzyme that has the MGL activity in Streptomyces for the first time. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Molecular passportization of clones of karelian birch using PCRwith semi-specific primers

Directory of Open Access Journals (Sweden)

Tatyana V Matveeva

2008-09-01

Full Text Available Using 4 clones of Karelian birch (Betula pendula Roth var caretica Merckl. from the collection of the karelian birch of the laboratory of genetics of Research Institute of Forest Genetics and Breeding, Voronezh, one normal tree of Betula pendula Roth, and one tree of B. pubescens Ehrh., collected from the nature, we have analyzed the possibility of application of PCR with semi-specific primers for molecular typing. We found primers with high percentage of polymorphic markers. These primers could be recommended for molecular typing of birch.

Transcriptome Analysis Revealed Changes of Multiple Genes Involved in Haliotis discus hannai Innate Immunity during Vibrio parahemolyticus Infection.

Science.gov (United States)

Nam, Bo-Hye; Jung, Myunghee; Subramaniyam, Sathiyamoorthy; Yoo, Seung-il; Markkandan, Kesavan; Moon, Ji-Young; Kim, Young-Ok; Kim, Dong-Gyun; An, Cheul Min; Shin, Younhee; Jung, Ho-jin; Park, Jun-hyung

2016-01-01

Abalone (Haliotis discus hannai) is one of the most valuable marine aquatic species in Korea, Japan and China. Tremendous exposure to bacterial infection is common in aquaculture environment, especially by Vibrio sp. infections. It's therefore necessary and urgent to understand the mechanism of H. discus hannai host defense against Vibrio parahemolyticus infection. However studies on its immune system are hindered by the lack of genomic resources. In the present study, we sequenced the transcriptome of control and bacterial challenged H. discus hannai tissues. Totally, 138 MB of reference transcriptome were obtained from de novo assembly of 34 GB clean bases from ten different libraries and annotated with the biological terms (GO and KEGG). A total of 10,575 transcripts exhibiting the differentially expression at least one pair of comparison and the functional annotations highlight genes related to immune response, cell adhesion, immune regulators, redox molecules and mitochondrial coding genes. Mostly, these groups of genes were dominated in hemocytes compared to other tissues. This work is a prerequisite for the identification of those physiological traits controlling H. discus hannai ability to survive against Vibrio infection.

Transcriptome Analysis Revealed Changes of Multiple Genes Involved in Haliotis discus hannai Innate Immunity during Vibrio parahemolyticus Infection

Science.gov (United States)

Nam, Bo-Hye; Jung, Myunghee; Subramaniyam, Sathiyamoorthy; Yoo, Seung-il; Markkandan, Kesavan; Moon, Ji-Young; Kim, Young-Ok; Kim, Dong-Gyun; An, Cheul Min; Shin, Younhee; Jung, Ho-jin; Park, Jun-hyung

2016-01-01

Abalone (Haliotis discus hannai) is one of the most valuable marine aquatic species in Korea, Japan and China. Tremendous exposure to bacterial infection is common in aquaculture environment, especially by Vibrio sp. infections. It’s therefore necessary and urgent to understand the mechanism of H. discus hannai host defense against Vibrio parahemolyticus infection. However studies on its immune system are hindered by the lack of genomic resources. In the present study, we sequenced the transcriptome of control and bacterial challenged H. discus hannai tissues. Totally, 138 MB of reference transcriptome were obtained from de novo assembly of 34 GB clean bases from ten different libraries and annotated with the biological terms (GO and KEGG). A total of 10,575 transcripts exhibiting the differentially expression at least one pair of comparison and the functional annotations highlight genes related to immune response, cell adhesion, immune regulators, redox molecules and mitochondrial coding genes. Mostly, these groups of genes were dominated in hemocytes compared to other tissues. This work is a prerequisite for the identification of those physiological traits controlling H. discus hannai ability to survive against Vibrio infection. PMID:27088873

Transcriptome Analysis Revealed Changes of Multiple Genes Involved in Haliotis discus hannai Innate Immunity during Vibrio parahemolyticus Infection.

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Bo-Hye Nam

Full Text Available Abalone (Haliotis discus hannai is one of the most valuable marine aquatic species in Korea, Japan and China. Tremendous exposure to bacterial infection is common in aquaculture environment, especially by Vibrio sp. infections. It's therefore necessary and urgent to understand the mechanism of H. discus hannai host defense against Vibrio parahemolyticus infection. However studies on its immune system are hindered by the lack of genomic resources. In the present study, we sequenced the transcriptome of control and bacterial challenged H. discus hannai tissues. Totally, 138 MB of reference transcriptome were obtained from de novo assembly of 34 GB clean bases from ten different libraries and annotated with the biological terms (GO and KEGG. A total of 10,575 transcripts exhibiting the differentially expression at least one pair of comparison and the functional annotations highlight genes related to immune response, cell adhesion, immune regulators, redox molecules and mitochondrial coding genes. Mostly, these groups of genes were dominated in hemocytes compared to other tissues. This work is a prerequisite for the identification of those physiological traits controlling H. discus hannai ability to survive against Vibrio infection.

Construction and characterization of HIV type 1 CRF07_BC infectious molecular clone from men who have sex with men.

Science.gov (United States)

Jiang, Yan-Ling; Bai, Wen-Wei; Qu, Fan-Wei; Ma, Hua; Jiang, Run-Sheng; Shen, Bao-Sheng

2016-03-01

This study aimed to investigate the biological characterization of HIV type 1 (HIV-1) CRF07_BC infection among men who have sex with men (MSM). From November 2011 to November 2013, a total of 66 blood samples were collected from MSM with acute HIV-1 infection with CRF07_BC subgroup strains. Deletion in the gag p6 region was detected by sequence alignment and comparative analysis. Peripheral blood mononuclear cells (PBMCs) of HNXX1301-1307 samples were separated by density gradient centrifugation. Nested polymerase chain reaction (nPCR) was used to amplify the viral DNA. The near full-length HIV-1 DNA products were ligated to the long terminal repeat (LTR) vector plasmid (07BCLTR) to construct a full-length HIV clone. The molecular clone was transfected into HEK-293T cells, TZM-b1 cells and patients' PBMCs. The pregenome of an infectious molecular clone of HIV-1 (pNL4-3) was amplified, and a subclone with CRF07_BC was developed to construct the full-length chimeric molecular clone pNL4-3/07BCLTR. Detection of p24 antigen and luciferase activity was used to measure the in vitro infectivity of pNL4-3/07BCLTR. Among the 66 MSM patients infected with CRF07_BC strains, deletion mutations of the Gag P6 proteins were found in 7 of 18CRF07_BC strains; deletion mutations of 2-13 amino acids in different regions were discovered in 6 strains; and the remaining 42 strains did not show deletions. Seven strains with amino acids deficiency in the P6 protein accounted for 27% of all strains and 75% of all deletion genotype strains. A total of 186 full-length molecular clones of CRF07_BC were constructed. There were 5, 9, 10 and 11 clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 that resulted in p24-positive supernatant when transfected into HEK-293T cells. Full-length clones of HNXX1302, HNXX1304, HNXX1305 and HNXX1306 showed slight infection in the transfected TZM-b1 cells, as judged by the fluorescence values of TZM-b1 cells 48h post-transfection. However, we were unable to

Characterization of nonprimate hepacivirus and construction of a functional molecular clone

DEFF Research Database (Denmark)

Scheel, Troels K H; Kapoor, Amit; Nishiuchi, Eiko

2015-01-01

Nonprimate hepacivirus (NPHV) is the closest known relative of hepatitis C virus (HCV) and its study could enrich our understanding of HCV evolution, immunity, and pathogenesis. High seropositivity is found in horses worldwide with ∼ 3% viremic. NPHV natural history and molecular virology remain...... circulating liver enzymes and mild hepatitis was observed, followed by viral clearance. This establishes the molecular components of a functional NPHV genome. Thus, NPHV appears to resemble HCV not only in genome structure but also in its ability to establish chronic infection with delayed seroconversion...... and hepatitis. This NPHV infectious clone and resulting acute phase sera will facilitate more detailed studies on the natural history, pathogenesis, and immunity of this novel hepacivirus in its natural host....

Cytogenetic analysis and chromosomal characteristics of the polymorphic 18S rDNA of Haliotis discus hannai from Fujian, China.

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Haishan Wang

Full Text Available We report on novel chromosomal characteristics of Haliotis discus hannai from a breeding population at Fujian, China. The karyotypes of H. discus hannai we obtained from an abalone farm include a common type 2n = 36 = 10M + 8SM (82% and two rare types 2n = 36 = 11M + 7SM (14% and 2n = 36 = 10M + 7SM + 1ST (4%. The results of silver staining showed that the NORs of H. discus hannai were usually located terminally on the long arms of chromosome pairs 14 and 17, NORs were also sometimes located terminally on the short arms of other chromosomes, either metacentric or submetacentric pairs. The number of Ag-nucleoli ranged from 2 to 8, and the mean number was 3.61 ± 0.93. Among the scored interphase cells, 41% had 3 detectable nucleoli and 37% had 4 nucleoli. The 18S rDNA FISH result is the first report of the location of 18S rDNA genes in H. discus hannai. The 18S rDNA locations were highly polymorphic in this species. Copies of the gene were observed in the terminal of long or/and short arms of submetacentric or/and metacentric chromosomes. Using FISH with probe for vertebrate-like telomeric sequences (CCCTAA3 displayed positive green FITC signals at telomere regions of all analyzed chromosome types. We found about 7% of chromosomes had breaks in prophase. A special form of nucleolus not previously described from H. discus hannai was observed in some interphase cells. It consists of many small silver-stained nucleoli gathered together to form a larger nucleolus and may correspond to prenucleolar bodies.

Molecular cloning and characterization of Izumo1 gene from bovine testis

OpenAIRE

Kim, Ekyune

2015-01-01

A well-characterized sperm specific protein of the Member of immunoglobulin superfamily, IZUMO1, has crucial role in fertilization by mediating sperm binding to the egg plasma membrane in the mouse. However little is known about IZUMO1 in bovine. Here, we describe the molecular cloning and expression analysis of bovine IZUMO1 (bIZUMO1). RT-PCR and Western blot analysis of the bovine tissues indicated that bIZUMO1 was specifically expressed in the testis and sperm, Furthermore, the result of o...

Ecology and life history of an Amazon floodplain cichlid: the discus fish Symphysodon (Perciformes: Cichlidae

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William G. R. Crampton

Full Text Available The discus fishes of the genus Symphysodon are popular ornamental cichlids that occur in floodplain lakes and flooded forests of the lowland Amazon Basin. These habitats are characterized by extreme seasonal fluctuations in the availability of food, shelter and dissolved oxygen, and also the densities of predators and parasites. Most aspects of discus biology are influenced by these fluctuating conditions. This paper reports an autoecological study of the western Amazonian discus S. haraldi (until recently classified as S. aequifasciatus. This species feeds predominantly on algal periphyton, fine organic detritus, plant matter, and small aquatic invertebrates. At high water it forages alone or in small groups in flooded forests. At low water it forms large aggregations in fallen tree crowns along lake margins. Breeding occurs at the beginning of the flood season, ensuring that the progeny are well grown before the next low water period. Symphysodon haraldi is an iteroparous partial spawner, reaches reproductive maturity within a year, and undertakes parental care of its eggs and larvae. The timing of spawning events, and/or the rate of brood survival, may be influenced by fluctuations in the flood level, resulting in a non-unimodal distribution of size classes for the subsequent 1+ cohort.

Bovine viral diarrhea virus: molecular cloning of genomic RNA and its diagnostic application

International Nuclear Information System (INIS)

Brock, K.V.

1987-01-01

Molecular cloning of a field isolate of bovine viral diarrhea virus (BVDV) strain 72 RNA was done in this study. The sensitivity and specificity of cloned cDNA sequences in hybridization assays with various BVDV strains were determined. cDNA was synthesized from polyadenylated BVDV RNA templates with oligo-dT primers, reverse transcriptase, and DNA polymerase I. The newly synthesized double-stranded BVDV cDNA was C-tailed with terminal deoxytransferase and annealed into G-tailed, Pst-1-cut pUC9 plasmid. Escherichia coli was transformed with the recombinant plasmids and a library of approximately 200 BVDV specific cDNA clones varying in length from 0.5 to 2.6 kilobases were isolated. The sensitivity and specificity of hybridization between the labelled cDNA and BVDV target sequences were determined. Cloned BVDV sequences were isolated from pUC9 plasmid DNA and labelled with 32 P by nick translation. The detection limit by dot blot hybridization assay was 20 pg of purified genomic BVDV RNA. cDNA hybridization probes were specific for all strains of BVDV tested, regardless of whether they were noncytopathic and cytopathic, but did not hybridize with heterologous bovine viruses tested. Probes did not hybridize with uninfected cell culture or cellular RNA. Hybridization probes were at least as sensitive as infectivity assays in detecting homologous virus

Biomecánica de la técnica de lanzamiento de disco. Influencia de la trayectoria del disco en la velocidad de liberación. Biomechanics of discus throw technique. The influence of the discus path on the velocity of release.

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Floría Martín, Pablo

2006-10-01

Full Text Available ResumenEl objetivo del estudio fue analizar la influencia de la trayectoria del disco, durante las dos últimas fases del lanzamiento, en su velocidad de liberación. Se analizaron 25 lanzamientos pertenecientes a 8 hombres y 5 mujeres, seleccionados por la Real Federación Española de Atletismo, que participaron en 5 competiciones. Se realizó un análisis cinemático tridimensional utilizando cámaras de cine de alta velocidad (100Hz. Las imágenes y los datos se trataron utilizando Fotogram-IBV y las variables biomecánicas de eficacia de latécnica se obtuvieron mediante la rutina de cálculo Biodisc.Para estudiar las asociaciones entre variables se aplicó el coeficiente de correlación de Pearson. Las variables altura máxima del disco (DISCOmax e incremento de la alturaentre la mínima y la de liberación (∆h_DISCOmin-DISCOlib guardan correlación significativa con la velocidad vertical del disco en el momento de la liberación (VvDISCOlib mientras que la altura mínima del disco (hDISCOmin fue significativa para mujeres y la altura en el instante de liberación(hDISCOlib lo fue para hombres, sugiriendo diferente estrategia para la consecución de la altura del disco necesaria para el vuelo. El ángulo del hombro derecho (Θ_HOMBd se halló relacionado con hDISCO max, hDISCO min y hDISCO lib mientras que el ángulo de la rodilla derecha(Θ_RODd se relacionó con un descenso del disco, necesario en mujeres, para aumentar el recorrido de aceleración. Por otro lado, ciertos aspectos técnicos utilizados por los entrenadores para la enseñanza y corrección de la técnica de sus atletas tienen una evidencia científica, por lo que se aconseja sean observados sistemáticamente.AbstractThe objective of this study was to analyze the influence of discus path on its release velocity during the last two phases of throwing event. It were analyzed 25 throwing belong to 8 men and 5 women selected for Royal Spanish Athletics Federation, in 5

Development of RAPD-SCAR markers for different Ganoderma species authentication by improved RAPD amplification and molecular cloning.

Science.gov (United States)

Fu, J J; Mei, Z Q; Tania, M; Yang, L Q; Cheng, J L; Khan, M A

2015-05-25

The sequence-characterized amplified region (SCAR) is a valuable molecular technique for the genetic identification of any species. This method is mainly derived from the molecular cloning of the amplified DNA fragments achieved from the random amplified polymorphic DNA (RAPD). In this study, we collected DNA from 10 species of Ganoderma mushroom and amplified the DNA using an improved RAPD technique. The amplified fragments were then cloned into a T-vector, and positive clones were screened, indentified, and sequenced for the development of SCAR markers. After designing PCR primers and optimizing PCR conditions, 4 SCAR markers, named LZ1-4, LZ2-2, LZ8-2, and LZ9-15, were developed, which were specific to Ganoderma gibbosum (LZ1-4 and LZ8-2), Ganoderma sinense (LZ2-2 and LZ8-2), Ganoderma tropicum (LZ8-2), and Ganoderma lucidum HG (LZ9-15). These 4 novel SCAR markers were deposited into GenBank with the accession Nos. KM391935, KM391936, KM391937, and KM391938, respectively. Thus, in this study we developed specific SCAR markers for the identification and authentication of different Ganoderma species.

Molecular cloning and expression of Corynebacterium glutamicum genes for amino acid synthesis in Escherichia coli cells

International Nuclear Information System (INIS)

Beskrovnaya, O.Yu.; Fonshtein, M.Yu.; Kolibaba, L.G.; Yankovskii, N.K.; Debabov, V.G.

1989-01-01

Molecular cloning of Corynebacterium glutamicum genes for threonine and lysine synthesis has been done in Escherichia coli cells. The clonal library of EcoRI fragments of chromosomal DNA of C. glutamicum was constructed on the plasmid vector λpSL5. The genes for threonine and lysine synthesis were identified by complementation of E. coli mutations in thrB and lysA genes, respectively. Recombinant plasmids, isolated from independent ThrB + clone have a common 4.1-kb long EcoRI DNA fragment. Hybrid plasmids isolated from LysA + transductants of E. coli have common 2.2 and 3.3 kb long EcoRI fragments of C. glutamicum DNA. The hybrid plasmids consistently transduced the markers thrB + and lysA + . The Southern hybridization analysis showed that the cloned DNA fragments hybridized with the fragments of identical length in C. glutamicum chromosomes

Comparative Transcriptome Analysis of Latex Reveals Molecular Mechanisms Underlying Increased Rubber Yield in Hevea brasiliensis Self-Rooting Juvenile Clones.

Science.gov (United States)

Li, Hui-Liang; Guo, Dong; Zhu, Jia-Hong; Wang, Ying; Chen, Xiong-Ting; Peng, Shi-Qing

2016-01-01

Rubber tree (Hevea brasiliensis) self-rooting juvenile clones (JCs) are promising planting materials for rubber production. In a comparative trial between self-rooting JCs and donor clones (DCs), self-rooting JCs exhibited better performance in rubber yield. To study the molecular mechanism associated with higher rubber yield in self-rooting JCs, we sequenced and comparatively analyzed the latex of rubber tree self-rooting JCs and DCs at the transcriptome level. Total raw reads of 34,632,012 and 35,913,020 bp were obtained from the library of self-rooting JCs and DCs, respectively, by using Illumina HiSeq 2000 sequencing technology. De novo assemblies yielded 54689 unigenes from the library of self-rooting JCs and DCs. Among 54689 genes, 1716 genes were identified as differentially expressed between self-rooting JCs and DCs via comparative transcript profiling. Functional analysis showed that the genes related to the mass of categories were differentially enriched between the two clones. Several genes involved in carbohydrate metabolism, hormone metabolism and reactive oxygen species scavenging were up-regulated in self-rooting JCs, suggesting that the self-rooting JCs provide sufficient molecular basis for the increased rubber yielding, especially in the aspects of improved latex metabolisms and latex flow. Some genes encoding epigenetic modification enzymes were also differentially expressed between self-rooting JCs and DCs. Epigenetic modifications may lead to gene differential expression between self-rooting JCs and DCs. These data will provide new cues to understand the molecular mechanism underlying the improved rubber yield of H. brasiliensis self-rooting clones.

FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

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Lu Jia

2011-10-01

Full Text Available Abstract Background Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process. Results Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA in a vector at any desired position. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp. Conclusion Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA.

Molecular cloning, expression analysis and sequence prediction of ...

African Journals Online (AJOL)

CCAAT/enhancer-binding protein beta as an essential transcriptional factor, regulates the differentiation of adipocytes and the deposition of fat. Herein, we cloned the whole open reading frame (ORF) of bovine C/EBPβ gene and analyzed its putative protein structures via DNA cloning and sequence analysis. Then, the ...

Isolation and characterization of a replication-competent molecular clone of an HIV-1 circulating recombinant form (CRF33_01B.

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Kok Keng Tee

Full Text Available A growing number of emerging HIV-1 recombinants classified as circulating recombinant forms (CRFs have been identified in Southeast Asia in recent years, establishing a molecular diversity of increasing complexity in the region. Here, we constructed a replication-competent HIV-1 clone for CRF33_01B (designated p05MYKL045.1, a newly identified recombinant comprised of CRF01_AE and subtype B. p05MYKL045.1 was reconstituted by cloning of the near full-length HIV-1 sequence from a newly-diagnosed individual presumably infected heterosexually in Kuala Lumpur, Malaysia. The chimeric clone, which contains the 5' LTR (long terminal repeat region of p93JP-NH1 (a previously isolated CRF01_AE infectious clone, showed robust viral replication in the human peripheral blood mononuclear cells. This clone demonstrated robust viral propagation and profound syncytium formation in CD4+, CXCR4-expressing human glioma NP-2 cells, indicating that p05MYKL045.1 is a CXCR4-using virus. Viral propagation, however, was not detected in various human T cell lines including MT-2, M8166, Sup-T1, H9, Jurkat, Molt-4 and PM1. p05MYKL045.1 appears to proliferate only in restricted host range, suggesting that unknown viral and/or cellular host factors may play a role in viral infectivity and replication in human T cell lines. Availability of a CRF33_01B molecular clone will be useful in facilitating the development of vaccine candidates that match the HIV-1 strains circulating in Southeast Asia.

Comparative Transcriptome Analysis of Latex Reveals Molecular Mechanisms Underlying Increased Rubber Yield in Hevea brasiliensis Self-Rooting Juvenile Clones

OpenAIRE

Li, Hui-Liang; Guo, Dong; Zhu, Jia-Hong; Wang, Ying; Chen, Xiong-Ting; Peng, Shi-Qing

2016-01-01

Rubber tree (Hevea brasiliensis) self-rooting juvenile clones (JCs) are promising planting materials for rubber production. In a comparative trial between self-rooting JCs and donor clones (DCs), self-rooting JCs exhibited better performance in rubber yield. To study the molecular mechanism associated with higher rubber yield in self-rooting JCs, we sequenced and comparatively analyzed the latex of rubber tree self-rooting JCs and DCs at the transcriptome level. Total raw reads of 34,632,012 ...

Animal cloning: problems and prospects.

Science.gov (United States)

Wells, D N

2005-04-01

An efficient animal cloning technology would provide many new opportunities for livestock agriculture, human medicine, and animal conservation. Nuclear cloning involves the production of animals that are genetically identical to the donor cells used in a technique known as nuclear transfer (NT). However, at present it is an inefficient process: in cattle, only around 6% of the embryos transferred to the reproductive tracts of recipient cows result in healthy, longterm surviving clones. Of concern are the high losses throughout gestation, during birth and in the post-natal period through to adulthood. Many of the pregnancy losses relate to failure of the placenta to develop and function correctly. Placental dysfunction may also have an adverse influence on postnatal health. These anomalies are probably due to incorrect epigenetic reprogramming of the donor genome following NT, leading to inappropriate patterns of gene expression during the development of clones. Whilst some physiological tests on surviving clones suggest normality, other reports indicate a variety of post-natal clone-associated abnormalities. This variability in outcome may reflect species-specific and/or cloning methodological differences. Importantly, to date it appears that these clone-associated phenotypes are not transmitted to offspring following sexual reproduction. This indicates that they represent epigenetic errors, rather than genetic errors, which are corrected during gametogenesis. Whilst this needs confirmation at the molecular level, it provides initial confidence in the first application of NT in agriculture, namely, the production of small numbers of cloned sires from genetically elite bulls, for natural mating, to effectively disseminate genetic gain. In addition to the animal welfare concerns with the technology, the underlying health of the animals and the consequential effect on food safety are critical aspects that require investigation to gain regulatory and consumer

Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone

International Nuclear Information System (INIS)

Adachi, A.; Gendelman, H.E.; Koenig, S.; Folks, T.; Willey, R.; Rabson, A.; Martin, M.A.

1986-01-01

The authors considered an infectious molecular clone of acquired immunodeficiency syndrome-associated retrovirus. Upon transfection, this clone directed the production of infectious virus particles in a wide variety of cells in addition to human T4 cells. The progeny, infectious virions, were synthesized in mouse, mink, monkey, and several human non-T cell lines, indicating the absence of any intracellular obstacle to viral RNA or protein production or assembly. During the course of these studies, a human colon carcinoma cell line, exquisitely sensitive to DNA transfection, was identified

Parental LTRs are important in a construct of a stable and efficient replication-competent infectious molecular clone of HIV-1 CRF08_BC.

Science.gov (United States)

Zhang, Qiwei; Zhang, Xiaomin; Wu, Hao; Seto, Donald; Zhang, Hao-Jie; Chen, Zhiwei; Wan, Chengsong; Zheng, Bo-Jian

2012-01-01

Circulating recombinant forms (CRFs) of HIV-1 have been identified in southern China in recent years. CRF08_BC is one of the most predominant subtypes circulating in China. In order to study HIV subtype biology and to provide a tool for biotechnological applications, the first full-length replication-competent infectious molecular clone harboring CRF08_BC is reported. The construction of this clone pBRGX indicates that a moderate-copy number vector is required for its amplification in E. coli. In addition, it is shown that the parental CRF08_BC LTRs are important for generating this efficient replication-competent infectious clone. These observations may aid in the construction of infectious clones from other subtypes. Both the pBRGX-derived virus and its parental isolate contain CCR5 tropism. Their full-length genomes were also sequenced, analyzed, compared and deposited in GenBank (JF719819 and JF719818, respectively). The availability of pBRGX as the first replication-competent molecular clone of CRF08_BC provides a useful tool for a wide range of studies of this newly emergent HIV subtype, including the development of HIV vaccine candidates, antiviral drug screening and drug resistance analysis.

Cloning Expeditions: Risky but Rewarding

Science.gov (United States)

2013-01-01

In the 1980s, a good part of my laboratory was using the then-new recombinant DNA techniques to clone and characterize many important cell surface membrane proteins: GLUT1 (the red cell glucose transporter) and then GLUT2 and GLUT4, the red cell anion exchange protein (Band 3), asialoglycoprotein receptor subunits, sucrase-isomaltase, the erythropoietin receptor, and two of the subunits of the transforming growth factor β (TGF-β) receptor. These cloned genes opened many new fields of basic research, including membrane insertion and trafficking of transmembrane proteins, signal transduction by many members of the cytokine and TGF-β families of receptors, and the cellular physiology of glucose and anion transport. They also led to many insights into the molecular biology of several cancers, hematopoietic disorders, and diabetes. This work was done by an exceptional group of postdocs and students who took exceptionally large risks in developing and using novel cloning technologies. Unsurprisingly, all have gone on to become leaders in the fields of molecular cell biology and molecular medicine. PMID:24061478

MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

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CLAUDIA TEREZIA SOCOL

2008-05-01

Full Text Available An efficient bacterial transformation system suitable for cloning the coding sequence of the ovine leptin gene in E. coli DH5α host cells using the pGEMT easy vector it is described in this paper. The necessity of producing leptin is based on the fact that the role of this molecule in the animal and human organism is still unknown, leptin not existing as commercial product on the Romanian market. The results obtained in the bacterial transformation, cloning, recombinant clones selection, control of the insertion experiments and DNA computational analysis represent the first steps in further genetic engineering experiments such as production of DNA libraries, DNA sequencing, protein expression, etc., for a further contribution in elucidating the role of leptin in the animal and human organism.

Genomic characterization and expression profiles upon bacterial infection of a novel cystatin B homologue from disk abalone (Haliotis discus discus).

Science.gov (United States)

Premachandra, H K A; Wan, Qiang; Elvitigala, Don Anushka Sandaruwan; De Zoysa, Mahanama; Choi, Cheol Young; Whang, Ilson; Lee, Jehee

2012-12-01

Cystatins are a large family of cysteine proteinase inhibitors which are involved in diverse biological and pathological processes. In the present study, we identified a gene related to cystatin superfamily, AbCyt B, from disk abalone Haliotis discus discus by expressed sequence tag (EST) analysis and BAC library screening. The complete cDNA sequence of AbCyt B is comprised of 1967 nucleotides with a 306 bp open reading frame (ORF) encoding for 101 amino acids. The amino acid sequence consists of a single cystatin-like domain, which has a cysteine proteinase inhibitor signature, a conserved Gly in N-terminal region, QVVAG motif and a variant of PW motif. No signal peptide, disulfide bonds or carbohydrate side chains were identified. Analysis of deduced amino acid sequence revealed that AbCyt B shares up to 44.7% identity and 65.7% similarity with the cystatin B genes from other organisms. The genomic sequence of AbCyt B is approximately 8.4 Kb, consisting of three exons and two introns. Phylogenetic tree analysis showed that AbCyt B was closely related to the cystatin B from pacific oyster (Crassostrea gigas) under the family 1.Functional analysis of recombinant AbCyt B protein exhibited inhibitory activity against the papain, with almost 84% inhibition at a concentration of 3.5 μmol/L. In tissue expression analysis, AbCyt B transcripts were expressed abundantly in the hemocyte, gill, mantle, and digestive tract, while weakly in muscle, testis, and hepatopancreas. After the immune challenge with Vibrio parahemolyticus, the AbCyt B showed significant (P<0.05) up-regulation of relative mRNA expression in gill and hemocytes at 24 and 6 h of post infection, respectively. These results collectively suggest that AbCyst B is a potent inhibitor of cysteine proteinases and is also potentially involved in immune responses against invading bacterial pathogens in abalone. Copyright © 2012 Elsevier Ltd. All rights reserved.

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

International Nuclear Information System (INIS)

Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

2015-01-01

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Woon, J. S. K., E-mail: jameswoon@siswa.ukm.edu.my; Murad, A. M. A., E-mail: munir@ukm.edu.my; Abu Bakar, F. D., E-mail: fabyff@ukm.edu.my [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor (Malaysia)

2015-09-25

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

Science.gov (United States)

Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

2015-09-01

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

Molecular cloning, genomic organization, and expression of a B-type (cricket-type) allatostatin preprohormone from Drosophila melanogaster

DEFF Research Database (Denmark)

Williamson, M; Lenz, C; Winther, A M

2001-01-01

and nonamidated C terminus. We have previously reported the structure of an A-type allatostatin preprohormone from the fruitfly Drosophila melanogaster. Here we describe the molecular cloning of a B-type prepro-allatostatin from Drosophila (DAP-B). DAP-B is 211 amino acid residues long and contains one copy each...

Recombination-assisted megaprimer (RAM) cloning

Science.gov (United States)

Mathieu, Jacques; Alvarez, Emilia; Alvarez, Pedro J.J.

2014-01-01

No molecular cloning technique is considered universally reliable, and many suffer from being too laborious, complex, or expensive. Restriction-free cloning is among the simplest, most rapid, and cost-effective methods, but does not always provide successful results. We modified this method to enhance its success rate through the use of exponential amplification coupled with homologous end-joining. This new method, recombination-assisted megaprimer (RAM) cloning, significantly extends the application of restriction-free cloning, and allows efficient vector construction with much less time and effort when restriction-free cloning fails to provide satisfactory results. The following modifications were made to the protocol:•Limited number of PCR cycles for both megaprimer synthesis and the cloning reaction to reduce error propagation.•Elimination of phosphorylation and ligation steps previously reported for cloning methods that used exponential amplification, through the inclusion of a reverse primer in the cloning reaction with a 20 base pair region of homology to the forward primer.•The inclusion of 1 M betaine to enhance both reaction specificity and yield. PMID:26150930

Multiple-clone infections of Plasmodium vivax: definition of a panel of markers for molecular epidemiology.

Science.gov (United States)

de Souza, Aracele M; de Araújo, Flávia C F; Fontes, Cor J F; Carvalho, Luzia H; de Brito, Cristiana F A; de Sousa, Taís N

2015-08-25

Plasmodium vivax infections commonly contain multiple genetically distinct parasite clones. The detection of multiple-clone infections depends on several factors, such as the accuracy of the genotyping method, and the type and number of the molecular markers analysed. Characterizing the multiplicity of infection has broad implications that range from population genetic studies of the parasite to malaria treatment and control. This study compared and evaluated the efficiency of neutral and non-neutral markers that are widely used in studies of molecular epidemiology to detect the multiplicity of P. vivax infection. The performance of six markers was evaluated using 11 mixtures of DNA with well-defined proportions of two different parasite genotypes for each marker. These mixtures were generated by mixing cloned PCR products or patient-derived genomic DNA. In addition, 51 samples of natural infections from the Brazil were genotyped for all markers. The PCR-capillary electrophoresis-based method was used to permit direct comparisons among the markers. The criteria for differentiating minor peaks from artifacts were also evaluated. The analysis of DNA mixtures showed that the tandem repeat MN21 and the polymorphic blocks 2 (msp1B2) and 10 (msp1B10) of merozoite surface protein-1 allowed for the estimation of the expected ratio of both alleles in the majority of preparations. Nevertheless, msp1B2 was not able to detect the majority of multiple-clone infections in field samples; it identified only 6 % of these infections. The merozoite surface protein-3 alpha and microsatellites (PvMS6 and PvMS7) did not accurately estimate the relative clonal proportions in artificial mixtures, but the microsatellites performed well in detecting natural multiple-clone infections. Notably, the use of a less stringent criterion to score rare alleles significantly increased the sensitivity of the detection of multi-clonal infections. Depending on the type of marker used, a considerable

Ab initio multiple cloning algorithm for quantum nonadiabatic molecular dynamics

Energy Technology Data Exchange (ETDEWEB)

Makhov, Dmitry V.; Shalashilin, Dmitrii V. [Department of Chemistry, University of Leeds, Leeds LS2 9JT (United Kingdom); Glover, William J.; Martinez, Todd J. [Department of Chemistry and The PULSE Institute, Stanford University, Stanford, California 94305, USA and SLAC National Accelerator Laboratory, Menlo Park, California 94025 (United States)

2014-08-07

We present a new algorithm for ab initio quantum nonadiabatic molecular dynamics that combines the best features of ab initio Multiple Spawning (AIMS) and Multiconfigurational Ehrenfest (MCE) methods. In this new method, ab initio multiple cloning (AIMC), the individual trajectory basis functions (TBFs) follow Ehrenfest equations of motion (as in MCE). However, the basis set is expanded (as in AIMS) when these TBFs become sufficiently mixed, preventing prolonged evolution on an averaged potential energy surface. We refer to the expansion of the basis set as “cloning,” in analogy to the “spawning” procedure in AIMS. This synthesis of AIMS and MCE allows us to leverage the benefits of mean-field evolution during periods of strong nonadiabatic coupling while simultaneously avoiding mean-field artifacts in Ehrenfest dynamics. We explore the use of time-displaced basis sets, “trains,” as a means of expanding the basis set for little cost. We also introduce a new bra-ket averaged Taylor expansion (BAT) to approximate the necessary potential energy and nonadiabatic coupling matrix elements. The BAT approximation avoids the necessity of computing electronic structure information at intermediate points between TBFs, as is usually done in saddle-point approximations used in AIMS. The efficiency of AIMC is demonstrated on the nonradiative decay of the first excited state of ethylene. The AIMC method has been implemented within the AIMS-MOLPRO package, which was extended to include Ehrenfest basis functions.

Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus polyhedrin gene and its variant forms

International Nuclear Information System (INIS)

Sinha-Datta, Uma; Chavali, Venkata Ramana Murthy; Ghosh, Ananta K.

2005-01-01

The segments 10 (S10) of the 11 double stranded RNA genomes from Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) encoding a novel polyhedrin polypeptide was converted to cDNA, cloned, and sequenced. Three cDNA clones consisting of 1502 (AmCPV10-1), 1120 (AmCPV10-2), and 1415 (AmCPV10-3) nucleotides encoding polyhedrin of 254, 339, and 319 amino acids with molecular masses of 29, 39, and 37 kDa, respectively, were obtained, and verified by Northern analysis. These clones showed 70-94% sequence identity among them but none with any sequences in databases. The expression of AmCPV10-1 cDNA encoded polyhedrin in Sf-9 cells was detected by immunoblot analysis and formation of polyhedra by electron microscopy, as observed in AmCPV-infected gut cells, but no expression of AmCPV10-2 or AmCPV10-3 cDNA was detected, indicating that during AmCPV replication, along with functional S10 RNA, some defective variant forms of S10 RNAs are packaged in virion particles

Automated cloning methods.; TOPICAL

International Nuclear Information System (INIS)

Collart, F.

2001-01-01

Argonne has developed a series of automated protocols to generate bacterial expression clones by using a robotic system designed to be used in procedures associated with molecular biology. The system provides plate storage, temperature control from 4 to 37 C at various locations, and Biomek and Multimek pipetting stations. The automated system consists of a robot that transports sources from the active station on the automation system. Protocols for the automated generation of bacterial expression clones can be grouped into three categories (Figure 1). Fragment generation protocols are initiated on day one of the expression cloning procedure and encompass those protocols involved in generating purified coding region (PCR)

Toward a molecular cytogenetic map for cultivated sunflower (Helianthus annuus L.) by landed BAC/BIBAC clones.

Science.gov (United States)

Feng, Jiuhuan; Liu, Zhao; Cai, Xiwen; Jan, Chao-Chien

2013-01-01

Conventional karyotypes and various genetic linkage maps have been established in sunflower (Helianthus annuus L., 2n = 34). However, the relationship between linkage groups and individual chromosomes of sunflower remains unknown and has considerable relevance for the sunflower research community. Recently, a set of linkage group-specific bacterial /binary bacterial artificial chromosome (BAC/BIBAC) clones was identified from two complementary BAC and BIBAC libraries constructed for cultivated sunflower cv. HA89. In the present study, we used these linkage group-specific clones (~100 kb in size) as probes to in situ hybridize to HA89 mitotic chromosomes at metaphase using the BAC-fluorescence in situ hybridization (FISH) technique. Because a characteristic of the sunflower genome is the abundance of repetitive DNA sequences, a high ratio of blocking DNA to probe DNA was applied to hybridization reactions to minimize the background noise. As a result, all sunflower chromosomes were anchored by one or two BAC/BIBAC clones with specific FISH signals. FISH analysis based on tandem repetitive sequences, such as rRNA genes, has been previously reported; however, the BAC-FISH technique developed here using restriction fragment length polymorphism (RFLP)-derived BAC/BIBAC clones as probes to apply genome-wide analysis is new for sunflower. As chromosome-specific cytogenetic markers, the selected BAC/BIBAC clones that encompass the 17 linkage groups provide a valuable tool for identifying sunflower cytogenetic stocks (such as trisomics) and tracking alien chromosomes in interspecific crosses. This work also demonstrates the potential of using a large-insert DNA library for the development of molecular cytogenetic resources.

Cloning arbuscule-related genes from mycorrhizas

DEFF Research Database (Denmark)

Burleigh, Stephen

2000-01-01

Until recently little was known about the identity of the genes expressed in the arbuscules of mycorrhizas, due in part to problems associated with cloning genes from the tissues of an obligate symbiont. However, the combination of advanced molecular techniques, innovative use of the materials...... available and fortuitous cloning has resulted in the recent identification of a number of arbuscule-related genes. This article provides a brief summary of the genes involved in arbuscule development, function and regulation, and the techniques used to study them. Molecular techniques include differential...

Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1

NARCIS (Netherlands)

Nielsen, Peter A; Beahm, Derek L; Giepmans, Ben N G; Baruch, Amos; Hall, James E; Kumar, Nalin M

2002-01-01

A novel human connexin gene (GJA11) was cloned from a genomic library. The open reading frame encoded a hypothetical protein of 294 amino acid residues with a predicted molecular mass of 31,933, hence referred to as connexin-31.9 (Cx31.9) or alpha 11 connexin. A clone in GenBank containing the

Molecular cloning of a functional allatostatin gut/brain receptor and an allatostatin preprohormone from the silkworm Bombyx mori

DEFF Research Database (Denmark)

Secher, Thomas; Lenz, C; Cazzamali, G

2001-01-01

in the DAR-1 and DAR-2 genes, showing that the three receptors are not only structurally but also evolutionarily related. Furthermore, we have cloned a Bombyx allatostatin preprohormone that contains eight different A-type allatostatins. Chinese hamster ovary cells permanently transfected with BAR DNA react......The cockroach-type or A-type allatostatins are inhibitory insect neuropeptides with the C-terminal sequence Tyr/Phe-X-Phe-Gly-Leu-NH(2). Here, we have cloned an A-type allatostatin receptor from the silkworm Bombyx mori (BAR). BAR is 361 amino acid residues long, has seven transmembrane domains....... Northern blots and quantitative reverse transcriptase-polymerase chain reaction of different larval tissues show that BAR mRNA is mainly expressed in the gut and to a much lesser extent in the brain. To our knowledge, this is the first report on the molecular cloning and functional expression of an insect...

DNA cloning: a practical approach. Volume 1

Energy Technology Data Exchange (ETDEWEB)

Glover, D M [ed.

1985-01-01

This book is written for the advanced molecular biologist who needs a detailed discussion of cloning technology. Topics of discussion include: genomic library cloning (size of a genomic library, screening methods, chromosome walking, host cell genetics, and general features of bacteriophage Iambda); use of gt10 and gt11 cDNA lambda vectors and general cDNA cloning; RNase H-Pol I cDNA synthesis; method of detecting fusion proteins produced in bacteria; pEMBL family of double-stranded plasmid vectors that can be used to generate single strands; Escherichia coli transformation; production of mutations in cloned sequences; and cloning in gram negative bacteria.

Molecular cloning and chromosome mapping of the human gene encoding protein phosphotyrosyl phosphatase 1B

International Nuclear Information System (INIS)

Brown-Shimer, S.; Johnson, K.A.; Bruskin, A.; Green, N.R.; Hill, D.E.; Lawrence, J.B.; Johnson, C.

1990-01-01

The inactivation of growth suppressor genes appears to play a major role in the malignant process. To assess whether protein phosphotyrosyl phosphatases function as growth suppressors, the authors have isolated a cDNA clone encoding human protein phosphotyrosyl phosphatase 1B for structural and functional characterization. The translation product deduced from the 1,305-nucleotide open reading frame predicts a protein containing 435 amino acids and having a molecular mass of 49,966 Da. The amino-terminal 321 amino acids deduced from the cDNA sequence are identical to the empirically determined sequence of protein phosphotyrosyl phosphatase 1B. A genomic clone has been isolated and used in an in situ hybridization to banded metaphase chromosomes to determine that the gene encoding protein phosphotyrosyl phosphatase 1B maps as a single-copy gene to the long arm of chromosome 20 in the region q13.1-q13.2

Genome sequence of pacific abalone (Haliotis discus hannai): the first draft genome in family Haliotidae.

Science.gov (United States)

Nam, Bo-Hye; Kwak, Woori; Kim, Young-Ok; Kim, Dong-Gyun; Kong, Hee Jeong; Kim, Woo-Jin; Kang, Jeong-Ha; Park, Jung Youn; An, Cheul Min; Moon, Ji-Young; Park, Choul Ji; Yu, Jae Woong; Yoon, Joon; Seo, Minseok; Kim, Kwondo; Kim, Duk Kyung; Lee, SaetByeol; Sung, Samsun; Lee, Chul; Shin, Younhee; Jung, Myunghee; Kang, Byeong-Chul; Shin, Ga-Hee; Ka, Sojeong; Caetano-Anolles, Kelsey; Cho, Seoae; Kim, Heebal

2017-05-01

Abalones are large marine snails in the family Haliotidae and the genus Haliotis belonging to the class Gastropoda of the phylum Mollusca. The family Haliotidae contains only one genus, Haliotis, and this single genus is known to contain several species of abalone. With 18 additional subspecies, the most comprehensive treatment of Haliotidae considers 56 species valid [ 1 ]. Abalone is an economically important fishery and aquaculture animal that is considered a highly prized seafood delicacy. The total global supply of abalone has increased 5-fold since the 1970s and farm production increased explosively from 50 mt to 103 464 mt in the past 40 years. Additionally, researchers have recently focused on abalone given their reported tumor suppression effect. However, despite the valuable features of this marine animal, no genomic information is available for the Haliotidae family and related research is still limited. To construct the H . discus hannai genome, a total of 580-G base pairs using Illumina and Pacbio platforms were generated with 322-fold coverage based on the 1.8-Gb estimated genome size of H . discus hannai using flow cytometry. The final genome assembly consisted of 1.86 Gb with 35 450 scaffolds (>2 kb). GC content level was 40.51%, and the N50 length of assembled scaffolds was 211 kb. We identified 29 449 genes using Evidence Modeler based on the gene information from ab initio prediction, protein homology with known genes, and transcriptome evidence of RNA-seq. Here we present the first Haliotidae genome, H . discus hannai , with sequencing data, assembly, and gene annotation information. This will be helpful for resolving the lack of genomic information in the Haliotidae family as well as providing more opportunities for understanding gastropod evolution. © The Authors 2017. Published by Oxford University Press.

Anticoagulant Activity and Structural Characterization of Polysaccharide from Abalone (Haliotis discus hannai Ino Gonad

Directory of Open Access Journals (Sweden)

Jun Zhao

2016-06-01

Full Text Available In this study, we aimed at characterizing the structure and the anticoagulant activity of a polysaccharide fraction (AGP33 isolated from the gonads of Haliotis discus hannai Ino. AGP33 was extracted by enzymatic hydrolysis and purified by ion-exchange and gel-filtration chromatography. The backbone fraction of AGP33 (BAGP33, which appeared to contain of mannose, glucose and galactose, was prepared by partial acid hydrolysis. According to methylation and nuclear magnetic resonance (NMR spectroscopy, the backbone of AGP33 was identified as mainly consisting of 1→3-linked, 1→4-linked, and 1→6-linked monosaccharides. AGP33 is a sulfated polysaccharide with sulfates occur at 3-O- and 4-O-positions. It prolonged thromboplastin time (APTT, thrombin time (TT and prothrombin time (PT compared to a saline control solution in a dosage-dependent manner. AGP33 exhibited an extension (p < 0.01 of APTT compared to the saline group at concentrations higher than 5 μg/mL. AGP33 exhibited higher anticoagulant activity than its desulfated product (AGP33-des and BAGP33. The results showed that polysaccharide with higher molecular weight and sulfate content demonstrated greater anticoagulant activity.

Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

International Nuclear Information System (INIS)

Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J.

1988-01-01

Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 10 6 recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria

Molecular cloning and expression analysis of a cDNAs encoding androgenic gland hormone precursors from two Porcellionidae species, Porcellio scaber and P. dilatatus

OpenAIRE

Ohira, Tsuyoshi; Hasegawa, Yuriko; Okuno, Atsuro; Nagasawa, Hiromichi

2003-01-01

Male sexual characteristics in Crustacea are induced by androgenic gland hormone (AGH), which is produced by the male-specific androgenic gland. Recently, AGH in the terrestrial isopod Armadillidium vulgare was characterized and its cDNA cloned, the first example in which the structure of AGH was elucidated. We report here the molecular cloning of cDNAs encoding AGH precursors from two additional terrestrial isopods, Porcellio scaber and P. dilatatus. cDNA fragments encoding Porcellio scaber ...

Molecular cloning of growth hormone encoding cDNA of Indian

Indian Academy of Sciences (India)

A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in ...

Molecular cloning of lupin leghemoglobin cDNA

DEFF Research Database (Denmark)

Konieczny, A; Jensen, E O; Marcker, K A

1987-01-01

Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... specific for nodules were selected by differential colony hybridization using 32P-labeled cDNA synthesized either from nodule poly(A)+ RNA or from poly(A)+ RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

Characterization of genic microsatellite markers derived from expressed sequence tags in Pacific abalone ( Haliotis discus hannai)

Science.gov (United States)

Li, Qi; Shu, Jing; Zhao, Cui; Liu, Shikai; Kong, Lingfeng; Zheng, Xiaodong

2010-01-01

Simple sequence repeat (SSR) markers were developed from the expressed sequence tags (ESTs) of Pacific abalone ( Haliotis discus hannai). Repeat motifs were found in 4.95% of the ESTs at a frequency of one repeat every 10.04 kb of EST sequences, after redundancy elimination. Seventeen polymorphic EST-SSRs were developed. The number of alleles per locus varied from 2-17, with an average of 6.8 alleles per locus. The expected and observed heterozygosities ranged from 0.159 to 0.928 and from 0.132 to 0.922, respectively. Twelve of the 17 loci (70.6%) were successfully amplified in H. diversicolor. Seventeen loci segregated in three families, with three showing the presence of null alleles (17.6%). The adequate level of variability and low frequency of null alleles observed in H. discus hannai, together with the high rate of transportability across Haliotis species, make this set of EST-SSR markers an important tool for comparative mapping, marker-assisted selection, and evolutionary studies, not only in the Pacific abalone, but also in related species.

Molecular cloning and characterization of recA-like gene from Schizosaccharomyces pombe

International Nuclear Information System (INIS)

Lee, J.S.; Kang, J.K.; Yoon, S.M.; Park, Y.; Yang, Y.K.; Kim, S.W.; Park, J.K.; Park, J.G.; Hong, S.H.; Park, S.D.

1996-01-01

We have previously purified and characterized a RecA-like protein from Schizosaccharomyces pombe (S. pombe). In the present study, we have cloned a gene encoding the RecA-like protein. The S. pombe recA-like gene was isolated by immunological screening of the expression library of S. pombe using anti-Escherichia coli (E. coli) RecA antibody as a probe. From 10(6) plaques screened, 6 putative clones were finally isolated. Five of the clones screened contained the same kinds of DNA inserts, as determined by crosshybridization analysis. Among the clones, TC-2 was selected for further studies. The pGEM3Zf(-)Delta 17 vector harboring the 4.3 kb DNA insert of TC-2 clone was capable of producing abeta-gal/RecA-like fusion protein, suggesting that the cloned gene encodes the RecA-like protein of S. pombe. It was also revealed by Southern hybridization analysis that the same DNA sequence as the cloned recA-like gene is located within the S. pombe chromosomal DNA. In addition, the cloned recA-like gene was transcribed into a 3.0 kb RNA transcript, as judged by Northern blot analysis. The level of the RNA transcript of recA-like gene was increased approximately 1.6 to 2.4-fold upon treatment with DNA damaging agents such as ultraviolet (UV)-light, methyl methanesulfonate (MMS), and mitomycin-C (MMC). This data suggests that the cloned S. pombe recA-like gene is slightly inducible to DNAdamage as in E. coli recA gene. These results suggest that an inducible repair mechanism analogous to that of E. coli may exist in fission yeast S. pombe

Molecular cloning and expression analysis of annexin A2 gene in sika deer antler tip

Directory of Open Access Journals (Sweden)

Yanling Xia

2018-04-01

Full Text Available Objective Molecular cloning and bioinformatics analysis of annexin A2 (ANXA2 gene in sika deer antler tip were conducted. The role of ANXA2 gene in the growth and development of the antler were analyzed initially. Methods The reverse transcriptase polymerase chain reaction (RT-PCR was used to clone the cDNA sequence of the ANXA2 gene from antler tip of sika deer (Cervus Nippon hortulorum and the bioinformatics methods were applied to analyze the amino acid sequence of Anxa2 protein. The mRNA expression levels of the ANXA2 gene in different growth stages were examined by real time reverse transcriptase polymerase chain reaction (real time RT-PCR. Results The nucleotide sequence analysis revealed an open reading frame of 1,020 bp encoding 339 amino acids long protein of calculated molecular weight 38.6 kDa and isoelectric point 6.09. Homologous sequence alignment and phylogenetic analysis indicated that the Anxa2 mature protein of sika deer had the closest genetic distance with Cervus elaphus and Bos mutus. Real time RT-PCR results showed that the gene had differential expression levels in different growth stages, and the expression level of the ANXA2 gene was the highest at metaphase (rapid growing period. Conclusion ANXA2 gene may promote the cell proliferation, and the finding suggested Anxa2 as an important candidate for regulating the growth and development of deer antler.

Molecular Cloning, Expression and Characterization of Pectin Methylesterase (CtPME) from Clostridium thermocellum.

Science.gov (United States)

Rajulapati, Vikky; Goyal, Arun

2017-05-01

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0-9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca 2+ or Mg 2+ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca 2+ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.

Molecular cloning, sequence analysis and phylogeny of first caudata g-type lysozyme in axolotl (Ambystoma mexicanum).

Science.gov (United States)

Yu, Haining; Gao, Jiuxiang; Lu, Yiling; Guang, Huijuan; Cai, Shasha; Zhang, Songyan; Wang, Yipeng

2013-11-01

Lysozymes are key proteins that play important roles in innate immune defense in many animal phyla by breaking down the bacterial cell-walls. In this study, we report the molecular cloning, sequence analysis and phylogeny of the first caudate amphibian g-lysozyme: a full-length spleen cDNA library from axolotl (Ambystoma mexicanum). A goose-type (g-lysozyme) EST was identified and the full-length cDNA was obtained using RACE-PCR. The axolotl g-lysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 184 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein are 21523.0 Da and 4.37, respectively. Expression of g-lysozyme mRNA is predominantly found in skin, with lower levels in spleen, liver, muscle, and lung. Phylogenetic analysis revealed that caudate amphibian g-lysozyme had distinct evolution pattern for being juxtaposed with not only anura amphibian, but also with the fish, bird and mammal. Although the first complete cDNA sequence for caudate amphibian g-lysozyme is reported in the present study, clones encoding axolotl's other functional immune molecules in the full-length cDNA library will have to be further sequenced to gain insight into the fundamental aspects of antibacterial mechanisms in caudate.

Effects of temperature and salinity on survival, growth and DNA methylation of juvenile Pacific abalone, Haliotis discus hannai Ino

Science.gov (United States)

Kong, Ning; Liu, Xiao; Li, Junyuan; Mu, Wendan; Lian, Jianwu; Xue, Yanjie; Li, Qi

2017-09-01

Temperature and salinity are two of the most potent abiotic factors influencing marine mollusks. In this study, we investigated the individual and combined effects of temperature and salinity on the survival and growth of juvenile Pacific abalone, Haliotis discus hannai Ino, and also examined the DNA methylation alteration that may underpin the phenotypic variation of abalone exposed to different rearing conditions. The single-factor data showed that the suitable ranges of temperature and salinity were 16-28°C at a constant salinity of 32, and 24-40 at a constant temperature of 20°C, respectively. The two-factor data indicated that both survival and growth were significantly affected by temperature, salinity and their interaction. The optimal temperature-salinity combination for juveniles was 23-25°C and 30-36. To explore environment-induced DNA methylation alteration, the methylation-sensitive amplified polymorphism (MSAP) technique was used to analyze the genomic methylation profiles of abalone reared in optimal and adverse conditions. Neither temperature nor salinity induced evident changes in the global methylation level, but 67 and 63 differentially methylated loci were identified in temperature and salinity treatments, respectively. The between-group eigen analysis also showed that both temperature and salinity could induce epigenetic differentiation in H. discus hannai Ino. The results of our study provide optimal rearing conditions for juvenile H. discus hannai Ino, and represent the first step toward revealing the epigenetic regulatory mechanism of abalone in response to thermal and salt stresses.

Molecular cloning and functional characterization of a rainbow trout liver Oatp

Energy Technology Data Exchange (ETDEWEB)

Steiner, Konstanze, E-mail: konstanze.steiner@uni-konstanz.de [University of Konstanz, Human- and Environmental Toxicology, 78464 Konstanz (Germany); Hagenbuch, Bruno, E-mail: bhagenbuch@kumc.edu [Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City 66160, KS (United States); Dietrich, Daniel R., E-mail: daniel.dietrich@uni-konstanz.de [University of Konstanz, Human- and Environmental Toxicology, 78464 Konstanz (Germany)

2014-11-01

Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrain fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772 bp containing a 2115 bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80 kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologues OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a K{sub m} value of 13.9 μM and 13.4 μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a K{sub m} value of 103 μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications. - Highlights: • A new Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. • rtOatp1d1 is predominantly expressed in the liver. • rtOatp1d1 displays multi-specific transport of endogenous and xenobiotic substrates. • rtOatp1d1 is a homologue of the OATP1A1, OATP1B1 and OATP1B3. • rtOatp1d1 is a microcystin (MC) transporter.

Molecular cloning and functional characterization of a rainbow trout liver Oatp

International Nuclear Information System (INIS)

Steiner, Konstanze; Hagenbuch, Bruno; Dietrich, Daniel R.

2014-01-01

Cyanobacterial blooms have an impact on the aquatic ecosystem due to the production of toxins (e.g. microcystins, MCs), which constrain fish health or even cause fish death. However the toxicokinetics of the most abundant toxin, microcystin-LR (MC-LR), are not yet fully understood. To investigate the uptake mechanism, the novel Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. The cDNA isolated from a clone library consisted of 2772 bp containing a 2115 bp open reading frame coding for a 705 aa protein with an approximate molecular mass of 80 kDa. This fish specific transporter belongs to the OATP1 family and has most likely evolved from a common ancestor of OATP1C1. Real time PCR analysis showed that rtOatp1d1 is predominantly expressed in the liver, followed by the brain while expression in other organs was not detectable. Transient transfection in HEK293 cells was used for further characterization. Like its human homologues OATP1A1, OATP1B1 and OATP1B3, rtOatp1d1 displayed multi-specific transport including endogenous and xenobiotic substrates. Kinetic analyses revealed a K m value of 13.9 μM and 13.4 μM for estrone-3-sulfate and methotrexate, respectively and a rather low affinity for taurocholate with a K m value of 103 μM. Furthermore, it was confirmed that rtOatp1d1 is a MC-LR transporter and therefore most likely plays a key role in the susceptibility of rainbow trout to MC intoxications. - Highlights: • A new Oatp1d1 in rainbow trout (rtOatp1d1) was cloned, identified and characterized. • rtOatp1d1 is predominantly expressed in the liver. • rtOatp1d1 displays multi-specific transport of endogenous and xenobiotic substrates. • rtOatp1d1 is a homologue of the OATP1A1, OATP1B1 and OATP1B3. • rtOatp1d1 is a microcystin (MC) transporter

Molecular cloning and characterization of pathogenesis-related ...

African Journals Online (AJOL)

use

2011-12-21

Dec 21, 2011 ... Available online at http://www.academicjournals.org/AJB ... November, 2011. We described the cloning and characterization of pathogenesis-related protein 5 gene in maize, named .... in two inbred lines was calculated using the ↵Ct method. .... Of the characterized PRs currently known, PR-1, PR-2,. PR-3 ...

[A review of the genomic and gene cloning studies in trees].

Science.gov (United States)

Yin, Tong-Ming

2010-07-01

Supported by the Department of Energy (DOE) of U.S., the first tree genome, black cottonwood (Populus trichocarpa), has been completely sequenced and publicly release. This is the milestone that indicates the beginning of post-genome era for forest trees. Identification and cloning genes underlying important traits are one of the main tasks for the post-genome-era tree genomic studies. Recently, great achievements have been made in cloning genes coordinating important domestication traits in some crops, such as rice, tomato, maize and so on. Molecular breeding has been applied in the practical breeding programs for many crops. By contrast, molecular studies in trees are lagging behind. Trees possess some characteristics that make them as difficult organisms for studying on locating and cloning of genes. With the advances in techniques, given also the fast growth of tree genomic resources, great achievements are desirable in cloning unknown genes from trees, which will facilitate tree improvement programs by means of molecular breeding. In this paper, the author reviewed the progress in tree genomic and gene cloning studies, and prospected the future achievements in order to provide a useful reference for researchers working in this area.

Molecular cloning and protein structure of a human blood group Rh polypeptide

International Nuclear Information System (INIS)

Cherif-Zahar, B.; Bloy, C.; Le Van Kim, C.; Blanchard, D.; Bailly, P.; Hermand, P.; Salmon, C.; Cartron, J.P.; Colin, Y.

1990-01-01

cDNA clones encoding a human blood group Rh polypeptide were isolated from a human bone marrow cDNA library by using a polymerase chain reaction-amplified DNA fragment encoding the known common N-terminal region of the Rh proteins. The entire primary structure of the Rh polypeptide has been deduced from the nucleotide sequence of a 1384-base-pair-long cDNA clone. Translation of the open reading frame indicates that the Rh protein is composed of 417 amino acids, including the initiator methionine, which is removed in the mature protein, lacks a cleavable N-terminal sequence, and has no consensus site for potential N-glycosylation. The predicted molecular mass of the protein is 45,500, while that estimated for the Rh protein analyzed in NaDodSO 4 /polyacrylamide gels is in the range of 30,000-32,000. These findings suggest either that the hydrophobic Rh protein behaves abnormally on NaDodSO 4 gels or that the Rh mRNA may encode a precursor protein, which is further matured by a proteolytic cleavage of the C-terminal region of the polypeptide. Hydropathy analysis and secondary structure predictions suggest the presence of 13 membrane-spanning domains, indicating that the Rh polypeptide is highly hydrophobic and deeply buried within the phospholipid bilayer. These results suggest that the expression of the Rh gene(s) might be restricted to tissues or cell lines expressing erythroid characters

Diffuse scattering and defect structure simulations a cook book using the program DISCUS

CERN Document Server

Neder, Reinhard B

2009-01-01

In recent years it has become apparent that knowing the average atomic structure of materials is insufficient to understand their properties. Diffuse scattering in addition to the Bragg scattering holds the key to learning about defects in materials, the topic of many recent books. What has been missing is a detailed step-by-step guide how to simulate disordered materials. The DISCUS cook book fills this need covering simple topics such as building a computer crystal to complextopic such as domain structures, stacking faults or using advanced refinement techniques to adjust parameters on a dis

Differentially-Expressed Genes Associated with Faster Growth of the Pacific Abalone, Haliotis discus hannai.

Science.gov (United States)

Choi, Mi-Jin; Kim, Gun-Do; Kim, Jong-Myoung; Lim, Han Kyu

2015-11-18

The Pacific abalone Haliotis discus hannai is used for commercial aquaculture in Korea. We examined the transcriptome of Pacific abalone Haliotis discus hannai siblings using NGS technology to identify genes associated with high growth rates. Pacific abalones grown for 200 days post-fertilization were divided into small-, medium-, and large-size groups with mean weights of 0.26 ± 0.09 g, 1.43 ± 0.405 g, and 5.24 ± 1.09 g, respectively. RNA isolated from the soft tissues of each group was subjected to RNA sequencing. Approximately 1%-3% of the transcripts were differentially expressed in abalones, depending on the growth rate. RT-PCR was carried out on thirty four genes selected to confirm the relative differences in expression detected by RNA sequencing. Six differentially-expressed genes were identified as associated with faster growth of the Pacific abalone. These include five up-regulated genes (including one specific to females) encoding transcripts homologous to incilarin A, perlucin, transforming growth factor-beta-induced protein immunoglobulin-heavy chain 3 (ig-h3), vitelline envelope zona pellucida domain 4, and defensin, and one down-regulated gene encoding tomoregulin in large abalones. Most of the transcripts were expressed predominantly in the hepatopancreas. The genes identified in this study will lead to development of markers for identification of high-growth-rate abalones and female abalones.

Differentially-Expressed Genes Associated with Faster Growth of the Pacific Abalone, Haliotis discus hannai

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Mi-Jin Choi

2015-11-01

Full Text Available The Pacific abalone Haliotis discus hannai is used for commercial aquaculture in Korea. We examined the transcriptome of Pacific abalone Haliotis discus hannai siblings using NGS technology to identify genes associated with high growth rates. Pacific abalones grown for 200 days post-fertilization were divided into small-, medium-, and large-size groups with mean weights of 0.26 ± 0.09 g, 1.43 ± 0.405 g, and 5.24 ± 1.09 g, respectively. RNA isolated from the soft tissues of each group was subjected to RNA sequencing. Approximately 1%–3% of the transcripts were differentially expressed in abalones, depending on the growth rate. RT-PCR was carried out on thirty four genes selected to confirm the relative differences in expression detected by RNA sequencing. Six differentially-expressed genes were identified as associated with faster growth of the Pacific abalone. These include five up-regulated genes (including one specific to females encoding transcripts homologous to incilarin A, perlucin, transforming growth factor-beta-induced protein immunoglobulin-heavy chain 3 (ig-h3, vitelline envelope zona pellucida domain 4, and defensin, and one down-regulated gene encoding tomoregulin in large abalones. Most of the transcripts were expressed predominantly in the hepatopancreas. The genes identified in this study will lead to development of markers for identification of high-growth-rate abalones and female abalones.

Proteomic profiling of eggs from a hybrid abalone and its parental lines: Haliotis discus hannai Ino and Haliotis gigantea.

Science.gov (United States)

Di, Guilan; Luo, Xuan; Huang, Miaoqin; Chen, Jun; Kong, Xianghui; Miao, Xiulian; Ke, Caihuan

2015-12-01

Proteomic analysis was performed on the eggs of hybrid abalone and their corresponding parental lines. A total of 915 ± 19 stained protein spots were detected from Haliotis discus hannai♀ × H. discus hannai♂ (DD), 935 ± 16 from H. gigantea♀ × H. gigantea♂ (GG) and 923 ± 13 from H. gigantea♀ × H. discus hannai♂ (GD). The spots from DD and GD were clustered together. The distance between DD and GG was maximal by hierarchical cluster analysis. A total of 112 protein gel spots were identified; of these, 59 were abalone proteins. The proteins were involved in major biological processes including energy metabolism, proliferation, apoptosis, signal transduction, immunity, lipid metabolism, electron carrier proteins, protein biosynthesis and decomposition, and cytoskeletal structure. Three of 20 differential expression protein spots involved in energy metabolism exhibited as upregulated in GD, 13 spots exhibited additivity, and four spots exhibited as downregulated in the offspring. Eleven protein spots were expressed at the highest level in DD. The proteins involved in stress responses included superoxide dismutase, peroxiredoxin 6, thioredoxin peroxidase and glutathione-S-transferase. Two of seven differential expression protein spots involved in response to stress exhibited as upregulated in GD, three exhibited additivity, and two exhibited as downregulated. These results might suggest that proteomic approaches are suitable for the analysis of hybrids and the functional prediction of abalone hybridization. © 2015 Stichting International Foundation for Animal Genetics.

Preparation, characterisation and use for antioxidant oligosaccharides of a cellulase from abalone (Haliotis discus hannai) viscera.

Science.gov (United States)

Tao, Zhi-Peng; Sun, Le-Chang; Qiu, Xu-Jian; Cai, Qiu-Feng; Liu, Guang-Ming; Su, Wen-Jin; Cao, Min-Jie

2016-07-01

In China, abalone (Haliotis discus hannai) production is growing annually. During industrial processing, the viscera, which are abundant of cellulase, are usually discarded or processed into low-value feedstuff. Thus, it is of interest to obtain cellulase from abalone viscera and investigate its application for preparation of functional oligosaccharides. A cellulase was purified from the hepatopancreas of abalone by ammonium sulfate precipitation and two-steps column chromatography. The molecular weight of the cellulase was 45 kDa on SDS-PAGE. Peptide mass fingerprinting analysis yielded 103 amino acid residues, which were identical to cellulases from other species of abalone. Substrate specificity analysis indicated that the cellulase is an endo-1,4-β-glucanase. Hydrolysis of seaweed Porphyra haitanensis polysaccharides by the enzyme produced oligosaccharides with degree of polymerisation of two to four, whose monosaccharide composition was 58% galactose, 4% glucose and 38% xylose. The oligosaccharides revealed 2,2'-diphenyl-1-picrylhydrazyl free radical as well as hydrogen peroxide scavenging activity. It is feasible and meaningful to utilise cellulase from the viscera of abalone for preparation of functional oligosaccharides. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

Molecular cloning and characterization of genes required for nucleotide excision repair in yeast

International Nuclear Information System (INIS)

Friedberg, E.C.

1987-01-01

Nucleotide excision repair in the yeast S. cerevisiae is a complex process which involves a large number of genes. At least five of these genes (RAD1, RAD2, RAD3, RAD4 and RAD10) are absolutely required for this process and mutations in any of these genes result in no detectable excision repair in vivo. In order to understand the function of these genes in DNA repair, the authors isolated a number of them by screening a yeast genomic library for recombinant plasmids which complement the phentoype of sensitivity to ultraviolet (UV) radiation imparted to mutant strains. A plasmid containing the RAD4 gene was isolated by an alternative strategy which will be discussed. The cloned genes have been extensively characterized. It has been determined that the RAD3 gene is essential for the viability of haploid yeast cells in the absence of DNA damage. The RAD2 gene is inducible by treatment of cells with a variety of DNA-damaging agents, including UV radiation and ionizing radiation. The RAD10 gene shares considerable amino acid sequence homology with a cloned gene involved in nucleotide excision repair in human cells. Yeast is a particularly versatile organism for studying gene function by molecular and genetic approaches and emphasis is placed on many of the techniques used in the present studies

Molecular cloning and characterization of the plasma membrane ...

African Journals Online (AJOL)

O. violaceus) was cloned. The full-length cDNA of O. violaceus gene (OvPIP) was 1314 bp and contained 1188 open reading frame encoding a protein of 395 amino acids. Homology analysis revealed that OvPIP strongly resembled other PIP ...

Molecular Cloning And Sequencing Of Disintegrin Like Domain ...

African Journals Online (AJOL)

Disintegrin-like domain was cloned and sequenced from Cerastes cerastes venom gland tissue. Nested RT-PCR was performed using initial primers designed based on the homology of disintegrins from Trimeresurus flavoviridis, Glodius halys , Agkistrodon halys and Trimeresurus macrosquamatus. The homology was ...

Construction and characterisation of a full-length infectious molecular clone from a fast replicating, X4-tropic HIV-1 CRF02.AG primary isolate

International Nuclear Information System (INIS)

Tebit, Denis M.; Zekeng, Leopold; Kaptue, Lazare; Kraeusslich, Hans-Georg; Herchenroeder, Ottmar

2003-01-01

Based on our previous analysis of HIV-1 isolates from Cameroon, we constructed a full-length infectious molecular clone from a primary isolate belonging to the CRF02.AG group of recombinant viruses which dominate the HIV-epidemic in West and Central Africa. The virus derived by transfection of the proviral clone pBD6-15 replicated with similar efficiency compared to its parental isolate and used CXCR4 as coreceptor as well. Furthermore, HIV-1 BD6-15 exhibited similar replication properties and virus yield as the reference B-type HIV-1 strain NL4-3. Sequence analysis revealed open reading frames for all structural and accessory genes apart from vpr. Phylogenetic and bootscanning analyses confirmed that BD6-15 clusters with CRF02.AG recombinant strains from West and Central Africa with similar cross-over points as described for the CRF02.AG prototype strain lbNG. Thus, pBD6-15 represents the first non-subtype B infectious molecular clone of a fast replicating, high producer, X4-tropic primary HIV-1 isolate, which had only been briefly passaged in primary cells

Molecular cloning, expression and characterization of Pru a 1, the major cherry allergen.

Science.gov (United States)

Scheurer, S; Metzner, K; Haustein, D; Vieths, S

1997-06-01

A high percentage of birch pollen allergic patients experiences food hypersensitivity reactions after ingestion of several fruits and vegetables. Previous work demonstrated common epitopes on an allergen of Mr 18,000 from sweet cherry (Prunus avium) and Bet v 1, the major allergen from birch pollen. N-terminal amino acid sequencing showed a sequence identity of 67% with Bet v 1. Here we report the cloning and cDNA sequencing of this cherry allergen. The entire deduced amino acid sequence described a protein of Mr 17,700 with 59.1% identity to Bet v 1. High degrees of identity in the range of 40 to 60% were also found with related allergens from other kinds of tree pollen and plant foods as well as with stress-induced proteins from food plants such as parsley, potato and soya. The coding DNA of the cherry protein was cloned into vector pET-16b and expressed in E. coli strain BL21(DE3) as a His-tag fusion protein. As shown by SDS-PAGE, the apparent molecular masses of the nonfusion protein and the natural allergen were identical. The fusion protein showed high IgE binding potency when sera from patients allergic to cherry were tested by immunoblotting and enzyme allergosorbent tests. Moreover, it cross-reacted strongly with IgE specific for the natural counterpart and for Bet v 1. The high biological activity of the recombinant fusion protein was further confirmed by the induction of a strong histamine release in basophils from cherry-allergic patients. Since sera from 17/19 of such patients contained IgE against this allergen it was classified as a major allergen and named Pru a 1. Recombinant Pru a 1 mimics most of the allergenic potency of cherry extract and hence could be a useful tool for studying the molecular and immunological properties of pollen related food allergens.

MOLECULAR DETECTION AND CLONING FOR RICKETTSIA-LIKE BACTERIA OF MILKY HAEMOLYMPH DISEASE OF SPINY LOBSTER Panulirus spp.

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Isti Koesharyani

2017-01-01

Full Text Available Spiny lobster (Panulirus homarus and Panulirus ornatus are important commodities for Indonesia. The aquaculture of lobster is susceptible for several diseases like parasite, fungi, bacteria, and virus. Among those diseases, milky haemolymph disease (MHD is often seen as a symptom to mass mortality occurred at lobster farms in Gerupuk Bay of Lombok. The purpose of this study was to determine the lobster diseases on cage culture in Gerupuk Bay of Lombok, West Nusa Tenggara. The study was undertaken from January to March 2015. Diseases status was determined by application of molecular plat-form, polymerase chain reaction (PCR with designation of specific primer for MHD (254F/R, 254F: 5’-CGA-GGA-CCA-GAG-ATG-GAC-CTT-3’ and 254R: 5’-GCT-CAT-TGT-CAC-CGC-CAT-TGT-3’ with PCR size product of 254 bp. and for cloned the pathogen was used TA-cloning Invitrogen for the DNA plasmid as positive control for other analysis. Several tissue samples i.e hepatopancreas, haemolymph, part of muscle hepatopancreas P. homarus and P. ornatus were taken from cage culture farms at Gerupuk Bay then preserved on 90% ethanol for further analysis by PCR and then the amplificated DNA were cloned into pCR®2.1 plasmid and transformed into competent E. coli. The result showed that almost all lobster samples from Gerupuk Bay were positive infected by MHD, as the results of PCR amplification whereas the band appeared at 254bp. Also MHD plasmid has been successfully cloned and will be used for further examination. Histopathologically in hepatopancreas infection have seen necrosis that contain numerous of rickettsia-like bacteria.

An Aspergillus oryzae acetyl xylan esterase: molecular cloning and characteristics of recombinant enzyme expressed in Pichia pastoris.

Science.gov (United States)

Koseki, Takuya; Miwa, Yozo; Akao, Takeshi; Akita, Osamu; Hashizume, Katsumi

2006-02-10

We screened 20,000 clones of an expressed sequence tag (EST) library from Aspergillus oryzae (http://www.nrib.go.jp/ken/EST/db/index.html) and obtained one cDNA clone encoding a protein with similarity to fungal acetyl xylan esterase. We also cloned the corresponding gene, designated as Aoaxe, from the genomic DNA. The deduced amino acid sequence consisted of a putative signal peptide of 31-amino acids and a mature protein of 276-amino acids. We engineered Aoaxe for heterologous expression in P. pastoris. Recombinant AoAXE (rAoAXE) was secreted by the aid of fused alpha-factor secretion signal peptide and accumulated as an active enzyme in the culture medium to a final level of 190 mg/l after 5 days. Purified rAoAXEA before and after treatment with endoglycosidase H migrated by SDS-PAGE with a molecular mass of 31 and 30 kDa, respectively. Purified rAoAXE displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. The recombinant enzyme catalyzed the release of acetic acid from birchwood xylan. No activity was detectable using methyl esters of ferulic, caffeic or sinapic acids. rAoAXE was thermolabile in comparison to other AXEs from Aspergillus.

Molecular cloning and characterization of strictosidine synthase, a ...

African Journals Online (AJOL)

Mitragynine is one of the most dominant indole alkaloids present in the leaves of Mitragyna speciosa, a species of Rubiaceae. This alkaloid is believed to be synthesized via condensation of the amino acid derivative, tryptamine and secologanine by the action of strictosidine synthase (STR). The cDNA clone encoding STR ...

Growth rates of Haliotis rufescens and Haliotis discus hannai in tank culture systems in southern Chile (41.5°S

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Alfonso Mardones

2013-11-01

Full Text Available The increased activity of aquaculture in Chile involves cultivation of salmonids, oysters mussels and other species such, and to a lesser extent species such as red abalone (Haliotis rufescens and Japanese abalone (Haliotis discus hannai. The aim of this study was to evaluate the growth rate of Haliotis rufescens and Haliotis discus hannai fed with different pellet based diets with Macrocystis sp. and Ulva sp., grown in ponds for 13 months. The results for both species denoted that there was an increase in length and biomass during experimental period, existing low growth rates during the austral winter (July-September and increase during the austral summer (December-January. Results are consistent with descriptions of literature that there is high rate of growth during the summer and using diet of brown algae. From the economic standpoint abalone farming would be an economically viable activity for local aquaculture, considering the water quality and food requirements.

Molecular cloning and characterization of glucose transporter 1 ...

African Journals Online (AJOL)

Glucose transporter type-1 (glut1) and citrate synthase plays crucial role in glucose transport and regulation of tricarboxylic acid cycle (TCA) cycle in mammalian energy metabolism. The present study was aimed to clone and characterize glut1 and citrate synthase cDNA in water buffalo (Bubalus bubalis). Total of 90 ...

Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata.

Science.gov (United States)

Pasion, S G; Hines, J C; Aebersold, R; Ray, D S

1992-01-01

A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.

Molecular cloning of transcripts induced by UV-radiation in rodent cells

International Nuclear Information System (INIS)

Fornace, A.J. Jr.; Mitchell, J.B.

1987-01-01

Several inducible DNA repair genes have been well characterized in bacteria. In eukaryotes including mammalian cells, there is increasing evidence that similar events may occur. Recently, the authors have shown that hybridization subtraction can be used to enrich for sequences induced only several fold by a particular cell treatment such as heat shock. Chinese hamster V79 cells were UV-irradiated with 17 Jm/sup -2/ and cDNA was synthesized from the polyadenylated (poly A) RNA. This ''UV'' cDNA was hybridized with a 3 fold excess of polyA RNA from unirradiated cells and the nonhybridizing cDNA was isolated. With this approach, UV-induced sequences were enriched over 20 fold. This enriched cDNA was cloned into a high copy number plasmid and a cDNA library was constructed. By RNA dot blot and northern analysis, 42 clones from this library were found to represent transcripts induced 3 to 25 fold by UV. The most common isolates were found to be metallothionein transcripts by DNA sequencing. The metallothionein transcripts were found to be induced 10 to 25 fold by UV with maximum induction at 4-8 h after 10 Jm/sup -2/. A similar approach was also used with a Chinese hamster ovary line which does not express metallothionein and multiple clones were isolated which represented transcripts induced 3-15 fold by UV. Except for the metallothionein clones, the other Chinese hamster cDNA clones have not been identified, but it is probable that the protein products of at least some of these transcripts play a role in the cellular response to UV damage

Molecular cloning and characterization of duck interleukin-17

Science.gov (United States)

Interleukin-17 (IL-17) belonging to the Th17 family is a proinflammatory cytokine produced by activated T cells. A 1034-bp cDNA encoding duck IL-17 (duIL-17) was cloned from ConA-activated splenic lymphocytes of ducks. The encoded protein, predicted to consisted of 169 amino acids, displayed a molec...

Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV.

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Benjamin Lamp

Full Text Available European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV, a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch's postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.

Molecular cloning, characterization and functional analysis of a 3 ...

African Journals Online (AJOL)

STORAGESEVER

2009-08-04

Aug 4, 2009 ... other plant HMGRs and contained 2 transmembrane domains and a catalytic domain. The potential significance ... used as animal feed. Therefore ... Table 1. Primers used in the cloning and analysis of JcHMGR gene. Primers.

Cloning of Plasmodium falciparum by single-cell sorting.

Science.gov (United States)

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-10-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. Copyright 2010 Elsevier Inc. All rights reserved.

Cloning of Plasmodium falciparum by single-cell sorting

Science.gov (United States)

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-01-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. PMID:20435038

DISCUS, Neutron Single to Double Scattering Ratio in Inelastic Scattering Experiment by Monte-Carlo

International Nuclear Information System (INIS)

Johnson, M.W.

1993-01-01

1 - Description of problem or function: DISCUS calculates the ratio of once-scattered to twice-scattered neutrons detected in an inelastic neutron scattering experiment. DISCUS also calculates the flux of once-scattered neutrons that would have been observed if there were no absorption in the sample and if, once scattered, the neutron would emerge without further re-scattering or absorption. Three types of sample geometry are used: an infinite flat plate, a finite flat plate or a finite length cylinder. (The infinite flat plate is included for comparison with other multiple scattering programs.) The program may be used for any sample for which the scattering law is of the form S(/Q/, omega). 2 - Method of solution: Monte Carlo with importance sampling is used. Neutrons are 'forced' both into useful angular trajectories, and useful energy bins. Biasing of the collision point according to the point of entry of the neutron into the sample is also utilised. The first and second order scattered neutron fluxes are calculated in independent histories. For twice-scattered neutron histories a square distribution in Q-omega space is used to sample the neutron coming from the first scattering event, whilst biasing is used for the second scattering event. (A square distribution is used so as to obtain reasonable inelastic-inelastic statistics.) 3 - Restrictions on the complexity of the problem: Unlimited number of detectors. Max. size of (Q, omega) matrix is 39*149. Max. number of points in momentum space for the scattering cross section is 199

Molecular Cloning Expression And Purification Studies With An ORF Of Mycobacterium Tuberculosis

Directory of Open Access Journals (Sweden)

Chiranjibi Chaudhary

2017-08-01

Full Text Available The study was initiated to develop a recombinant strain for expression and production of large scale protein and to develop its purification protocol. The MRAORF-X was amplified from the genomic DNA of M. tuberculosis H37Ra. The amplicon was successfully cloned in a cloning vector pGEM-T Easy and transformed in cloning host DH5amp945. Recombinant clones were identified by blue-white screening and insert presence was confirmed by restriction digestion of plasmid isolated from white colonies. Expression vector pET32a was used for protein expression. The recombinant plasmid was transformed into expression host BL21 and protein expression was checked by SDS-PAGE. The desired protein was approximately 60 kDa in size including tags. The purification protocol was established for purification from inclusion bodies. The purity of purified protein was assessed by SDS-PAGE gel run and presence of a single band at 60 kDa suggested that the inclusion bodies were a good source of purified protein.

Immune-mediated bone marrow failure syndromes of progenitor and stem cells: molecular analysis of cytotoxic T cell clones.

Directory of Open Access Journals (Sweden)

Ramon Tiu

2007-03-01

Full Text Available The unique structure of the T cell receptor (TCR enables molecular identification of individual T cell clones and provides an unique opportunity for the design of molecular diagnostic tests based on the structure of the rearranged TCR chain e.g., using the TCR CDR3 region. Initially, clonal T cell malignancies, including T cell large granular lymphocyte leukemia (T-LGL, mucosis fungoides and peripheral T cell lymphoma were targets for the TCR-based analytic assays such as detection of clonality by T-gamma rearrangement using y-chain-specific PCR or Southern Blotting. Study of these disorders facilitated further analytic concepts and application of rational methods of TCR analysis to investigations of polyclonal T cell-mediated diseases. In hematology, such conditions include graft versus host disease (GvHD and immune-mediated bone marrow failure syndromes. In aplastic anemia (AA, myelodysplastic syndrome (MDS or paroxysmal nocturnal hemoglobinuria (PNH, cytotoxic T cell responses may be directed against certain antigens located on stem or more lineage-restricted progenitor cells in single lineage cytopenias. The nature of the antigenic targets driving polyclonal CTL responses remains unclear. Novel methods of TCR repertoire analysis, include VB flow cytometry, peptide-specific tetramer staining, in vitro stimulation assays and TCR CDR3-specific PCR. Such PCR assay can be either VB family-specific or multiplexed for all VB families. Amplified products can be characterized and quantitated to facilitate detection of the most immunodominant clonotypes. Such clonotypes may serve as markers for the global polyclonal T cell response. Identification of these clonotypes can be performed in blood and tissue biopsy material by various methods. Once immunodominant clonotypes corresponding to pathogenic CTL clones are identified they can serve as surrogate markers for the activity of the pathophysiologic process or even indicate the presence of specific

Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA.

OpenAIRE

Adachi, A; Sakai, K; Kitamura, N; Nakanishi, S; Niwa, O; Matsuyama, M; Ishimoto, A

1984-01-01

The highly oncogenic erythroleukemia-inducing Friend mink cell focus-inducing (MCF) virus was molecularly cloned in phage lambda gtWES.lambda B, and the DNA sequences of the env gene and the long terminal repeat were determined. The nucleotide sequences of Friend MCF virus and Friend spleen focus-forming virus were quite homologous, supporting the hypothesis that Friend spleen focus-forming virus might be generated via Friend MCF virus from an ecotropic Friend virus mainly by some deletions. ...

Cloning and Sequence Analysis of Vibrio halioticoli Genes Encoding Three Types of Polyguluronate Lyase.

Science.gov (United States)

Sugimura; Sawabe; Ezura

2000-01-01

The alginate lyase-coding genes of Vibrio halioticoli IAM 14596(T), which was isolated from the gut of the abalone Haliotis discus hannai, were cloned using plasmid vector pUC 18, and expressed in Escherichia coli. Three alginate lyase-positive clones, pVHB, pVHC, and pVHE, were obtained, and all clones expressed the enzyme activity specific for polyguluronate. Three genes, alyVG1, alyVG2, and alyVG3, encoding polyguluronate lyase were sequenced: alyVG1 from pVHB was composed of a 1056-bp open reading frame (ORF) encoding 352 amino acid residues; alyVG2 gene from pVHC was composed of a 993-bp ORF encoding 331 amino acid residues; and alyVG3 gene from pVHE was composed of a 705-bp ORF encoding 235 amino acid residues. Comparison of nucleotide and deduced amino acid sequences among AlyVG1, AlyVG2, and AlyVG3 revealed low homologies. The identity value between AlyVG1 and AlyVG2 was 18.7%, and that between AlyVG2 and AlyVG3 was 17.0%. A higher identity value (26.0%) was observed between AlyVG1 and AlyVG3. Sequence comparison among known polyguluronate lyases including AlyVG1, AlyVG2, and AlyVG3 also did not reveal an identical region in these sequences. However, AlyVG1 showed the highest identity value (36.2%) and the highest similarity (73.3%) to AlyA from Klebsiella pneumoniae. A consensus region comprising nine amino acid (YFKAGXYXQ) in the carboxy-terminal region previously reported by Mallisard and colleagues was observed only in AlyVG1 and AlyVG2.

Identification of cDNA clones expressing immunodiagnostic antigens from Trichinella spiralis

International Nuclear Information System (INIS)

Zarlenga, D.; Gamble, H.R.

1987-01-01

A cDNA expression library was built in lambda gt11 phage using poly A mRNA isolated from Trichinella spiralis muscle stage larvae. This library was screened with rabbit antibodies to parasite excretory-secretory (ES) products and greater than 180 clones were isolated. Thirteen clones producing highly immunogenic protein antigens were plaque purified and rescreened with pig antisera to T.spiralis, Trichuris suis or Ascaris suum to identify clones producing epitopes specific to T.spiralis ES products, only. Two clones, TsAc-2 and TsAc-8, which displayed strong interactions with pig antisera to T. spiralis were lysogenized in E. coli Y1089 and the protein extracted. Western blots of the crude fusion proteins revealed molecular weights of 133 kD and 129 kD, respectively. Northern blot analysis of total RNA with 32 P labelled cDNA:lambda gt11 probes indicated single RNA transcripts for each clone with molecular sizes corresponding to 800-850 nucleotides. dscDNA inserts were estimated by southern blot analysis to be 500 bp and 340 bp, respectively, with no cross-hybridization observed between the cloned sequences. Dot blots using pig sera to screen crude fusion protein preparations, total bacterial protein (negative controls) and crude worm extract or ES products from T.spiralis, T.suis and A.suum (positive controls) corroborated the specificity and sensitivity of these clones as potential diagnostic antigens for swine trichinellosis

Molecular cloning and characterization of a cDNA encoding ...

African Journals Online (AJOL)

enoh

2012-03-29

Mar 29, 2012 ... Cloning and Functional. Expression of Cycloartenol Synthases from Mangrove Species. Rhizophora stylosa Griff. And Kandelia candel (L.) Druce. Biosci. Biotechnol. Biochem. 71(7): 1788-1792. Felsenstein J (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 39:783-791 ...

Construction of recombinant DNA clone for bovine viral diarrhea virus

International Nuclear Information System (INIS)

Yeo, S.G.; Cho, H.J.; Masri, S.A.

1992-01-01

Molecular cloning was carried out on the Danish strain of bovine viral diarrhea virus (BVDV) to construct strategy for the diagnostic tools and effective vaccine of BVD afterwards. A recombinant DNA clone (No. 29) was established successfully from cDNA for viral RNA tailed with adenine homopolymer at 3 -end. 32 P-labeled DNA probes of 300~1, 800bp fragments, originating from the clone 29, directed specific DNA-RNA hybridization results with BVDV RNA. Recombinant DNA of the clone 29 was about 5,200bp representing 41.6% of the full length of Danish strain's RNA, and restriction sites were recognized for EooR I, Sst I, Hind III and Pst I restriction enzymes in the DNA fragment

Spread of carbapenem-resistant international clones of Acinetobacter baumannii in Turkey and Azerbaijan: a collaborative study

NARCIS (Netherlands)

Ahmed, S.S.; Alp, E.; Ulu-Kilic, A.; Dinc, G.; Aktas, Z.; Ada, B.; Bagirova, F.; Baran, I.; Ersoy, Y.; Esen, S.; Guven, T.G.; Hopman, J.; Hosoglu, S.; Koksal, F.; Parlak, E.; Yalcin, A.N.; Yilmaz, G.; Voss, A.; Melchers, W.J.

2016-01-01

Epidemic clones of Acinetobacter baumannii, described as European clones I, II, and III, are associated with hospital epidemics throughout the world. We aimed to determine the molecular characteristics and genetic diversity between European clones I, II, and III from Turkey and Azerbaijan. In this

Cloning

Science.gov (United States)

Cloning describes the processes used to create an exact genetic replica of another cell, tissue or organism. ... named Dolly. There are three different types of cloning: Gene cloning, which creates copies of genes or ...

What is Cloning?

Science.gov (United States)

Donate Home Cloning What is Cloning What is Cloning Clones are organisms that are exact genetic copies. ... clones made through modern cloning technologies. How Is Cloning Done? Many people first heard of cloning when ...

Genetic Characterization of Five Hatchery Populations of the Pacific Abalone (Haliotis discus hannai Using Microsatellite Markers

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Jeong-In Myeong

2011-07-01

Full Text Available The Pacific abalone, Haliotis discus hannai, is a popular food in Eastern Asia. Aquacultural production of this species has increased because of recent resource declines, the growing consumption, and ongoing government-operated stock release programs. Therefore, the genetic characterization of hatchery populations is necessary to maintain the genetic diversity of this species and to develop more effective aquaculture practices. We analyzed the genetic structures of five cultured populations in Korea using six microsatellite markers. The number of alleles per locus ranged from 15 to 64, with an average of 23.5. The mean observed and expected heterozygosities were 0.797 and 0.904, respectively. The inbreeding coefficient FIS ranged from 0.054 to 0.184 (mean FIS = 0.121 ± 0.056. The genetic differentiation across all populations was low but significant (overall FST = 0.009, P < 0.01. Pairwise multilocus FST tests, estimates of genetic distance, and phylogenetic and principal component analyses did not show a consistent relationship between geographic and genetic distances. These results could reflect extensive aquaculture, the exchange of breeds and eggs between hatcheries and/or genetic drift due to intensive breeding practices. Thus, for optimal resource management, the genetic variation of hatchery stocks should be monitored and inbreeding controlled within the abalone stocks that are being released every year. This genetic information will be useful for the management of both H. discus hannai fisheries and the aquaculture industry.

Molecular cloning and functional characterization of avian interleukin-19

Science.gov (United States)

The present study describes the cloning and functional characterization of avian interleukin (IL)-19, a cytokine that, in mammals, alters the balance of Th1 and Th2 cells in favor of the Th2 phenotype. The full-length avian IL-19 gene, located on chromosome 26, was amplified from LPS-stimulated chi...

Molecular characterization of resistance to Rifampicin in an emerging hospital-associated Methicillin-resistant Staphylococcus aureus clone ST228, Spain

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Liñares Josefina

2010-03-01

Full Text Available Abstract Background Methicillin-resistant S. aureus (MRSA has been endemic in Hospital Universitari de Bellvitge, Barcelona, since 1990. During the 1990-95 period the Iberian clone (ST-247; SCCmec-I was dominant. Isolates of clonal complex 5 (ST-125; SCCmec-IV gradually replaced the Iberian clone from 1996 to 2003. A new multiresistant MRSA phenotype showing rifampicin resistance emerged in 2004 and rapidly increased from 25% in 2004 to 45% in 2006. The aims of this study were i the molecular characterisation of rifampicin resistant MRSA isolates, ii the study of the rifampicin resistance expression by disk diffusion, microdilution and E-test, and iii the analysis of the rpoB gene mutations involved in rifampicin resistance. Results A sample of representative 108 rifampicin-resistant MRSA isolates belonged to a single PFGE genotype, ST-228, SCCmec type I and spa type t041. Of 108 isolates, 104 (96% had a low-level rifampicin resistance (MICs, 2 to 4 mg/L and 4 a high-level rifampicin resistance (MICs, 128 - ≥ 256 mg/L. Disk diffusion and E-test methods failed to identify a low-level rifampicin resistance in 20 and 12 isolates, respectively. A low-level rifampicin resistance was associated with amino acid substitution 481His/Asn in the beta-subunit of RNA polymerase. Isolates with a high-level rifampicin resistance carried additional mutations in the rpoB gene. Conclusions The emergence of MRSA clone ST228-SCCmecI, related to the Southern Germany clone, involved a therapeutical challenge for treating serious MRSA infections. Decreased susceptibility to rifampicin in MRSA strains of ST228-SCCmecI was associated with one or two specific mutations in the rpoB gene. One fifth of isolates with low-level rifampicin-resistance were missed by the diffusion methods.

Molecular cloning and functional analysis of the gene encoding ...

African Journals Online (AJOL)

Here we report for the first time the cloning of a full-length cDNA encoding GGPPS (Jc-GGPPS) from Jatropha curcas L. The full-length cDNA was 1414 base pair (bp), with an 1110-bp open reading frame (ORF) encoding a 370- amino-acids polypeptide. Bioinformatic analysis revealed that Jc-GGPPS is a member of the ...

Climbing Mount Efficiency--small steps, not giant leaps towards higher cloning success in farm animals.

Science.gov (United States)

Oback, Björn

2008-07-01

Despite more than a decade of research efforts, farm animal cloning by somatic cell nuclear transfer (SCNT) is still frustratingly inefficient. Inefficiency manifests itself at different levels, which are currently not well integrated. At the molecular level, it leads to widespread genetic, epigenetic and transcriptional aberrations in cloned embryos. At the organismal level, these genome-wide abnormalities compromise development of cloned foetuses and offspring. Specific molecular defects need to be causally linked to specific cloned phenotypes, in order to design specific treatments to correct them. Cloning efficiency depends on the ability of the nuclear donor cell to be fully reprogrammed into an embryonic state and the ability of the enucleated recipient cell to carry out the reprogramming reactions. It has been postulated that reprogrammability of the somatic donor cell epigenome is influenced by its differentiation status. However, direct comparisons between cells of divergent differentiation status within several somatic lineages have found no conclusive evidence for this. Choosing somatic stem cells as donors has not improved cloning efficiency, indicating that donor cell type may be less critical for cloning success. Different recipient cells, on the other hand, vary in their reprogramming ability. In bovine, using zygotes instead of oocytes has increased cloning success. Other improvements in livestock cloning efficiency include better coordinating donor cell type with cell cycle stage and aggregating cloned embryos. In the future, it will be important to demonstrate if these small increases at every step are cumulative, adding up to an integrated cloning protocol with greatly improved efficiency.

Clone DB: an integrated NCBI resource for clone-associated data

Science.gov (United States)

Schneider, Valerie A.; Chen, Hsiu-Chuan; Clausen, Cliff; Meric, Peter A.; Zhou, Zhigang; Bouk, Nathan; Husain, Nora; Maglott, Donna R.; Church, Deanna M.

2013-01-01

The National Center for Biotechnology Information (NCBI) Clone DB (http://www.ncbi.nlm.nih.gov/clone/) is an integrated resource providing information about and facilitating access to clones, which serve as valuable research reagents in many fields, including genome sequencing and variation analysis. Clone DB represents an expansion and replacement of the former NCBI Clone Registry and has records for genomic and cell-based libraries and clones representing more than 100 different eukaryotic taxa. Records provide details of library construction, associated sequences, map positions and information about resource distribution. Clone DB is indexed in the NCBI Entrez system and can be queried by fields that include organism, clone name, gene name and sequence identifier. Whenever possible, genomic clones are mapped to reference assemblies and their map positions provided in clone records. Clones mapping to specific genomic regions can also be searched for using the NCBI Clone Finder tool, which accepts queries based on sequence coordinates or features such as gene or transcript names. Clone DB makes reports of library, clone and placement data on its FTP site available for download. With Clone DB, users now have available to them a centralized resource that provides them with the tools they will need to make use of these important research reagents. PMID:23193260

Research Article Molecular cloning and mRNA expression pattern of ...

Indian Academy of Sciences (India)

SAMSUNG

homologue from the brain of Misgurnus anguillicaudatus using homologous cloning and ... developmental processes, including sex determination, embryonic stem cell ..... anguillicaudatus, possibly aiding in unravelling reproductive biology ... significant difference (p < 0.05) between female and male in the same tissue.

Cloning and sequencing of Lol pI, the major allergenic protein of rye-grass pollen.

Science.gov (United States)

Griffith, I J; Smith, P M; Pollock, J; Theerakulpisut, P; Avjioglu, A; Davies, S; Hough, T; Singh, M B; Simpson, R J; Ward, L D

1991-02-25

We have isolated a full length cDNA clone encoding the major glycoprotein allergen Lol pI. The clone was selected using a combination of immunological screening of a cDNA expression library and PCR amplification of Lol pI-specific transcripts. Lol pI expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in sera of grass pollen-allergic subjects. Northern analysis has shown that the Lol pI transcripts are expressed only in pollen of rye-grass. Molecular cloning of Lol pI provides a molecular genetic approach to study the structure-function relationship of allergens.

Distribution and Molecular Characterization of Acinetobacter baumannii International Clone II Lineage in Japan.

Science.gov (United States)

Matsui, Mari; Suzuki, Masato; Suzuki, Masahiro; Yatsuyanagi, Jun; Watahiki, Masanori; Hiraki, Yoichi; Kawano, Fumio; Tsutsui, Atsuko; Shibayama, Keigo; Suzuki, Satowa

2018-02-01

Multidrug-resistant (MDR) Acinetobacter spp. have been globally disseminated in association with the successful clonal lineage Acinetobacter baumannii international clone II (IC II). Because the prevalence of MDR Acinetobacter spp. in Japan remains very low, we characterized all Acinetobacter spp. ( n = 866) from 76 hospitals between October 2012 and March 2013 to describe the entire molecular epidemiology of Acinetobacter spp. The most prevalent species was A. baumannii ( n = 645; 74.5%), with A. baumannii IC II ( n = 245) accounting for 28.3% of the total. Meropenem-resistant isolates accounted for 2.0% ( n = 17) and carried IS Aba1-bla OXA-23-like ( n = 10), bla IMP ( n = 4), or IS Aba1-bla OXA-51-like ( n = 3). Multilocus sequence typing of 110 representative A. baumannii isolates revealed the considerable prevalence of domestic sequence types (STs). A. baumannii IC II isolates were divided into the domestic sequence type 469 (ST469) ( n = 18) and the globally disseminated STs ST208 ( n = 14) and ST219 ( n = 4). ST469 isolates were susceptible to more antimicrobial agents, while ST208 and ST219 overproduced the intrinsic AmpC β-lactamase. A. baumannii IC II and some A. baumannii non-IC II STs (e.g., ST149 and ST246) were associated with fluoroquinolone resistance. This study revealed that carbapenem-susceptible A. baumannii IC II was moderately disseminated in Japan. The low prevalence of acquired carbapenemase genes and presence of domestic STs could contribute to the low prevalence of MDR A. baumannii A similar epidemiology might have appeared before the global dissemination of MDR epidemic lineages. In addition, fluoroquinolone resistance associated with A. baumannii IC II may provide insight into the significance of A. baumannii epidemic clones. Copyright © 2018 American Society for Microbiology.

Molecular cloning of a Bangladeshi strain of very virulent infectious bursal disease virus of chickens and its adaptation in tissue culture by site-directed mutagenesis

International Nuclear Information System (INIS)

Islam, M.R.; Raue, R.; Mueller, H.

2005-01-01

Full-length cDNA of both genome segments of a Bangladeshi strain of very virulent infectious bursal disease virus (BD 3/99) were cloned in plasmid vectors along with the T7 promoter tagged to the 5'-ends. Mutations were introduced in the cloned cDNA to bring about two amino acid exchanges (Q253H and A284T) in the capsid protein VP2. Transfection of primary chicken embryo fibroblast cells with RNA transcribed in vitro from the full-length cDNA resulted in the formation of mutant infectious virus particles that grow in tissue culture. The pathogenicity of this molecularly-cloned, tissue-culture- adapted virus (BD-3tc) was tested in commercial chickens. The parental wild-type strain, BD 3/99, was included for comparison. The subclinical course of the disease and delayed bursal atrophy in BD-3tc-inoculated birds suggested that these amino acid substitutions made BD-3tc partially attenuated. (author)

Molecular cloning and functional characterization of borneol dehydrogenase from the glandular trichomes of Lavandula x intermedia.

Science.gov (United States)

Sarker, Lukman S; Galata, Mariana; Demissie, Zerihun A; Mahmoud, Soheil S

2012-12-15

Several varieties of Lavandula x intermedia (lavandins) are cultivated for their essential oils (EOs) for use in cosmetic, hygiene and personal care products. These EOs are mainly constituted of monoterpenes including camphor, which contributes an off odor reducing the olfactory appeal of the oil. We have recently constructed a cDNA library from the glandular trichomes (the sites of EO synthesis) of L. x intermedia plants. Here, we describe the cloning of a borneol dehydrogenase cDNA (LiBDH) from this library. The 780 bp open reading frame of the cDNA encoded a 259 amino acid short chain alcohol dehydrogenase with a predicted molecular mass of ca. 27.5 kDa. The recombinant LiBDH was expressed in Escherichia coli, purified by Ni-NTA agarose affinity chromatography, and functionally characterized in vitro. The bacterially produced enzyme specifically converted borneol to camphor as the only product with K(m) and k(cat) values of 53 μM and 4.0 × 10(-4) s(-1), respectively. The LiBDH transcripts were specifically expressed in glandular trichomes of mature flowers indicating that like other Lavandula monoterpene synthases the expression of this gene is regulated in a tissue-specific manner. The cloning of LiBDH has far reaching implications in improving the quality of Lavandula EOs through metabolic engineering. Copyright © 2012 Elsevier Inc. All rights reserved.

Molecular cloning and functional analysis of the follicle-stimulating ...

African Journals Online (AJOL)

A 762 bp fragment of the 5'-flanking region of the FSHR gene from the Jintang black goat was cloned. The putative initial transcript site was the A at 681 bp and there were 7 putative cis-acting elements and 3 AT-rich regions. The sequence of the FSHR promoter from the Jintang black goat is 99.34% homology to Capra ...

Molecular cloning, characterization and developmental expression of porcine β-synuclein

DEFF Research Database (Denmark)

Larsen, Knud; Frandsen, Pernille Munk; Madsen, Lone Bruhn

2010-01-01

The synuclein family includes three known proteins: alpha-synuclein, beta-synuclein and gamma-synuclein. beta-Synuclein inhibits the aggregation of alpha-synuclein, a protein involved in Parkinson's disease. We have cloned and characterized the cDNA sequence for porcine beta-synuclein (SNCB) from...

Molecular cloning of the Coch gene of guinea pig inner ear and its expression analysis in cultured fibrocytes of the spiral ligament.

Science.gov (United States)

Li, Lishu; Ikezono, Tetsuo; Sekine, Kuwon; Shindo, Susumu; Matsumura, Tomohiro; Pawankar, Ruby; Ichimiya, Issei; Yagi, Toshiaki

2010-08-01

We have cloned guinea pig Coch cDNA and the sequence information will be useful for future molecular study combined with physiological experiments. Proper Coch gene expression appears to be dependent on the unique extracellular micro-environment of the inner ear in vivo. These results provide insight into the Coch gene expression and its regulation. To characterize the guinea pig Coch gene, we performed molecular cloning and expression analysis in the inner ear and cultured fibrocytes of the spiral ligament. The Coch cDNA was isolated using RACE. Cochlin isofoms were studied by Western blot using three different types of mammalian inner ear. The cochlear fibrocytes were cultured and characterized by immunostaining. Coch gene expression in the fibrocytes was investigated and the influence of cytokine stimulation was evaluated. The full-length 1991 bp Coch cDNA that encodes a 553 amino acid protein was isolated. The sequence had significant homology with other mammals, and the sizes of the Cochlin isoforms were identical. In the cultured fibrocytes, Coch mRNA was expressed in a very small amount and the isoform production was different, compared with the results in vivo. Cytokine stimulation did not alter the level of mRNA expression or isoform formation.

Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

Science.gov (United States)

Throop, Andrea L.; LaBaer, Joshua

2015-01-01

The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088

[Molecular cloning, expression and characterization of lysine decarboxylase gene of endophytic fungus Shiraia sp. Slf14 from Huperzia serrata].

Science.gov (United States)

Peng, Silu; Yang, Huilin; Zhu, Du; Zhang, Zhibin; Yan, Riming; Wang, Ya

2016-04-14

Huperzine A (HupA) was approved as a drug for the treatment of Alzheimer's disease. The HupA biosynthetic pathway was started from lysine decarboxylase (LDC), which catalyzes lysine to cadaverine. In this study, we cloned and expressed an LDC gene from a HupA-producing endophytic fungus, and tested LDC activities. An endophytic fungus Shiraia sp. Slf14 from Huperzia serrata was used. LDC gene was obtained by RT-PCR, and cloned into pET-22b(+) and pET-32a(+) vectors to construct recombinant plasmids pET- 22b-LDC and pET-32a-LDC. These two recombinant plasmids were transformed into E. coli BL21, cultured for 8 h at 24 °C, 200 r/min with 1×10–3 mol/L IPTG into medium to express the LDC proteins, respectively. LDC proteins were purified by Ni2+ affinity chromatography. Catalytic activities were measured by Thin Layer Chromatography. At last, the physicochemical properties and structures of these two LDCs were obtained by bioinformatics software. LDC and Trx-LDC were expressed in E. coli BL21 successfully. SDS-PAGE analysis shows that the molecular weight of LDC and Trx-LDC were 24.4 kDa and 42.7 kDa respectively, which are consistent with bioinformatics analysis. In addition, TLC analysis reveals that both LDC and Trx-LDC had catalytic abilities. This work can provide fundamental data for enriching LDC molecular information and reveal the HupA biosynthetic pathway in endophytic fungi.

Molecular cloning of chicken metallothionein. Deduction of the complete amino acid sequence and analysis of expression using cloned cDNA

Energy Technology Data Exchange (ETDEWEB)

Wei, D; Andrews, G K

1988-01-25

A cDNA library was constructed using RNA isolated from the livers of chickens which had been treated with zinc. This library was screened with a RNA probe complementary to mouse metallothionein-I (MT), and eight chicken MT cDNA clones were obtained. All of the cDNA clones contained nucleotide sequences homologous to regions of the longest (375 bp) cDNA clone. The latter contained an open reading frame of 189 bp, and the deduced amino acid sequence indicates a protein of 63 amino acids of which 20 are cysteine residues. Amino acid composition and partial amino acid sequence analyses of purified chicken MT protein agreed with the amino acid composition and sequence deduced from the cloned cDNA. Amino acid sequence comparison establish that chicken MT shares extensive homology with mammalian MTs. Southern blot analysis of chicken DNA indicates that the chicken MT gene is not a part of a large family of related sequences, but rather is likely to be a unique gene sequence. In the chicken liver, levels of chicken MT mRNA were rapidly induced by metals (Cd/sup 2 +/, Zn/sup 2 +/, Cu/sup 2 +/), glucocorticoids and lipopolysaccharide. MT mRNA was present in low levels in embryonic liver and increased to high levels during the first week after hatching before decreasing again to the basal levels found in adult liver. The results of this study establish that MT is highly conserved between birds and mammals and is regulated in the chicken by agents which also regulate expression of mammalian MT genes. However, in contrast to the mammals, the results suggest the existence of a single isoform of MT in the chicken.

Unified Approach to Universal Cloning and Phase-Covariant Cloning

OpenAIRE

Hu, Jia-Zhong; Yu, Zong-Wen; Wang, Xiang-Bin

2008-01-01

We analyze the problem of approximate quantum cloning when the quantum state is between two latitudes on the Bloch's sphere. We present an analytical formula for the optimized 1-to-2 cloning. The formula unifies the universal quantum cloning (UQCM) and the phase covariant quantum cloning.

Molecular Cloning and Characterization of Carbonic Anhydrase XII from Pufferfish (Takifugu rubripes

Directory of Open Access Journals (Sweden)

Kanij Rukshana Sumi

2018-03-01

Full Text Available In this study, an 1888-bp carbonic anhydrase XII (CA XII sequence was cloned from the brain of the pufferfish, Takifugu rubripes. The cloned sequence contained a coding region of 1470-bp, which was predicted to translate into a protein of 490 amino acid residues. The predicted protein showed between 68–56% identity with the large yellow croaker (Larimichthys crocea, tilapia (Oreochromis niloticus, and Asian arowana (Scleropages formosus CA XII proteins. It also exhibited 36% and 53% identity with human CA II and CA XII, respectively. The cloned sequence contained a 22 amino acid NH2-terminal signal sequence and three Asn-Xaa-Ser/Thr sequons, among which one was potentially glycosylated. Four cysteine residues were also identified (Cys-21, Cys-201, Cys-355, and Cys-358, two of which (Cys-21 and Cys-201 could potentially form a disulfide bond. A 22-amino acid COOH-terminal cytoplasmic tail containing a potential site for phosphorylation by protein kinase A was also found. The cloned sequence might be a transmembrane protein, as predicted from in silico and phylogenetic analyses. The active site analysis of the predicted protein showed that its active site residues were highly conserved with tilapia CA XII protein. Homology modeling of the pufferfish CA XII was done using the crystal structure of the extracellular domain of human carbonic anhydrase XII at 1.55 Å resolution as a template. Semi-quantitative reverse transcription (RT-PCR, quantitative PCR (q-PCR, and in situ hybridization confirmed that pufferfish CA XII is highly expressed in the brain.

Proteomic analysis of pancreas derived from adult cloned pig

International Nuclear Information System (INIS)

Chae, Jung-Il; Cho, Young Keun; Cho, Seong-Keun; Kim, Jin-Hoi; Han, Yong-Mahn; Koo, Deog-Bon; Lee, Kyung-Kwang

2008-01-01

The potential medical applications of animal cloning include xenotransplantation, but the complex molecular cascades that control porcine organ development are not fully understood. Still, it has become apparent that organs derived from cloned pigs may be suitable for transplantation into humans. In this study, we examined the pancreas of an adult cloned pig developed through somatic cell nuclear transfer (SCNT) using two-dimensional electrophoresis (2-DE) and Western blotting. Proteomic analysis revealed 69 differentially regulated proteins, including such apoptosis-related species as annexins, lamins, and heat shock proteins, which were unanimously upregulated in the SCNT sample. Among the downregulated proteins in SCNT pancreas were peroxiredoxins and catalase. Western blot results indicate that several antioxidant enzymes and the anti-apoptotic protein were downregulated in SCNT pancreas, whereas several caspases were upregulated. Together, these data suggest that the accumulation of reactive oxygen species (ROS) in the pancreas of an adult cloned pig leads to apoptosis

Molecular Characterization of Methicillin Resistant Staphylococcus aureus Strains Isolated from Intensive Care Units in Iran: ST22-SCCmec IV/t790 Emerges as the Major Clone.

Science.gov (United States)

Goudarzi, Mehdi; Goudarzi, Hossein; Sá Figueiredo, Agnes Marie; Udo, Edet E; Fazeli, Maryam; Asadzadeh, Mohammad; Seyedjavadi, Sima Sadat

2016-01-01

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in different patient populations is a major public health concern. This study determined the prevalence and distribution of circulating molecular types of MRSA in hospitalized patients in ICU of hospitals in Tehran. A total of 70 MRSA isolates were collected from patients in eight hospitals. Antimicrobial resistance patterns were determined using the disk diffusion method. The presence of toxin encoding genes and the vancomycin resistance gene were determined by PCR. The MRSA isolates were further analyzed using multi-locus sequence, spa, SCCmec, and agr typing. The MRSA prevalence was 93.3%. Antimicrobial susceptibility testing revealed a high resistance rate (97.1%) to ampicillin and penicillin. The rate of resistance to the majority of antibiotics tested was 30% to 71.4%. Two isolates belonging to the ST22-SCCmec IV/t790 clone (MIC ≥ 8 μg/ml) had intermediate resistance to vancomycin. The majority of MRSA isolates (24.3%) were associated with the ST22-SCCmec IV/t790 clone; the other MRSA clones were ST859-SCCmec IV/t969 (18.6%), ST239-SCCmec III/t037 (17.1%), and ST291-SCCmec IV/t030 (8.6%). The circulating MRSA strains in Iranian hospitals were genetically diverse with a relatively high prevalence of the ST22-SCCmec IV/t790 clone. These findings support the need for future surveillance studies on MRSA to better elucidate the distribution of existing MRSA clones and detect emergence of new MRSA clones.

Molecular and Biological Characterization of an Isolate of Cucumber mosaic virus from Glycine soja by Generating its Infectious Full-genome cDNA Clones

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Mi Sa Vo Phan

2014-06-01

Full Text Available Molecular and biological characteristics of an isolate of Cucumber mosaic virus (CMV from Glycine soja (wild soybean, named as CMV-209, was examined in this study. Comparison of nucleotide sequences and phylogenetic analyses of CMV-209 with the other CMV strains revealed that CMV-209 belonged to CMV subgroup I. However, CMV-209 showed some genetic distance from the CMV strains assigned to subgroup IA or subgroup IB. Infectious full-genome cDNA clones of CMV-209 were generated under the control of the Cauliflower mosaic virus 35S promoter. Infectivity of the CMV-209 clones was evaluated in Nicotiana benthamiana and various legume species. Our assays revealed that CMV-209 could systemically infect Glycine soja (wild soybean and Pisum sativum (pea as well as N. benthamiana, but not the other legume species.

Molecular cloning and restriction analysis of EcoRI-fragments of Vicia faba rDNA

International Nuclear Information System (INIS)

Yakura, Kimitaka; Tanifuji, Shigeyuki.

1983-01-01

EcoRI-fragments of Vicia faba rDNA were cloned in plasmid pBR325. Southern blot hybridization of BamHI-digests of these cloned plasmids and Vicia genomic DNA led to the determination of relative positions of BamHI sites in the rDNA and the physical map that had been tentatively made is corrected. (author)

Molecular cloning and characterization of RGA1 encoding a G protein alpha subunit from rice (Oryza sativa L. IR-36).

Science.gov (United States)

Seo, H S; Kim, H Y; Jeong, J Y; Lee, S Y; Cho, M J; Bahk, J D

1995-03-01

A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein alpha subunit as a probe from a rice (Oryza sativa L. IR-36) seedling cDNA library from roots and leaves. Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa. The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein alpha subunits. A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region. The RGA1 protein contains all the consensus regions of G protein alpha subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin. The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 microM [adenylate-32P] NAD and activated cholera toxin. Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome. Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.

Molecular Cloning and Characterization of Growth Factor Receptor Bound-Protein in Clonorchis sinensis

Science.gov (United States)

Bai, Xuelian; Lee, Ji-Yun; Kim, Tae Im; Dai, Fuhong; Lee, Tae-Jin; Hong, Sung-Jong

2014-01-01

Background Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2) is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2) from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. Methodology/Principal Findings A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. Conclusion Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths. PMID:24454892

Molecular cloning and characterization of growth factor receptor bound-protein in Clonorchis sinensis.

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Xuelian Bai

Full Text Available BACKGROUND: Clonorchis sinensis causes clonorchiasis, a potentially serious disease. Growth factor receptor-bound protein 2 (Grb2 is a cytosolic protein conserved among animals and plays roles in cellular functions such as meiosis, organogenesis and energy metabolism. In the present study, we report first molecular characters of growth factor receptor bound-protein (CsGrb2 from C. sinensis as counter part of Grb2 from animals and its possible functions in development and organogenesis of C. sinensis. METHODOLOGY/PRINCIPAL FINDINGS: A CsGrb2 cDNA clone retrieved from the C. sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure. Recombinant CsGrb2 was bacterially produced and purified to homogeneity. Native CsGrb2 with estimated molecular weight was identified from C. sinensis adult extract by western blotting using a mouse immune serum to recombinant CsGrb2. CsGrb2 transcripts was more abundant in the metacercariae than in the adults. Immunohistochemical staining showed that CsGrb2 was localized to the suckers, mesenchymal tissues, sperms in seminal receptacle and ovary in the adults, and abundantly expressed in most organs of the metacercariae. Recombinant CsGrb2 was evaluated to be little useful as a serodiagnostic reagent for C. sinesis human infections. CONCLUSION: Grb2 protein found in C. sinensis was conserved among animals and suggested to play a role in the organogenesis, energy metabolism and mitotic spermatogenesis of C. sinensis. These findings from C. sinensis provide wider understanding on diverse function of Grb2 in lower animals such as platyhelminths.

Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the Metagenome

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Liu Yu

2008-12-01

, purified and characterized. The molecular mass of the native enzyme was approximately 31 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of the Pye3 indicated molecular mass of 31 kDa and 31.5 kDa, respectively, suggesting that the Pye3 is a monomer. The purified Pye3 not only degraded all pyrethroid pesticides tested, but also hydrolyzed ρ-nitrophenyl esters of medium-short chain fatty acids, indicating that the Pye3 is an esterase with broader specificity. The Km values for trans-Permethrin and cis-permethrin are 0.10 μM and 0.18 μM, respectively, and these catalytic properties were superior to carboxylesterases from resistant insects and mammals. The catalytic activity of the Pye3 was strongly inhibited by Hg2+, Ag+, ρ-chloromercuribenzoate, whereas less pronounced effect was observed in the presence of divalent cations, the chelating agent EDTA and phenanthroline. Conclusion A novel pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, the broader substrate specificities and higher activity of the pyrethroid-hydrolyzing esterase (Pye3 make it an ideal candidate for in situ for detoxification of pyrethroids where they cause environmental contamination problems. Consequently, metagenomic DNA clone library offers possibilities to discover novel bio-molecules through the expression of genes from uncultivated bacteria.

Thermostable, salt tolerant, wide pH range novel chitobiase from Vibrio parahemolyticus: isolation, characterization, molecular cloning, and expression.

Science.gov (United States)

Zhu, B C; Lo, J Y; Li, Y T; Li, S C; Jaynes, J M; Gildemeister, O S; Laine, R A; Ou, C Y

1992-07-01

A chitobiase gene from Vibrio parahemolyticus was cloned into plasmid pUC18 in Escherichia coli strain DH5 alpha. The plasmid construct, pC120, contained a 6.4 kb Vibrio DNA insert. The recombinant gene expressed chitobiase [EC 3.2.1.30] activity similar to that found in the native Vibrio. The enzyme was purified by ion exchange, hydroxylapatite and gel permeation chromatographies, and exhibited an apparent molecular weight of 80 kDa on SDS-polyacrylamide gel electrophoresis. Chitobiose and 6 more substrates, including beta-N-acetyl galactosamine glycosides, were hydrolyzed by the recombinant chitobiase, indicating its putative classification as an hexosaminidase [EC 3.2.1.52]. The enzyme was resistant to denaturation by 2 M NaCl, thermostable at 45 degrees C and active over a very unusual (for glycosyl hydrolases) pH range, from 4 to 10. The purified cloned chitobiase gave 4 closely focussed bands on an isoelectric focusing gel, at pH 4 to 6.5. The N-terminal 43 amino acid sequence shows no homology with other proteins in commercial databanks or in the literature, and from its N-terminal sequence, appears to be a novel protein, unrelated in sequence to chitobiases from other Vibrios reported and unrelated to hexosaminidases from other organisms.

Enzyme free cloning for high throughput gene cloning and expression

NARCIS (Netherlands)

de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

2006-01-01

Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning

Molecular cloning and characterization of Fasciola gigantica thioredoxin-glutathione reductase.

Science.gov (United States)

Changklungmoa, Narin; Kueakhai, Pornanan; Sangpairoj, Kant; Chaichanasak, Pannigan; Jaikua, Wipaphorn; Riengrojpitak, Suda; Sobhon, Prasert; Chaithirayanon, Kulathida

2015-06-01

The Fasciola gigantica thioredoxin-glutathione reductase (FgTGR) gene is a fusion between thioredoxin reductase (TR) and a glutaredoxin (Grx) gene. FgTGR was cloned by polymerase chain reaction (PCR) from adult complementary DNA (cDNA), and its sequences showed two isoforms, i.e., the cytosolic and mitochondrial FgTGR. Cytosolic FgTGR (cytFgTGR) was composed of 2370 bp, and its peptide had no signal sequence and hence was not a secreted protein. Mitochondrial FgTGR (mitFgTGR) was composed of 2506 bp with a signal peptide of 43 amino acids; therefore, it was a secreted protein. The putative cytFgTGR and mitFgTGR peptides comprised of 598 and 641 amino acids, respectively, with a molecular weight of 65.8 kDa for cytFgTGR and mitFgTGR, with a conserved sequence (CPYC) of TR, and ACUG and CVNVGC of Grx domains. The recombinant FgTGR (rFgTGR) was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTGR). The FgTGR protein expression, estimated by indirect ELISA using the rabbit anti-rFgTGR as probe, showed high levels of expression in eggs, and 2- and 4-week-old juveniles and adults. The rFgTGR exhibited specific activities in the 5,5'-dithiobis (2-nitro-benzoic acid) (DTNB) reductase assay for TR activity and in β-hydroxyethul disulfide (HED) for Grx activity. When analyzed by immunoblotting and immunohistochemistry, rabbit anti-rFgTGR reacted with natural FgTGR at a molecular weight of 66 kDa from eggs, whole body fraction (WB) of metacercariae, NEJ, 2- and 4-week-old juveniles and adults, and the tegumental antigen (TA) of adult. The FgTGR protein was expressed at high levels in the tegument of 2- and 4-week-old juveniles. The FgTGR may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or a drug target.

Alternative Splicing Profile and Sex-Preferential Gene Expression in the Female and Male Pacific Abalone Haliotis discus hannai.

Science.gov (United States)

Kim, Mi Ae; Rhee, Jae-Sung; Kim, Tae Ha; Lee, Jung Sick; Choi, Ah-Young; Choi, Beom-Soon; Choi, Ik-Young; Sohn, Young Chang

2017-03-09

In order to characterize the female or male transcriptome of the Pacific abalone and further increase genomic resources, we sequenced the mRNA of full-length complementary DNA (cDNA) libraries derived from pooled tissues of female and male Haliotis discus hannai by employing the Iso-Seq protocol of the PacBio RSII platform. We successfully assembled whole full-length cDNA sequences and constructed a transcriptome database that included isoform information. After clustering, a total of 15,110 and 12,145 genes that coded for proteins were identified in female and male abalones, respectively. A total of 13,057 putative orthologs were retained from each transcriptome in abalones. Overall Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyzed in each database showed a similar composition between sexes. In addition, a total of 519 and 391 isoforms were genome-widely identified with at least two isoforms from female and male transcriptome databases. We found that the number of isoforms and their alternatively spliced patterns are variable and sex-dependent. This information represents the first significant contribution to sex-preferential genomic resources of the Pacific abalone. The availability of whole female and male transcriptome database and their isoform information will be useful to improve our understanding of molecular responses and also for the analysis of population dynamics in the Pacific abalone.

Alternative Splicing Profile and Sex-Preferential Gene Expression in the Female and Male Pacific Abalone Haliotis discus hannai

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Mi Ae Kim

2017-03-01

Full Text Available In order to characterize the female or male transcriptome of the Pacific abalone and further increase genomic resources, we sequenced the mRNA of full-length complementary DNA (cDNA libraries derived from pooled tissues of female and male Haliotis discus hannai by employing the Iso-Seq protocol of the PacBio RSII platform. We successfully assembled whole full-length cDNA sequences and constructed a transcriptome database that included isoform information. After clustering, a total of 15,110 and 12,145 genes that coded for proteins were identified in female and male abalones, respectively. A total of 13,057 putative orthologs were retained from each transcriptome in abalones. Overall Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG pathways analyzed in each database showed a similar composition between sexes. In addition, a total of 519 and 391 isoforms were genome-widely identified with at least two isoforms from female and male transcriptome databases. We found that the number of isoforms and their alternatively spliced patterns are variable and sex-dependent. This information represents the first significant contribution to sex-preferential genomic resources of the Pacific abalone. The availability of whole female and male transcriptome database and their isoform information will be useful to improve our understanding of molecular responses and also for the analysis of population dynamics in the Pacific abalone.

A Seminar on Human Cloning: Cloning in Reproductive Medicine

OpenAIRE

Illmensee, Karl

2001-01-01

This review article summarizes the historical development of mammalian cloning, presents current advances and presumed risk factors in the field of reproductive cloning, discusses possible clinical applications of therapeutic and diagnostic cloning and outlines prospective commercial trends in pharmacytical cloning. Predictable progress in biotechnology and stem cell engineering should prove to be advantageous for patients' health and for novel benefits in reproductive and regenerative medicine.

A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning and expression.

Science.gov (United States)

Goffner, D; Van Doorsselaere, J; Yahiaoui, N; Samaj, J; Grima-Pettenati, J; Boudet, A M

1998-03-01

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. In a previous study, an atypical form of CAD (CAD 1) was identified in Eucalyptus gunnii [12]. We report here the molecular cloning and characterization of the corresponding cDNA, CAD 1-5, which encodes this novel aromatic alcohol dehydrogenase. The identity of CAD 1-5 was unambiguously confirmed by sequence comparison of the cDNA with peptide sequences derived from purified CAD 1 protein and by functional expression of CAD 1 recombinant protein in Escherichia coli. Both native and recombinant CAD 1 exhibit high affinity towards lignin precursors including 4-coumaraldehyde and coniferaldehyde, but they do not accept sinapaldehyde. Moreover, recombinant CAD 1 can also utilize a wide range of aromatic substrates including unsubstituted and substituted benzaldehydes. The open reading frame of CAD 1-5 encodes a protein with a calculated molecular mass of 35,790 Da and an isoelectric point of 8.1. Although sequence comparisons with proteins in databases revealed significant similarities with dihydroflavonol-4-reductases (DFR; EC 1.1.1.219) from a wide range of plant species, the most striking similarity was found with cinnamoyl-CoA reductase (CCR; EC 1.2.1.44), the enzyme which directly precedes CAD in the lignin biosynthetic pathway. RNA blot analysis and immunolocalization experiments indicated that CAD 1 is expressed in both lignified and unlignified tissues/cells. Based on the catalytic activity of CAD 1 in vitro and its localization in planta, CAD 1 may function as an 'alternative' enzyme in the lignin biosynthetic pathway. However, additional roles in phenolic metabolism are not excluded.

Molecular Cloning and Expression of a Novel Gene Related to ...

African Journals Online (AJOL)

A new legume lectin gene, designated as SmL1, was cloned from Salvia miltiorrhiza Bunge, a famous traditional Chinese medicinal plant. The cDNA of SmL1 was 919 bp in length and contained an 822 bp open reading frame (ORF) encoding a putative lectin precursor with two legume lectin domains. The deduced SML1 ...

Somatic cell nuclear transfer cloning: practical applications and current legislation.

Science.gov (United States)

Niemann, H; Lucas-Hahn, A

2012-08-01

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved. © 2012 Blackwell Verlag GmbH.

Molecular Cloning and Characterization of PnbHLH1 Transcription Factor in Panax notoginseng

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Xiang Zhang

2017-07-01

Full Text Available Panax notoginseng has been extensively used as a traditional Chinese medicine. In the current study, molecular cloning and characterization of PnbHLH1 transcription factor were explored in Panax notoginseng. The full length of the PnbHLH1 gene obtained by splicing was 1430 bp, encoding 321 amino acids. Prokaryotic expression vector pET-28a-PnbHLH1 was constructed and transferred into the BL21 prokaryotic expression strain. An electrophoretic mobility shift assay of PnbHLH1 protein binding to E-box cis-acting elements verified that PnbHLH1 belonged to the bHLH class transcription factor which could interact with the promoter region of the E-box core sequence. The expression levels of key genes involved in the biosynthesis of triterpenoid saponins in PnbHLH1 transgenic cells were higher than those in the wild cells. Similarly, the total saponin contents were increased in the PnbHLH1 transgenic cell lines compared with the wild cell lines. Such results suggest that the PnbHLH1 transcription factor is a positive regulator in the biosynthesis of triterpenoid saponins in Panax notoginseng.

Cloning of observables

OpenAIRE

Ferraro, Alessandro; Galbiati, Matteo; Paris, Matteo G. A.

2005-01-01

We introduce the concept of cloning for classes of observables and classify cloning machines for qubit systems according to the number of parameters needed to describe the class under investigation. A no-cloning theorem for observables is derived and the connections between cloning of observables and joint measurements of noncommuting observables are elucidated. Relationships with cloning of states and non-demolition measurements are also analyzed.

Molecular Cloning and Expression Analysis of Cu/Zn SOD Gene from Gynura bicolor DC.

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Xin Xu

2017-01-01

Full Text Available Superoxide dismutase is an important antioxidant enzyme extensively existing in eukaryote, which scavenges reactive oxygen species (ROS and plays an essential role in stress tolerance of higher plants. A full-length cDNA encoding Cu/Zn SOD was cloned from leaves of Gynura bicolor DC. by rapid amplification of cDNA ends (RACE. The full-length cDNA of Cu/Zn SOD is 924 bp and has a 681 bp open reading frame encoding 227 amino acids. Bioinformatics analysis revealed that belonged to the plant SOD super family. Cu/Zn SODs of the Helianthus annuus, Mikania micrantha, and Solidago canadensis var. scabra all have 86% similarity to the G. bicolor Cu/Zn SOD. Analysis of the expression of Cu/Zn SOD under different treatments revealed that Cu/Zn SOD was a stress-responsive gene, especially to 1-MCP. It indicates that the Cu/Zn SOD gene would be an important gene in the resistance to stresses and will be helpful in providing evidence for future research on underlying molecular mechanism and choosing proper postharvest treatments for G. bicolor.

Molecular epidemiology of Escherichia coli producing extended-spectrum {beta}-lactamases in Lugo (Spain): dissemination of clone O25b:H4-ST131 producing CTX-M-15.

Science.gov (United States)

Blanco, Miguel; Alonso, Maria Pilar; Nicolas-Chanoine, Marie-Hélène; Dahbi, Ghizlane; Mora, Azucena; Blanco, Jesús E; López, Cecilia; Cortés, Pilar; Llagostera, Montserrat; Leflon-Guibout, Véronique; Puentes, Beatriz; Mamani, Rosalía; Herrera, Alexandra; Coira, María Amparo; García-Garrote, Fernando; Pita, Julia María; Blanco, Jorge

2009-06-01

Having shown that the Xeral-Calde Hospital in Lugo (Spain) has been concerned by Escherichia coli clone O25:H4-ST131 producing CTX-M-15 (Nicolas-Chanoine et al. J Antimicrob Chemother 2008; 61: 273-81), the present study was carried out to evaluate the prevalence of this clone among the extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates and also to molecularly characterize the E. coli isolates producing ESBL other than CTX-M-15. In the first part of this study, 105 ESBL-producing E. coli isolates (February 2006 to March 2007) were characterized with regard to ESBL enzymes, serotypes, virulence genes, phylogenetic groups, multilocus sequence typing (MLST) and PFGE. In the second part of this study, 249 ESBL-producing E. coli isolates (April 2007 to May 2008) were investigated only for the detection of clone O25b:H4-ST131 producing CTX-M-15 using a triplex PCR developed in this study and based on the detection of the new operon afa FM955459 and the targets rfbO25b and 3' end of the bla(CTX-M-15) gene. Of the 105 ESBL-producing E. coli isolates, 60 (57.1%) were positive for CTX-M-14, 23 (21.9%) for CTX-M-15, 10 (9.5%) for SHV-12 and 7 (6.7%) for CTX-M-32. Serotypes, virulence genes, phylogenetic groups and molecular typing by PFGE demonstrated high homogeneity within those producing CTX-M-15 and high diversity within E. coli producing CTX-M-14 and other ESBLs. By PFGE, CTX-M-15-producing E. coli isolates O25b:H4 belonging to the phylogenetic group B2 and MLST profile ST131 were grouped in the same cluster. The epidemic strain of clone O25b:H4-ST131 represented 23.1%, 22.5% and 20.0% of all ESBL-producing E. coli isolated in 2006, 2007 and 2008, respectively. CTX-M-type ESBLs, primarily CTX-M-14 and CTX-M-15, have emerged as the predominant types of ESBL produced by E. coli isolates in Lugo. In view of the reported findings, long-term care facilities for elderly people may represent a significant reservoir for E. coli clone O25b:H4-ST131 producing CTX

Cloning of observables

International Nuclear Information System (INIS)

Ferraro, Alessandro; Galbiati, Matteo; Paris, Matteo G A

2006-01-01

We introduce the concept of cloning for classes of observables and classify cloning machines for qubit systems according to the number of parameters needed to describe the class under investigation. A no-cloning theorem for observables is derived and the connections between cloning of observables and joint measurements of noncommuting observables are elucidated. Relationships with cloning of states and non-demolition measurements are also analysed. (letter to the editor)

Cloning of the cDNA for human 12-lipoxygenase

International Nuclear Information System (INIS)

Izumi, T.; Hoshiko, S.; Radmark, O.; Samuelsson, B.

1990-01-01

A full-length cDNA clone encoding 12-lipoxygenase was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

CERN Document Server

Foulon, V; Croes, K; Waelkens, E

1999-01-01

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl- CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon-carbon bond cleavage during à-oxidation of 3- methyl-branched fatty acids

cDNA, genomic cloning and sequence analysis of ribosomal protein ...

African Journals Online (AJOL)

enoh

2012-03-13

Mar 13, 2012 ... cDNA and the genomic sequence of RPS4X were cloned successfully from ... S4 genes plays a role in Turner syndrome; however, this ..... Project of Educational Committee of Sichuan Province ... Molecular biology of the cell.

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.

Science.gov (United States)

Weger-Lucarelli, James; Duggal, Nisha K; Bullard-Feibelman, Kristen; Veselinovic, Milena; Romo, Hannah; Nguyen, Chilinh; Rückert, Claudia; Brault, Aaron C; Bowen, Richard A; Stenglein, Mark; Geiss, Brian J; Ebel, Gregory D

2017-01-01

Zika virus (ZIKV; family Flaviviridae, genus Flavivirus) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available that lack significant attenuation, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system based on a 2015 isolate from Puerto Rico (PRVABC59). We generated a two-plasmid infectious clone system from which infectious virus was rescued that replicates in human and mosquito cells with growth kinetics representative of wild-type ZIKV. Infectious clone-derived virus initiated infection and transmission rates in Aedes aegypti mosquitoes comparable to those of the primary isolate and displayed similar pathogenesis in AG129 mice. This infectious clone system provides a valuable resource to the research community to explore ZIKV molecular biology, vaccine development, antiviral development, diagnostics, vector competence, and disease pathogenesis. ZIKV is a rapidly spreading mosquito-borne pathogen that has been linked to Guillain-Barré syndrome in adults and congenital microcephaly in developing fetuses and infants. ZIKV can also be sexually transmitted. The viral molecular determinants of any of these phenotypes are not well understood. There is no reverse genetics system available for the current epidemic virus that will allow researchers to study ZIKV immunity, develop novel vaccines, or develop antiviral drugs. Here we provide a novel infectious clone system generated from a recent ZIKV isolated from a patient infected in Puerto Rico. This infectious clone produces virus with in vitro and in vivo characteristics similar to those of the primary isolate, providing a critical tool to study ZIKV infection and disease. Copyright © 2016 American Society for Microbiology.

Babesia bovis clones: biochemical and enzymatic characterization

International Nuclear Information System (INIS)

Rodriguez Camarillo, S.D.

1985-01-01

Studies were undertaken to generate additional knowledge of the biochemistry of Babesia bovis. A modified in vitro culture technique used for cloning B. bovis. This technique included a low oxygen concentration atmosphere (2%, O 2 , 5% CO 2 , 93% N 2 ) and 4 mm fluid level. Cultures initiated with one infected erythrocyte were maintained until parasitemias of positive wells reached 2% parasitemia. Primary clones were obtained and from these, nine clones were recloned twice and used for subsequent studies. A procedure was developed to concentrate and separate B. bovis merozoites and infected erythrocytes by Percoll density gradients. Merozoites separated at 1.087 g/ml specific density, whereas infected erythrocytes separated at 1.121 g/ml. Viability of purified parasites was not affected. Agarose gel electrophoresis was used to identify metabolic enzyme in B. bovis and B. bigemina. The enzymes LDH, GDH, GPI and HK were detected in both species. Molecular analysis by one and two-dimensional gel electrophoresis of proteins metabolically labeled with 35 S-methionine indicated that two clones, derived from the same field strain, were similar but not identical to the parent. Fewer proteins were observed in the parental strain. Growth of two 60-Co irradiated B. bovis clones indicated a dose-effect relationship. Growth of parasites exposed for the longest period was initially retarded but returned to normal growth after two or three subcultures. Cultures exposed for shorter periods were unaffected with respect to the rate of growth. Analysis of electrophoretic mobility of metabolic enzyme showed a change in migration pattern

Phenotypic and molecular characterization of Neisseria gonorrhoeae isolates from Slovenia, 2006-12: rise and fall of the multidrug-resistant NG-MAST genogroup 1407 clone?

Science.gov (United States)

Jeverica, Samo; Golparian, Daniel; Matičič, Mojca; Potočnik, Marko; Mlakar, Boštjan; Unemo, Magnus

2014-06-01

To determine the phenotypic and molecular characteristics of Neisseria gonorrhoeae isolates obtained between 2006 and 2012 in Slovenia. Gonococcal isolates obtained between 2006 and 2012 in Slovenia (n = 194) were investigated with Etest for susceptibility to cefixime, ceftriaxone, penicillin, ciprofloxacin, azithromycin, tetracycline, gentamicin and spectinomycin. All isolates were examined with N. gonorrhoeae multiantigen sequence typing for molecular epidemiology and sequencing of the major extended-spectrum cephalosporin (ESC) resistance determinants (penA, mtrR and penB) was performed. The overall prevalence of decreased susceptibility or resistance to cefixime and ceftriaxone (MIC ≥0.125 mg/L) was 11% and 5%, respectively. The decreased susceptibility or resistance showed an epidemic peak in 2011 (33% for cefixime and 11% for ceftriaxone), decreasing to 6% and 4%, respectively, in 2012. ST1407 (9% of isolates), ST21 (6%) and ST225 (6%) were the most common sequence types (STs) during 2006-12. Genogroup G1407 (ST1407 most prevalent ST), an internationally spread clone with decreased susceptibility or resistance to ESCs, was most prevalent (48%) in 2009. However, the G1407 prevalence then declined: in 2010, 30%; in 2011, 28%; and in 2012, 8%. Instead, in 2012 the ESC- and ciprofloxacin-susceptible G21 was the predominant genogroup (26%). The prevalence of gonococcal resistance to ESCs in Slovenia has been high, but fluctuating. Fortunately, in 2012 some ESC- and ciprofloxacin-susceptible clones, such as genogroups G21, G1195 and G2992, appeared to have mainly replaced the multidrug-resistant G1407 clone, a replacement also seen in several European countries. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The Clone Factory

Science.gov (United States)

Stoddard, Beryl

2005-01-01

Have humans been cloned? Is it possible? Immediate interest is sparked when students are asked these questions. In response to their curiosity, the clone factory activity was developed to help them understand the process of cloning. In this activity, students reenact the cloning process, in a very simplified simulation. After completing the…

Molecular cloning of the human gene for von Willebrand factor and identification of the transcription initiation site

International Nuclear Information System (INIS)

Collins, C.J.; Underdahl, J.P.; Levene, R.B.; Ravera, C.P.; Morin, M.J.; Dombalagian, M.J.; Ricca, G.; Livingston, D.M.; Lynch, D.C.

1987-01-01

A series of overlapping cosmid genomic clones have been isolated that contain the entire coding unit of the human gene for van Willebrand factor (vWf), a major component of the hemostatic system. The cloned segments span ≅ 175 kilobases of human DNA sequence, and hybridization analysis suggest that the vWf coding unit is ≅150 kilobases in length. Within one of these clones, the vWF transcription initiation site has been mapped and a portion of the vWf promoter region has been sequenced, revealing a typical TATA box, a downstream CCAAT box, and a perfect downstream repeat of the 8 base pairs containing the transcription start site. Sequencing of a segment of another genomic clone has revealed the vWF translation termination codon. Where tested, comparative restriction analysis of cloned and chromosomal DNA segments strongly suggests that no major alterations occurred during cloning and that there is only one complete copy of the vWf gene in the human haploid genome. Similar analyses of DNA from vWf-producing endothelial cells and nonexpressing leukocytes suggest that vWf gene expression is not accompanied by gross genomic rearrangements. In addition, there is significant homology of C-terminal coding sequences among the vWf genes of several vertebrate species

Simple and versatile molecular method of copy-number measurement using cloned competitors.

Directory of Open Access Journals (Sweden)

Hyun-Kyoung Kim

Full Text Available Variations and alterations of copy numbers (CNVs and CNAs carry disease susceptibility and drug responsiveness implications. Although there are many molecular methods to measure copy numbers, sensitivity, reproducibility, cost, and time issues remain. In the present study, we were able to solve those problems utilizing our modified real competitive PCR method with cloned competitors (mrcPCR. First, the mrcPCR for ERBB2 copy number was established, and the results were comparable to current standard methods but with a shorter assay time and a lower cost. Second, the mrcPCR assays for 24 drug-target genes were established, and the results in a panel of NCI-60 cells were comparable to those from real-time PCR and microarray. Third, the mrcPCR results for FCGR3A and the FCGR3B CNVs were comparable to those by the paralog ratio test (PRT, but without PRT's limitations. These results suggest that mrcPCR is comparable to the currently available standard or the most sensitive methods. In addition, mrcPCR would be invaluable for measurement of CNVs in genes with variants of similar structures, because combination of the other methods is not necessary, along with its other advantages such as short assay time, small sample amount requirement, and applicability to all sequences and genes.

Functional cDNA expression cloning: Pushing it to the limit

Science.gov (United States)

OKAYAMA, Hiroto

2012-01-01

The 1970s and the following decade are the era of the birth and early development of recombinant DNA technologies, which have entirely revolutionized the modern life science by providing tools that enable us to know the structures of genes and genomes and to dissect their components and understand their functions at the molecular and submolecular levels. One major objective of the life sciences is to achieve molecular and chemical understandings of the functions of genes and their encoded proteins, which are responsible for the manifestation of all biological phenomena in organisms. In the early 1980s, I developed, together with Paul Berg, a new technique that enables the cloning of full-length complementary DNAs (cDNAs) on the basis of their functional expression in a given cell of interest. I review the development, application and future implications in the life sciences of this gene-cloning technique. PMID:22450538

Molecular cloning, sequence analysis and homology modeling of the first caudata amphibian antifreeze-like protein in axolotl (Ambystoma mexicanum).

Science.gov (United States)

Zhang, Songyan; Gao, Jiuxiang; Lu, Yiling; Cai, Shasha; Qiao, Xue; Wang, Yipeng; Yu, Haining

2013-08-01

Antifreeze proteins (AFPs) refer to a class of polypeptides that are produced by certain vertebrates, plants, fungi, and bacteria and which permit their survival in subzero environments. In this study, we report the molecular cloning, sequence analysis and three-dimensional structure of the axolotl antifreeze-like protein (AFLP) by homology modeling of the first caudate amphibian AFLP. We constructed a full-length spleen cDNA library of axolotl (Ambystoma mexicanum). An EST having highest similarity (∼42%) with freeze-responsive liver protein Li16 from Rana sylvatica was identified, and the full-length cDNA was subsequently obtained by RACE-PCR. The axolotl antifreeze-like protein sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 93 amino acids. The calculated molecular mass and the theoretical isoelectric point (pl) of this mature protein were 10128.6 Da and 8.97, respectively. The molecular characterization of this gene and its deduced protein were further performed by detailed bioinformatics analysis. The three-dimensional structure of current AFLP was predicted by homology modeling, and the conserved residues required for functionality were identified. The homology model constructed could be of use for effective drug design. This is the first report of an antifreeze-like protein identified from a caudate amphibian.

Molecular cloning and characterization of a group 3 LEA gene from ...

African Journals Online (AJOL)

Late embryogenesis-abundant (LEA) protein is one of the components involved in desiccation tolerance (DT) by maintaining cellular structures in the dry state. In this study, a member of the group 3 LEA, MwLEA1, was cloned from Mongolian wheatgrass (Agropyron mongolium Keng) based on a homologous sequence from ...

Molecular cloning and variation of ω-gliadin genes from a somatic ...

Indian Academy of Sciences (India)

repetitive domain, which hampers cloning. Further analysis of ω-gliadins at the DNA level would provide more informa- tion to define the evolution and function of this gene family. (Hassania et al. 2008). ∗For correspondence. E-mail: fanguo2002@sdu.edu.cn. Agropyron elongatum (Host) Nevishi (syn. Thinopyrum ponticum ...

Molecular cloning and characterization of an acetylcholinesterase cDNA in the brown planthopper, Nilaparvata lugens.

Science.gov (United States)

Yang, Zhifan; Chen, Jun; Chen, Yongqin; Jiang, Sijing

2010-01-01

A full cDNA encoding an acetylcholinesterase (AChE, EC 3.1.1.7) was cloned and characterized from the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae). The complete cDNA (2467 bp) contains a 1938-bp open reading frame encoding 646 amino acid residues. The amino acid sequence of the AChE deduced from the cDNA consists of 30 residues for a putative signal peptide and 616 residues for the mature protein with a predicted molecular weight of 69,418. The three residues (Ser242, Glu371, and His485) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds are completely conserved, and 10 out of the 14 aromatic residues lining the active site gorge of the AChE are also conserved. Northern blot analysis of poly(A)+ RNA showed an approximately 2.6-kb transcript, and Southern blot analysis revealed there likely was just a single copy of this gene in N. lugens. The deduced protein sequence is most similar to AChE of Nephotettix cincticeps with 83% amino acid identity. Phylogenetic analysis constructed with 45 AChEs from 30 species showed that the deduced N. lugens AChE formed a cluster with the other 8 insect AChE2s. Additionally, the hypervariable region and amino acids specific to insect AChE2 also existed in the AChE of N. lugens. The results revealed that the AChE cDNA cloned in this work belongs to insect AChE2 subgroup, which is orthologous to Drosophila AChE. Comparison of the AChEs between the susceptible and resistant strains revealed a point mutation, Gly185Ser, is likely responsible for the insensitivity of the AChE to methamidopho in the resistant strain.

pClone: Synthetic Biology Tool Makes Promoter Research Accessible to Beginning Biology Students

Science.gov (United States)

Eckdahl, Todd; Cronk, Brian; Andresen, Corinne; Frederick, Paul; Huckuntod, Samantha; Shinneman, Claire; Wacker, Annie; Yuan, Jason

2014-01-01

The Vision and Change report recommended genuine research experiences for undergraduate biology students. Authentic research improves science education, increases the number of scientifically literate citizens, and encourages students to pursue research. Synthetic biology is well suited for undergraduate research and is a growing area of science. We developed a laboratory module called pClone that empowers students to use advances in molecular cloning methods to discover new promoters for use by synthetic biologists. Our educational goals are consistent with Vision and Change and emphasize core concepts and competencies. pClone is a family of three plasmids that students use to clone a new transcriptional promoter or mutate a canonical promoter and measure promoter activity in Escherichia coli. We also developed the Registry of Functional Promoters, an open-access database of student promoter research results. Using pre- and posttests, we measured significant learning gains among students using pClone in introductory biology and genetics classes. Student posttest scores were significantly better than scores of students who did not use pClone. pClone is an easy and affordable mechanism for large-enrollment labs to meet the high standards of Vision and Change. PMID:26086659

Molecular Cloning and Pharmacological Properties of an Acidic PLA2 from Bothrops pauloensis Snake Venom

Science.gov (United States)

Ferreira, Francis Barbosa; Gomes, Mário Sérgio Rocha; Naves de Souza, Dayane Lorena; Gimenes, Sarah Natalie Cirilo; Castanheira, Letícia Eulalio; Borges, Márcia Helena; Rodrigues, Renata Santos; Yoneyama, Kelly Aparecida Geraldo; Homsi Brandeburgo, Maria Inês; Rodrigues, Veridiana M.

2013-01-01

In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A2 (PLA2) isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA2-TXI, was purified by four chromatographic steps and represents 2.4% of the total snake venom protein content. BpPLA2-TXI is a monomeric protein with a molecular mass of 13.6 kDa, as demonstrated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis and its theoretical isoelectric point was 4.98. BpPLA2-TXI was catalytically active and showed some pharmacological effects such as inhibition of platelet aggregation induced by collagen or ADP and also induced edema and myotoxicity. BpPLA2-TXI displayed low cytotoxicity on TG-180 (CCRF S 180 II) and Ovarian Carcinoma (OVCAR-3), whereas no cytotoxicity was found in regard to MEF (Mouse Embryonic Fibroblast) and Sarcoma 180 (TIB-66). The N-terminal sequence of forty-eight amino acid residues was determined by Edman degradation. In addition, the complete primary structure of 122 amino acids was deduced by cDNA from the total RNA of the venom gland using specific primers, and it was significantly similar to other acidic D49 PLA2s. The phylogenetic analyses showed that BpPLA2-TXI forms a group with other acidic D49 PLA2s from the gender Bothrops, which are characterized by a catalytic activity associated with anti-platelet effects. PMID:24304676

Molecular Cloning and Expression of Pro J 1: A New Allergen of Prosopis Juliflora Pollen.

Science.gov (United States)

Dousti, Fatemeh; Assarehzadegan, Mohammad-Ali; Morakabati, Payam; Khosravi, Gholam Reza; Akbari, Bahareh

2016-04-01

Pollen from mesquite (Prosopis juliflora) is one of the important causes of immediate hypersensitivity reactions in the arid and semi-arid regions of the world. The aim of present study is to produce and purify the recombinant form of allergenic Ole e 1-like protein from the pollen of this allergenic tree. Immunological and cross-inhibition assays were performed for the evaluation of IgE-binding capacity of purified recombinant protein. For molecular cloning, the coding sequence of the mesquite Ole e 1-like protein was inserted into pTZ57R/T vector and expressed in Escherichia coli using the vector pET-21b(+). After purification of the recombinant protein, its immunoreactivity was analysed by in vitro assays using sera from twenty one patients with an allergy to mesquite pollen. The purified recombinant allergen was a member of Ole e 1-like protein family and consisted of 150 amino acid residues, with a predicted molecular mass of 16.5 kDa and a calculated isoelectric point (pI) of 4.75. Twelve patients (57.14%) had significant specific IgE levels for this recombinant allergen. Immunodetection and inhibition assays indicated that the purified recombinant allergen might be the same as that in the crude extract. Herein, we introduce an important new allergen from P. juliflora pollen (Pro j 1), which is a member of the Ole e 1-like protein family and exhibits significant identity and similarity to other allergenic members of this family.

Molecular cloning and biological characterization of the human excision repair gene ERCC-3

International Nuclear Information System (INIS)

Weeda, G.; van Ham, R.C.; Masurel, R.; Westerveld, A.; Odijk, H.; de Wit, J.; Bootsma, D.; van der Eb, A.J.; Hoeijmakers, J.H.

1990-01-01

In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion

Multipartite asymmetric quantum cloning

International Nuclear Information System (INIS)

Iblisdir, S.; Gisin, N.; Acin, A.; Cerf, N.J.; Filip, R.; Fiurasek, J.

2005-01-01

We investigate the optimal distribution of quantum information over multipartite systems in asymmetric settings. We introduce cloning transformations that take N identical replicas of a pure state in any dimension as input and yield a collection of clones with nonidentical fidelities. As an example, if the clones are partitioned into a set of M A clones with fidelity F A and another set of M B clones with fidelity F B , the trade-off between these fidelities is analyzed, and particular cases of optimal N→M A +M B cloning machines are exhibited. We also present an optimal 1→1+1+1 cloning machine, which is an example of a tripartite fully asymmetric cloner. Finally, it is shown how these cloning machines can be optically realized

Purification of cold-shock-like proteins from Stigmatella aurantiaca - molecular cloning and characterization of the cspA gene.

Science.gov (United States)

Stamm, I; Leclerque, A; Plaga, W

1999-09-01

Prominent low-molecular-weight proteins were isolated from vegetative cells of the myxobacterium Stigmatella aurantiaca and were found to be members of the cold-shock protein family. A first gene of this family (cspA) was cloned and sequenced. It encodes a protein of 68 amino acid residues that displays up to 71% sequence identity with other bacterial cold-shock(-like) proteins. A cysteine residue within the RNP-2 motif is a peculiarity of Stigmatella CspA. A cspA::(Deltatrp-lacZ) fusion gene construct was introduced into Stigmatella by electroporation, a method that has not been used previously for this strain. Analysis of the resultant transformants revealed that cspA transcription occurs at high levels during vegetative growth at 20 and 32 degrees C, and during fruiting body formation.

Cloning and characterization of the gsk gene encoding guanosine kinase of Escherichia coli

DEFF Research Database (Denmark)

Harlow, Kenneth W.; Nygaard, Per; Hove-Jensen, Bjarne

1995-01-01

The Escherichia coli gsk gene encoding guanosine kinase was cloned from the Kohara gene library by complementation of the E. coli gsk-1 mutant allele. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 433 amino acids with a molecular mass of 48,113 Da. Minicell...

Molecular cloning, characterization, and overexpression of a novel [Fe]-hydrogenase isolated from a high rate of hydrogen producing Enterobacter cloacae IIT-BT 08

International Nuclear Information System (INIS)

Mishra, Jayshree; Khurana, Seema; Kumar, Narendra; Ghosh, Ananta K.; Das, Debabrata

2004-01-01

Degenerate primers were designed from the conserved zone of hydA structural gene encoding for catalytic subunit of [Fe]-hydrogenase of different hydrogen producing bacteria. A 750 bp of PCR product was amplified by using the above-mentioned degenerate primers and genomic DNA of Enterobacter cloacae IIT-BT 08 as template. The amplified PCR product was cloned and sequenced. The sequence showed the presence of an ORF of 450 bp with significant similarity (40%) with C-terminal end of the conserved zone (H-cluster) of [Fe]- hydrogenase. hydA ORF was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in a non-hydrogen producing Escherichia coli BL-21 to produce a GST-fusion protein of a calculated molecular mass of about 42.1 kDa. Recombinant protein was purified and specifically recognized by anti-GST monoclonal antibody through Western blot. Southern hybridization confirmed the presence of this gene in E. cloacae IIT-BT 08 genome. In vitro hydrogenase assay with the overexpressed hydrogenase enzyme showed that it is catalytically active upon anaerobic adaptation. In vivo hydrogenase assay confirmed the presence of H 2 gas in the gas mixture obtained from the batch culture of recombinant E. coli BL-21. A tentative molecular mechanism has been proposed about the transfer of electron from electron donor to H-cluster without the mediation of the F-cluster

Distinct forms of the β subunit of GTP-binding regulatory proteins identified by molecular cloning

International Nuclear Information System (INIS)

Fong, H.K.W.; Amatruda, T.T. III; Birren, B.W.; Simon, M.I.

1987-01-01

Two distinct β subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as β 1 and β 1 subunits. The bovine transducin β subunit (β 1 ) has been cloned previously. The author now isolated and analyzed cDNA clones that encode the β 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue M/sub r/ 37,329 Β 2 protein is 90% identical with β 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine β 2 subunit is 1.7 kilobases in length. It is expressed at lower levels than β 1 subunit mRNA in all tissues examined. The β 1 and β 2 messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that β 1 and β 2 are encoded by separate genes. The amino acid sequences for the bovine and human β 2 subunit are identical, as are the amino acid sequences for the bovine and human β 1 subunit. This evolutionary conservation suggests that the two β subunits have different roles in the signal transduction process

Molecular cloning of L-methylmalonyl-CoA mutase: Gene transfer and analysis of mut cell lines

International Nuclear Information System (INIS)

Ledley, F.D.; Lumetta, M.; Nguyen, P.N.; Kolhouse, J.F.; Allen, R.H.

1988-01-01

L-Methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) is a mitochondrial adenosylcobalamin-requiring enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. This enzyme is deficient in methylmalonic acidemia, an often fatal disorder of organic acid metabolism. Antibody against human placental MCM was used to screen human placenta and liver cDNA expression libraries for MCM cDNA clones. One clone expressed epitopes that could affinity-purify antibodies against MCM. A cDNA corresponding in length to the mRNA was obtained and introduced into COS cells by DNA-mediated gene transfer. Cells transformed with this clone expressed increased levels of MCM enzymatic activity. RNA blot analysis of cells genetically deficient in MCM indicates that several deficient cell lines have a specific decrease in the amount of hybridizable mRNA. These data confirm the authenticity of the MCM cDNA clone, establish the feasibility of constituting MCM activity by gene transfer for biochemical analysis and gene therapy, and provide a preliminary picture of the genotypic spectrum underlying MCM deficiency

Molecular cloning, sequencing and structural studies of granulocyte-macrophage colony-stimulating factor (GM-CSF) from Indian water buffalo (Bubalus bubalis)

KAUST Repository

Sugumar, Thennarasu; Ganesan, Pugalenthi; Harishankar, Murugesan; Dhinakar Raj, Gopal

2013-01-01

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is essential for growth and development of progenitors of granulocytes and monocytes/macrophages. In this study, we report molecular cloning, sequencing and characterization of GM-CSF from Indian water buffalo, Bubalus bubalis. In addition, we performed sequence and structural analysis for buffalo GM-CSF. Buffalo GM-CSF has been compared with 17 mammalian GM-CSFs using multiple sequence alignment and phylogenetic tree. Three-dimensional model for buffalo GM-CSF and human receptor complex was built using homology modelling to study cross-reactivity between two species. Detailed analysis was performed to study GM-CSF interface and various interactions at the interface. © 2013 John Wiley & Sons Ltd.

Molecular cloning, sequencing and structural studies of granulocyte-macrophage colony-stimulating factor (GM-CSF) from Indian water buffalo (Bubalus bubalis)

KAUST Repository

Sugumar, Thennarasu

2013-06-25

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is essential for growth and development of progenitors of granulocytes and monocytes/macrophages. In this study, we report molecular cloning, sequencing and characterization of GM-CSF from Indian water buffalo, Bubalus bubalis. In addition, we performed sequence and structural analysis for buffalo GM-CSF. Buffalo GM-CSF has been compared with 17 mammalian GM-CSFs using multiple sequence alignment and phylogenetic tree. Three-dimensional model for buffalo GM-CSF and human receptor complex was built using homology modelling to study cross-reactivity between two species. Detailed analysis was performed to study GM-CSF interface and various interactions at the interface. © 2013 John Wiley & Sons Ltd.

Identifying more epidemic clones during a hospital outbreak of multidrug-resistant Acinetobacter baumannii.

Directory of Open Access Journals (Sweden)

Matthieu Domenech de Cellès

Full Text Available Infections caused by multidrug-resistant bacteria are a major concern in hospitals. Current infection-control practices legitimately focus on hygiene and appropriate use of antibiotics. However, little is known about the intrinsic abilities of some bacterial strains to cause outbreaks. They can be measured at a population level by the pathogen's transmission rate, i.e. the rate at which the pathogen is transmitted from colonized hosts to susceptible hosts, or its reproduction number, counting the number of secondary cases per infected/colonized host. We collected data covering a 20-month surveillance period for carriage of multidrug-resistant Acinetobacter baumannii (MDRAB in a surgery ward. All isolates were subjected to molecular fingerprinting, and a cluster analysis of profiles was performed to identify clonal groups. We then applied stochastic transmission models to infer transmission rates of MDRAB and each MDRAB clone. Molecular fingerprinting indicated that 3 clonal complexes spread in the ward. A first model, not accounting for different clones, quantified the level of in-ward cross-transmission, with an estimated transmission rate of 0.03/day (95% credible interval [0.012-0.049] and a single-admission reproduction number of 0.61 [0.30-1.02]. The second model, accounting for different clones, suggested an enhanced transmissibility of clone 3 (transmission rate 0.047/day [0.018-0.091], with a single-admission reproduction number of 0.81 [0.30-1.56]. Clones 1 and 2 had comparable transmission rates (respectively, 0.016 [0.001-0.045], 0.014 [0.001-0.045]. The method used is broadly applicable to other nosocomial pathogens, as long as surveillance data and genotyping information are available. Building on these results, more epidemic clones could be identified, and could lead to follow-up studies dissecting the functional basis for variation in transmissibility of MDRAB lineages.

Cloning and characterization of a y-type inactive HMW glutenin ...

African Journals Online (AJOL)

The high molecular weight glutenin subunits (HMW-GS) are key factors of the breadmaking quality of common wheat flour. In the present study, one unexpressed 1By gene from Triticum durum cultivar youmangbingmai was cloned and characterized. The results indicated that the silenced 1By gene in youmangbingmai ...

A novel approach to sequence validating protein expression clones with automated decision making

Directory of Open Access Journals (Sweden)

Mohr Stephanie E

2007-06-01

Full Text Available Abstract Background Whereas the molecular assembly of protein expression clones is readily automated and routinely accomplished in high throughput, sequence verification of these clones is still largely performed manually, an arduous and time consuming process. The ultimate goal of validation is to determine if a given plasmid clone matches its reference sequence sufficiently to be "acceptable" for use in protein expression experiments. Given the accelerating increase in availability of tens of thousands of unverified clones, there is a strong demand for rapid, efficient and accurate software that automates clone validation. Results We have developed an Automated Clone Evaluation (ACE system – the first comprehensive, multi-platform, web-based plasmid sequence verification software package. ACE automates the clone verification process by defining each clone sequence as a list of multidimensional discrepancy objects, each describing a difference between the clone and its expected sequence including the resulting polypeptide consequences. To evaluate clones automatically, this list can be compared against user acceptance criteria that specify the allowable number of discrepancies of each type. This strategy allows users to re-evaluate the same set of clones against different acceptance criteria as needed for use in other experiments. ACE manages the entire sequence validation process including contig management, identifying and annotating discrepancies, determining if discrepancies correspond to polymorphisms and clone finishing. Designed to manage thousands of clones simultaneously, ACE maintains a relational database to store information about clones at various completion stages, project processing parameters and acceptance criteria. In a direct comparison, the automated analysis by ACE took less time and was more accurate than a manual analysis of a 93 gene clone set. Conclusion ACE was designed to facilitate high throughput clone sequence

Cytokinin oxidase/dehydrogenase genes in barley and wheat. Cloning and heterologous expression

Czech Academy of Sciences Publication Activity Database

Galuszka, P.; Frébortová, Jitka; Werner, T.; Yamada, M.; Strnad, Miroslav; Schmülling, T.; Frébort, I.

2004-01-01

Roč. 271, č. 20 (2004), s. 3990-4002 ISSN 0014-2956 Institutional research plan: CEZ:AV0Z5038910 Keywords : cereals * cloning * cytokinin oxidase/dehydrogenase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.260, year: 2004

Molecular Characterization of Kastamonu Garlic: An Economically Important Garlic Clone in Turkey

Science.gov (United States)

This study was conducted to assess genetic relationship of Kastamonu garlic, which is very popular in Turkey due to its high quality features, along with some previously characterized garlic clones collected from different regions of the world using AFLP and locus specific DNA markers. UPGMA cluste...

Molecular Cloning and Pharmacological Properties of an Acidic PLA2 from Bothrops pauloensis Snake Venom

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Francis Barbosa Ferreira

2013-12-01

Full Text Available In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A2 (PLA2 isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA2-TXI, was purified by four chromatographic steps and represents 2.4% of the total snake venom protein content. BpPLA2-TXI is a monomeric protein with a molecular mass of 13.6 kDa, as demonstrated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF analysis and its theoretical isoelectric point was 4.98. BpPLA2-TXI was catalytically active and showed some pharmacological effects such as inhibition of platelet aggregation induced by collagen or ADP and also induced edema and myotoxicity. BpPLA2-TXI displayed low cytotoxicity on TG-180 (CCRF S 180 II and Ovarian Carcinoma (OVCAR-3, whereas no cytotoxicity was found in regard to MEF (Mouse Embryonic Fibroblast and Sarcoma 180 (TIB-66. The N-terminal sequence of forty-eight amino acid residues was determined by Edman degradation. In addition, the complete primary structure of 122 amino acids was deduced by cDNA from the total RNA of the venom gland using specific primers, and it was significantly similar to other acidic D49 PLA2s. The phylogenetic analyses showed that BpPLA2-TXI forms a group with other acidic D49 PLA2s from the gender Bothrops, which are characterized by a catalytic activity associated with anti-platelet effects.

Creating libraries for commercial yeast strains through miniaturization of cloning and transformations using the BioRAPTR FRD Microfluidic workstation

Science.gov (United States)

The ability to miniaturize molecular reactions can lead to significant cost savings when creating libraries of thousands of clones. For this application Beckman Coulter partnered with the USDA to provide a low-volume automated solution for library cloning for use in the development of yeast strains...

Human therapeutic cloning (NTSC): applying research from mammalian reproductive cloning.

Science.gov (United States)

French, Andrew J; Wood, Samuel H; Trounson, Alan O

2006-01-01

Human therapeutic cloning or nuclear transfer stem cells (NTSC) to produce patient-specific stem cells, holds considerable promise in the field of regenerative medicine. The recent withdrawal of the only scientific publications claiming the successful generation of NTSC lines afford an opportunity to review the available research in mammalian reproductive somatic cell nuclear transfer (SCNT) with the goal of progressing human NTSC. The process of SCNT is prone to epigenetic abnormalities that contribute to very low success rates. Although there are high mortality rates in some species of cloned animals, most surviving clones have been shown to have normal phenotypic and physiological characteristics and to produce healthy offspring. This technology has been applied to an increasing number of mammals for utility in research, agriculture, conservation, and biomedicine. In contrast, attempts at SCNT to produce human embryonic stem cells (hESCs) have been disappointing. Only one group has published reliable evidence of success in deriving a cloned human blastocyst, using an undifferentiated hESC donor cell, and it failed to develop into a hESC line. When optimal conditions are present, it appears that in vitro development of cloned and parthenogenetic embryos, both of which may be utilized to produce hESCs, may be similar to in vitro fertilized embryos. The derivation of ESC lines from cloned embryos is substantially more efficient than the production of viable offspring. This review summarizes developments in mammalian reproductive cloning, cell-to-cell fusion alternatives, and strategies for oocyte procurement that may provide important clues facilitating progress in human therapeutic cloning leading to the successful application of cell-based therapies utilizing autologous hESC lines.

Geotemporal analysis of Neisseria meningitidis clones in the United States: 2000-2005.

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Ann E Wiringa

Full Text Available The detection of meningococcal outbreaks relies on serogrouping and epidemiologic definitions. Advances in molecular epidemiology have improved the ability to distinguish unique Neisseria meningitidis strains, enabling the classification of isolates into clones. Around 98% of meningococcal cases in the United States are believed to be sporadic.Meningococcal isolates from 9 Active Bacterial Core surveillance sites throughout the United States from 2000 through 2005 were classified according to serogroup, multilocus sequence typing, and outer membrane protein (porA, porB, and fetA genotyping. Clones were defined as isolates that were indistinguishable according to this characterization. Case data were aggregated to the census tract level and all non-singleton clones were assessed for non-random spatial and temporal clustering using retrospective space-time analyses with a discrete Poisson probability model.Among 1,062 geocoded cases with available isolates, 438 unique clones were identified, 78 of which had ≥2 isolates. 702 cases were attributable to non-singleton clones, accounting for 66.0% of all geocoded cases. 32 statistically significant clusters comprised of 107 cases (10.1% of all geocoded cases were identified. Clusters had the following attributes: included 2 to 11 cases; 1 day to 33 months duration; radius of 0 to 61.7 km; and attack rate of 0.7 to 57.8 cases per 100,000 population. Serogroups represented among the clusters were: B (n = 12 clusters, 45 cases, C (n = 11 clusters, 27 cases, and Y (n = 9 clusters, 35 cases; 20 clusters (62.5% were caused by serogroups represented in meningococcal vaccines that are commercially available in the United States.Around 10% of meningococcal disease cases in the U.S. could be assigned to a geotemporal cluster. Molecular characterization of isolates, combined with geotemporal analysis, is a useful tool for understanding the spread of virulent meningococcal clones and patterns of

Molecular cloning, expression, and in silico structural analysis of guinea pig IL-17.

Science.gov (United States)

Dirisala, Vijaya R; Jeevan, Amminikutty; Ramasamy, Suresh K; McMurray, David N

2013-11-01

Interleukin-17A (IL-17A) is a potent proinflammatory cytokine and the signature cytokine of Th17 cells, a subset which is involved in cytokine and chemokine production, neutrophil recruitment, promotion of T cell priming, and antibody production. IL-17 may play an important role in tuberculosis and other infectious diseases. In preparation for investigating its role in the highly relevant guinea pig model of pulmonary tuberculosis, we cloned guinea pig IL-17A for the first time. The complete coding sequence of the guinea pig IL-17A gene (477 nucleotides; 159 amino acids) was subcloned into a prokaryotic expression vector (pET-30a) resulting in the expression of a 17 kDa recombinant guinea pig IL-17A protein which was confirmed by mass spectrometry analysis. Homology modeling of guinea pig IL-17A revealed that the three-dimensional structure resembles that of human IL-17A. The secondary structure predicted for this protein showed the presence of one extra helix in the N-terminal region. The expression profile of IL-17A was analyzed quantitatively in spleen, lymph node, and lung cells from BCG-vaccinated guinea pigs by real-time PCR. The guinea pig IL-17A cDNA and its recombinant protein will serve as valuable tools for molecular and immunological studies in the guinea pig model of pulmonary TB and other human diseases.

Molecular cloning and expression of the IL-10 gene from guinea pigs.

Science.gov (United States)

Dirisala, Vijaya R; Jeevan, Amminikutty; Bix, Gregory; Yoshimura, Teizo; McMurray, David N

2012-04-25

The Guinea pig (Cavia porcellus) is one of the most relevant small animals for modeling human tuberculosis (TB) in terms of susceptibility to low dose aerosol infection, the organization of granulomas, extrapulmonary dissemination and vaccine-induced protection. It is also considered to be a gold standard for a number of other infectious and non-infectious diseases; however, this animal model has a major disadvantage due to the lack of readily available immunological reagents. In the present study, we successfully cloned a cDNA for the critical Th2 cytokine, interleukin-10 (IL-10), from inbred Strain 2 guinea pigs using the DNA sequence information provided by the genome project. The complete open reading frame (ORF) consists of 537 base pairs which encodes a protein of 179 amino acids. This cDNA sequence exhibited 87% homology with human IL-10. Surprisingly, it showed only 84% homology with the previously published IL-10 sequence from the C4-deficient (C4D) guinea pig, leading us to clone IL-10 cDNA from the Hartley strain of guinea pig. The IL-10 gene from the Hartley strain showed 100% homology with the IL-10 sequence of Strain 2 guinea pigs. In order to validate the only published IL-10 sequence existing in Genbank reported from C4D guinea pigs, genomic DNA was isolated from tissues of C4D guinea pigs. Amplification with various sets of primers showed that the IL-10 sequence reported from C4D guinea pigs contained numerous errors. Hence the IL-10 sequence that is being reported by us replaces the earlier sequence making our IL-10 sequence to be the first one accurate from guinea pig. Recombinant guinea pig IL-10 proteins were subsequently expressed in both prokaryotic and eukaryotic cells, purified and were confirmed by N-terminal sequencing. Polyclonal anti-IL-10 antibodies were generated in rabbits using the recombinant IL-10 protein expressed in this study. Taken together, our results indicate that the DNA sequence information provided by the genome project

[Research progress on the cloning of Mendel's gene in pea (Pisum sativum L.) and its application in genetics teaching].

Science.gov (United States)

He, Feng-Hua; Zhu, Bi-Yan; Gao, Feng; Li, Shao-Shan; Li, Niang-Hui

2013-07-01

One hundred and fifty years ago, Gregor Mendel investigated the segregation of seven traits in pea (Pisum sativum) and established the law of segregation and the law of independent assortment in genetics. After the two laws of genetics were rediscovered in 1900, the seven traits have been extensively investigated in the fields of plant physiology and biochemistry as well as in the cell and molecular levels. Recently, with the development of molecular technology in genetics, four genes for seed shape (R), stem length (Le), cotyledon colour (I), and flower colour (A) have been cloned and sequenced; and another three genes for immature pod colour (Gp), fasciation (Fa) and pod form (V) have been located in the linkage groups, respectively. The identification and cloning of the four Mendel's genes will help deeply understand the basic concept of gene in many respects: like the diversity of gene function, the different origins for gene mutation in molecular level, and the molecular nature of a dominant gene or a recessive gene. In teaching of genetics, the introduction of most recent research advancements of cloning of Mendel's genes to the students and the interpretation of the Mendel's laws in molecular level will help students promote their learning interests in genetics and help students grasp the whole content from classical genetics to molecular genetics and the developmental direction of this subject.

Coding sequence of human rho cDNAs clone 6 and clone 9

Energy Technology Data Exchange (ETDEWEB)

Chardin, P; Madaule, P; Tavitian, A

1988-03-25

The authors have isolated human cDNAs including the complete coding sequence for two rho proteins corresponding to the incomplete isolates previously described as clone 6 and clone 9. The deduced a.a. sequences, when compared to the a.a. sequence deduced from clone 12 cDNA, show that there are in human at least three highly homologous rho genes. They suggest that clone 12 be named rhoA, clone 6 : rhoB and clone 9 : rhoC. RhoA, B and C proteins display approx. 30% a.a. identity with ras proteins,. mainly clustered in four highly homologous internal regions corresponding to the GTP binding site; however at least one significant difference is found; the 3 rho proteins have an Alanine in position corresponding to ras Glycine 13, suggesting that rho and ras proteins might have slightly different biochemical properties.

Ethical issues in animal cloning.

Science.gov (United States)

Fiester, Autumn

2005-01-01

The issue of human reproductive cloning has recently received a great deal attention in public discourse. Bioethicists, policy makers, and the media have been quick to identify the key ethical issues involved in human reproductive cloning and to argue, almost unanimously, for an international ban on such attempts. Meanwhile, scientists have proceeded with extensive research agendas in the cloning of animals. Despite this research, there has been little public discussion of the ethical issues raised by animal cloning projects. Polling data show that the public is decidedly against the cloning of animals. To understand the public's reaction and fill the void of reasoned debate about the issue, we need to review the possible objections to animal cloning and assess the merits of the anti-animal cloning stance. Some objections to animal cloning (e.g., the impact of cloning on the population of unwanted animals) can be easily addressed, while others (e.g., the health of cloned animals) require more serious attention by the public and policy makers.

Optimally cloned binary coherent states

DEFF Research Database (Denmark)

Mueller, C. R.; Leuchs, G.; Marquardt, Ch

2017-01-01

their quantum-optimal clones. We analyze the Wigner function and the cumulants of the clones, and we conclude that optimal cloning of binary coherent states requires a nonlinearity above second order. We propose several practical and near-optimal cloning schemes and compare their cloning fidelity to the optimal...

Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

Science.gov (United States)

Džunková, Mária; D'Auria, Giuseppe; Pérez-Villarroya, David; Moya, Andrés

2012-01-01

Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb) cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs) with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.

Novel glucose dehydrogenase from Mucor prainii: Purification, characterization, molecular cloning and gene expression in Aspergillus sojae.

Science.gov (United States)

Satake, Ryoko; Ichiyanagi, Atsushi; Ichikawa, Keiichi; Hirokawa, Kozo; Araki, Yasuko; Yoshimura, Taro; Gomi, Keiko

2015-11-01

Glucose dehydrogenase (GDH) is of interest for its potential applications in the field of glucose sensors. To improve the performance of glucose sensors, GDH is required to have strict substrate specificity. A novel flavin adenine dinucleotide (FAD)-dependent GDH was isolated from Mucor prainii NISL0103 and its enzymatic properties were characterized. This FAD-dependent GDH (MpGDH) exhibited high specificity toward glucose. High specificity for glucose was also observed even in the presence of saccharides such as maltose, galactose and xylose. The molecular masses of the glycoforms of GDH ranged from 90 to 130 kDa. After deglycosylation, a single 80 kDa band was observed. The gene encoding MpGDH was cloned and expressed in Aspergillus sojae. The apparent kcat and Km values of recombinant enzyme for glucose were found to be 749.7 s(-1) and 28.3 mM, respectively. The results indicated that the characteristics of MpGDH were suitable for assaying blood glucose levels. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Molecular cloning and expression analysis of turnip (Brassica rapa var. rapa sucrose transporter gene family

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Yuanyuan Liu

2017-06-01

Full Text Available In higher plants, sugars (mainly sucrose are produced by photosynthetically assimilated carbon in mesophyll cells of leaves and translocated to heterotrophic organs to ensure plant growth and development. Sucrose transporters, or sucrose carriers (SUCs, play an important role in the long-distance transportation of sucrose from source organs to sink organs, thereby affecting crop yield and quality. The identification, characterization, and molecular function analysis of sucrose transporter genes have been reported for monocot and dicot plants. However, no relevant study has been reported on sucrose transporter genes in Brassica rapa var. rapa, a cruciferous root crop used mainly as vegetables and fodder. We identified and cloned 12 sucrose transporter genes from turnips, named BrrSUC1.1 to BrrSUC6.2 according to the SUC gene sequences of B. rapa pekinensis. We constructed a phylogenetic tree and analyzed conserved motifs for all 12 sucrose transporter genes identified. Real-time quantitative polymerase chain reaction was conducted to understand the expression levels of SUC genes in different tissues and developmental phases of the turnip. These findings add to our understanding of the genetics and physiology of sugar transport during taproot formation in turnips.

Molecular cloning and expression of nanos in the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae).

Science.gov (United States)

Ogaugwu, Christian E; Wimmer, Ernst A

2013-01-01

The gene nanos (nos) is a maternal-effect gene that plays an important role in posterior patterning and germ cell development in early stage embryos. nos is known from several diverse insect species, but has so far not been described for any Tephritid fruit fly. Here, we report the molecular cloning and expression pattern of the nos orthologous gene, Ccnos, in the Mediterranean fruit fly Ceratitis capitata, which is a destructive pest of high agricultural importance. CcNOS contains 398 amino acids and has a C-terminal region with two conserved CCHC zinc-binding motifs known to be essential for NOS function. Transcripts of Ccnos were confirmed by in situ hybridization to be maternally-derived and localized to the posterior pole of early stage embryos. Regulatory regions of nos have been employed in genetic engineering in some dipterans such as Drosophila and mosquitoes. Given the similarity in spatial and temporal expression between Ccnos and nos orthologs from other dipterans, its regulatory regions will be valuable to generate additional genetic tools that can be applied for engineering purposes to improve the fight against this devastating pest. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular cloning and expression of bovine kappa-casein in Escherichia coli

International Nuclear Information System (INIS)

Kang, Y.C.; Richardson, T.

1988-01-01

A cDNA library was constructed using poly(A) + RNA from bovine mammary gland. This cDNA library of 6000 clones was screened employing colony hybridization using 32 P-labelled oligonucleotide probes and restriction endonuclease mapping. The cDNA from the selected plasmid, pKR76, was sequenced using the dideoxy-chain termination method. The cDNA insert of pKR76 carries the full-length sequence, which codes for mature kappa-casein protein. The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence with three exceptions; the reported pyroglutamic acid at position 1, tyrosine at position 35, and aspartic acid at position 81 are, respectively, a glutamine, a histidine, and an asparagine in the clone containing pKR76. The MspI-, NlaIV-cleaved fragment (630 base pair) from the kappa-casein cDNA insert has been subcloned into expression vectors pUC18 and pKK233-2, which contain a lac promoter and a trc promoter, respectively. Escherichia coli cells carrying the recombinant expression plasmids were shown to produce kappa-casein protein having the expected mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and being recognized by specific antibodies raised against natural bovine kappa-casein

Geology, physical properties, and surface effects at Discus Thrower Site, Yucca Flat, Nevada Test Site

International Nuclear Information System (INIS)

Carr, W.J.; Miller, C.H.; Dodge, H.W. Jr.

1975-01-01

Geologic studies in connection with Project Discus Thrower have furnished detailed stratigraphic and structural information about northwestern Yucca Flat, Nevada Test Site. The Paleozoic rocks consist of a lower carbonate sequence, argillite of the Eleana Formation, and an upper carbonate sequence. The distribution of these rocks suggests that both top and bottom of the Eleana are structural contacts, probably thrusts or reverse faults. The overlying tuff includes several units recognized in the subsurface, such as the Fraction Tuff and tuff of Redrock Valley. Other units recognized include bedded tuff associated with the Grouse Canyon Member of Belted Range Tuff, and the Rainier Mesa and Ammonia Tanks Members of the Timber Mountain Tuff. The Timber Mountain and Grouse Canyon are extensively altered to montmorillonite (a swelling clay), possibly as a result of ponding of alkaline water. The overlying alluvium locally contains at the base a clayey, tuffaceous sandstone

Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

Directory of Open Access Journals (Sweden)

Mária Džunková

Full Text Available Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.

The topsy-turvy cloning law.

Science.gov (United States)

Brassington, Iain; Oultram, Stuart

2011-03-01

In debates about human cloning, a distinction is frequently drawn between therapeutic and reproductive uses of the technology. Naturally enough, this distinction influences the way that the law is framed. The general consensus is that therapeutic cloning is less morally problematic than reproductive cloning--one can hold this position while holding that both are morally unacceptable--and the law frequently leaves the way open for some cloning for the sake of research into new therapeutic techniques while banning it for reproductive purposes. We claim that the position adopted by the law has things the wrong way around: if we accept a moral distinction between therapeutic and reproductive cloning, there are actually more reasons to be morally worried about therapeutic cloning than about reproductive cloning. If cloning is the proper object of legal scrutiny, then, we ought to make sure that we are scrutinising the right kind of clone.

Biological Parameters and Molecular Markers of Clone CL Brener - The Reference Organism of the Trypanosoma cruzi Genome Project

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Bianca Zingales

1997-11-01

Full Text Available Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. Some biological parameters of CL Brener were determined: (a the doubling time of epimastigote forms cultured in liver infusion-tryptose (LIT medium at 28oC is 58±13 hr; (b differentiation of epimastigotes to metacyclic trypomastigotes is obtained by incubation in LIT-20% Grace´s medium; (c trypomastigotes infect mammalian cultured cells and perform the complete intracellular cycle at 33 and 37oC; (d blood forms are highly infective to mice; (e blood forms are susceptible to nifurtimox and benznidazole. The molecular typing of CL Brener has been determined: (a isoenzymatic profiles are characteristic of zymodeme ZB; (b PCR amplification of a 24Sa ribosomal RNA sequence indicates it belongs to T. cruzi lineage 1; (c schizodeme, randomly amplified polymorphic DNA (RAPD and DNA fingerprinting analyses were performed

Molecular Cloning and Characterization of a Broad Substrate Terpenoid Oxidoreductase from Artemisia annua

NARCIS (Netherlands)

Ryden, Anna-Margareta; Ruyter-Spira, Carolien; Litjens, Ralph; Takahashi, Shunji; Quax, Wim; Osada, Hiroyuki; Bouwmeester, Harro; Kayser, Oliver

From Artemisia annua L., a new oxidoreductase (Red 1) was cloned, sequenced and functionally characterized. Through bioinformatics, heterologous protein expression and enzyme substrate conversion assays, the elucidation of the enzymatic capacities of Red1 was achieved. Red1 acts on monoterpenoids,

Molecular cloning and characterization of a broad substrate terpenoid oxidoreductase from Artemisia annua.

NARCIS (Netherlands)

Ryden, A.M.; Ruyter-Spira, C.P.; Litjens, R.; Takahashi, S.; Quax, W.J.; Osada, H.; Bouwmeester, H.J.; Kayser, O.

2010-01-01

From Artemisia annua L., a new oxidoreductase (Red 1) was cloned, sequenced and functionally characterized. Through bioinformatics, heterologous protein expression, and enzyme substrate conversion assays, the elucidation of the enzymatic capacities of Red1 was achieved. Red1 acts on monoterpenoids,

Academic Cloning.

Science.gov (United States)

Sikula, John P.; Sikula, Andrew F.

1980-01-01

The authors define "cloning" as an integral feature of all educational systems, citing teaching practices which reward students for closely reproducing the teacher's thoughts and/or behaviors and administrative systems which tend to promote like-minded subordinates. They insist, however, that "academic cloning" is not a totally…

Molecular Cloning, Characterization, and Expression of Cuc m 2, a Major Allergen in Cucumis melo

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Mojtaba Sankian

2013-05-01

Full Text Available Background: Several studies reported the clinical features of IgE-mediated hypersensitivity after ingestion of melon. Melon allergy is a common IgE-mediated fruit allergy in Iran. This prompted us to investigate immunochemical and molecular properties of the major allergen in melon fruit, to compare the IgE-binding capacity of the natural protein with the recombinant allergen, and to determine cross-reactivity of the major allergen with closely-related allergens from other plants displaying clinical cross-reactivity with melon. Methods: Identification and molecular characterization of the major melon allergen were performed using IgE immunoblotting, allergen-specific ELISA, affinity-based purifications, cross-inhibition assays, cloning, and expression of the allergen in Escherichia coli. Results: Melon profilin was identified and isolated as a major IgE-binding component and designated as Cuc m 2. Sequencing corresponding cDNA revealed an open reading frame of 363 bp coding for 131 amino acid residues and two fragments of 171 bp and 383 bps for the 5’and 3’ UTRs, respectively. Significant cross-reactivity was found between melon profilin and Cynodon dactylon, tomato, peach, and grape profilins in cross-inhibition assays. Although the highest degree of amino acid identity was revealed with watermelon profilin, there was no significant cross-reactivity between melon and watermelon profilins. Conclusion: Melon profilin is the major IgE-binding component in melon extract, and the recombinant and natural forms exhibited similar IgE-binding capacities. A part of the fruit-fruit and pollen-fruit cross-reactions could be explained by the presence of this conserved protein; however, sequence homology provides insufficient information to predict IgE cross-reactivity of profilins.

Human terminal deoxyribonucleotidyltransferase: molecular cloning and structural analysis of the gene and 5' flanking region

International Nuclear Information System (INIS)

Riley, L.K.; Morrow, J.K.; Danton, M.J.; Coleman, M.S.

1988-01-01

Human terminal deoxyribonucleotidyltransferase cDNA contains an open reading frame of 1530 base pairs (bp) corresponding to a protein containing 510 amino acids. The encoded protein is a template-independent DNA polymerase found only in a restricted population of normal and malignant prelymphocytes. To begin to investigate the genetic elements responsible for the tissue-specific expression of terminal deoxyribonucleotidyltransferase, genomic clones, containing the entire human gene were isolated and characterized. Initially, cDNA clones were isolated from a library generated from the human lymphoblastoid cell line, MOLT-4R. A cDNA clone containing the entire coding region of the protein was used to isolate a series of overlapping clones from two human genomic libraries. The gene comprises 11 exons and 10 introns and spans 49.4 kilobases. The 5' flanking region (709 bp) including exon 1 was sequenced. Several putative transcription initiation sites were mapped. Within 500 nucleotides of the translation start site, a series of promoter elements was detected. TATA and CAAT sequences, respectively, were found to start at nucleotides -185 and -204, -328 and -370, and -465 and -505. Start sites were found for a cyclic AMP-dependent promoter analog at nucleotide -121, an eight-base sequence corresponding to the IgG promoter enhancer (cd) at nucleotide -455, and an analog of the IgG promoter (pd) at nucleotide -159. These findings suggest that transcripts coding for terminal deoxyribonucleotidyltransferase may be variable in length and that transcription may be influenced by a variety of genetic elements

pYEMF, a pUC18-derived XcmI T-vector for efficient cloning of PCR products.

Science.gov (United States)

Gu, Jingsong; Ye, Chunjiang

2011-03-01

A 1330-bp DNA sequence with two XcmI cassettes was inserted into pUC18 to construct an efficient XcmI T-vector parent plasmid, pYEMF. The large size of the inserted DNA fragment improved T-vector cleavage efficiency, and guaranteed good separation of the molecular components after restriction digestion. The pYEMF-T-vector generated from parent plasmid pYEMF permits blue/white colony screening; cloning efficiency analysis showed that most white colonies (>75%) were putative transformants which carried the cloning product. The sequence analysis and design approach presented here will facilitate applications in the fields of molecular biology and genetic engineering.

MOLECULAR GENE CLONING OF NICOTINE-DEHIDROGENASE FROM THE pAO1 MEGAPLASMID OF ARTHROBACTER NICOTINOVORANS

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Andreea Andrei

2013-10-01

Full Text Available 6-hydroxi-L-nicotine (6HNic has an important potential as a drug for neuro-degenerative disorders and a  suitable simple and reliable method for obtaining contaminant-free 6HNic preparations is required. Here, we envision the in-vitro production of 6HNic by using purified nicotine-dehydrogenase (NDH followed by HPLC or capillary electrophoresis techniques and we focus on the isolation and cloning of the three genes coding the NDH enzyme.  A PCR protocol was established for easy amplification and the DNA fragment containing the ndhLSM genes was directionally cloned into the pART2 vector.

A single-copy galK promoter cloning vector suitable for cloning strong promoters

DEFF Research Database (Denmark)

Dandanell, Gert; Court, Donald L.; Hammer, Karin

1986-01-01

We report the construction of lambda galK promoter cloning vectors for cloning and characterization of strong promoters. This phage, which contains a unique HindIII cloning site, was applied to the cloning and analysis of transcription initiations of the regulatory region of the deo-operon of...

Cloning and analysis of the mouse Fanconi anemia group a cDNA and an overlapping penta zinc finger cDNA

NARCIS (Netherlands)

Wong, JCY; Alon, N; Norga, K; Kruyt, FAE; Youssoufian, H; Buchwald, M

2000-01-01

Despite the cloning of four disease-associated genes for Fanconi anemia (FA), the molecular pathogenesis of FA remains largely unknown. To study FA complementation group A using the mouse as a mode I system, we cloned and characterized the mouse homolog of the human FANCA cDNA, The mouse cDNA

Molecular cloning and transcriptional analysis of a NPY receptor-like in common Chinese cuttlefish Sepiella japonica

Science.gov (United States)

Yang, Jingwen; Xu, Yuchao; Xu, Ke; Ping, Hongling; Shi, Huilai; Lü, Zhenming; Wu, Changwen; Wang, Tianming

2017-08-01

Neuropeptide Y (NPY) has a pivotal role in the regulation of many physiological processes. In this study, the gene encoding a NPY receptor-like from the common Chinese cuttlefish Sepiella japonica (SjNPYR-like) was identified and characterized. The full-length SjNPYR-like cDNA was cloned containing a 492-bp of 5' untranslated region (UTR), 1 182 bp open reading frame (ORF) encoding a protein of 393 amino acid residues, and 228 bp of 3' UTR. The putative protein was predicted to have a molecular weight of 45.54 kDa and an isoelectric point (pI) of 8.13. By informatic analyses, SjNPYR-like was identified as belonging to the class A G protein coupled receptor (GPCR) family (the rhodopsin-type). The amino acid sequence contained 12 potential phosphorylation sites and five predicted N-linked glycosylation sites. Multiple sequence alignment and 3D structure modeling were conducted to clarify SjNPYR bioinformatics characteristics. Phylogenetic analysis identifies it as an NPYR with identity of 33% to Lymnaea stagnalis NPFR. Transmembrane properties of SjNPYR-like were demonstrated in vitro using HEK293 cells and the pEGFP-N1 plasmid. Relative quantification of SjNPYR-like mRNA level confirmed a high level expression and broad distribution of SjNPYR - like in various tissues of female S. japonica. In addition, the transcriptional profile of SjNPYR - like in the brain, liver, and ovary during gonadal development was analyzed. The results provide basic understanding on the molecular characteristics of SjNPYR-like and its potentially physical functions.

Molecular cloning and expression of a novel trehalose synthase gene from Enterobacter hormaechei

Directory of Open Access Journals (Sweden)

Yue Ming

2009-06-01

Full Text Available Abstract Background Trehalose synthase (TreS which converts maltose to trehalose is considered to be a potential biocatalyst for trehalose production. This enzymatic process has the advantage of simple reaction and employs an inexpensive substrate. Therefore, new TreS producing bacteria with suitable enzyme properties are expected to be isolated from extreme environment. Results Six TreS producing strains were isolated from a specimen obtained from soil of the Tibetan Plateau using degenerate PCR. A novel treS gene from Enterobacter hormaechei was amplified using thermal asymmetric interlaced PCR. The gene contained a 1626 bp open reading frame encoding 541 amino acids. The gene was expressed in Escherichia coli, and the recombinant TreS was purified and characterized. The purified TreS had a molecular mass of 65 kDa and an activity of 18.5 U/mg. The optimum temperature and pH for the converting reaction were 37°C and 6, respectively. Hg2+, Zn2+, Cu2+and SDS inhibited the enzyme activity at different levels whereas Mn2+ showed an enhancing effect by 10%. Conclusion In this study, several TreS producing strains were screened from a source of soil bacteria. The characterization of the recombinant TreS of Enterobacter hormaechei suggested its potential application. Consequently, a strategy for isolation of TreS producing strains and cloning of novel treS genes from natural sources was demonstrated.

Changes in the gut microbiota of cloned and non-cloned control pigs during development of obesity: gut microbiota during development of obesity in cloned pigs.

Science.gov (United States)

Pedersen, Rebecca; Andersen, Anders Daniel; Mølbak, Lars; Stagsted, Jan; Boye, Mette

2013-02-07

Obesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs. non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non-cloned control pigs (n= 6) was investigated biweekly over a period of 136 days, by terminal restriction fragment length polymorphism (T-RFLP) and quantitative real time PCR (qPCR). A positive correlation was observed between body-weight at endpoint and percent body-fat in cloned (r=0.9, Pmicrobiota between the cloned pigs or between cloned and non-cloned control pigs. Body-weight correlated positively with the relative abundance of Firmicutes in both cloned (r=0.37; Pgut microbiota in neither the obese nor the lean state. Diet-induced obesity was associated with an increase in the relative abundance of Firmicutes over time. Our results suggest that cloned pigs are not a more suitable animal model for gut microbiota-obesity related studies than non-cloned pigs. This study is the first to evaluate if cloned pigs provide a better animal model than conventional pigs in diet-intervention, obesity and gut microbiota research.

Evolution of Soybean mosaic virus-G7 molecularly cloned genome in Rsv1-genotype soybean results in emergence of a mutant capable of evading Rsv1-mediated recognition

International Nuclear Information System (INIS)

Hajimorad, M.R.; Eggenberger, A.L.; Hill, J.H.

2003-01-01

Plant resistance (R) genes direct recognition of pathogens harboring matching avirluent signals leading to activation of defense responses. It has long been hypothesized that under selection pressure the infidelity of RNA virus replication together with large population size and short generation times results in emergence of mutants capable of evading R-mediated recognition. In this study, the Rsv1/Soybean mosaic virus (SMV) pathosystem was used to investigate this hypothesis. In soybean line PI 96983 (Rsv1), the progeny of molecularly cloned SMV strain G7 (pSMV-G7) provokes a lethal systemic hypersensitive response (LSHR) with up regulation of a defense-associated gene transcript (PR-1). Serial passages of a large population of the progeny in PI 96983 resulted in emergence of a mutant population (vSMV-G7d), incapable of provoking either Rsv1-mediated LSHR or PR-1 protein gene transcript up regulation. An infectious clone of the mutant (pSMV-G7d) was synthesized whose sequences were very similar but not identical to the vSMV-G7d population; however, it displayed a similar phenotype. The genome of pSMV-G7d differs from parental pSMV-G7 by 17 substitutions, of which 10 are translationally silent. The seven amino acid substitutions in deduced sequences of pSMV-G7d differ from that of pSMV-G7 by one each in P1 proteinase, helper component-proteinase, and coat protein, respectively, and by four in P3. To the best of our knowledge, this is the first demonstration in which experimental evolution of a molecularly cloned plant RNA virus resulted in emergence of a mutant capable of evading an R-mediated recognition

Quantum cloning machines and the applications

Energy Technology Data Exchange (ETDEWEB)

Fan, Heng, E-mail: hfan@iphy.ac.cn [Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190 (China); Collaborative Innovation Center of Quantum Matter, Beijing 100190 (China); Wang, Yi-Nan; Jing, Li [School of Physics, Peking University, Beijing 100871 (China); Yue, Jie-Dong [Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190 (China); Shi, Han-Duo; Zhang, Yong-Liang; Mu, Liang-Zhu [School of Physics, Peking University, Beijing 100871 (China)

2014-11-20

No-cloning theorem is fundamental for quantum mechanics and for quantum information science that states an unknown quantum state cannot be cloned perfectly. However, we can try to clone a quantum state approximately with the optimal fidelity, or instead, we can try to clone it perfectly with the largest probability. Thus various quantum cloning machines have been designed for different quantum information protocols. Specifically, quantum cloning machines can be designed to analyze the security of quantum key distribution protocols such as BB84 protocol, six-state protocol, B92 protocol and their generalizations. Some well-known quantum cloning machines include universal quantum cloning machine, phase-covariant cloning machine, the asymmetric quantum cloning machine and the probabilistic quantum cloning machine. In the past years, much progress has been made in studying quantum cloning machines and their applications and implementations, both theoretically and experimentally. In this review, we will give a complete description of those important developments about quantum cloning and some related topics. On the other hand, this review is self-consistent, and in particular, we try to present some detailed formulations so that further study can be taken based on those results.

Quantum cloning machines and the applications

International Nuclear Information System (INIS)

Fan, Heng; Wang, Yi-Nan; Jing, Li; Yue, Jie-Dong; Shi, Han-Duo; Zhang, Yong-Liang; Mu, Liang-Zhu

2014-01-01

No-cloning theorem is fundamental for quantum mechanics and for quantum information science that states an unknown quantum state cannot be cloned perfectly. However, we can try to clone a quantum state approximately with the optimal fidelity, or instead, we can try to clone it perfectly with the largest probability. Thus various quantum cloning machines have been designed for different quantum information protocols. Specifically, quantum cloning machines can be designed to analyze the security of quantum key distribution protocols such as BB84 protocol, six-state protocol, B92 protocol and their generalizations. Some well-known quantum cloning machines include universal quantum cloning machine, phase-covariant cloning machine, the asymmetric quantum cloning machine and the probabilistic quantum cloning machine. In the past years, much progress has been made in studying quantum cloning machines and their applications and implementations, both theoretically and experimentally. In this review, we will give a complete description of those important developments about quantum cloning and some related topics. On the other hand, this review is self-consistent, and in particular, we try to present some detailed formulations so that further study can be taken based on those results

Molecular cloning and characterization of arginine kinase gene of Toxocara canis

OpenAIRE

Sahu, Shivani; Samanta, S.; Harish, D. R.; Sudhakar, N. R.; Raina, O. K.; Shantaveer, S. B.; Madhu, D. N.; Kumar, Ashok

2013-01-01

Toxocara canis is an important gastrointestinal nematode of dogs and also a causative agent of visceral larva migrans in humans. Arginine kinase (AK) gene is one of the important biomolecule of phosphagen kinase of T. canis which is emerging as an exciting novel diagnostic target in toxocarosis. The present study was carried out to clone and characterize AK gene of T. canis for future utilization as a diagnostic molecule. Total RNA was extracted from intact adult worms and reverse transcripti...

Introductory group theory and its application to molecular structure

CERN Document Server

Ferraro, John R

1975-01-01

The success of the first edition of this book has encouraged us to revise and update it. In the second edition we have attempted to further clarify por­ tions of the text in reference to point symmetry, keeping certain sections and removing others. The ever-expanding interest in solids necessitates some discussion on space symmetry. In this edition we have expanded the discus­ sion on point symmetry to include space symmetry. The selection rules in­ clude space group selection rules (for k = 0). Numerous examples are pro­ vided to acquaint the reader with the procedure necessary to accomplish this. Recent examples from the literature are given to illustrate the use of group theory in the interpretation of molecular spectra and in the determination of molecular structure. The text is intended for scientists and students with only a limited theoretical background in spectroscopy. For this reason we have presented detailed procedures for carrying out the selection rules and normal coor­ dinate treatment of ...

Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome b5 reductase

International Nuclear Information System (INIS)

Yubisui, T.; Naitoh, Y.; Zenno, S.; Tamura, M.; Takeshita, M.; Sakaki, Y.

1987-01-01

A cDNA coding for human liver NADH-cytochrome b 5 reductase was cloned from a human liver cDNA library constructed in phage λgt11. The library was screened by using an affinity-purified rabbit antibody against NADH-cytochrome b 5 reductase of human erythrocytes. A cDNA about 1.3 kilobase pairs long was isolated. By using the cDNA as a probe, another cDNA (pb 5 R141) of 1817 base pairs was isolated that hybridized with a synthetic oligonucleotide encoding Pro-Asp-Ile-Lys-Tyr-Pro, derived from the amino acid sequence at the amino-terminal region of the enzyme from human erythrocytes. Furthermore, by using the pb 5 R141 as a probe, cDNA clones having more 5' sequence were isolated from a human placenta cDNA library. The amino acid sequences deduced from the nucleotide sequences of these cDNA clones overlapped each other and consisted of a sequence that completely coincides with that of human erythrocytes and a sequence of 19 amino acid residues extended at the amino-terminal side. The latter sequence closely resembles that of the membrane-binding domain of steer liver microsomal enzyme

[Nuclear transfer and therapeutic cloning].

Science.gov (United States)

Xu, Xiao-Ming; Lei, An-Min; Hua, Jin-Lian; Dou, Zhong-Ying

2005-03-01

Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.

Stable MSAP markers for the distinction of Vitis vinifera cv Pinot noir clones.

Science.gov (United States)

Ocaña, Juan; Walter, Bernard; Schellenbaum, Paul

2013-11-01

Grapevine is one of the most economically important fruit crops. Molecular markers have been used to study grapevine diversity. For instance, simple sequence repeats are a powerful tool for identification of grapevine cultivars, while amplified fragment length polymorphisms have shown their usefulness in intra-varietal diversity studies. Other techniques such as sequence-specific amplified polymorphism are based on the presence of mobile elements in the genome, but their detection lies upon their activity. Relevant attention has been drawn toward epigenetic sources of variation. In this study, a set of Vitis vinifera cv Pinot noir clones were analyzed using the methylation-sensitive amplified polymorphism technique with isoschizomers MspI and HpaII. Nine out of fourteen selective primer combinations were informative and generated two types of polymorphic fragments which were categorized as "stable" and "unstable." In total, 23 stable fragments were detected and they discriminated 92.5 % of the studied clones. Detected stable polymorphisms were either common to several clones, restricted to a few clones or unique to a single clone. The identification of these stable epigenetic markers will be useful in clonal diversity studies. We highlight the relevance of stable epigenetic variation in V. vinifera clones and analyze at which level these markers could be applicable for the development of forthright techniques for clonal distinction.

Probabilistic cloning of equidistant states

International Nuclear Information System (INIS)

Jimenez, O.; Roa, Luis; Delgado, A.

2010-01-01

We study the probabilistic cloning of equidistant states. These states are such that the inner product between them is a complex constant or its conjugate. Thereby, it is possible to study their cloning in a simple way. In particular, we are interested in the behavior of the cloning probability as a function of the phase of the overlap among the involved states. We show that for certain families of equidistant states Duan and Guo's cloning machine leads to cloning probabilities lower than the optimal unambiguous discrimination probability of equidistant states. We propose an alternative cloning machine whose cloning probability is higher than or equal to the optimal unambiguous discrimination probability for any family of equidistant states. Both machines achieve the same probability for equidistant states whose inner product is a positive real number.

Quantum cloning and signaling

International Nuclear Information System (INIS)

Simon, C.; Weihs, G.; Zeilinger, A.

1999-01-01

We discuss the close connections between cloning of quantum states and superluminal signaling. We present an optimal universal cloning machine based on stimulated emission recently proposed by the authors. As an instructive example, we show how a scheme for superluminal communication based on this cloning machine fails. (Authors)

Analysis of the ergosterol biosynthesis pathway cloning, molecular characterization and phylogeny of lanosterol 14α-demethylase (ERG11 gene of Moniliophthora perniciosa

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Geruza de Oliveira Ceita

2014-12-01

Full Text Available The phytopathogenic fungus Moniliophthora perniciosa (Stahel Aime & Philips-Mora, causal agent of witches' broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11 that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR. Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.

A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate.

Science.gov (United States)

Santos, Jefferson J S; Cordeiro, Marli T; Bertani, Giovani R; Marques, Ernesto T A; Gil, Laura H V G

2014-12-01

Dengue infections represent one of the most prevalent arthropod-borne diseases worldwide, causing a wide spectrum of clinical outcomes. Engineered infectious clone is an important tool to study Dengue virus (DENV) biology. Functional full-length cDNA clones have been constructed for many positive-strand RNA viruses and have provided valuable tools for studying the molecular mechanisms involved in viral genome replication, virion assembly, virus pathogenesis and vaccine development. We report herein the successful development of an infectious clone from a primary Brazilian isolate of dengue virus 3 (DENV3) of the genotype III. Using a two-plasmid strategy, DENV3 genome was divided in two parts and cloned separately into a yeast-bacteria shuttle vector. All plasmids were assembled in yeast by homologous recombination technique and a full-length template for transcription was obtained by in vitro ligation of the two parts of the genome. Transcript-derived DENV3 is infectious upon transfection into BHK-21 cells and in vitro characterization confirmed its identity. Growth kinetics of transcript-derived DENV3 was indistinguishable from wild type DENV3. This system is a powerful tool that will help shed light on molecular features of DENV biology, as the relationship of specific mutations and DENV pathogenesis.

Human cloning and child welfare.

Science.gov (United States)

Burley, J; Harris, J

1999-01-01

In this paper we discuss an objection to human cloning which appeals to the welfare of the child. This objection varies according to the sort of harm it is expected the clone will suffer. The three formulations of it that we will consider are: 1. Clones will be harmed by the fearful or prejudicial attitudes people may have about or towards them (H1); 2. Clones will be harmed by the demands and expectations of parents or genotype donors (H2); 3. Clones will be harmed by their own awareness of their origins, for example the knowledge that the genetic donor is a stranger (H3). We will show why these three versions of the child welfare objection do not necessarily supply compelling reasons to ban human reproductive cloning. The claim that we will develop and defend in the course of our discussion is that even if it is the case that a cloned child will suffer harms of the type H1-H3, it is none the less permissible to conceive by cloning so long as these cloning-induced welfare deficits are not such as to blight the existence of the resultant child, whoever this may be. PMID:10226914

Interclonal variations in the molecular karyotype of Trypanosoma cruzi: chromosome rearrangements in a single cell-derived clone of the G strain.

Science.gov (United States)

Lima, Fabio Mitsuo; Souza, Renata Torres; Santori, Fábio Rinaldo; Santos, Michele Fernandes; Cortez, Danielle Rodrigues; Barros, Roberto Moraes; Cano, Maria Isabel; Valadares, Helder Magno Silva; Macedo, Andréa Mara; Mortara, Renato Arruda; da Silveira, José Franco

2013-01-01

Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure.

Social behavior and kin discrimination in a mixed group of cloned and non cloned heifers (Bos taurus).

Science.gov (United States)

Coulon, M; Baudoin, C; Abdi, H; Heyman, Y; Deputte, B L

2010-12-01

For more than ten years, reproductive biotechnologies using somatic cell nuclear transfer have made possible the production of cloned animals in various domestic and laboratory species. The influence of the cloning process on offspring characteristics has been studied in various developmental aspects, however, it has not yet been documented in detail for behavioral traits. Behavioral studies of cloned animals have failed to show clear inter-individual differences associated with the cloning process. Preliminary results showed that clones favor each other's company. Preferential social interactions were observed among cloned heifers from the same donor in a mixed herd that also included cloned heifers and control heifers produced by artificial insemination (AI). These results suggest behavioral differences between cloned and non-cloned animals and similarities between clones from the same donor. The aim of the present study was to replicate and to extend these previous results and to study behavioral and cognitive mechanisms of this preferential grouping. We studied a group composed of five cloned heifers derived from the same donor cow, two cloned heifers derived from another donor cow, and AI heifers. Cloned heifers from the same donor were more spatially associated and interacted more between themselves than with heifers derived from another donor or with the AI individuals. This pattern indicates a possible kin discrimination in clones. To study this process, we performed an experiment (using an instrumental conditioning procedure with food reward) of visual discrimination between images of heads of familiar heifers, either related to the subjects or not. The results showed that all subjects (AI and cloned heifers) discriminated between images of familiar cloned heifers produced from the same donor and images of familiar unrelated heifers. Cattle discriminated well between images and used morphological similarities characteristic of cloned related heifers. Our

Molecular identification of disk abalone (Haliotis discus discus) tetraspanin 33 and CD63: Insights into potent players in the disk abalone host defense system.

Science.gov (United States)

Priyathilaka, Thanthrige Thiunuwan; Bathige, S D N K; Herath, H M L P B; Lee, Sukkyoung; Lee, Jehee

2017-10-01

Tetraspanins are a superfamily of transmembrane proteins involved in a diverse range of physiological processes including differentiation, adhesion, signal transduction, cell motility, and immune responses. In the present study, two tetraspanins, CD63 and tetraspanin 33 (TSPAN33) from disk abalone (AbCD63 and AbTSPAN33), were identified and characterized at the molecular level. The coding sequences for AbCD63 and AbTSPAN33 encoded polypeptides of 234 and 290 amino acids (aa) with predicted molecular mass of 25.3 and 32.5 kDa, respectively. The deduced AbCD63 and AbTSPAN33 protein sequences were also predicted to have a typical tetraspanin domain architecture, including four transmembrane domains (TM), short N- and C- terminal regions, a short intracellular loop, as well as a large and small extracellular loop. A characteristic CCG motif and cysteine residues, which are highly conserved across CD63 and TSPAN33 proteins of different species, were present in the large extracellular loop of both abalone tetraspanins. Phylogenetic analysis revealed that the AbCD63 and AbTSPAN33 clustered in the invertebrate subclade of tetraspanins, thus exhibiting a close relationship with tetraspanins of other mollusks. The AbCD63 and AbTSPAN33 mRNA transcripts were detected at early embryonic development stages of disk abalone with significantly higher amounts at the trochophore stage, suggesting the involvement of these proteins in embryonic development. Both AbCD63 and AbTSPAN33 were ubiquitously expressed in all the tissues of unchallenged abalones analyzed, with the highest expression levels found in hemocytes. Moreover, significant induction of AbCD63 and AbTSPAN33 mRNA expression was observed in immunologically important tissues, such as hemocytes and gills, upon stimulation with live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia virus), and two potent immune stimulators [polyinosinic:polycytidylic acid (poly I:C) and

Optimally cloned binary coherent states

Science.gov (United States)

Müller, C. R.; Leuchs, G.; Marquardt, Ch.; Andersen, U. L.

2017-10-01

Binary coherent state alphabets can be represented in a two-dimensional Hilbert space. We capitalize this formal connection between the otherwise distinct domains of qubits and continuous variable states to map binary phase-shift keyed coherent states onto the Bloch sphere and to derive their quantum-optimal clones. We analyze the Wigner function and the cumulants of the clones, and we conclude that optimal cloning of binary coherent states requires a nonlinearity above second order. We propose several practical and near-optimal cloning schemes and compare their cloning fidelity to the optimal cloner.

Three concepts of cloning in human beings.

Science.gov (United States)

Cui, Ke-Hui

2005-07-01

Human cloning, organ cloning and tissue cloning are various types of cloning that occur at different levels with different methodologies. According to three standards of terminology for an embryo (fertilization through germ cells, development in the uterus and having the potential to produce a human life), tissue cloning and type I organ cloning will not produce an embryo. In contrast, human cloning and type II organ cloning will produce an embryo. Thus, only non-germinal tissue cloning and type I organ cloning are beyond the ethical question and will not change human beings as a species. Using cloned tissues to make new tissues or organs is promising for the future of medicine.

Local cloning of CAT states

International Nuclear Information System (INIS)

Rahaman, Ramij

2011-01-01

In this Letter we analyze the (im)possibility of the exact cloning of orthogonal three-qubit CAT states under local operation and classical communication (LOCC) with the help of a restricted entangled state. We also classify the three-qubit CAT states that can (not) be cloned under LOCC restrictions and extend the results to the n-qubit case. -- Highlights: → We analyze the (im)possibility of exact cloning of orthogonal CAT states under LOCC. → We also classify the set of CAT states that can(not) be cloned by LOCC. → No set of orthogonal CAT states can be cloned by LOCC with help of similar CAT state. → Any two orthogonal n-qubit GHZ-states can be cloned by LOCC with help of a GHZ state.

Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

Science.gov (United States)

Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

2013-02-01

Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a

Molecular characterization of a Leishmania donovani cDNA clone with similarity to human 20S proteasome a-type subunit

DEFF Research Database (Denmark)

Christensen, C B; Jørgensen, L; Jensen, A T

2000-01-01

Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L....... donovanii poly(A(+))mRNA indicated the isolation of a full length cDNA clone with a transcript size of 1.9 kb. The expressed recombinant LePa fusion protein induced proliferation of peripheral blood mononuclear cells in one out of seven patients who had suffered from visceral leishmaniasis. Plasma from 16...

Molecular cloning and sequence of the B880 holochrome gene from Rhodospirillum rubrum

International Nuclear Information System (INIS)

Anon.

1986-01-01

Restriction fragments of genomic Rhodospirillum rubrum DNA were selected according to size by electrophoresis followed by hybridization with [ 32 P]mRNA encoding the two B880 holochrome polypeptides. The fragments were cloned into Escherchia coli C600 with plasmid pBR327 as a vector. The clones were selected by colony hybridization with 32 P-holochrome-mRNA and counter selected by hybridization with Rs. rubrum ribosomal RNA, a minor contaminant of the mRNA preparation. Chimeric plasmid pRR22 was shown to contain the B880 genes by hybrid selection of B880 holochrome-mRNA. A restriction map of its 2.2-kilobase insert and the sequence of a 430 base pair fragment thereof is reported. Genes α and β are nearly contiguous, indicating that they are transcribed as a single operon. The predicted amino acid sequences coincide with the sequences of the α and β polypeptides established in other laboratories, except for additional C-terminal tails of 10 and 13 amino acid residues, respectively

Molecular Cloning and Sequence Analysis of a Phenylalanine Ammonia-Lyase Gene from Dendrobium

Science.gov (United States)

Cai, Yongping; Lin, Yi

2013-01-01

In this study, a phenylalanine ammonia-lyase (PAL) gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748) has 2,458 bps and contains a complete open reading frame (ORF) of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum. PMID:23638048

Lessons learned from cloning dogs.

Science.gov (United States)

Kim, M J; Oh, H J; Kim, G A; Park, J E; Park, E J; Jang, G; Ra, J C; Kang, S K; Lee, B C

2012-08-01

The aim of this article is to review dog cloning research and to suggest its applications based on a discussion about the normality of cloned dogs. Somatic cell nuclear transfer was successfully used for production of viable cloned puppies despite limited understanding of in vitro dog embryo production. Cloned dogs have similar growth characteristics to those born from natural fertilization, with no evidence of serious adverse effects. The offspring of cloned dogs also have similar growth performance and health to those of naturally bred puppies. Therefore, cloning in domestic dogs can be applied as an assisted reproductive technique to conserve endangered species, to treat sterile canids or aged dogs, to improve reproductive performance of valuable individuals and to generate disease model animals. © 2012 Blackwell Verlag GmbH.

Consumers' attitudes toward consumption of cloned beef. The impact of exposure to technological information about animal cloning.

Science.gov (United States)

Aizaki, Hideo; Sawada, Manabu; Sato, Kazuo

2011-10-01

Novel food technologies, such as cloning, have been introduced into the meat production sector; however, their use is not widely supported by many consumers. This study was designed to assess whether Japanese consumers' attitudes toward consumption of cloned beef (specifically, beef derived from bovine embryo and somatic cell-cloned cattle) would change after they were provided with technological information on animal cloning through a web-based survey. The results revealed that most respondents did not discriminate between their attitudes toward the consumption of the two types of cloned beef, and that most respondents did not change their attitudes toward cloned beef after receiving the technological information. The respondents' individual characteristics, including their knowledge about the food safety of cloned beef and their basic knowledge about animal cloning, influenced the likelihood of a change in their attitudes after they received the information. In conclusion, some consumers might become less uncomfortable about the consumption of cloned beef by the straightforward provision of technological information about animal cloning; however, most consumers are likely to maintain their attitudes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Cloning and characterization of an epoxide hydrolase-encoding gene from Rhodotorula glutinis

NARCIS (Netherlands)

Visser, H.; Vreugdenhil, S.; Bont, de J.A.M.; Verdoes, J.C.

2000-01-01

We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of

Effects of dietary menadione on the activity of antioxidant enzymes in abalone, Haliotis discus hannai Ino

Science.gov (United States)

Fu, Jinghua; Xu, Wei; Mai, Kangsen; Zhang, Wenbing; Feng, Xiuni; Liufu, Zhiguo

2012-01-01

A 240-day growth experiment in a re-circulating water system was conducted to investigate the effects of dietary menadione on the growth and antioxidant responses of abalone Haliotis discus hannai Ino. Triplicate groups of juvenile abalone (initial weight: 1.19 ± 0.01 g; shell length: 19.23 ± 0.01 mm) were fed to satiation with 3 semi-purified diets containing 0, 10, and 1 000 mg menadione sodium bisulfite (MSB)/kg, respectively. Results show that there were no significant differences in the rate of weight gain or in the daily increment in shell length of abalone among different treatments. Activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) in viscera were significantly decreased with dietary menadione. However, activities of these enzymes except for GPX in muscle were increased. Therefore, antioxidant responses of abalone were increased in muscle and decreased in viscera by dietary menadione.

Acute toxicity of live and decomposing green alga Ulva ( Enteromorpha) prolifera to abalone Haliotis discus hannai

Science.gov (United States)

Wang, Chao; Yu, Rencheng; Zhou, Mingjiang

2011-05-01

From 2007 to 2009, large-scale blooms of green algae (the so-called "green tides") occurred every summer in the Yellow Sea, China. In June 2008, huge amounts of floating green algae accumulated along the coast of Qingdao and led to mass mortality of cultured abalone and sea cucumber. However, the mechanism for the mass mortality of cultured animals remains undetermined. This study examined the toxic effects of Ulva ( Enteromorpha) prolifera, the causative species of green tides in the Yellow Sea during the last three years. The acute toxicity of fresh culture medium and decomposing algal effluent of U. prolifera to the cultured abalone Haliotis discus hannai were tested. It was found that both fresh culture medium and decomposing algal effluent had toxic effects to abalone, and decomposing algal effluent was more toxic than fresh culture medium. The acute toxicity of decomposing algal effluent could be attributed to the ammonia and sulfide presented in the effluent, as well as the hypoxia caused by the decomposition process.

Anti-allergic effects of a nonameric peptide isolated from the intestine gastrointestinal digests of abalone (Haliotis discus hannai) in activated HMC-1 human mast cells.

Science.gov (United States)

Ko, Seok-Chun; Lee, Dae-Sung; Park, Won Sun; Yoo, Jong Su; Yim, Mi-Jin; Qian, Zhong-Ji; Lee, Chang-Min; Oh, Junghwan; Jung, Won-Kyo; Choi, Il-Whan

2016-01-01

The aim of the present study was to examine whether the intestine gastrointestinal (GI) digests of abalone [Haliotis discus hannai (H. discus hannai)] modulate inflammatory responses and to elucidate the mechanisms involved. The GI digests of the abalone intestines were fractionated into fractions I (>10 kDa), II (5-10 kDa) and Ⅲ (abalone intestine GI digests (AIGIDs), fraction Ⅲ inhibited the passive cutaneous anaphylaxis (PCA) reaction in mice. Subsequently, a bioactive peptide [abalone intestine GI digest peptide (AIGIDP)] isolated from fraction Ⅲ was determined to be 1175.2 Da, and the amino acid sequence was found to be PFNQGTFAS. We noted that the purified nonameric peptide (AIGIDP) attenuated the phorbol‑12‑myristate 13-acetate plus calcium ionophore A23187 (PMACI)-induced histamine release and the production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in human mast cells (HMC-1 cells). In addition, we also noted that AIGIDP inhibited the PMACI‑induced activation of nuclear factor‑κB (NF-κB) by suppressing IκBα phosphorylation and that it suppressed the production of cytokines by decreasing the phosphorylation of JNK. The findings of our study indicate that AIGIDP exerts a modulatory, anti-allergic effect on mast cell-mediated inflammatory diseases.

Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption

Science.gov (United States)

2013-01-01

Background The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker. Results We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered. Conclusions Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain. PMID:23497512

Molecular Cloning and Sequencing of Hemoglobin-Beta Gene of Channel Catfish, Ictalurus Punctatus Rafinesque

Science.gov (United States)

: Hemoglobin-y gene of channel catfish , lctalurus punctatus, was cloned and sequenced . Total RNA from head kidneys was isolated, reverse transcribed and amplified . The sequence of the channel catfish hemoglobin-y gene consists of 600 nucleotides . Analysis of the nucleotide sequence reveals one o...

Molecular biology of human muscle disease

Energy Technology Data Exchange (ETDEWEB)

Dunne, P.W.; Epstein, H.F. (Baylor Coll. of Medicine, Houston, TX (United States))

1991-01-01

The molecular revolution that is transforming the entire biomedical field has had far-reaching impact in its application to inherited human muscle disease. The gene for Duchenne muscular dystrophy was one of the first cloned without knowledge of the defective protein product. This success was based upon the availability of key chromosomal aberrations that provided molecular landmarks for the disease locus. Subsequent discoveries regarding the mode of expression for this gene, the structure and localization of its protein product dystrophin, and molecular diagnosis of affected and carrier individuals constitute a paradigm for investigation of human genetics. Finding the gene for myotonic muscular dystrophy is requiring the brute force approach of cloning several million bases of DNA, identifying expressed sequences, and characterizing candidate genes. The gene that causes hypertrophic cardiomyopathy has been found serendipitously to be one of the genetic markers on chromosome 14, the {beta} myosin heavy chain.

Molecular Cloning, Expression Analysis, and Functional Characterization of the H(+)-Pyrophosphatase from Jatropha curcas.

Science.gov (United States)

Yang, Yumei; Luo, Zhu; Zhang, Mengru; Liu, Chang; Gong, Ming; Zou, Zhurong

2016-04-01

H(+)-pyrophosphatase (H(+)-PPase) is a primary pyrophosphate (PPi)-energized proton pump to generate electrochemical H(+) gradient for ATP production and substance translocations across membranes. It plays an important role in stress adaptation that was intensively substantiated by numerous transgenic plants overexpressing H(+)-PPases yet devoid of any correlated studies pointing to the elite energy plant, Jatropha curcas. Herein, we cloned the full length of J. curcas H(+)-PPase (JcVP1) complementary DNA (cDNA) by reverse transcription PCR, based on the assembled sequence of its ESTs highly matched to Hevea brasiliensis H(+)-PPase. This gene encodes a polypeptide of 765 amino acids that was predicted as a K(+)-dependent H(+)-PPase evolutionarily closest to those of other Euphorbiaceae plants. Many cis-regulatory elements relevant to environmental stresses, molecular signals, or tissue-specificity were identified by promoter prediction within the 1.5-kb region upstream of JcVP1 coding sequence. Meanwhile, the responses of JcVP1 expression to several common abiotic stresses (salt, drought, heat, cold) were characterized with a considerable accordance with the inherent stress tolerance of J. curcas. Moreover, we found that the heterologous expression of JcVP1 could significantly improve the salt tolerance in both recombinant Escherichia coli and Saccharomyces cerevisiae, and this effect could be further fortified in yeast by N-terminal addition of a vacuole-targeting signal peptide from the H(+)-PPase of Trypanosoma cruzi.

Molecular cloning of human protein 4.2: A major component of the erythrocyte membrane

International Nuclear Information System (INIS)

Sung, L.A.; Chien, Shu; Lambert, K.; Chang, Longsheng; Bliss, S.A.; Bouhassira, E.E.; Nagel, R.L.; Schwartz, R.S.; Rybicki, A.C.

1990-01-01

Protein 4.2 (P4.2) comprises ∼5% of the protein mass of human erythrocyte (RBC) membranes. Anemia occurs in patients with RBCs deficient in P4.2, suggesting a role for this protein in maintaining RBC stability and integrity. The authors now report the molecular cloning and characterization of human RBC P4.2 cDNAs. By immunoscreening a human reticulocyte cDNA library and by using the polymerase chain reaction, two cDNA sequences of 2.4 and 2.5 kilobases (kb) were obtained. These cDNAs differ only by a 90-base-air insert in the longer isoform located three codons downstream from the putative initiation site. The 2.4- and 2.5-kb cDNAs predict proteins of ∼77 and ∼80 kDa, respectively, and the authenticity was confirmed by sequence identity with 46 amino acids of three cyanogen bromide-cleaved peptides of P4.2. Northern blot analysis detected a major 2.4-kb RNA species in reticulocytes. Isolation of two P4.2 cDNAs implies existence of specific regulation of P4.2 expression in human RBCs. Human RBC P4.2 has significant homology with human factor XIII subunit a and guinea pig liver transglutaminase. Sequence alignment of P4.2 with these two transglutaminases, however, revealed that P4.2 lacks the critical cysteine residue required for the enzymatic crosslinking of substrates

Cloning, killing, and identity.

Science.gov (United States)

McMahan, J

1999-01-01

One potentially valuable use of cloning is to provide a source of tissues or organs for transplantation. The most important objection to this use of cloning is that a human clone would be the sort of entity that it would be seriously wrong to kill. I argue that entities of the sort that you and I essentially are do not begin to exist until around the seventh month of fetal gestation. Therefore to kill a clone prior to that would not be to kill someone like you or me but would be only to prevent one of us from existing. And even after one of us begins to exist, the objections to killing it remain comparatively weak until its psychological capacities reach a certain level of maturation. These claims support the permissibility of killing a clone during the early stages of its development in order to use its organs for transplantation. PMID:10226909

Cloning-free CRISPR

NARCIS (Netherlands)

Arbab, Mandana; Srinivasan, Sharanya; Hashimoto, Tatsunori; Geijsen, Niels; Sherwood, Richard I.

2015-01-01

We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each

Molecular Cloning and Characterization of Four Genes Encoding Ethylene Receptors Associated with Pineapple (Ananas comosus L.) Flowering.

Science.gov (United States)

Li, Yun-He; Wu, Qing-Song; Huang, Xia; Liu, Sheng-Hui; Zhang, Hong-Na; Zhang, Zhi; Sun, Guang-Ming

2016-01-01

Exogenous ethylene, or ethephon, has been widely used to induce pineapple flowering, but the molecular mechanism behind ethephon induction is still unclear. In this study, we cloned four genes encoding ethylene receptors (designated AcERS1a, AcERS1b, AcETR2a, and AcETR2b). The 5' flanking sequences of these four genes were also cloned by self-formed adaptor PCR and SiteFinding-PCR, and a group of putative cis-acting elements was identified. Phylogenetic tree analysis indicated that AcERS1a, AcERS1b, AcETR2a, and AcETR2b belonged to the plant ERS1s and ETR2/EIN4-like groups. Quantitative real-time PCR showed that AcETR2a and AcETR2b (subfamily 2) were more sensitive to ethylene treatment compared with AcERS1a and AcERS1b (subfamily 1). The relative expression of AcERS1b, AcETR2a, and AcETR2b was significantly increased during the earlier period of pineapple inflorescence formation, especially at 1-9 days after ethylene treatment (DAET), whereas AcERS1a expression changed less than these three genes. In situ hybridization results showed that bract primordia (BP) and flower primordia (FP) appeared at 9 and 21 DAET, respectively, and flowers were formed at 37 DAET. AcERS1a, AcERS1b, AcETR2a, and AcETR2b were mainly expressed in the shoot apex at 1-4 DAET; thereafter, with the appearance of BP and FP, higher expression of these genes was found in these new structures. Finally, at 37 DAET, the expression of these genes was mainly focused in the flower but was also low in other structures. These findings indicate that these four ethylene receptor genes, especially AcERS1b, AcETR2a, and AcETR2b, play important roles during pineapple flowering induced by exogenous ethephon.

Distinct herpesvirus resistances and immune responses of three gynogenetic clones of gibel carp revealed by comprehensive transcriptomes.

Science.gov (United States)

Gao, Fan-Xiang; Wang, Yang; Zhang, Qi-Ya; Mou, Cheng-Yan; Li, Zhi; Deng, Yuan-Sheng; Zhou, Li; Gui, Jian-Fang

2017-07-24

immune responses of three gynogenetic gibel carp clones to herpesvirus infection by comprehensive transcriptomes. These differential DEUs, immune-related pathways and IFN system genes identified from susceptible and resistant clones will be beneficial to marker-assisted selection (MAS) breeding or molecular module-based resistance breeding in gibel carp.

Molecular cloning and sequence of cDNA encoding the plasma membrane proton pump (H+-ATPase) of Arabidopsis thaliana

International Nuclear Information System (INIS)

Harper, J.F.; Surowy, T.K.; Sussman, M.R.

1989-01-01

In plants, the transport of solutes across the plasma membrane is driven by a proton pump (H + -ATPase) that produces an electric potential and pH gradient. The authors isolated and sequenced a full-length cDNA clone that encodes this enzyme in Arabidopsis thaliana. The protein predicted from its nucleotide sequence encodes 959 amino acids and has a molecular mass of 104,207 Da. The plant protein shows structural features common to a family of cation-translocating ATPases found in the plasma membrane of prokaryotic and eukaryotic cells, with the greatest overall identity in amino acid sequence (36%) to the H + -ATPase observed in the plasma membrane of fungi. The structure predicted from a hydropathy plant contains at least eight transmembrane segments, with most of the protein (73%) extending into the cytoplasm and only 5% of the residues exposed on the external surface. Unique features of the plant enzyme include diverged sequences at the amino and carboxyl termini as well as greater hydrophilic character in three extracellular loops

Molecular cloning and nucleotide sequence of cDNA for human liver arginase

International Nuclear Information System (INIS)

Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

1987-01-01

Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in λ gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated λ hARG6 and λ hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying λ hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes

Molecular cloning and expression of the calmodulin gene from guinea pig hearts.

Science.gov (United States)

Feng, Rui; Liu, Yan; Sun, Xuefei; Wang, Yan; Hu, Huiyuan; Guo, Feng; Zhao, Jinsheng; Hao, Liying

2015-06-01

The aim of the present study was to isolate and characterize a complementary DNA (cDNA) clone encoding the calmodulin (CaM; GenBank accession no. FJ012165) gene from guinea pig hearts. The CaM gene was amplified from cDNA collected from guinea pig hearts and inserted into a pGEM®-T Easy vector. Subsequently, CaM nucleotide and protein sequence similarity analysis was conducted between guinea pigs and other species. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to investigate the CaM 3 expression patterns in different guinea pig tissues. Sequence analysis revealed that the CaM gene isolated from the guinea pig heart had ∼90% sequence identity with the CaM 3 genes in humans, mice and rats. Furthermore, the deduced peptide sequences of CaM 3 in the guinea pig showed 100% homology to the CaM proteins from other species. In addition, the RT-PCR results indicated that CaM 3 was widely and differentially expressed in guinea pigs. In conclusion, the current study provided valuable information with regard to the cloning and expression of CaM 3 in guinea pig hearts. These findings may be helpful for understanding the function of CaM3 and the possible role of CaM3 in cardiovascular diseases.

Reversibility of continuous-variable quantum cloning

International Nuclear Information System (INIS)

Filip, Radim; Marek, Petr; Fiurasek, Jaromir

2004-01-01

We analyze a reversibility of optimal Gaussian 1→2 quantum cloning of a coherent state using only local operations on the clones and classical communication between them and propose a feasible experimental test of this feature. Performing Bell-type homodyne measurement on one clone and anticlone, an arbitrary unknown input state (not only a coherent state) can be restored in the other clone by applying appropriate local unitary displacement operation. We generalize this concept to a partial reversal of the cloning using only local operations and classical communication (LOCC) and we show that this procedure converts the symmetric cloner to an asymmetric cloner. Further, we discuss a distributed LOCC reversal in optimal 1→M Gaussian cloning of coherent states which transforms it to optimal 1→M ' cloning for M ' < M. Assuming the quantum cloning as a possible eavesdropping attack on quantum communication link, the reversibility can be utilized to improve the security of the link even after the attack

Molecular cloning of the human eosinophil-derived neurotoxin: A member of the ribonuclease gene family

International Nuclear Information System (INIS)

Rosenberg, H.F.; Tenen, D.G.; Ackerman, S.J.

1989-01-01

The authors have isolated a 725-base-pair cDNA clone for human eosinophil-derived neurotoxin (EDN). EDN is a distinct cationic protein of the eosinophil's large specific granule known primarily for its ability to induce ataxia, paralysis, and central nervous system cellular degeneration in experimental animals (Gordon phenomenon). The open reading frame encodes a 134-amino acid mature polypeptide with a molecular mass of 15.5 kDa and a 27-residue amino-terminal hydrophobic leader sequence. The sequence of the mature polypeptide is identical to that reported for human urinary ribonuclease, and to the amino-terminal sequence of human liver ribonuclease; the cDNA encodes a tryptophan in position 7. Both EDN and the related granule protein, eosinophil cationic protein, have ribonucleolytic activity; sequence similarities among EDN, eosinophil cationic protein, ribonucleases from liver, urine, and pancreas, and angiogenin define a ribonuclease multigene family. mRNA encoding EDN was detected in uninduced HL-60 cells and was up-regulated in cells induced toward eosinophilic differentiation with B-cell growth factor 2/interleukin 5 and toward neutrophilic differentiation with dimethyl sulfoxide. EDN mRNA was detected in mature neutrophils even though EDN-like neurotoxic activity is not found neutrophil extracts. These results suggest that neutrophils contain a protein that is closely related or identical to EDN

Human cloning. Fact or fiction

International Nuclear Information System (INIS)

Abushama, Mandy D.; Ahmed, Badreldeen I.

2003-01-01

Cloning is the production of one or more individual plants or animals that are genetically identical to other plant, animal or human. Scientists even demonstrated that they were able to clone frog tadpoles from frog embryonic cells using nuclear transfer.Many animals have been cloned from adult cells using nuclear transfer. Somatic cell nuclear transfer which refers to the transfer of the nucleous from a somatic cell to an egg cell. Article further deals with benefits and misuses of human cloning

Molecular cloning and sequence analysis of a phenylalanine ammonia-lyase gene from dendrobium.

Directory of Open Access Journals (Sweden)

Qing Jin

Full Text Available In this study, a phenylalanine ammonia-lyase (PAL gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748 has 2,458 bps and contains a complete open reading frame (ORF of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum.

Cloning and heterologous expression of a novel insecticidal gene (tccC1) from Xenorhabdus nematophilus strain

International Nuclear Information System (INIS)

Joo Lee, Pom; Ahn, Ji-Young; Kim, Yang-Hoon; Wook Kim, Seung; Kim, Ji-Yeon; Park, Jae-Sung; Lee, Jeewon

2004-01-01

We have identified and cloned a novel toxin gene (tccC1/xptB1) from Xenorhabdus nematophilus strain isolated from Korea-specific entomophagous nematode Steinernema glaseri MK. The DNA sequence of cloned toxin gene (3048 bp) has an open reading frame encoding 1016 amino acids with a predicted molecular mass of 111058 Da. The toxin sequence shares 50-96% identical amino acid residues with the previously reported tccC1 cloned from X. nematophilus (AJ308438), Photorhabdus luminescens W14 (AF346499) P. luminescens TTO1 (BX571873), and Yersinia pestis CO92 (NC 0 03143). The toxin gene was successfully expressed in Escherichia coli, and the recombinant toxin protein caused a rapid cessation in mortality of Galleria mellonella larvae (80% death of larvae within 2 days). Conclusively, the heterologous expression of the novel gene tccC1 cloned into E. coli plasmid vector produced recombinant toxin with high insecticidal activity

Local cloning of two product states

International Nuclear Information System (INIS)

Ji Zhengfeng; Feng Yuan; Ying Mingsheng

2005-01-01

Local quantum operations and classical communication (LOCC) put considerable constraints on many quantum information processing tasks such as cloning and discrimination. Surprisingly, however, discrimination of any two pure states survives such constraints in some sense. We show that cloning is not that lucky; namely, probabilistic LOCC cloning of two product states is strictly less efficient than global cloning. We prove our result by giving explicitly the efficiency formula of local cloning of any two product states

Structured Review of Code Clone Literature

NARCIS (Netherlands)

Hordijk, W.T.B.; Ponisio, Laura; Wieringa, Roelf J.

2008-01-01

This report presents the results of a structured review of code clone literature. The aim of the review is to assemble a conceptual model of clone-related concepts which helps us to reason about clones. This conceptual model unifies clone concepts from a wide range of literature, so that findings

Porcine insulin receptor substrate 4 (IRS4) gene: cloning, polymorphism and association study

Czech Academy of Sciences Publication Activity Database

Masopust, Martin; Vykoukalová, Z.; Knoll, Aleš; Bartenschlager, H.; Mileham, A.; Deeb, N.; Rohrer, G. A.; Čepica, Stanislav

2010-01-01

Roč. 38, - (2010), 2611-2617 ISSN 0301-4851 R&D Projects: GA ČR GA523/07/0353; GA ČR GAP502/10/1216 Institutional research plan: CEZ:AV0Z50450515 Keywords : PCR cloning * Polymorphism * IRS4 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.875, year: 2010

Multiple-clone activation of hypnozoites is the leading cause of relapse in Plasmodium vivax infection.

Directory of Open Access Journals (Sweden)

Flávia Carolina F de Araujo

Full Text Available BACKGROUND: Plasmodium vivax infection is characterized by a dormant hepatic stage, the hypnozoite that is activated at varying periods of time after clearance of the primary acute blood-stage, resulting in relapse. Differentiation between treatment failure and new infections requires characterization of initial infections, relapses, and clone multiplicity in vivax malaria infections. METHODOLOGY/PRINCIPAL FINDINGS: Parasite DNA obtained from primary/relapse paired blood samples of 30 patients with P. vivax infection in Brazil was analyzed using 10 molecular markers (8 microsatellites and MSP-1 blocks 2 and 10. Cloning of PCR products and genotyping was used to identify low-frequency clones of parasites. We demonstrated a high frequency of multiple-clone infections in both primary and relapse infections. Few alleles were identified per locus, but the combination of these alleles produced many haplotypes. Consequently, the majority of parasites involved in relapse showed haplotypes that were distinct from those of primary infections. Plasmodium vivax relapse was characterized by temporal variations in the predominant parasite clones. CONCLUSIONS/SIGNIFICANCE: The high rate of low frequency alleles observed in both primary and relapse infections, along with temporal variation in the predominant alleles, might be the source of reported heterologous hypnozoite activation. Our findings complicate the concept of heterologous activation, suggesting the involvement of undetermined mechanisms based on host or environmental factors in the simultaneous activation of multiple clones of hypnozoites.

Human cloning: can it be made safe?

Science.gov (United States)

Rhind, Susan M; Taylor, Jane E; De Sousa, Paul A; King, Tim J; McGarry, Michelle; Wilmut, Ian

2003-11-01

There are continued claims of attempts to clone humans using nuclear transfer, despite the serious problems that have been encountered in cloning other mammals. It is known that epigenetic and genetic mechanisms are involved in clone failure, but we still do not know exactly how. Human reproductive cloning is unethical, but the production of cells from cloned embryos could offer many potential benefits. So, can human cloning be made safe?

Molecular markers shared by diverse apomictic Pennisetum species.

Science.gov (United States)

Lubbers, E L; Arthur, L; Hanna, W W; Ozias-Akins, P

1994-11-01

Two molecular markers, a RAPD (randomly amplified polymorphic DNA) and a RFLP/STS (restriction fragment length polymorphism/sequence-tagged site), previously were found associated with apomictic reproductive behavior in a backcross population produced to transfer apomixis from Pennisetum squamulatum to pearl millet. The occurrence of these molecular markers in a range of 29 accessions of Pennisetum comprising 11 apomictic and 8 sexual species was investigated. Both markers were specific for apomictic species in Pennisetum. The RFLP/STS marker, UGT 197, was found to be associated with all taxa that displayed apomictic reproductive behavior except those in section Brevivalvula. Neither UGT197 nor the cloned RAPD fragment OPC-04600 hybridized with any sexually reproducing representatives of the genus. The cloned C04600 was associated with 3 of the 11 apomictic species, P. ciliare, P. massaicum, and P. squamulatum. UGT197 was more consistently associated with apomictic reproductive behavior than OPC04600 or cloned C04600, thus it could be inferred that UGT197 is more closely linked to the gene(s) for apomixis than the cloned C04600. The successful use of these probes to survey other Pennisetum species indicates that apomixis is a trait that can be followed across species by using molecular means. This technique of surveying species within a genus will be useful in determining the relative importance of newly isolated markers and may facilitate the identification of the apomixis gene(s).

Gene cloning: exploring cotton functional genomics and genetic improvement

Institute of Scientific and Technical Information of China (English)

Diqiu LIU; Xianlong ZHANG

2008-01-01

Cotton is the most important natural fiber plant in the world. The genetic improvement of the quality of the cotton fiber and agricultural productivity is imperative under the situation of increasing consumption and rapid development of textile technology. Recently, the study of cotton molecular biology has progressed greatly. A lot of specifically or preferentially expressed cotton fiber genes were cloned and analyzed. On the other hand, identification of stress response genes expressed in cotton was performed by other research groups. The major stress factors were studied including the wilt pathogens Verticillium dahliae, Fusarium oxy-sporum f. sp. vasinfectum, bacterial blight, root-knot nematode, drought, and salt stress. What is more, a few genes related to the biosynthesis of gossypol, other sesquiterpene phytoalexins and the major seed oil fatty acids were isolated from cotton. In the present review, we focused on the major advances in cotton gene cloning and expression profiling in the recent years.

Probabilistic cloning and deleting of quantum states

International Nuclear Information System (INIS)

Feng Yuan; Zhang Shengyu; Ying Mingsheng

2002-01-01

We construct a probabilistic cloning and deleting machine which, taking several copies of an input quantum state, can output a linear superposition of multiple cloning and deleting states. Since the machine can perform cloning and deleting in a single unitary evolution, the probabilistic cloning and other cloning machines proposed in the previous literature can be thought of as special cases of our machine. A sufficient and necessary condition for successful cloning and deleting is presented, and it requires that the copies of an arbitrarily presumed number of the input states are linearly independent. This simply generalizes some results for cloning. We also derive an upper bound for the success probability of the cloning and deleting machine

Asymmetric quantum cloning machines

International Nuclear Information System (INIS)

Cerf, N.J.

1998-01-01

A family of asymmetric cloning machines for quantum bits and N-dimensional quantum states is introduced. These machines produce two approximate copies of a single quantum state that emerge from two distinct channels. In particular, an asymmetric Pauli cloning machine is defined that makes two imperfect copies of a quantum bit, while the overall input-to-output operation for each copy is a Pauli channel. A no-cloning inequality is derived, characterizing the impossibility of copying imposed by quantum mechanics. If p and p ' are the probabilities of the depolarizing channels associated with the two outputs, the domain in (√p,√p ' )-space located inside a particular ellipse representing close-to-perfect cloning is forbidden. This ellipse tends to a circle when copying an N-dimensional state with N→∞, which has a simple semi-classical interpretation. The symmetric Pauli cloning machines are then used to provide an upper bound on the quantum capacity of the Pauli channel of probabilities p x , p y and p z . The capacity is proven to be vanishing if (√p x , √p y , √p z ) lies outside an ellipsoid whose pole coincides with the depolarizing channel that underlies the universal cloning machine. Finally, the tradeoff between the quality of the two copies is shown to result from a complementarity akin to Heisenberg uncertainty principle. (author)

Effective and efficient model clone detection

DEFF Research Database (Denmark)

Störrle, Harald

2015-01-01

Code clones are a major source of software defects. Thus, it is likely that model clones (i.e., duplicate fragments of models) have a significant negative impact on model quality, and thus, on any software created based on those models, irrespective of whether the software is generated fully...... automatically (“MDD-style”) or hand-crafted following the blueprint defined by the model (“MBSD-style”). Unfortunately, however, model clones are much less well studied than code clones. In this paper, we present a clone detection algorithm for UML domain models. Our approach covers a much greater variety...... of model types than existing approaches while providing high clone detection rates at high speed....

Wildlife conservation and reproductive cloning.

Science.gov (United States)

Holt, William V; Pickard, Amanda R; Prather, Randall S

2004-03-01

Reproductive cloning, or the production of offspring by nuclear transfer, is often regarded as having potential for conserving endangered species of wildlife. Currently, however, low success rates for reproductive cloning limit the practical application of this technique to experimental use and proof of principle investigations. In this review, we consider how cloning may contribute to wildlife conservation strategies. The cloning of endangered mammals presents practical problems, many of which stem from the paucity of knowledge about their basic reproductive biology. However, situations may arise where resources could be targeted at recovering lost or under-represented genetic lines; these could then contribute to the future fitness of the population. Approaches of this type would be preferable to the indiscriminate generation of large numbers of identical individuals. Applying cloning technology to non-mammalian vertebrates may be more practical than attempting to use conventional reproductive technologies. As the scientific background to cloning technology was pioneered using amphibians, it may be possible to breed imminently threatened amphibians, or even restore extinct amphibian species, by the use of cloning. In this respect species with external embryonic development may have an advantage over mammals as developmental abnormalities associated with inappropriate embryonic reprogramming would not be relevant.

Molecular cloning and immunochemical characterization of a novel major Japanese cedar pollen allergen belonging to the aspartic protease family.

Science.gov (United States)

Ibrahim, Ahmed Ragaa Nour; Kawamoto, Seiji; Aki, Tsunehiro; Shimada, Yayoi; Rikimaru, Satoshi; Onishi, Nobukazu; Babiker, Elfadil Elfadl; Oiso, Isao; Hashimoto, Kunihiko; Hayashi, Takaharu; Ono, Kazuhisa

2010-01-01

Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis in Japan. Protease activity in the pollen grains may trigger pro-allergic responses but no such proteases have yet been identified as pollen allergens. We report the molecular cloning and immunochemical characterization of a novel C. japonica pollen allergen belonging to the aspartic protease family. We focused on the C. japonica pollen allergen spot No. 63 (CPA63, 47.5% IgE binding frequency) on our 2-dimensional IgE immunoblot map. The internal amino acid sequences were determined using time-of-flight mass spectrometry. Full-length cpa63 cDNA was cloned by rapid amplification of cDNA ends (RACE)-PCR. Recombinant CPA63 (r-CPA63) was expressed using the baculovirus-insect cell culture system and its IgE binding capacity was analyzed by enzyme-linked immunosorbent assay (ELISA). The proteolytic activity of r-CPA63 was also assessed using a putative mature enzyme produced upon autolysis. cpa63 cDNA encoded a 472 amino acid polypeptide showing about 40% sequence identity to members of the plant atypical aspartic protease family. ELISA showed that r-CPA63 was recognized by IgE antibodies in the serum of 58% (18/31) of Japanese cedar pollinosis patients. We also demonstrated an aspartic protease-like enzyme activity of the putative mature r-CPA63. We have identified the first plant aspartic protease allergen from Japanese cedar pollen. The availability of the CPA63 sequence and its recombinant allergen production system are useful not only for pharmaceutical applications but also for further examination of the role of protease activity in the pathogenesis of cedar pollinosis. 2010 S. Karger AG, Basel.

Evaluation of cloned cells, animal model, and ATRA sensitivity of human testicular yolk sac tumor

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Zhao Junfeng

2012-03-01

Full Text Available Abstract The testicular yolk sac tumor (TYST is the most common neoplasm originated from germ cells differentiated abnormally, a major part of pediatric malignant testicular tumors. The present study aimed at developing and validating the in vitro and vivo models of TYST and evaluating the sensitivity of TYST to treatments, by cloning human TYST cells and investigating the histology, ultra-structure, growth kinetics and expression of specific proteins of cloned cells. We found biological characteristics of cloned TYST cells were similar to the yolk sac tumor and differentiated from the columnar to glandular-like or goblet cells-like cells. Chromosomes for tumor identification in each passage met nature of the primary tumor. TYST cells were more sensitive to all-trans-retinoic acid which had significantly inhibitory effects on cell proliferation. Cisplatin induced apoptosis of TYST cells through the activation of p53 expression and down-regulation of Bcl- expression. Thus, we believe that cloned TYST cells and the animal model developed here are useful to understand the molecular mechanism of TYST cells and develop potential therapies for human TYST.

Toward positional cloning of Fhb1, a major QTL for Fusarium head blight resistance in wheat

Czech Academy of Sciences Publication Activity Database

Liu, S. X.; Pumphrey, M. O.; Gill, B. S.; Trick, H. N.; Zhang, J. X.; Doležel, Jaroslav; Chalhoub, B.; Anderson, J. A.

2008-01-01

Roč. 36, suppl. B (2008), s. 195-201 ISSN 0133-3720 Institutional research plan: CEZ:AV0Z50380511 Keywords : map-based cloning * Fusarium head blight * Fhb1 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.190, year: 2007

Purification, characterization and molecular cloning of alpha-2-macroglobulin in cobia, Rachycentron canadum.

Science.gov (United States)

Chuang, Wen-Hsiao; Liu, Ping-Chung; Hung, Chia-Yu; Lee, Kuo-Kau

2014-12-01

Alpha-2-macroglobulin (α-2-M) is a broad spectrum protease inhibitor which is abundant in the plasma of vertebrates and several invertebrates. The α-2-M was purified from cobia (Rachycentron canadum) plasma by a four-step procedure: poly ethylene glycol fractionation, affinity chromatography, hydrophobic interaction chromatography and ion exchange chromatography on Fast Protein liquid chromatography system in the present study. It migrated as one protein band with a molecular mass of about 360 kDa in the native state, whereas in SDS-PAGE it was about 180 kDa under non-reducing condition. This result revealed that the native protein was a dimer. In addition, it was cleaved into two different fragments of molecular mass about 93 and 87 kDa when reduced by dithiothreitol (DTT). The anti-protease activity of the purified α-2-M was apparently decreased as temperature elevated above 50 °C. The α-2-M exhibited highest protease inhibitory activity at pH 9. The results indicate that the α-2-M is a heat-labile and alkaline protease inhibitor. The purified α-2-M exhibited more than 50% protease inhibitory activity against extracellular products (ECP) of Vibrio alginolytius isolated from diseased cobia. It seems that the protease activities in ECP may be affected by the plasma α-2-M. The protease inhibitory activities of cobia plasma or purified α-2-M were decreased when incubated with 10 mM methylamine for 30 min. The α-2-M cDNA consisted of 4611 bp with an open reading frame of 4374 bp had been cloned from cobia liver. This sequence contained thioester domain (GCGEQ) and thirteen predicted N-linked glycosylation sites. In addition, the amino acid sequence of thioester domain and genes of adjacent regions of cobia α-2-M were further compared with sequences of known fish species in GenBank. The unweighted pair group method using arithmetic average (UPGMA) was employed to construct the phylogenetic trees of α-2-M among different fish species (freshwater fish, sea

Islamic perspectives on human cloning.

Science.gov (United States)

Sadeghi, Mahmoud

2007-01-01

The present paper seeks to assess various views from Islamic jurists relating to human cloning, which is one of the controversial topics in the recent past. Taking Islamic jurisprudence principles, such as the rule of necessity for self preservation and respect for human beings, the rule of la darar wa la dirar ('the necessity to refrain from causing harm to oneself and others') and the rule of usr wa haraj, one may indicate that if human cloning could not be prohibited, as such, it could still be opposed because it gives way to various harmful consequences, which include family disorder, chaos in the clone's family relationships, physical and mental diseases for clones and suffering of egg donors and surrogate mothers. However with due attention to the fact that the reasons behind the prohibition of abortion only restrict the destruction of human embryos in their post-implantation stages, human cloning for biomedical research and exploitation of stem cells from cloned embryos at the blastocyst stage for therapeutic purposes would be acceptable.

Quantum cloning machines for equatorial qubits

International Nuclear Information System (INIS)

Fan Heng; Matsumoto, Keiji; Wang Xiangbin; Wadati, Miki

2002-01-01

Quantum cloning machines for equatorial qubits are studied. For the case of a one to two phase-covariant quantum cloning machine, we present the networks consisting of quantum gates to realize the quantum cloning transformations. The copied equatorial qubits are shown to be separable by using Peres-Horodecki criterion. The optimal one to M phase-covariant quantum cloning transformations are given

Cloning and expression analysis of two dehydrodolichyl diphosphate synthase genes from Tripterygium wilfordii

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Lin-Hui Gao

2018-01-01

Full Text Available Objective: To clone and investigate two dehydrodolichyl diphosphate synthase genes of Tripterygium wilfordii by bioinformatics and tissue expression analysis. Materials and Methods: According to the T. wifordii transcriptome database, specific primers were designed to clone the TwDHDDS1 and TwDHDDS2 genes via PCR. Based on the cloned sequences, protein structure prediction, multiple sequence alignment and phylogenetic tree construction were performed. The expression levels of the genes in different tissues of T. wilfordii were measured by real-time quantitative PCR. Results: The TwDHDDS1 gene encompassed a 873 bp open reading frame (ORF and encoded a protein of 290 amino acids. The calculated molecular weight of the translated protein was about 33.46 kDa, and the theoretical isoelectric point (pI was 8.67. The TwDHDDS2 encompassed a 768 bp ORF, encoding a protein of 255 amino acids with a calculated molecular weight of about 21.19 kDa, and a theoretical isoelectric point (pI of 7.72. Plant tissue expression analysis indicated that TwDHDDS1 and TwDHDDS2 both have relatively ubiquitous expression in all sampled organ tissues, but showed the highest transcription levels in the stems. Conclusions: The results of this study provide a basis for further functional studies of TwDHDDS1 and TwDHDDS2. Most importantly, these genes are promising genetic targets for the regulation of the biosynthetic pathways of important bioactive terpenoids such as triptolide.

A Gateway MultiSite recombination cloning toolkit.

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Lena K Petersen

Full Text Available The generation of DNA constructs is often a rate-limiting step in conducting biological experiments. Recombination cloning of single DNA fragments using the Gateway system provided an advance over traditional restriction enzyme cloning due to increases in efficiency and reliability. Here we introduce a series of entry clones and a destination vector for use in two, three, and four fragment Gateway MultiSite recombination cloning whose advantages include increased flexibility and versatility. In contrast to Gateway single-fragment cloning approaches where variations are typically incorporated into model system-specific destination vectors, our Gateway MultiSite cloning strategy incorporates variations in easily generated entry clones that are model system-independent. In particular, we present entry clones containing insertions of GAL4, QF, UAS, QUAS, eGFP, and mCherry, among others, and demonstrate their in vivo functionality in Drosophila by using them to generate expression clones including GAL4 and QF drivers for various trp ion channel family members, UAS and QUAS excitatory and inhibitory light-gated ion channels, and QUAS red and green fluorescent synaptic vesicle markers. We thus establish a starter toolkit of modular Gateway MultiSite entry clones potentially adaptable to any model system. An inventory of entry clones and destination vectors for Gateway MultiSite cloning has also been established (www.gatewaymultisite.org.

Molecular Cloning, Expression and Characterization of Plasmid Encoding Rhomboid 4 (ROM4 of Tachyzoite of Toxoplasma gondii RH Strain

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Mohammad Taghi RAHIMI

2017-12-01

Full Text Available AbstractBackground: The objective of this study was to clone, express and characterize the gene encoding rhomboid 4 (ROM4 proteins, a vital gene in surface adhesion and host cell invasion process of tachyzoite of T. gondii in an appropriate expression vector and eukaryotic cell for production of recombinant protein.Methods: Toxoplasma RNA was isolated from tachyzoites (RH strain and complementary DNA was synthesized. Oligonucleotide primer pair was designed based on Toxoplasma ROM4 gene sequence with XhoI and EcoRI restriction sites at 5´ end of forward and reverse primers, respectively. ROM4 gene was amplified by PCR, cloned into pTG19-T vector and the recombinant plasmid was sequenced. The gene was subcloned into pcDNA3 plasmid and expressed in CHO cells as eukaryotic cell. SDS-PAGE and western blotting were performed for protein determination and verification.Results: Cloning of ROM4 gene in pTG19-T vector was confirmed by colony-PCR and enzymatic digestion. The results of enzymatic digestion and gene sequencing confirmed successful cloning and subcloning procedures. The nucleotide sequence of the cloned ROM4 gene showed 99% homology compared to the corresponding sequences of original gene. SDS-PAGE and western blotting analyses of the purified protein revealed a single band having expected size of 65 kDa.Conclusion: This eukaryotic expression system is an appropriate system for high-level recombinant protein production of ROM4 gene from T. gondii tachyzoites used as antigenic component for serological assay and vaccine development.

Methodology of molecular diagnose of the Dashen Mosaic Virus for the certification of commercial clones of dashen in vitro plants

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José E. González Rámirez

2005-01-01

Full Text Available The quick propagation of Dasheen clones (Xanthosoma spp and (Colocasia esculenta L. through biotechnical techniques has generated a great demand of free of diseases lines, especially to the Dasheen Mosaic Virus, this pathogen, belonging to the potivirus group, is the most important viral disease that affect the crop leading up to 40% of yield losses. The UM-ELISA diagnostic useded in massive certification programs, with big advantages over other kinds of analysis, has a limit of sensibility that can allow escape of contaminated vegetal material. With the introduction of molecular techniques of diagnostic small viral concentrations can be detected in vitroplants. In the present work, the detection of the Dasheen Mosaic Virus using the technique of reverse transcription and polymerase chain reaction is carried out. The established methodology was validated and lines of in vitro plants of dasheen were certified to be used in the micropropagation in biofactories. Xanthosoma Key words: Colocasia, DMV, healthy plants, RT-PCR,

Update on the First Cloned Dog and Outlook for Canine Cloning.

Science.gov (United States)

Jang, Goo; Lee, ByeongChun

2015-10-01

As man's best friend, dogs have an important position in human society. Ten years ago, we reported the first cloned dog, and his birth has raised various scientific issues, such as those related to health, reproduction, and life span. He has developed without any unique health issues. In this article, we summarize and present perspectives on canine cloning.

Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster

DEFF Research Database (Denmark)

Lenz, C; Williamson, M; Grimmelikhuijzen, C J

2000-01-01

We (C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 269, 91-96) and others (N. Birgül et al. (1999) EMBO J. 18, 5892-5900) have recently cloned a Drosophila receptor that was structurally related to the mammalian galanin receptors, but turned out to be a receptor for a Drosophila peptide bel...

Molecular cloning of a K+ channel from the malaria parasite Plasmodium falciparum

DEFF Research Database (Denmark)

Ellekvist, Peter; Ricke, Christina Høier; Litman, Thomas

2004-01-01

In most living cells, K(+) channels are important for the generation of the membrane potential and for volume regulation. The parasite Plasmodium falciparum, which causes malignant malaria, must be able to deal with large variations in the ambient K(+) concentration: it is exposed to high...... concentrations of K(+) when inside the erythrocyte and low concentrations when in plasma. In the recently published genome of P. falciparum, we have identified a gene, pfkch1, encoding a potential K(+) channel, which to some extent resembles the big-conductance (BK) K(+) channel. We have cloned the approximately...

Molecular characterization and clonal diversity of meticillin-resistant Staphylococcus aureus isolated from the community in Spain: emergence of clone sequence type 72.

Science.gov (United States)

Potel, C; Rey, S; Otero, S; Rubio, J; Álvarez, M

2016-08-01

Sequence type 72 meticillin-resistant Staphylococcus aureus (ST72 MRSA) was recently detected in our hospital. Although in Europe this clone is rarely isolated, it is the leading cause of community-associated MRSA infections in Korea, spreading also into hospitals, where it has also emerged as the main MRSA clone recovered from raw meat. We studied MRSA isolated from outpatients in Spain during a nine-year period. More than 70% of the isolates belonged to predominant clones found in hospitals. There was a significant increase in the ST72 prevalence. It appears that boundaries of dominance among MRSA clones have become blurred, demanding continuous surveillance. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Therapeutic and reproductive cloning: a critique.

Science.gov (United States)

Bowring, Finn

2004-01-01

This article is a critical examination of the science and ethics of human cloning. It summarises the key scientific milestones in the development of nuclear transplantation, explains the importance of cloning to research into the medical potential of embryonic stem cells, and discusses the well-worn distinction between 'therapeutic' and 'reproductive' cloning. Suggesting that this distinction will be impossible to police, it goes on to consider the ethics of full human cloning. It is concluded that it represents an unacceptable form of parental despotism, and that the genetic engineering and cloning of future human beings will fracture the foundations of modern humanism.

Novel cloning machine with supplementary information

International Nuclear Information System (INIS)

Qiu Daowen

2006-01-01

Probabilistic cloning was first proposed by Duan and Guo. Then Pati established a novel cloning machine (NCM) for copying superposition of multiple clones simultaneously. In this paper, we deal with the novel cloning machine with supplementary information (NCMSI). For the case of cloning two states, we demonstrate that the optimal efficiency of the NCMSI in which the original party and the supplementary party can perform quantum communication equals that achieved by a two-step cloning protocol wherein classical communication is only allowed between the original and the supplementary parties. From this equivalence, it follows that NCMSI may increase the success probabilities for copying. Also, an upper bound on the unambiguous discrimination of two nonorthogonal pure product states is derived. Our investigation generalizes and completes the results in the literature

Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum

International Nuclear Information System (INIS)

Tanaka, K.; Satokata, I.; Ogita, Z.; Uchida, T.; Okada, Y.

1989-01-01

For isolation of the gene responsible for xeroderma pigmentosum (XP) complementation group A, plasmid pSV2gpt and genomic DNA from a mouse embryo were cotransfected into XP2OSSV cells, a group-A XP cell line. Two primary UV-resistant XP transfectants were isolated from about 1.6 X 10(5) pSV2gpt-transformed XP colonies. pSV2gpt and genomic DNA from the primary transfectants were again cotransfected into XP2OSSV cells and a secondary UV-resistant XP transfectant was obtained by screening about 4.8 X 10(5) pSV2gpt-transformed XP colonies. The secondary transfectant retained fewer mouse repetitive sequences. A mouse gene that complements the defect of XP2OSSV cells was cloned into an EMBL3 vector from the genome of a secondary transfectant. Transfections of the cloned DNA also conferred UV resistance on another group-A XP cell line but not on XP cell lines of group C, D, F, or G. Northern blot analysis of poly(A)+ RNA with a subfragment of cloned mouse DNA repair gene as the probe revealed that an approximately 1.0 kilobase mRNA was transcribed in the donor mouse embryo and secondary transfectant, and approximately 1.0- and approximately 1.3-kilobase mRNAs were transcribed in normal human cells, but none of these mRNAs was detected in three strains of group-A XP cells. These results suggest that the cloned DNA repair gene is specific for group-A XP and may be the mouse homologue of the group-A XP human gene

Artificial cloning of domestic animals.

Science.gov (United States)

Keefer, Carol L

2015-07-21

Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research.

[Scientific ethics of human cloning].

Science.gov (United States)

Valenzuela, Carlos Y

2005-01-01

True cloning is fission, budding or other types of asexual reproduction. In humans it occurs in monozygote twinning. This type of cloning is ethically and religiously good. Human cloning can be performed by twinning (TWClo) or nuclear transfer (NTClo). Both methods need a zygote or a nuclear transferred cell, obtained in vitro (IVTec). They are under the IVTec ethics. IVTecs use humans (zygotes, embryos) as drugs or things; increase the risk of malformations; increase development and size of abnormalities and may cause long-term changes. Cloning for preserving extinct (or almost extinct) animals or humans when sexual reproduction is not possible is ethically valid. The previous selection of a phenotype in human cloning violates some ethical principles. NTClo for reproductive or therapeutic purposes is dangerous since it increases the risk for nucleotide or chromosome mutations, de-programming or re-programming errors, aging or malignancy of the embryo cells thus obtained.

Molecular epidemiology and emergence of worldwide epidemic clones of Neisseria meningitidis in Taiwan

Directory of Open Access Journals (Sweden)

Chang Hsiu-Li

2006-02-01

30 STs suggested the serogroup B isolates be assigned into 5 clonally related groups/ clonal complexes and 7 unique clones. The ST-41/44 complex/Lineage 3, and the ST-3439 and ST-3200 groups represented 79% of the serogroup B meningococci. In contrast, isolates within serogroups A, serogroup W135 (and C, and serogroup Y, respectively, simply belonged to ST-7, ST-11, and ST-23 clones. Conclusion Our data suggested that serogroup B isolates were derived from several distinct lineages, most of which could either be indigenous or were introduced into Taiwan a long time ago. The serogroup A, W135 (and C, and Y isolates, respectively, belonged to the ST-7, ST-11, and ST-23, and the represented clones that are currently the major circulating clones in the world and are introduced into Taiwan more recently. The emergence of serogroup A, C and Y strains contributed partly to the increase in cases of meningococcal disease in 2001–2002.

The ethics of human reproductive cloning.

Science.gov (United States)

Strong, Carson

2005-03-01

This article addresses the question of whether human reproductive cloning could be ethically justifiable in at least some cases involving infertile couples who would choose cloning as a way to have a genetically related child. At present, the risk of congenital anomalies constitutes a compelling argument against human reproductive cloning. The article explores whether reproductive cloning could be ethically justifiable if, at some future time, cloning becomes possible without an elevated risk of anomalies. It is argued that freedom to use cloning is a form of procreative freedom and, as such, deserves respect. All of the objections that have been raised against human reproductive cloning fall under three main categories: those that appeal to the interests of the child, those based on consequences for society, and those arising from teleological views. Objections that appeal to the child's interests are, in turn, of two main kinds: consequentialist and deontological. All of these types of objections are examined, and it is found that each involves serious problems that prevent it from being a reasonable objection in the context of the infertility cases considered. It is concluded that human reproductive cloning would be ethically justifiable in at least some cases involving infertile couples, provided that it could be performed without an elevated risk of anomalies.

Therapeutic cloning: The ethical limits

International Nuclear Information System (INIS)

Whittaker, Peter A.

2005-01-01

A brief outline of stem cells, stem cell therapy and therapeutic cloning is given. The position of therapeutic cloning with regard to other embryonic manipulations - IVF-based reproduction, embryonic stem formation from IVF embryos and reproductive cloning - is indicated. The main ethically challenging stages in therapeutic cloning are considered to be the nuclear transfer process including the source of eggs for this and the destruction of an embryo to provide stem cells for therapeutic use. The extremely polarised nature of the debate regarding the status of an early human embryo is noted, and some potential alternative strategies for preparing immunocompatible pluripotent stem cells are indicated

Economical quantum cloning in any dimension

International Nuclear Information System (INIS)

Durt, Thomas; Fiurasek, Jaromir; Cerf, Nicolas J.

2005-01-01

The possibility of cloning a d-dimensional quantum system without an ancilla is explored, extending on the economical phase-covariant cloning machine for qubits found in Phys. Rev. A 60, 2764 (1999). We prove the impossibility of constructing an economical version of the optimal universal 1→2 cloning machine in any dimension. We also show, using an ansatz on the generic form of cloning machines, that the d-dimensional 1→2 phase-covariant cloner, which optimally clones all balanced superpositions with arbitrary phases, can be realized economically only in dimension d=2. The used ansatz is supported by numerical evidence up to d=7. An economical phase-covariant cloner can nevertheless be constructed for d>2, albeit with a slightly lower fidelity than that of the optimal cloner requiring an ancilla. Finally, using again an ansatz on cloning machines, we show that an economical version of the 1→2 Fourier-covariant cloner, which optimally clones the computational basis and its Fourier transform, is also possible only in dimension d=2

Analysis of nuclear reprogramming in cloned miniature pig embryos by expression of Oct-4 and Oct-4 related genes

International Nuclear Information System (INIS)

Lee, Eugine; Lee, So Hyun; Kim, Sue

2006-01-01

Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P < 0.05) in cloned hatched blastocysts derived from fibroblasts and many of fibroblast-derived clones failed to reactivate at least one of the tested genes, while most of the germ cell clones and control embryos correctly expressed these genes. In conclusion, our results suggest that the reprogramming of fibroblast-derived cloned embryos is highly aberrant and this improper reprogramming could be one reason of the early lethality and post-implantation anomalies of somatic cell-derived clones

Molecular cloning of a seed specific multifunctional RFO synthase/ galactosylhydrolase in Arabidopsis thaliana

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Roman eGangl

2015-09-01

Full Text Available Stachyose is among the raffinose family oligosaccharides one of the major water-soluble carbohydrates next to sucrose in seeds of a number of plant species. Especially in leguminous seeds, e.g. chickpea, stachyose is reported as the major component. In contrast to their ambiguous potential as essential source of carbon for germination, raffinose family oligosaccharides are indigestible for humans and can contribute to diverse abdominal disorders.In the genome of Arabidopsis thaliana, six putative raffinose synthase genes are reported, whereas little is known about these putative raffinose synthases and their biochemical characteristics or their contribution to the raffinose family oligosaccharide physiology in A. thaliana.In this paper, we report on the molecular cloning, functional expression in Escherichia coli and purification of recombinant AtRS4 from A. thaliana and the biochemical characterisation of the putative stachyose synthase (AtSTS, At4g01970 as a raffinose and high affinity stachyose synthase (Km for raffinose 259.2 ± 21.15 µM as well as stachyose and galactinol specific galactosylhydrolase. A T-DNA insertional mutant in the AtRS4 gene was isolated. Only sqPCR from WT siliques showed a specific transcriptional AtRS4 PCR product. Metabolite measurements in seeds of ΔAtRS4 mutant plants revealed a total loss of stachyose in ΔAtRS4 mutant seeds. We conclude that AtRS4 is the only stachyose synthase in the genome of A. thaliana that AtRS4 represents a key regulation mechanism in the raffinose family oligosaccharide physiology of A. thaliana due to its multifunctional enzyme activity and that AtRS4 is possibly the second seed specific raffinose synthase beside AtRS5, which is responsible for Raf-accumulation under abiotic stress.

No-cloning theorem on quantum logics

International Nuclear Information System (INIS)

Miyadera, Takayuki; Imai, Hideki

2009-01-01

This paper discusses the no-cloning theorem in a logicoalgebraic approach. In this approach, an orthoalgebra is considered as a general structure for propositions in a physical theory. We proved that an orthoalgebra admits cloning operation if and only if it is a Boolean algebra. That is, only classical theory admits the cloning of states. If unsharp propositions are to be included in the theory, then a notion of effect algebra is considered. We proved that an atomic Archimedean effect algebra admitting cloning operation is a Boolean algebra. This paper also presents a partial result, indicating a relation between the cloning on effect algebras and hidden variables.

Phase-covariant quantum cloning of qudits

International Nuclear Information System (INIS)

Fan Heng; Imai, Hiroshi; Matsumoto, Keiji; Wang, Xiang-Bin

2003-01-01

We study the phase-covariant quantum cloning machine for qudits, i.e., the input states in a d-level quantum system have complex coefficients with arbitrary phase but constant module. A cloning unitary transformation is proposed. After optimizing the fidelity between input state and single qudit reduced density operator of output state, we obtain the optimal fidelity for 1 to 2 phase-covariant quantum cloning of qudits and the corresponding cloning transformation

Cloning and characterization of the rec2 gene of Ustilago maydis

International Nuclear Information System (INIS)

Bauchwitz, R.P.; Holloman, W.K.

1989-01-01

The authors are exploring the molecular basis for genetic recombination using the corn smut fungus Ustilago maydis, from which the first two eucaryotic DNA repair and recombination mutants, rec1 and rec2, were described. Cells mutant at the rec2 locus are unable to repair lethal damage to their DNA from UV and X irradiation or from chemical alkylating agents such as N-methyl-nitrosoguanidine. Rec2 mutants retain only a residual level of DNA-damage inducible mitotic recombination, and are unable to complete meiosis. Using an autonomously replicating plasmid vector for Ustilago, they established the first nonintegrating plasmid library of the Ustilago genome. The rec2 locus was cloned by complementation of the rec2 mutation in vivo. One clone was found to restore all of the deficient activities. Although this rec2 complementing clone is present on a multicopy plasmid, the authors observed that it fully restored but did not further increase the fifty-fold inducibility of heteroallelic recombination at the nitrate reductase and inositol loci of rec2 or wild type cells. Northern blot analysis using the rec2 complementing clone revealed three UV inducible transcripts, one of which is absent in a rec2 mutant strain. This transcript organization resembles that of the yeast rad10 and the human ERCC-1 genes (MCB 9:1794), but sequence obtained to date from rec2 does not show homology with these genes. They have also observed that the rec2 mutation may alter the level of homologous integration of transformed DNA markers. Integration of a Leu1 complementing plasmid by Scott Fotheringham of the lab has shown that while much of plasmid integration in wild type Ustilago is nonhomologous, integration in at least some rec2 strains is entirely homologous. They are using the cloned rec2 gene to confirm that rec2 is indeed involved in altering the level of homologous integration in Ustilago, and if so, they plan to clone a mammalian analogue of rec2

History of the molecular biology of cytomegaloviruses.

Science.gov (United States)

Stinski, Mark F

2014-01-01

The history of the molecular biology of cytomegaloviruses from the purification of the virus and the viral DNA to the cloning and expression of the viral genes is reviewed. A key genetic element of cytomegalovirus (the CMV promoter) contributed to our understanding of eukaryotic cell molecular biology and to the development of lifesaving therapeutic proteins. The study of the molecular biology of cytomegaloviruses also contributed to the development of antivirals to control the viral infection.

Effect of TH-lines and clones on the growth and differentiation of B cell clones in microculture.

Science.gov (United States)

Kotloff, D B; Cebra, J J

1988-02-01

Antibody isotype expression by B cell clones was analyzed using in vitro microcultures containing low numbers of hapten-gelatin-enriched B cells and higher numbers of hemocyanin-specific helper T cell lines or clones. Twenty-eight to sixty-three percent of clones grown in microculture with haptenated hemocyanin and T cells from established lines expressed IgG and/or IgA isotypes in random mixtures, almost always accompanied by IgM. Helper T cells from hemocyanin-specific clones also supported the expression of non-IgM isotypes by the B cell clones, suggesting that a single specificity of T cell can provide sufficient growth and differentiation factors for the display of isotype switching. A positive correlation between the antibody output of clones and the expression of non-IgM isotypes indicated that the switching process may be associated with cell division. Although memory B cells that give clones expressing IgG and/or IgA in the absence of IgM are also enriched on haptenated gelatin, they are not stimulable under conditions of this microculture assay.

Molecular cloning of cDNA for rat brain metallothionein-2 and regulation of its gene expression

Energy Technology Data Exchange (ETDEWEB)

Saijoh, Kiyofumi; Sumino, Kimiaki [Department of Public Health, Kobe University School of Medicine (Japan); Kuno, Takayoshi; Shuntoh, Hisato; Tanaka, Chikako [Department of Pharmacology, Kobe University of Medicine (Japan)

1989-01-01

A rat brain metallothionein-II (MT-II) complementary DNA (cDNA) clone was isolated from a cDNA plasmid library, which was prepared from non-treated rat brain mRNA, by a colony screening procedure using /sup 32/P-labeled synthetic oligonucleotide probes. It is deduced that the clone encodes for a protein of 61 amino acids comprising 20 cysteines, which is highly homologous to MT-IIs in other species. Northern blot analysis demonstrated major mRNA species in the brain, liver and kidneys (approximately 350 b in size), which is induced in response to dexamethasone, zinc, cadmium and mercury but not to methyl mercury. These findings confirm that MT-II genes are expressed and regulated both by steroid and heavy metals in the brain as well as in peripheral organs. (author).

Molecular cloning of cDNA for rat brain metallothionein-2 and regulation of its gene expression

International Nuclear Information System (INIS)

Saijoh, Kiyofumi; Sumino, Kimiaki; Kuno, Takayoshi; Shuntoh, Hisato; Tanaka, Chikako

1989-01-01

A rat brain metallothionein-II (MT-II) complementary DNA (cDNA) clone was isolated from a cDNA plasmid library, which was prepared from non-treated rat brain mRNA, by a colony screening procedure using 32 P-labeled synthetic oligonucleotide probes. It is deduced that the clone encodes for a protein of 61 amino acids comprising 20 cysteines, which is highly homologous to MT-IIs in other species. Northern blot analysis demonstrated major mRNA species in the brain, liver and kidneys (approximately 350 b in size), which is induced in response to dexamethasone, zinc, cadmium and mercury but not to methyl mercury. These findings confirm that MT-II genes are expressed and regulated both by steroid and heavy metals in the brain as well as in peripheral organs. (author)

Molecular cloning and expression of gene encoding aromatic amino acid decarboxylase in 'Vidal blanc' grape berries.

Science.gov (United States)

Pan, Qiu-Hong; Chen, Fang; Zhu, Bao-Qing; Ma, Li-Yan; Li, Li; Li, Jing-Ming

2012-04-01

The pleasantly fruity and floral 2-phenylethanol are a dominant aroma compound in post-ripening 'Vidal blanc' grapes. However, to date little has been reported about its synthetic pathway in grapevine. In the present study, a full-length cDNA of VvAADC (encoding aromatic amino acid decarboxylase) was firstly cloned from the berries of 'Vidal blanc', an interspecific hybrid variety of Vitis vinifera × Vitis riparia. This sequence encodes a complete open reading frame of 482 amino acids with a calculated molecular mass of 54 kDa and isoelectric point value (pI) of 5.73. The amino acid sequence deduced shared about 79% identity with that of aromatic L: -amino acid decarboxylases (AADCs) from tomato. Real-time PCR analysis indicated that VvAADC transcript abundance presented a small peak at 110 days after full bloom and then a continuous increase at the berry post-ripening stage, which was consistent with the accumulation of 2-phenylethanol, but did not correspond to the trends of two potential intermediates, phenethylamine and 2-phenylacetaldehyde. Furthermore, phenylalanine still exhibited a continuous increase even in post-ripening period. It is thus suggested that 2-phenylethanol biosynthetic pathway mediated by AADC exists in grape berries, but it has possibly little contribution to a considerable accumulation of 2-phenylethanol in post-ripening 'Vidal blanc' grapes.

Human reproductive cloning: a conflict of liberties.

Science.gov (United States)

Havstad, Joyce C

2010-02-01

Proponents of human reproductive cloning do not dispute that cloning may lead to violations of clones' right to self-determination, or that these violations could cause psychological harms. But they proceed with their endorsement of human reproductive cloning by dismissing these psychological harms, mainly in two ways. The first tactic is to point out that to commit the genetic fallacy is indeed a mistake; the second is to invoke Parfit's non-identity problem. The argument of this paper is that neither approach succeeds in removing our moral responsibility to consider and to prevent psychological harms to cloned individuals. In fact, the same commitment to personal liberty that generates the right to reproduce by means of cloning also creates the need to limit that right appropriately. Discussion of human reproductive cloning ought to involve a careful and balanced consideration of both the relevant aspects of personal liberty - the parents' right to reproductive freedom and the cloned child's right to self-determination.

Changes in the gut microbiota of cloned and non-cloned control pigs during development of obesity: gut microbiota during development of obesity in cloned pigs

DEFF Research Database (Denmark)

Pedersen, Rebecca; Andersen, Anders Daniel; Mølbak, Lars

2013-01-01

Background Obesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model...... suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs....... non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non...

Probabilistic cloning of three symmetric states

International Nuclear Information System (INIS)

Jimenez, O.; Bergou, J.; Delgado, A.

2010-01-01

We study the probabilistic cloning of three symmetric states. These states are defined by a single complex quantity, the inner product among them. We show that three different probabilistic cloning machines are necessary to optimally clone all possible families of three symmetric states. We also show that the optimal cloning probability of generating M copies out of one original can be cast as the quotient between the success probability of unambiguously discriminating one and M copies of symmetric states.

Molecular cloning of a cDNA and chromosomal localization of a human theta-class glutathione S-transferase gene (GSTT2) to chromosome 22

Energy Technology Data Exchange (ETDEWEB)

Tan, K.L.; Baker, R.T.; Board, P.G. [Australian National Univ., Canberra (Australia)] [and others

1995-01-20

Until recently the Theta-class glutathione S-transferases (GSTs) were largely overlooked due to their low activity with the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) and their failure to bind to immobilized glutathione affinity matrices. Little is known about the number of genes in this class. Recently, Pemble et al. reported the cDNA cloning of a human Theta-class GST, termed GSTT1. In this study, we describe the molecular cloning of a cDNA encoding a second human Theta-class GST (GSTT2) from a {lambda}gt11 human liver 5{prime}-stretch cDNA library. The encoded protein contains 244 amino acids and has 78.3% sequence identity with the rat subunit 12 and only 55.0% identity with human GSTT1. GSTT2 has been mapped to chromosome 22 by somatic cell hybrid analysis. The precise position of the gene was localized to subband 22q11.2 by in situ hybridization. The absence of other regions of hybridization suggests that there are no closely related sequences (e.g., reverse transcribed pseudogenes) scattered throughout the genome and that if there are closely related genes, they must be clustered near GSTT2. Southern blot analysis of human DNA digested with BamHI shows that the size of the GSTT2 gene is relatively small, as the coding sequence falls within a 3.6-kb BamHI fragment. 35 refs., 6 figs.

HIGH-THROUGHPUT IDENTIFICATION OF THE PREDOMINANT MALARIA PARASITE CLONE IN COMPLEX BLOOD STAGE INFECTIONS USING A MULTI-SNP MOLECULAR HAPLOTYPING ASSAY

Science.gov (United States)

COLE-TOBIAN, JENNIFER L.; ZIMMERMAN, PETER A.; KING, CHRISTOPHER L.

2013-01-01

Individuals living in malaria endemic areas are often infected with multiple parasite clones. Currently used single nucleotide polymorphism (SNP) genotyping methods for malaria parasites are cumbersome; furthermore, few methods currently exist that can rapidly determine the most abundant clone in these complex infections. Here we describe an oligonucleotide ligation assay (OLA) to distinguish SNPs in the Plasmodium vivax Duffy binding protein gene (Pvdbp) at 14 polymorphic residues simultaneously. Allele abundance is determined by the highest mean fluorescent intensity of each allele. Using mixtures of plasmids encoding known haplotypes of the Pvdbp, single clones of P. vivax parasites from infected Aotus monkeys, and well-defined mixed infections from field samples, we were able to identify the predominant Pvdbp genotype with > 93% accuracy when the dominant clone is twice as abundant as a lesser genotype and > 97% of the time if the ratio was 5:1 or greater. Thus, the OLA can accurately, reproducibly, and rapidly determine the predominant parasite haplotype in complex blood stage infections. PMID:17255222

BIOETHICS AND HUMAN CLONING

Directory of Open Access Journals (Sweden)

Željko Kaluđerović

2011-12-01

Full Text Available In this paper the authors analyze the process of negotiating and beginning of the United Nations Declaration on Human Cloning as well as the paragraphs of the very Declaration. The negotiation was originally conceived as a clear bioethical debate that should have led to a general agreement to ban human cloning. However, more often it had been discussed about human rights, cultural, civil and religious differences between people and about priorities in case of eventual conflicts between different value systems. In the end, a non-binding Declaration on Human Cloning had been adopted, full of numerous compromises and ambiguous formulations, that relativized the original intention of proposer states. According to authors, it would have been better if bioethical discussion and eventual regulations on cloning mentioned in the following text had been left over to certain professional bodies, and only after the public had been fully informed about it should relevant supranational organizations have taken that into consideration.

cDNA cloning and mRNA expression of heat shock protein 70 gene ...

African Journals Online (AJOL)

In this study, the full-length heat shock protein 70 of Tegillarca granosa was cloned from cDNA library by rapid amplification of cDNA end (RACE). The open reading frame (ORF) of heat shock protein 70 was 1968 bp, and it encoded a protein of 655 amino acids with a predicted molecular weight of 71.48 kDa and an ...

Molecular biology - Part I: Techniques, terminology, and concepts

International Nuclear Information System (INIS)

Brown, J. Martin

1996-01-01

Purpose/Objective: One of the barriers to understanding modern molecular biology is the lack of a clear understanding of the relevant terminology, techniques, and concepts. This refresher course is intended to address these deficiencies starting from a basic level. The lecture will cover many of the common uses of recombinant DNA, including gene cloning and manipulation. The goal is to enable the nonspecialist to increase his or her understanding of molecular biology in order to more fully enjoy reading current publications and/or listening seminars. Radiation biologists trying to understand a little more molecular biology should also benefit. The following concepts will be among those explained and illustrated: restriction endonucleases, gel electrophoresis, gene cloning, use of vectors such as plasmids, bacteriophage, cosmids and viruses, cDNA and genomic libraries, Southern, Northern, and Western blotting, fluorescent in situ hybridization, polymerase chain reaction (PCR), gel retardation, and reporter gene assays

Methods for plant molecular biology

National Research Council Canada - National Science Library

Weissbach, Arthur; Weissbach, Herbert

1988-01-01

.... Current techniques to carry out plant cell culture and protoplast formation are described as are methods for gene and organelle transfer. The detection of DNA and RNA viruses by molecular probes or ELISA assays and the cloning and transcription of viral RNA complete the volume.

Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula.

Science.gov (United States)

Zhu, Yue; Peng, Qingzhong; Li, Kegang; Xie, De-Yu

2018-04-10

Vitis bellula is a new grape crop in southern China. Berries of this species are rich in antioxidative anthocyanins and proanthocyanidins. This study reports cloning and functional characterization of a cDNA encoding a V. bellula dihydroflavonol reductase (VbDFR) involved in the biosynthesis of anthocyanins and proanthocyanidins. A cDNA including 1014 bp was cloned from young leaves and its open reading frame (ORF) was deduced encoding 337 amino acids, highly similar to V. vinifera DFR (VvDFR). Green florescence protein fusion and confocal microscopy analysis determined the cytosolic localization of VbDFR in plant cells. A soluble recombinant VbDFR was induced and purified from E. coli for enzyme assay. In the presence of NADPH, the recombinant enzyme catalyzed dihydrokaempferol (DHK) and dihydroquercetin (DHQ) to their corresponding leucoanthocyanidins. The VbDFR cDNA was introduced into tobacco plants via Agrobacterium -mediated transformation. The overexpression of VbDFR increased anthocyanin production in flowers. Anthocyanin hydrolysis and chromatographic analysis revealed that transgenic flowers produced pelargonidin and delphinidin, which were not detected in control flowers. These data demonstrated that the overexpression of VbDFR produced new tobacco anthocyanidins. In summary, all data demonstrate that VbDFR is a useful gene to provide three types of substrates for metabolic engineering of anthocyanins and proanthocyanidins in grape crops and other crops.

Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula

Directory of Open Access Journals (Sweden)

Yue Zhu

2018-04-01

Full Text Available Vitis bellula is a new grape crop in southern China. Berries of this species are rich in antioxidative anthocyanins and proanthocyanidins. This study reports cloning and functional characterization of a cDNA encoding a V. bellula dihydroflavonol reductase (VbDFR involved in the biosynthesis of anthocyanins and proanthocyanidins. A cDNA including 1014 bp was cloned from young leaves and its open reading frame (ORF was deduced encoding 337 amino acids, highly similar to V. vinifera DFR (VvDFR. Green florescence protein fusion and confocal microscopy analysis determined the cytosolic localization of VbDFR in plant cells. A soluble recombinant VbDFR was induced and purified from E. coli for enzyme assay. In the presence of NADPH, the recombinant enzyme catalyzed dihydrokaempferol (DHK and dihydroquercetin (DHQ to their corresponding leucoanthocyanidins. The VbDFR cDNA was introduced into tobacco plants via Agrobacterium-mediated transformation. The overexpression of VbDFR increased anthocyanin production in flowers. Anthocyanin hydrolysis and chromatographic analysis revealed that transgenic flowers produced pelargonidin and delphinidin, which were not detected in control flowers. These data demonstrated that the overexpression of VbDFR produced new tobacco anthocyanidins. In summary, all data demonstrate that VbDFR is a useful gene to provide three types of substrates for metabolic engineering of anthocyanins and proanthocyanidins in grape crops and other crops.

Molecular cloning and characterization of a Streptococcus sanguis DNase necessary for repair of DNA damage induced by UV light and methyl methanesulfonate

International Nuclear Information System (INIS)

Lindler, L.E.; Macrina, F.L.

1987-01-01

We developed a method for cloning cellular nucleases from streptococci. Recombinant lambda gt11 bacteriophage containing streptococcal nuclease determinants were identified by the production of pink plaques on toluidine blue O DNase plates. We used this technique to clone a 3.2-kilobase-pair EcoRI fragment with DNase activity from the chromosome of Streptococcus sanguis. The locus was designated don (DNase one) and could be subcloned and stably maintained on plasmid vectors in Escherichia coli. Minicell analyses of various subclones of the don locus allowed us to determine the coding region and size of the Don nuclease in E. coli. The don gene product had an apparent molecular mass of 34 kilodaltons and degraded native DNA most efficiently, with lesser activity against denatured DNA and no detectable activity against RNA. S. sanguis don deletion mutants were constructed by transformation of competent cells with in vitro-prepared plasmid constructs. S. sanguis don deletion mutants retained normal transformation frequencies for exogenously added donor DNA. However, when compared with Don+ wild-type cells, these mutants were hypersensitive to DNA damage induced by UV light and methyl methanesulfonate. An S. sanguis don-specific DNA probe detected homology to chromosomal DNA isolated from Streptococcus pneumoniae and Streptococcus mutans Bratthall serogroups d and g. Our results suggested that the don locus was the S. sanguis allele of the previously described S. pneumoniae major exonuclease and was involved in repair of DNA damage. Furthermore, hybridization studies suggested that the don locus was conserved among species of oral streptococci

Molecular Cloning and Function of FAS/APO1 Associated Protein in Breast Cancer.

Science.gov (United States)

1996-06-01

Ariyama T, Abe T, Druck T, Ohta M, Huebner K, Yanagisawa J, Reed JC, Sato T: PTPN13, a Fas-associated protein tyrosine phosphatase, is located on...20. Yang, Q., and Tonks, N. K. (1991). Isolation of a cDNA clone encoding a human protein-tyrosine phosphatase with homology 7. Huebner, K., Druck , T...Acad. Sci. U.S.A. 91, 7477 (1994). Res. 53, 1945 (1993).(Fig. 3D ). In contrast to Jurkat cells which 13. The original description of PTP-BAS (12

Molecular Cloning, Expression, and In Silico Structural Analysis of Guinea Pig IL-17

OpenAIRE

Dirisala, Vijaya R.; Jeevan, Amminikutty; Ramasamy, Suresh K.; McMurray, David N.

2013-01-01

Interleukin-17A (IL-17A) is a potent proinflammatory cytokine and the signature cytokine of Th17 cells, a subset which is involved in cytokine and chemokine production, neutrophil recruitment, promotion of T cell priming, and antibody production. IL-17 may play an important role in tuberculosis and other infectious diseases. In preparation for investigating its role in the highly relevant guinea pig model of pulmonary tuberculosis, we cloned guinea pig IL-17A for the first time. The complete ...

The First Human Cloned Embryo.

Science.gov (United States)

Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

2002-01-01

Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

Molecular cloning, expression, and characterization of mouse amine N-sulfotransferases

International Nuclear Information System (INIS)

Takahashi, Saki; Sakakibara, Yoichi; Mishiro, Emi; Kouriki, Haruna; Nobe, Rika; Kurogi, Katsuhisa; Yasuda, Shin; Liu, M.-C.; Suiko, Masahito

2008-01-01

By searching the GenBank database, we recently identified a novel mouse cytosolic sulfotransferase (SULT) cDNA (IMAGE Clone ID 679629) and a novel mouse SULT gene (LOC 215895). Sequence analysis revealed that both mouse SULTs belong to the cytosolic SULT3 gene family. The recombinant form of these two newly identified SULTs, designated SULT3A1 and SULT3A2, were expressed using the pGEX-4T-1 glutathione S-transferase fusion system and purified from transformed BL21 Escherichia coli cells. Both purified SULT3A1 and SULT3A2 exhibited strong amine N-sulfonating activities toward 1-naphthylamine among a variety of endogenous and xenobiotic compounds tested as substrates. Kinetic constants of the sulfation of 1-naphthylamine and 1-naphthol by these two enzymes were determined. Collectively, these results imply that these two amine-sulfonating SULT3s may play essential roles in the metabolism and detoxification of aromatic amine compounds in the body

Molecular cloning, expression, and characterization of four novel thermo-alkaliphilic enzymes retrieved from a metagenomic library.

Science.gov (United States)

Maruthamuthu, Mukil; van Elsas, Jan Dirk

2017-01-01

Enzyme discovery is a promising approach to aid in the deconstruction of recalcitrant plant biomass in an industrial process. Novel enzymes can be readily discovered by applying metagenomics on whole microbiomes. Our goal was to select, examine, and characterize eight novel glycoside hydrolases that were previously detected in metagenomic libraries, to serve biotechnological applications with high performance. Here, eight glycosyl hydrolase family candidate genes were selected from metagenomes of wheat straw-degrading microbial consortia using molecular cloning and subsequent gene expression studies in Escherichia coli. Four of the eight enzymes had significant activities on either p NP-β-d-galactopyranoside, p NP-β-d-xylopyranoside, p NP-α-l-arabinopyranoside or p NP-α-d-glucopyranoside. These proteins, denoted as proteins 1, 2, 5 and 6, were his-tag purified and their nature and activities further characterized using molecular and activity screens with the p NP-labeled substrates. Proteins 1 and 2 showed high homologies with (1) a β-galactosidase (74%) and (2) a β-xylosidase (84%), whereas the remaining two (5 and 6) were homologous with proteins reported as a diguanylate cyclase and an aquaporin, respectively. The β-galactosidase- and β-xylosidase-like proteins 1 and 2 were confirmed as being responsible for previously found thermo-alkaliphilic glycosidase activities of extracts of E. coli carrying the respective source fosmids. Remarkably, the β-xylosidase-like protein 2 showed activities with both p NP-Xyl and p NP-Ara in the temperature range 40-50 °C and pH range 8.0-10.0. Moreover, proteins 5 and 6 showed thermotolerant α-glucosidase activity at pH 10.0. In silico structure prediction of protein 5 revealed the presence of a potential "GGDEF" catalytic site, encoding α-glucosidase activity, whereas that of protein 6 showed a "GDSL" site, encoding a 'new family' α-glucosidase activity. Using a rational screening approach, we identified and

Skewed X-inactivation in cloned mice

International Nuclear Information System (INIS)

Senda, Sho; Wakayama, Teruhiko; Yamazaki, Yukiko; Ohgane, Jun; Hattori, Naka; Tanaka, Satoshi; Yanagimachi, Ryuzo; Shiota, Kunio

2004-01-01

In female mammals, dosage compensation for X-linked genes is accomplished by inactivation of one of two X chromosomes. The X-inactivation ratio (a percentage of the cells with inactivated maternal X chromosomes in the whole cells) is skewed as a consequence of various genetic mutations, and has been observed in a number of X-linked disorders. We previously reported that phenotypically normal full-term cloned mouse fetuses had loci with inappropriate DNA methylation. Thus, cloned mice are excellent models to study abnormal epigenetic events in mammalian development. In the present study, we analyzed X-inactivation ratios in adult female cloned mice (B6C3F1). Kidneys of eight naturally produced controls and 11 cloned mice were analyzed. Although variations in X-inactivation ratio among the mice were observed in both groups, the distributions were significantly different (Ansary-Bradley test, P < 0.01). In particular, 2 of 11 cloned mice showed skewed X-inactivation ratios (19.2% and 86.8%). Similarly, in intestine, 1 of 10 cloned mice had a skewed ratio (75.7%). Skewed X-inactivation was observed to various degrees in different tissues of different individuals, suggesting that skewed X-inactivation in cloned mice is the result of secondary cell selection in combination with stochastic distortion of primary choice. The present study is the first demonstration that skewed X-inactivation occurs in cloned animals. This finding is important for understanding both nuclear transfer technology and etiology of X-linked disorders

Comparison of polysaccharides of Haliotis discus hannai and Volutharpa ampullacea perryi by PMP-HPLC-MS(n) analysis upon acid hydrolysis.

Science.gov (United States)

Wang, Hongxu; Zhao, Jun; Li, Dongmei; Wen, Chengrong; Liu, Haiman; Song, Shuang; Zhu, Beiwei

2015-10-13

Haliotis discus hannai Ino (Haliotis) is a highly valued marine shellfish, and it is sometimes replaced by another cheaper Gastropoda mollusk, Volutharpa ampullacea perryi (Volutharpa). Polysaccharides from pleopods, viscera and gonads of these two gastropods were compared by analyzing the mono- and di-saccharides in their acid hydrolysates using high performance liquid chromatography-mass spectrometry (HPLC-MS(n)) after 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization. Disaccharide analysis revealed the distribution of uronic acid-containing polysaccharides (UACPs) in the biological samples. GlcA-(1 → 2)-Man, GlcA-(1 → 3)-GalN, and another disaccharide consisting of a hexuronic acid linked to a hexose were found in the hydrolysates, which indicated the existence of AGSP (abalone gonad sulfated polysaccharide) with the backbone composed of → 2)-α-Man(1 → 4)-β-GlcA(1 → repeating unit, AAP (abalone glycosaminoglycan-like polysaccharide) with the backbone of → 3)-GalNAc-(1 → 2)-GlcA-(1 → 3)-GalNAc-(1 → 4)-GlcA-(1 → repeating unit, and unidentified DS1P containing a hexuronic acid linked to a hexose unit, respectively. As shown by extracted ion chromatograms (XICs), AAP was the only UACP found in pleopods of the two gastropods; gonads and viscera of Haliotis contained DS1P and AGSP, while those of Volutharpa contained DS1P, AGSP as well as AAP. Monosaccharides in the acid hydrolysates were demonstrated in XICs by extracting their corresponding PMP derivative quasi-molecular ions one by one, and the results indicated the similar conclusion to the disaccharide analysis. Therefore, it could be concluded that polysaccharides from pleopods of the two gastropods are very similar, while those from their viscera and gonads differ greatly. Copyright © 2015 Elsevier Ltd. All rights reserved.

Species-specific challenges in dog cloning.

Science.gov (United States)

Kim, G A; Oh, H J; Park, J E; Kim, M J; Park, E J; Jo, Y K; Jang, G; Kim, M K; Kim, H J; Lee, B C

2012-12-01

Somatic cell nuclear transfer (SCNT) is now an established procedure used in cloning of several species. SCNT in dogs involves multiple steps including the removal of the nuclear material, injection of a donor cell, fusion, activation of the reconstructed oocytes and finally transfer to a synchronized female recipient. There are therefore many factors that contribute to cloning efficiency. By performing a retrospective analysis of 2005-2012 published papers regarding dog cloning, we define the optimum procedure and summarize the specific feature for dog cloning. © 2012 Blackwell Verlag GmbH.

Assessment of the genetic diversity of natural rubber tree clones of the SINCHI Institutes clone collection, using of morphological descriptors

International Nuclear Information System (INIS)

Quesada Mendez, Isaac; Quintero Barrera, Lorena; Aristizabal, Fabio A; Rodriguez Acuna, Olga

2011-01-01

Genetic diversity of natural rubber clones of the in SINCHI Institute’s clone collection was assessed. Clones of Hevea brasiliensis (Willd. ex Adr. De Juss.) Muell.Arg., Hevea spp. (H. brasiliensis x H. benthamiana), and three more species of Hevea genus are a part of the collection. Seventy-two materials were characterized with twenty-eight morphological descriptors. They were later used to generate a similarity matrix through the analysis of multi-categorical variables, and to obtain clusters based on the matrix. A low variability between clones of H. brasiliensis and H. spp. was observed, presumably because of the direct descendants of most of the materials from crosses of parental PB 80, PB 5/51, PB 49 and Tjir, exception made of clone GU 1410. Clustering between some materials product of exclusive cross of PB series, a group between clones descendants of parental clones PB 86, and clustering between descendants of parental clones PB 5/51, were observed. Clones from other species of Hevea differ from this big group.

Molecular characterization of two sweepoviruses from China and evaluation of the infectivity of cloned SPLCV-JS in Nicotiana benthamiana.

Science.gov (United States)

Bi, Huiping; Zhang, Peng

2012-03-01

Sweepoviruses are important begomoviruses that infect Ipomoea plants worldwide and cause sweet potato yield losses and cultivar decline. Two sweepoviruses, sweet potato leaf curl virus-Jiangsu (SPLCV-JS) and sweet potato leaf curl China virus-Zhejiang (SPLCCNV-ZJ), were cloned from diseased sweet potato plants collected in the Jiangsu and Zhejiang provinces of China. Sequence characterization and phylogenetic analysis demonstrated that both are typical monopartite begomoviruses and have close relationships to several reported SPLCV and SPLCCNV isolates, respectively, from Asian countries. Analysis of the protein alignments and subcellular localizations of the six SPLCV-JS proteins was also conducted to verify their putative functions. In Nicotiana benthamiana, an infectivity assay of the infectious SPLCV-JS clone resulted in mild symptoms and weak viral DNA accumulation. Interestingly, SPLCV-JS, together with a heterologous betasatellite DNA (tomato yellow leaf curl China virus isolate Y10 [TYLCCNV-Y10] DNA-β), showed a synergistic effect on enhanced symptom severity and viral DNA accumulation. This is the first reported infectious SPLCV clone.

Molecular cloning and sequence analysis of hamster CENP-A cDNA

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Valdivia Manuel M

2002-05-01

Full Text Available Abstract Background The centromere is a specialized locus that mediates chromosome movement during mitosis and meiosis. This chromosomal domain comprises a uniquely packaged form of heterochromatin that acts as a nucleus for the assembly of the kinetochore a trilaminar proteinaceous structure on the surface of each chromatid at the primary constriction. Kinetochores mediate interactions with the spindle fibers of the mitotic apparatus. Centromere protein A (CENP-A is a histone H3-like protein specifically located to the inner plate of kinetochore at active centromeres. CENP-A works as a component of specialized nucleosomes at centromeres bound to arrays of repeat satellite DNA. Results We have cloned the hamster homologue of human and mouse CENP-A. The cDNA isolated was found to contain an open reading frame encoding a polypeptide consisting of 129 amino acid residues with a C-terminal histone fold domain highly homologous to those of CENP-A and H3 sequences previously released. However, significant sequence divergence was found at the N-terminal region of hamster CENP-A that is five and eleven residues shorter than those of mouse and human respectively. Further, a human serine 7 residue, a target site for Aurora B kinase phosphorylation involved in the mechanism of cytokinesis, was not found in the hamster protein. A human autoepitope at the N-terminal region of CENP-A described in autoinmune diseases is not conserved in the hamster protein. Conclusions We have cloned the hamster cDNA for the centromeric protein CENP-A. Significant differences on protein sequence were found at the N-terminal tail of hamster CENP-A in comparison with that of human and mouse. Our results show a high degree of evolutionary divergence of kinetochore CENP-A proteins in mammals. This is related to the high diverse nucleotide repeat sequences found at the centromere DNA among species and support a current centromere model for kinetochore function and structural

Similaridade genética entre clones de seringueira (Hevea brasiliensis, por meio de marcadores RAPD Genetic similarity among rubber tree (Hevea brasiliensis clones using RAPD markers

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Karine Cristina Bicalho

2008-10-01

Full Text Available A seringueira [Hevea brasiliensis (Willd. ex. Adr. de Juss Muell.-Arg.] é uma espécie nativa da região amazônica e compreende a maior fonte produtora de borracha natural do mundo. Na busca de condições mais favoráveis ao cultivo, além da busca pela auto-suficiência na produção de borracha natural, o cultivo da seringueira migrou para outras regiões do país. Objetivou-se, com o presente trabalho, estimar a similaridade genética de genótipos de seringueira, provenientes de regiões distintas do país, Lavras-MG (UFLA e Campinas-SP (IAC, por meio de marcadores moleculares RAPD. A análise foi efetuada em 41 indivíduos, representados por 17 genótipos diferentes, com base em 19 primers, que geraram 121 fragmentos polimórficos. Os dados foram analisados utilizando o software NTSYS-pc - 2.1, por meio do coeficiente de Dice e pelo método das médias (UPGMA. A similaridade genética entre o material analisado variou de 0,56 a 1,00. Na análise do dendrograma, foram observados 18 grupos. Os clones (RRIM600, GT1, PB235, PL PIM e FX2261, utilizados em diferentes repetições, foram idênticos, quando comparados entre si, entretanto o mesmo não foi observado para os clones identificados como RRIM 701. Os resultados obtidos sugerem que o material avaliado na UFLA é o mesmo implantado no IAC, exceto o RRIM 701, mostrando uma ampla variabilidade genética, disponível para estudos e propagação da cultura.The rubber tree [Hevea brasiliensis (Willd. ex. Adr. de Juss Muell.-Arg.] is a native species from Amazon region, and represents the biggest source of natural rubber in the world.. However, the rubber tree culture has had an expansion to other brazilian regions, in search of more favorable conditions for its cultivation and self-sufficiency in natural rubber. The aim of this work was to estimate genetic similarity among rubber tree clones, from different Brazilian regions, Lavras (UFLA and Campinas (IAC, by using RAPD molecular markers

Serine esterase and hemolytic activity in human cloned cytotoxic T lymphocytes

OpenAIRE

1988-01-01

Target cell lysis by most murine cytotoxic T lymphocytes appears to be mediated by a complement (C9)-like protein called perforin, contained in high-density cytoplasmic granules. These granules also contain high levels of serine esterase activity, which may also play a role in cytolysis. Analysis of 17 cloned human cytotoxic T lymphocytes revealed the presence of serine esterase that is very similar to its murine counterpart in substrate and inhibitor specificities, pH optimum, and molecular ...

Unified universal quantum cloning machine and fidelities

Energy Technology Data Exchange (ETDEWEB)

Wang Yinan; Shi Handuo; Xiong Zhaoxi; Jing Li; Mu Liangzhu [School of Physics, Peking University, Beijing 100871 (China); Ren Xijun [School of Physics and Electronics, Henan University, Kaifeng 4750011 (China); Fan Heng [Institute of Physics, Chinese Academy of Sciences, Beijing 100190 (China)

2011-09-15

We present a unified universal quantum cloning machine, which combines several different existing universal cloning machines together, including the asymmetric case. In this unified framework, the identical pure states are projected equally into each copy initially constituted by input and one half of the maximally entangled states. We show explicitly that the output states of those universal cloning machines are the same. One importance of this unified cloning machine is that the cloning procession is always the symmetric projection, which reduces dramatically the difficulties for implementation. Also, it is found that this unified cloning machine can be directly modified to the general asymmetric case. Besides the global fidelity and the single-copy fidelity, we also present all possible arbitrary-copy fidelities.

Survival of Skin Graft between Transgenic Cloned Dogs and Non-Transgenic Cloned Dogs

Science.gov (United States)

Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Choi, Jin; Park, Jung Eun; Park, Eun Jung; Lim, Sang Hyun; Yoon, Byung Il; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

2014-01-01

Whereas it has been assumed that genetically modified tissues or cells derived from somatic cell nuclear transfer (SCNT) should be accepted by a host of the same species, their immune compatibility has not been extensively explored. To identify acceptance of SCNT-derived cells or tissues, skin grafts were performed between cloned dogs that were identical except for their mitochondrial DNA (mtDNA) haplotypes and foreign gene. We showed here that differences in mtDNA haplotypes and genetic modification did not elicit immune responses in these dogs: 1) skin tissues from genetically-modified cloned dogs were successfully transplanted into genetically-modified cloned dogs with different mtDNA haplotype under three successive grafts over 63 days; and 2) non-transgenic cloned tissues were accepted into transgenic cloned syngeneic recipients with different mtDNA haplotypes and vice versa under two successive grafts over 63 days. In addition, expression of the inserted gene was maintained, being functional without eliciting graft rejection. In conclusion, these results show that transplanting genetically-modified tissues into normal, syngeneic or genetically-modified recipient dogs with different mtDNA haplotypes do not elicit skin graft rejection or affect expression of the inserted gene. Therefore, therapeutically valuable tissue derived from SCNT with genetic modification might be used safely in clinical applications for patients with diseased tissues. PMID:25372489

Knowledge and attitudes toward human cloning in Israel.

Science.gov (United States)

Barnoy, Sivia; Ehrenfeld, Malka; Sharon, Rina; Tabak, Nili

2006-04-01

The success of mammal cloning in 1997 has brought the issue of human cloning into public discussion. Human cloning has several aspects and potential applications for use in both reproductive and non-reproductive matters. The aim of this study was to evaluate the knowledge and attitudes toward human cloning in Israel. Data from 120 respondents (68 health professionals and 52 non-health professionals), all Jewish, Hebrew speaking with at least 15 years of education each, were collected using two questionnaires that dealt with knowledge and attitudes toward human cloning. Results showed that although health professionals had significantly more knowledge that non-health professionals, all respondents had poor knowledge about cloning. No difference in attitudes was found between the groups. Most respondents opposed human cloning, but more positive attitudes toward non-reproductive cloning were found. The results are discussed in the context of the deficit model. The findings indicate a need to provide information about human cloning to allow people to form their attitudes based on factual knowledge.

Reproductive cloning combined with genetic modification.

Science.gov (United States)

Strong, C

2005-11-01

Although there is widespread opposition to reproductive cloning, some have argued that its use by infertile couples to have genetically related children would be ethically justifiable. Others have suggested that lesbian or gay couples might wish to use cloning to have genetically related children. Most of the main objections to human reproductive cloning are based on the child's lack of unique nuclear DNA. In the future, it may be possible safely to create children using cloning combined with genetic modifications, so that they have unique nuclear DNA. The genetic modifications could be aimed at giving such children genetic characteristics of both members of the couple concerned. Thus, cloning combined with genetic modification could be appealing to infertile, lesbian, or gay couples who seek genetically related children who have genetic characteristics of both members. In such scenarios, the various objections to human reproductive cloning that are based on the lack of genetic uniqueness would no longer be applicable. The author argues that it would be ethically justifiable for such couples to create children in this manner, assuming these techniques could be used safely.

Human cloning and 'posthuman' society.

Science.gov (United States)

Blackford, Russell

2005-01-01

Since early 1997, when the creation of Dolly the sheep by somatic cell nuclear transfer was announced in Nature, numerous government reports, essays, articles and books have considered the ethical problems and policy issues surrounding human reproductive cloning. In this article, I consider what response a modern liberal society should give to the prospect of human cloning, if it became safe and practical. Some opponents of human cloning have argued that permitting it would place us on a slippery slope to a repugnant future society, comparable to that portrayed in Aldous Huxley's novel, Brave New World. I conclude that, leaving aside concerns about safety, none of the psychological or social considerations discussed in this article provides an adequate policy justification for invoking the state's coercive powers to prevent human cloning.

Molecular cloning and evolutionary analysis of the GJA1 (connexin43) gene from bats (Chiroptera).

Science.gov (United States)

Wang, Li; Li, Gang; Wang, Jinhong; Ye, Shaohui; Jones, Gareth; Zhang, Shuyi

2009-04-01

Gap junction protein connexin43 (Cx43), encoded by the GJA1 gene, is the most abundant connexin in the cardiovascular system and was reported as a crucial factor maintaining cardiac electrical conduction, as well as having a very important function in facilitating the recycling of potassium ions from hair cells in the cochlea back into the cochlear endolymph during auditory transduction processes. In mammals, bats are the only taxon possessing powered flight, placing exceptional demand on many organismal processes. To meet the demands of flying, the hearts of bats show many specialties. Moreover, ultrasonic echolocation allows bat species to orientate and often detect and locate food in darkness. In this study, we cloned the full-length coding region of GJA1 gene from 12 different species of bats and obtained orthologous sequences from other mammals. We used the maximum likelihood method to analyse the evolution of GJA1 gene in mammals and the lineage of bats. Our results showed this gene is much conserved in mammals, as well as in bats' lineage. Compared with other mammals, we found one private amino acid substitution shared by bats, which is located on the inner loop domain, as well as some species-specific amino acid substitutions. The evolution rate analyses showed the signature of purifying selection on not only different classification level lineages but also the different domains and amino acid residue sites of this gene. Also, we suggested that GJA1 gene could be used as a good molecular marker to do the phylogenetic reconstruction.

Molecular cloning and expression analysis of a zebrafish novel zinc finger protein gene rnf141

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Wenqian Deng

2009-01-01

Full Text Available ZNF230 is a novel zinc finger gene cloned by our laboratory. In order to understand the potential functions of this gene in vertebrate development, we cloned the zebrafish orthologue of human ZNF230, named rnf141. The cDNA fragment of rnf141 was obtained by rapid amplification of cDNA ends (RACE. The open reading frame (ORF encodes a polypeptide of 222 amino acids which shares 75.65% identity with the human ZNF230. RT-PCR analysis in zebrafish embryo and adult tissues revealed that rnf141 transcripts are maternally derived and that rnf141 mRNA has a broad distribution. Zygotic rnf141 message is strongly localized in the central nervous system, as shown by whole-mount in situ hybridization. Knockdown and over expression of rnf141 can induce abnormal phenotypes, including abnormal development of brain, as well as yolk sac and axis extendsion. Marker gene analysis showed that rnf141 may play a role in normal dorsoventral patterning of zebrafish embryos, suggesting that rnf141 may have a broad function during early development of vertebrates.

Molecular epidemiology of Panton-Valentine leukocidin-positive Staphylococcus aureus in Spain: emergence of the USA300 clone in an autochthonous population.

Science.gov (United States)

Blanco, Raquel; Tristan, Anne; Ezpeleta, Guillermo; Larsen, Anders Rhod; Bes, Michèle; Etienne, Jérôme; Cisterna, Ramon; Laurent, Frédéric

2011-01-01

We characterized all of the Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus isolates collected between 2005 and 2008 in the Bilbao, Spain, area. For the first time, the USA300 clone is reported as predominant among PVL-positive clones in a European autochthonous population, requiring active monitoring of the incidence of USA300 in Spain and throughout Europe.

U.S. consumers attitudes toward farm animal cloning.

Science.gov (United States)

Brooks, Kathleen R; Lusk, Jayson L

2011-10-01

In January 2008, the United States Food and Drug Administration concluded "meat and milk from cattle, swine, and goat clones or their offspring are as safe to eat as food we eat from those species now" (U.S. FDA, 2010). However, cloning remains a very controversial topic. A web-based survey administered by Knowledge Networks was used to determine U.S. consumers' awareness of and attitudes toward meat and milk from cloned cattle. Findings reveal consumers do not differentiate much between products from cloned animals and products from non-cloned animals. Overall consumers are concerned that animal cloning is an unnatural process and that it will lead to human cloning. Copyright © 2011 Elsevier Ltd. All rights reserved.

Local circulating clones of Staphylococcus aureus in Ecuador.

Science.gov (United States)

Zurita, Jeannete; Barba, Pedro; Ortega-Paredes, David; Mora, Marcelo; Rivadeneira, Sebastián

The spread of pandemic Staphylococcus aureus clones, mainly methicillin-resistant S. aureus (MRSA), must be kept under surveillance to assemble an accurate, local epidemiological analysis. In Ecuador, the prevalence of the USA300 Latin American variant clone (USA300-LV) is well known; however, there is little information about other circulating clones. The aim of this work was to identify the sequence types (ST) using a Multiple-Locus Variable number tandem repeat Analysis 14-locus genotyping approach. We analyzed 132 S. aureus strains that were recovered from 2005 to 2013 and isolated in several clinical settings in Quito, Ecuador. MRSA isolates composed 46.97% (62/132) of the study population. Within MRSA, 37 isolates were related to the USA300-LV clone (ST8-MRSA-IV, Panton-Valentine Leukocidin [PVL] +) and 10 were related to the Brazilian clone (ST239-MRSA-III, PVL-). Additionally, two isolates (ST5-MRSA-II, PVL-) were related to the New York/Japan clone. One isolate was related to the Pediatric clone (ST5-MRSA-IV, PVL-), one isolate (ST45-MRSA-II, PVL-) was related to the USA600 clone, and one (ST22-MRSA-IV, PVL-) was related to the epidemic UK-EMRSA-15 clone. Moreover, the most prevalent MSSA sequence types were ST8 (11 isolates), ST45 (8 isolates), ST30 (8 isolates), ST5 (7 isolates) and ST22 (6 isolates). Additionally, we found one isolate that was related to the livestock associated S. aureus clone ST398. We conclude that in addition to the high prevalence of clone LV-ST8-MRSA-IV, other epidemic clones are circulating in Quito, such as the Brazilian, Pediatric and New York/Japan clones. The USA600 and UK-EMRSA-15 clones, which were not previously described in Ecuador, were also found. Moreover, we found evidence of the presence of the livestock associated clone ST398 in a hospital environment. Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

Characterization of the Brazilian endemic clone of methicillin-resistant Staphylococcus aureus (MRSA from hospitals throughout Brazil

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Geraldo A. Oliveira

Full Text Available The objective of this study was to characterize patterns of the Brazilian endemic clone of methicillin-resistant Staphylococcus aureus (MRSA from hospitals throughout Brazil. We studied 83 MRSA strains isolated from patients hospitalized in 27 public and private hospitals in 19 cities located in 14 Brazilian states from September, 1995, to June, 1997. The MRSA strains were typed using antibiograms, bacteriophage typing and pulsed field gel electrophoresis (PFGE. The analysis of genomic DNA by PFGE showed that 65 isolates presented the same PFGE pattern. This pattern was present in all of the hospitals studied indicating the presence of an endemic MRSA clone widely disseminated throughout Brazilian hospitals (BEC. All isolates belonging to the BEC proved to be resistant to ciprofloxacin, erythromycin, lincomycin, trimethoprim-sulphamethoxazole, and tetracycline. Variable susceptibility to these drugs was found only in isolates belonging to clones other than the BEC. The results show that, among MRSA, the BEC is common in Brazil. The best method for mapping changes in the frequency of this clone among MRSA is pulsed field gel electrophoresis. Use of molecular mapping is an important tool for monitoring the spread of potentially dangerous microbes.

Molecular cloning and characterization of Aspergillus nidulans cyclophilin B.

Science.gov (United States)

Joseph, J D; Heitman, J; Means, A R

1999-06-01

Cyclophilins are an evolutionarily conserved family of proteins which serve as the intracellular receptors for the immunosuppressive drug cyclosporin A. Here we report the characterization of the first cyclophilin cloned from the filamentous fungus Aspergillus nidulans (CYPB). Sequence analysis of the cypB gene predicts an encoded protein with highest homology to the murine cyclophilin B protein. The sequence similarity includes an N-terminal sequence predicted to target the protein to the endoplasmic reticulum (ER) as well as a C-terminal sequence predicted to retain the mature protein in the ER. The bacterially expressed hexa-histidine tagged protein displays peptidyl-prolyl isomerase activity which is inhibited by cyclosporin A. In the presence of cyclosporin A, the expressed protein also inhibits purified calcineurin. When the endogenous cypB gene was disrupted and placed under the control of the regulatable alcohol dehydrogenase promoter, the strain demonstrated no detectable growth phenotype under conditions which induce or repress cypB transcription. Induction or repression of the cypB gene also did not effect sensitivity of A. nidulans to cyclosporin A. cypB mRNA levels were significantly elevated under severe heat shock conditions, indicating a possible role for the A. nidulans cyclophilin B protein during growth in high stress environments. Copyright 1999 Academic Press.

Local cloning of entangled states

International Nuclear Information System (INIS)

Gheorghiu, Vlad; Yu Li; Cohen, Scott M.

2010-01-01

We investigate the conditions under which a set S of pure bipartite quantum states on a DxD system can be locally cloned deterministically by separable operations, when at least one of the states is full Schmidt rank. We allow for the possibility of cloning using a resource state that is less than maximally entangled. Our results include that: (i) all states in S must be full Schmidt rank and equally entangled under the G-concurrence measure, and (ii) the set S can be extended to a larger clonable set generated by a finite group G of order |G|=N, the number of states in the larger set. It is then shown that any local cloning apparatus is capable of cloning a number of states that divides D exactly. We provide a complete solution for two central problems in local cloning, giving necessary and sufficient conditions for (i) when a set of maximally entangled states can be locally cloned, valid for all D; and (ii) local cloning of entangled qubit states with nonvanishing entanglement. In both of these cases, we show that a maximally entangled resource is necessary and sufficient, and the states must be related to each other by local unitary 'shift' operations. These shifts are determined by the group structure, so need not be simple cyclic permutations. Assuming this shifted form and partially entangled states, then in D=3 we show that a maximally entangled resource is again necessary and sufficient, while for higher-dimensional systems, we find that the resource state must be strictly more entangled than the states in S. All of our necessary conditions for separable operations are also necessary conditions for local operations and classical communication (LOCC), since the latter is a proper subset of the former. In fact, all our results hold for LOCC, as our sufficient conditions are demonstrated for LOCC, directly.

Molecular Cloning and Functional Expression of a Δ9- Fatty Acid Desaturase from an Antarctic Pseudomonas sp. A3

Science.gov (United States)

Garba, Lawal; Mohamad Ali, Mohd Shukuri; Oslan, Siti Nurbaya; Rahman, Raja Noor Zaliha Raja Abd

2016-01-01

Fatty acid desaturase enzymes play an essential role in the synthesis of unsaturated fatty acids. Pseudomonas sp. A3 was found to produce a large amount of palmitoleic and oleic acids after incubation at low temperatures. Using polymerase Chain Reaction (PCR), a novel Δ9- fatty acid desaturase gene was isolated, cloned, and successfully expressed in Escherichia coli. The gene was designated as PA3FAD9 and has an open reading frame of 1,185 bp which codes for 394 amino acids with a predicted molecular weight of 45 kDa. The activity of the gene product was confirmed via GCMS, which showed a functional putative Δ9-fatty acid desaturase capable of increasing the total amount of cellular unsaturated fatty acids of the E. coli cells expressing the gene. The results demonstrate that the cellular palmitoleic acids have increased two-fold upon expression at 15°C using only 0.1 mM IPTG. Therefore, PA3FAD9 from Pseudomonas sp.A3 codes for a Δ9-fatty acid desaturase-like protein which was actively expressed in E. coli. PMID:27494717

Quantum cloning of mixed states in symmetric subspaces

International Nuclear Information System (INIS)

Fan Heng

2003-01-01

Quantum-cloning machine for arbitrary mixed states in symmetric subspaces is proposed. This quantum-cloning machine can be used to copy part of the output state of another quantum-cloning machine and is useful in quantum computation and quantum information. The shrinking factor of this quantum cloning achieves the well-known upper bound. When the input is identical pure states, two different fidelities of this cloning machine are optimal

Cloning of a quantum measurement

International Nuclear Information System (INIS)

Bisio, Alessandro; D'Ariano, Giacomo Mauro; Perinotti, Paolo; Sedlak, Michal

2011-01-01

We analyze quantum algorithms for cloning of a quantum measurement. Our aim is to mimic two uses of a device performing an unknown von Neumann measurement with a single use of the device. When the unknown device has to be used before the bipartite state to be measured is available we talk about 1→2 learning of the measurement, otherwise the task is called 1→2 cloning of a measurement. We perform the optimization for both learning and cloning for arbitrary dimension d of the Hilbert space. For 1→2 cloning we also propose a simple quantum network that achieves the optimal fidelity. The optimal fidelity for 1→2 learning just slightly outperforms the estimate and prepare strategy in which one first estimates the unknown measurement and depending on the result suitably prepares the duplicate.

Cloning of a quantum measurement

Energy Technology Data Exchange (ETDEWEB)

Bisio, Alessandro; D' Ariano, Giacomo Mauro; Perinotti, Paolo; Sedlak, Michal [QUIT Group, Dipartimento di Fisica ' ' A. Volta' ' and INFN, via Bassi 6, I-27100 Pavia (Italy); QUIT Group, Dipartimento di Fisica ' ' A. Volta' ' via Bassi 6, I-27100 Pavia (Italy) and Institute of Physics, Slovak Academy of Sciences, Dubravska cesta 9, SK-845 11 Bratislava (Slovakia)

2011-10-15

We analyze quantum algorithms for cloning of a quantum measurement. Our aim is to mimic two uses of a device performing an unknown von Neumann measurement with a single use of the device. When the unknown device has to be used before the bipartite state to be measured is available we talk about 1{yields}2 learning of the measurement, otherwise the task is called 1{yields}2 cloning of a measurement. We perform the optimization for both learning and cloning for arbitrary dimension d of the Hilbert space. For 1{yields}2 cloning we also propose a simple quantum network that achieves the optimal fidelity. The optimal fidelity for 1{yields}2 learning just slightly outperforms the estimate and prepare strategy in which one first estimates the unknown measurement and depending on the result suitably prepares the duplicate.

Pathogenicity of a Very Virulent Strain of Marek's Disease Herpesvirus Cloned as Infectious Bacterial Artificial Chromosomes

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Lorraine P. Smith

2011-01-01

Full Text Available Bacterial artificial chromosome (BAC vectors containing the full-length genomes of several herpesviruses have been used widely as tools to enable functional studies of viral genes. Marek's disease viruses (MDVs are highly oncogenic alphaherpesviruses that induce rapid-onset T-cell lymphomas in chickens. Oncogenic strains of MDV reconstituted from BAC clones have been used to examine the role of viral genes in inducing tumours. Past studies have demonstrated continuous increase in virulence of MDV strains. We have previously reported on the UK isolate C12/130 that showed increased virulence features including lymphoid organ atrophy and enhanced tropism for the central nervous system. Here we report the construction of the BAC clones (pC12/130 of this strain. Chickens were infected with viruses reconstituted from the pC12/130 clones along with the wild-type virus for the comparison of the pathogenic properties. Our studies show that BAC-derived viruses induced disease similar to the wild-type virus, though there were differences in the levels of pathogenicity between individual viruses. Generation of BAC clones that differ in the potential to induce cytolytic disease provide the opportunity to identify the molecular determinants of increased virulence by direct sequence analysis as well as by using reverse genetics approaches on the infectious BAC clones.

Effects of dietary carbohydrates sources on lipids compositions in abalone, Haliotis discus hannai Ino

Science.gov (United States)

Wang, Weifang; Mai, Kangsen; Zhang, Wenbing; Xu, Wei; Ai, Qinghui; Yao, Chunfeng; Li, Huitao

2009-09-01

A study was conducted to evaluate the effects of dietary carbohydrates on triglyceride, cholesterol and fatty acid concentrations in abalone, Haliotis discus hannai Ino. Six semi-purified diets with different carbohydrates (dextrin, heat-treated wheat starch, wheat starch, corn starch, tapioca starch and potato starch, respectively), all containing a carbohydrate level of 33.5%, were fed to abalone (initial shell length: 29.98 mm ± 0.09 mm; initial weight: 3.42 g ± 0.02 g) for 24 weeks in a recirculation system. The results indicate that serum triglyceride concentrations were significantly ( P abalone fed with dextrin, heat-treated wheat starch and wheat starch than those fed with corn starch, and serum cholesterol concentrations were significantly ( P abalone fed with dextrin, heat-treated wheat starch than those fed with corn starch. Fatty acid C20:4n-6 in the foot muscles were significantly ( P abalone fed with dextrin than those fed with wheat starch, corn starch, tapioca starch and potato starch. Fatty acid C20:4n-6 in hepatopancreas was significantly ( P abalone fed with heat-treated wheat starch than those fed with corn starch, tapioca starch and potato starch. Fatty acid C22:6n-3 in the foot muscles were significantly ( P abalone fed with dextrin and heat-treated wheat starch than those fed with wheat starch and potato starch.

Human embryo cloning prohibited in Hong Kong.

Science.gov (United States)

Liu, Athena

2005-12-01

Since the birth of Dolly (the cloned sheep) in 1997, debates have arisen on the ethical and legal questions of cloning-for-biomedical-research (more commonly termed "therapeutic cloning") and of reproductive cloning using human gametes. Hong Kong enacted the Human Reproductive Technology Ordinance (Cap 561) in 2000. Section 15(1)(e) of this Ordinance prohibits the "replacing of the nucleus of a cell of an embryo with a nucleus taken from any other cell," i.e., nucleus substitution. Section 15(1)(f) prohibits the cloning of any embryo. The scope of the latter, therefore, is arguably the widest, prohibiting all cloning techniques such as cell nucleus replacement, embryo splitting, parthenogenesis, and cloning using stem cell lines. Although the Human Reproductive Technology Ordinance is not yet fully operative, this article examines how these prohibitions may adversely impact on basic research and the vision of the Hong Kong scientific community. It concludes that in light of recent scientific developments, it is time to review if the law offers a coherent set of policies in this area.

Cloning the entanglement of a pair of quantum bits

International Nuclear Information System (INIS)

Lamoureux, Louis-Philippe; Navez, Patrick; Cerf, Nicolas J.; Fiurasek, Jaromir

2004-01-01

It is shown that any quantum operation that perfectly clones the entanglement of all maximally entangled qubit pairs cannot preserve separability. This 'entanglement no-cloning' principle naturally suggests that some approximate cloning of entanglement is nevertheless allowed by quantum mechanics. We investigate a separability-preserving optimal cloning machine that duplicates all maximally entangled states of two qubits, resulting in 0.285 bits of entanglement per clone, while a local cloning machine only yields 0.060 bits of entanglement per clone

Human cloning: Eastern Mediterranean Region perspective.

Science.gov (United States)

Abdur Rab, M; Khayat, M H

2006-01-01

Recent advances in genomics and biotechnology have ushered in a new era in health development. Therapeutic cloning possesses enormous potential for revolutionizing medical and therapeutic techniques. Cloning technology, however, is perceived as having the potential for reproductive cloning, which raises serious ethical and moral concerns. It is important that the Islamic countries come to a consensus on this vital issue. Developing science and technology for better health is a religious and moral obligation. There is an urgent need for Muslim scholars to discuss the issue of stem cell research and cloning rationally; such dialogue will not only consider the scientific merits but also the moral, ethical and legal implications.

T cell epitopes on the 36K and 65K Mycobacterium leprae antigens defined by human T cell clones

NARCIS (Netherlands)

van Schooten, W. C.; Ottenhoff, T. H.; Klatser, P. R.; Thole, J.; de Vries, R. R.; Kolk, A. H.

1988-01-01

To identify the molecular localization and specificity of Mycobacterium leprae antigenic determinants inducing T cell activation, we studied the reactivity of M. leprae-reactive T cell clones from two tuberculoid leprosy patients towards a battery of different mycobacterial strains and purified

Radiation-induced aneusomic clones in bone marrow of rats

International Nuclear Information System (INIS)

Kohno, Sei-Ichi; Ishihara, Takaaki

1976-01-01

Wistar rats 3 months old were given a single whole-body X-irradiation with 700 R. They were killed 9.3 months, on average, after irradiation. From the bone marrows of the 23 irradiated rats, 54 clones of cells with radiation-induced chromosome abnormalities ranging from 3.3 to 78.3% in size were obtained. Karyotype analysis at the banding level showed that 43 out of the 54 clones had balanced chromosome constitutions and that the remaining 11 clones were unbalanced. The 43 balanced clones consisted of 33 clones with reciprocal translocations, 6 with inversions and 4 with both translocations and inversions. The 11 unbalanced clones were made up of 7 aneuploid clones and 4 pseudo-diploid clones. Of the 54 clones, 15 were large with frequencies of more than 25%. Contrary to general belief that cells with unbalanced chromosome constitutions have less capacity to proliferate than those with balanced ones, 8 of the 15 large clones, especially all, except 1, of the largest 6 clones were unbalanced, either aneuploid or pseudo-diploid

Molecular cloning and characterization of the full-length cDNA encoding the tree shrew (tupaia belangeri) CD28

Science.gov (United States)

Huang, Xiaoyan; Yan, Yan; Wang, Sha; Wang, Qinying; Shi, Jian; Shao, Zhanshe; Dai, Jiejie

2017-11-01

CD28 is one of the most important co-stimulatory molecules expressed by naive and primed T cells. The tree shrews (Tupaia belangeri), as an ideal animal model for analyzing mechanism of human diseases receiving extensive attentions, demands essential research tools, in particular in the study of cellular markers and monoclonal antibodies for immunological studies. However, little is known about tree shrew CD28 (tsCD28) until now. In this study, a 663 bp of the full-length CD28 cDNA, encoding a polypeptide of 220 amino acids was cloned from tree shrew spleen lymphocytes. The nucleotide sequence of the tsCD28 showed 85%, 76%, and 75% similarities with human, rat, and mouse, respectively, which showed the affinity relationship between tree shrew and human is much closer than between human and rodents. The open reading frame (ORF) sequence of tsCD28 gene was predicted to be in correspondence with the signal sequence, immunoglobulin variable-like (IgV) domain, transmembrane domain and cytoplasmic tail, respectively.We also analyzed its molecular characteristics with other mammals by using biology software such as Clustal W 2.0 and so forth. Our results showed that tsCD28 contained many features conserved in CD28 genes from other mammals, including conserved signal peptide and glycosylation sites, and several residues responsible for binding to the CD28R, and the tsCD28 amino acid sequence were found a close genetic relationship with human and monkey. The crystal structure and surface charge revealed most regions of tree shrew CD28 molecule surface charges are similar as human. However, compared with human CD28 (hCD28) regions, in some areas, the surface positive charge of tsCD28 was less than hCD28, which may affect antibody binding. The present study is the first report of cloning and characterization of CD28 in tree shrew. This study provides a theoretical basis for the further study the structure and function of tree shrew CD28 and utilize tree shrew as an effective

Porcine ubiquitin-like 5 (UBL5) gene: genomic organization, polymorphisms, mRNA cloning, splicing variants and association study

Czech Academy of Sciences Publication Activity Database

Masopust, Martin; Weisz, Filip; Bartenschlager, H.; Knoll, A.; Vykoukalová, Z.; Geldermann, H.; Čepica, Stanislav

2014-01-01

Roč. 41, č. 4 (2014), s. 2353-2362 ISSN 0301-4851 R&D Projects: GA ČR GAP502/10/1216 Institutional support: RVO:67985904 Keywords : pig * UBL5 * PCR cloning Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.024, year: 2014

PlasmaDNA: a free, cross-platform plasmid manipulation program for molecular biology laboratories

Directory of Open Access Journals (Sweden)

Rainy Jeffrey

2007-09-01

Full Text Available Abstract Background Most molecular biology experiments, and the techniques associated with this field of study, involve a great deal of engineering in the form of molecular cloning. Like all forms of engineering, perfect information about the starting material is crucial for successful completion of design and strategies. Results We have generated a program that allows complete in silico simulation of the cloning experiment. Starting with a primary DNA sequence, PlasmaDNA looks for restriction sites, open reading frames, primer annealing sequences, and various common domains. The databases are easily expandable by the user to fit his most common cloning needs. PlasmaDNA can manage and graphically represent multiple sequences at the same time, and keeps in memory the overhangs at the end of the sequences if any. This means that it is possible to virtually digest fragments, to add the digestion products to the project, and to ligate together fragments with compatible ends to generate the new sequences. Polymerase Chain Reaction (PCR fragments can also be virtually generated using the primer database, automatically adding to the fragments any 5' extra sequences present in the primers. Conclusion PlasmaDNA is a program available both on Windows and Apple operating systems, designed to facilitate molecular cloning experiments by building a visual map of the DNA. It then allows the complete planning and simulation of the cloning experiment. It also automatically updates the new sequences generated in the process, which is an important help in practice. The capacity to maintain multiple sequences in the same file can also be used to archive the various steps and strategies involved in the cloning of each construct. The program is freely available for download without charge or restriction.

Human Cloning

National Research Council Canada - National Science Library

Johnson, Judith A; Williams, Erin D

2006-01-01

.... Scientists in other labs, including Harvard University and the University of California at San Francisco, intend to produce cloned human embryos in order to derive stem cells for medical research...

Molecular cloning and characterization of human papilloma virus DNA derived from a laryngeal papilloma.

OpenAIRE

Gissmann, L; Diehl, V; Schultz-Coulon, H J; zur Hausen, H

1982-01-01

Papilloma virus DNA from a laryngeal papilloma was cloned in phage lambda L 47 and characterized after cleavage with different restriction enzymes. Hybridization with the DNAs of human papilloma virus types 1, 2, 3, 4, 5, and 8 showed no homology under stringent hybridization conditions. Human papilloma virus type 6 DNA, however, was partially identical to laryngeal papilloma virus DNA; different restriction enzyme fragments hybridizing with the other DNA were identified on each genome. The d...

Molecular cloning and functional characterization of the diapause hormone receptor in the corn earworm Helicoverpa zea

Science.gov (United States)

The diapause hormone (DH) in the heliothine moth has shown its activity in termination of pupal diapause, while the orthology in the silkworm is known to induce embryonic diapause. In the current study, we cloned the diapause hormone receptor from the corn earworm Helicoverpa zea (HzDHr) and tested ...

"Goodbye Dolly?" The ethics of human cloning.

Science.gov (United States)

Harris, J

1997-01-01

The ethical implications of human clones have been much alluded to, but have seldom been examined with any rigour. This paper examines the possible uses and abuses of human cloning and draws out the principal ethical dimensions, both of what might be done and its meaning. The paper examines some of the major public and official responses to cloning by authorities such as President Clinton, the World Health Organisation, the European parliament, UNESCO, and others and reveals their inadequacies as foundations for a coherent public policy on human cloning. The paper ends by defending a conception of reproductive rights of "procreative autonomy" which shows human cloning to be not inconsistent with human rights and dignity. PMID:9451604

Chorioallantoic placenta defects in cloned mice

International Nuclear Information System (INIS)

Wakisaka-Saito, Noriko; Kohda, Takashi; Inoue, Kimiko; Ogonuki, Narumi; Miki, Hiromi; Hikichi, Takafusa; Mizutani, Eiji; Wakayama, Teruhiko; Kaneko-Ishino, Tomoko; Ogura, Atsuo; Ishino, Fumitoshi

2006-01-01

Somatic cell nuclear transfer technology has been applied to produce live clones successfully in several mammalian species, but the success rates are very low. In mice, about half of the nuclear transfer embryos undergo implantation, but very few survive to term. We undertook detailed histological analyses of placentas from cloned mouse embryos generated from cumulus cells at 10.5 dpc of pregnancy, by which stage most clones have terminated their development. At 10.5 dpc, the extraembryonic tissues displayed several defined histological patterns, each reflecting their stage of developmental arrest. The most notable abnormality was the poor development of the spongiotrophoblast layer of diploid cells. This is in contrast to the placental hyperplasia frequently observed in somatic clones at 12.5 dpc or later stages. A variety of structural abnormalities were also observed in the embryos. Both placental and embryonic defects likely contribute to the low success rate of the mouse clones

HTP-OligoDesigner: An Online Primer Design Tool for High-Throughput Gene Cloning and Site-Directed Mutagenesis.

Science.gov (United States)

Camilo, Cesar M; Lima, Gustavo M A; Maluf, Fernando V; Guido, Rafael V C; Polikarpov, Igor

2016-01-01

Following burgeoning genomic and transcriptomic sequencing data, biochemical and molecular biology groups worldwide are implementing high-throughput cloning and mutagenesis facilities in order to obtain a large number of soluble proteins for structural and functional characterization. Since manual primer design can be a time-consuming and error-generating step, particularly when working with hundreds of targets, the automation of primer design process becomes highly desirable. HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent gene cloning and site-directed mutagenesis and a Tm calculator for quick queries.

Molecular cloning of a novel bioH gene from an environmental metagenome encoding a carboxylesterase with exceptional tolerance to organic solvents

DEFF Research Database (Denmark)

Shi, Yuping; Pan, Yingjie; Li, Bailin

2013-01-01

with a strong potential in industrial applications. CONCLUSIONS: This study constituted the first investigation of a novel bioHx gene in a biotin biosynthetic gene cluster cloned from an environmental metagenome. The bioHx gene was successfully cloned, expressed and characterized. The results demonstrated...... that BioHx is a novel carboxylesterase, displaying distinct biochemical properties with strong application potential in industry. Our results also provided the evidence for the effectiveness of functional metagenomic approach for identifying novel bioH genes from complex ecosystem.......ABSTRACT: BACKGROUND: BioH is one of the key enzymes to produce the precursor pimeloyl-ACP to initiate biotin biosynthesis de novo in bacteria. To date, very few bioH genes have been characterized. In this study, we cloned and identified a novel bioH gene, bioHx, from an environmental metagenome...

Public perceptions of farm animal cloning in Europe

DEFF Research Database (Denmark)

Lassen, Jesper

This report presents a picture of European opinion on farm animal cloning. In the report, both agricultural and biomedical applications of farm animal cloning are considered. With the arrival of Dolly, animal cloning became an integral part of the biotech debate, but this debate did not isolate...... animal cloning as a single issue....

Lovely clone of coconuts

Energy Technology Data Exchange (ETDEWEB)

Branton, R.; Blake, J.

1983-05-01

It has taken over 10 years research and development to clone oil palms and coconut palms successfully. Unilever has recently built a tissue culture factory in England with a potential capacity for producing half a million clonal oil palms a year for export. Research on the cloning of coconut palms is reported here. Cloned palms may increase yields from oil palms by 20 to 30 percent and yields from coconut could be as high as five-fold over unselected stock. Improved yields would not only increase the yield of oil and copra but also the harvests of husk and shell which are immense potential sources of energy; the 1978 Philippine harvest of over 12 million nuts is equivalent in terms of energy to 3.8 billion litres of petrol (31 x 10/sup 12/ kcal).

Ethical issues regarding human cloning: a nursing perspective.

Science.gov (United States)

Dinç, Leyla

2003-05-01

Advances in cloning technology and successful cloning experiments in animals raised concerns about the possibility of human cloning in recent years. Despite many objections, this is not only a possibility but also a reality. Human cloning is a scientific revolution. However, it also introduces the potential for physical and psychosocial harm to human beings. From this point of view, it raises profound ethical, social and health related concerns. Human cloning would have an impact on the practice of nursing because it could result in the creation of new physiological and psychosocial conditions that would require nursing care. The nursing profession must therefore evaluate the ethics of human cloning, in particular the potential role of nurses. This article reviews the ethical considerations of reproductive human cloning, discusses the main reasons for concern, and reflects a nursing perspective regarding this issue.

[Mystery and problems of cloning].

Science.gov (United States)

Nikitin, V A

2010-01-01

The attention of investigators is attracted to the fact that, in spite of great efforts in mammalian cloning, advances that have been made in this area of research are not great, and cloned animals have developmental pathologies often incompatible with life and/or reproduction ability. It is yet not clear what technical or biological factors underlie this, and how they are connected or interact with each other, which is more realistic strategically. There is a great number of articles dealing with the influence of cloning with the nuclear transfer on genetic and epigenetic reprogramming of donor cells. At the same time we can see the practical absence of analytical investigations concerning the technology of cloning as such, its weak points, and possible sources of cellular trauma in the course of microsurgery of nuclear transfer or twinning. This article discusses step by step several nuclear transfer techniques and the methods of dividing early preimplanted embryos for twinning with the aim to reveal possible sources of cell damage during micromanipulation that may have negative influence on the development of cloned organisms. Several new author's technologies based on the study of cell biophysical characteristics are described, which allow one to avoid cellular trauma during manipulation and minimize the possibility of cell damage at any rate.

Quantum cloning without external control

International Nuclear Information System (INIS)

Chiara, G. de; Fazio, R.; Macchiavello, C.; Montangero, S.; Palma, G.M.

2005-01-01

Full text: In this work we present an approach to quantum cloning with unmodulated spin networks. The cloner is realized by a proper design of the network and a choice of the coupling between the qubits. We show that in the case of phase covariant cloner the XY coupling gives the best results. In the 1 → 2 cloning we find that the value for the fidelity of the optimal cloner is achieved, and values comparable to the optimal ones in the general N → M case can be attained. If a suitable set of network symmetries are satisfied, the output fidelity of the clones does not depend on the specific choice of the graph. We show that spin network cloning is robust against the presence of static imperfections. Moreover, in the presence of noise, it outperforms the conventional approach. In this case the fidelity exceeds the corresponding value obtained by quantum gates even for a very small amount of noise. Furthermore we show how to use this method to clone qutrits and qudits. By means of the Heisenberg coupling it is also possible to implement the universal cloner although in this case the fidelity is 10 % off that of the optimal cloner. (author)

Generation of phase-covariant quantum cloning

International Nuclear Information System (INIS)

Karimipour, V.; Rezakhani, A.T.

2002-01-01

It is known that in phase-covariant quantum cloning, the equatorial states on the Bloch sphere can be cloned with a fidelity higher than the optimal bound established for universal quantum cloning. We generalize this concept to include other states on the Bloch sphere with a definite z component of spin. It is shown that once we know the z component, we can always clone a state with a fidelity higher than the universal value and that of equatorial states. We also make a detailed study of the entanglement properties of the output copies and show that the equatorial states are the only states that give rise to a separable density matrix for the outputs

LAMININS IN COLORECTAL CANCER: EXPRESSION, FUNCTION, PROGNOSTIC POWER AND MOLECULAR MECHANISMS

Directory of Open Access Journals (Sweden)

S. A. Rodin

2017-01-01

Full Text Available Extracellular matrix (ECM proteins are a major component of the tumor stroma. Laminins emerge as one of the main families of ECM proteins with signaling properties. Apart from the structural function, laminins and products of their degradation affect survival and differentiation of cancer cells, motility of cancer and stromal cells, angiogenesis, invasion into distant organs, and other aspects of cancer development. Here, we discus expression of laminins in colorectal cancer (CRC, studying of laminin functions in in vitro and in vivo models of CRC, and using laminins as prognostic markers of CRC. Recently, we have reported a new approach to assessing prognostic power using classifiers constructed from sets of laminin genes. The method allows for accurate prognosis of CRC and provides additional information that may suggest possible molecular mechanisms of laminin function in CRC progression.

Dose dependency of the frequency of micronucleated binucleated clone cells and of division related median clone sizes difference. Pt. 2

International Nuclear Information System (INIS)

Hagemann, G,; Kreczik, A.; Treichel, M.

1996-01-01

Following irradiation of the progenitor cells the clone growth of CHO cells decreases as a result of cell losses. Lethally acting expressions of micronuclei are produced by heritable lethal mutations. The dependency of the frequency of micronucleated binucleated clone cells and of the median clone sizes difference on the radiation dose was measured and compared to non-irradiated controls. Using the cytokinesis-block-micronucleus-method binucleated cells with micronuclei were counted as ratio of all binucleated cells within a clone size distribution. This ratio (shortened: micronucleus yield) was determined for all clone size distributions, which had been exposed to different irradiation doses and incubation times. The micronucleus yields were compared to the corresponding median clone sizes differences. The micronucleus yield is linearly dependent on the dose and is independent of the incubation time. The same holds true for the division related median clone sizes difference, which as a result is also linearly dependent on the micronucleus yield. Due to the inevitably errors of the cell count of micronucleated binucleated cells, an automatic measurement of the median clone sizes differences is the preferred method for evaluation of cellular radiation sensitivity for heritable lethal mutations. This value should always be determined in addition, if clone survival fractions are used as predictive test because it allows for an estimation of the remission probability of surviving cells. (orig.) [de

Molecular cloning and characterization of the recA gene of Pseudomonas aeruginosa PAO

Energy Technology Data Exchange (ETDEWEB)

Kokjohn, T.A.; Miller, R.V.

1985-08-01

The recA gene of Pseudomonas aeruginosa PAO has been isolated and introduced into Escherichia coli K-12. Resistance to killing by UV irradiation was restored in several RecA-E. coli K-12 hosts by the P. aeruginosa gene, as was resistance to methyl methanesulfonate. Recombination proficiency was also restored, as measured by HfrH-mediated conjugation and by the ability to propagate Fec-phage lambda derivatives. The cloned P. aeruginosa recA gene restored both spontaneous and mitomycin C-stimulated induction of lambda prophage in lysogens of a recA strain of E. coli K-12.

Biodiversity versus cloning

International Nuclear Information System (INIS)

Jaramillo T, Jose Hernan

1998-01-01

The announcement has been made on the cloning of mice in these days and he doesn't stop to miss, because the world lives a stage where conscience of the protection is creating that should be given to the biodiversity. It is known that alone we won't subsist and the protection of the means and all that contains that environment is of vital importance for the man. But it is also known that the vegetables and animal transgenic that they come to multiply the species have appeared that we prepare. The transgenic has been altered genetically, for substitution of one or more genes of other species, inclusive human genes. This represents an improvement compared with the investigations that gave origin to the cloning animal. But it is necessary to notice that to it you arrived through the cloning. This year 28 million hectares have been sowed in cultivations of transgenic seeds and there is around 700 bovine transgenic whose milk contains a necessary protein in the treatment of the man's illnesses

Molecular cloning and sequence analysis of growth hormone cDNA of Neotropical freshwater fish Pacu (Piaractus mesopotamicus

Directory of Open Access Journals (Sweden)

Janeth Silva Pinheiro

2008-01-01

Full Text Available RT-PCR was used for amplifying Piaractus mesopotamicus growth hormone (GH cDNA obtained from mRNA extracted from pituitary cells. The amplified fragment was cloned and the complete cDNA sequence was determined. The cloned cDNA encompassed a sequence of 543 nucleotides that encoded a polypeptide of 178 amino acids corresponding to mature P. mesopotamicus GH. Comparison with other GH sequences showed a gap of 10 amino acids localized in the N terminus of the putative polypeptide of P. mesopotamicus. This same gap was also observed in other members of the family. Neighbor-joining tree analysis with GH sequences from fishes belonging to different taxonomic groups placed the P. mesopotamicus GH within the Otophysi group. To our knowledge, this is the first GH sequence of a Neotropical characiform fish deposited in GenBank.

Quantitative discrimination of Aggregatibacter actinomycetemcomitans highly leukotoxic JP2 clone from non-JP2 clones in diagnosis of aggressive periodontitis.

Science.gov (United States)

Yoshida, Akihiro; Ennibi, Oum-Keltoum; Miyazaki, Hideo; Hoshino, Tomonori; Hayashida, Hideaki; Nishihara, Tatsuji; Awano, Shuji; Ansai, Toshihiro

2012-10-11

Aggregatibacter actinomycetemcomitans is the etiological agent of periodontitis, and there is a strong association between clone JP2 and aggressive periodontitis in adolescents of African descent. The JP2 clone has an approximately 530-bp deletion (∆530) in the promoter region of the lkt/ltx gene, which encodes leukotoxin, and this clone has high leukotoxic activity. Therefore, this clone is very important in aggressive periodontitis. To diagnose this disease, culture methods and conventional PCR techniques are used. However, quantitative detection based on qPCR for the JP2 clone has not been developed due to genetic difficulties. In this study, we developed a qPCR-based quantification method specific to the JP2 clone. Based on our analysis of the DNA sequence of the lkt/ltx gene and its flanking region, we designed a reverse primer specific for the ∆530 deletion border sequence and developed a JP2-specific PCR-based quantification method using this primer. We also analyzed the DNA sequence of the ∆530 locus and found it to be highly conserved (97-100%) among 17 non-JP2 strains. Using the ∆530 locus, we designed a qPCR primer-probe set specific to non-JP2 clones. Next, we determined the numbers of JP2 and non-JP2 clone cells in the periodontal pockets of patients with aggressive periodontitis. The JP2-specific primers specifically amplified the genomic DNA of the A. actinomycetemcomitans JP2 clone and did not react with other bacterial DNA, whereas the non-JP2 specific primers reacted only with A. actinomycetemcomitans non-JP2 clones. Samples from the 88 periodontal sites in the 11 patients with aggressive periodontitis were analyzed. The bacterial cell numbers in 88 periodontal sites ranged from 0 to 4.8 × 10(8) (mean 1.28 × 10(7)) for JP2 clones and from 0 to 1.6 × 10(6) for non-JP2 clones (mean 1.84 × 10(5)). There were significant differences in the JP2 cell number between a clinical attachment level (CAL) ≤6 mm and a level ≥7 mm (p clones. This

Cloning and superluminal signaling£

Indian Academy of Sciences (India)

Cloning; cloning fidelity; superluminal signaling; state discrimination. PACS No. 03.65.Bz. 1. .... The possibility of superluminal signaling in quantum mechanics stems from the concept .... quantum mechanics and relativity [13]. .... [13] A Shimony, in Foundations of quantum mechanics in the light of new technology edited by.

Cloning Mice.

Science.gov (United States)

Ogura, Atsuo

2017-08-01

Viable and fertile mice can be generated by somatic nuclear transfer into enucleated oocytes, presumably because the transplanted somatic cell genome becomes reprogrammed by factors in the oocyte. The first somatic cloned offspring of mice were obtained by directly injecting donor nuclei into recipient enucleated oocytes. When this method is used (the so-called Honolulu method of somatic cell nuclear transfer [SCNT]), the donor nuclei readily and completely condense within the enucleated metaphase II-arrested oocytes, which contain high levels of M-phase-promoting factor (MPF). It is believed that the condensation of the donor chromosomes promotes complete reprogramming of the donor genome within the mouse oocytes. Another key to the success of mouse cloning is the use of blunt micropipettes attached to a piezo impact-driving micromanipulation device. This system saves a significant amount of time during the micromanipulation of oocytes and thus minimizes the loss of oocyte viability in vitro. For example, a group of 20 oocytes can be enucleated within 10 min by an experienced operator. This protocol is composed of seven parts: (1) preparing micropipettes, (2) setting up the enucleation and injection micropipettes, (3) collecting and enucleating oocytes, (4) preparing nucleus donor cells, (5) injecting donor nuclei, (6) activating embryos and culturing, and (7) transferring cloned embryos. © 2017 Cold Spring Harbor Laboratory Press.

[Cloning and gene expression in lactic acid bacteria].

Science.gov (United States)

Bondarenko, V M; Beliavskaia, V A

2000-01-01

The possibility of using the genera Lactobacillus and Lactococcus as vector representatives is widely discussed at present. The prospects of the construction of recombinant bacteria are closely connected with the solution of a number of problems: the level of the transcription of cloned genes, the effectiveness of the translation of heterologous mRNA, the stability of protein with respect to bacterial intracellular proteases, the method by protein molecules leave the cell (by secretion or as the result of lysis). To prevent segregation instability, the construction of vector molecules on the basis of stable cryptic plasmids found in wild strains of lactic acid bacteria was proposed. High copying plasmids with low molecular weight were detected in L. plantarum and L. pentosus strains. Several plasmids with molecular weights of 1.7, 1.8 and 2.3 kb were isolated from bacterial cells to be used as the basis for the construction of vector molecules. Genes of chloramphenicol- and erythromycin-resistance from Staphylococcus aureus plasmids were used as marker genes ensuring cell transformation. The vector plasmids thus constructed exhibited high transformation activity in the electroporation of different strains, including L. casei, L. plantarum, L. acidophilus, L. fermentum and L. brevis which could be classified with the replicons of a wide circle of hosts. But the use of these plasmids was limited due to the risk of the uncontrolled dissemination of recombinant plasmids. L. acidophilus were also found to have strictly specific plasmids with good prospects of being used as the basis for the creation of vectors, incapable of dissemination. In addition to the search of strain-specific plasmids, incapable of uncontrolled gene transmission, the use of chromosome-integrated heterologous genes is recommended in cloning to ensure the maximum safety.

Construction of an infectious plasmid clone of Muscovy duck parvovirus by TA cloning and creation of a partially attenuated strain.

Science.gov (United States)

Yen, T-Y; Li, K-P; Ou, S-C; Shien, J-H; Lu, H-M; Chang, P-C

2015-01-01

Muscovy duck parvovirus (MDPV) infection is a highly contagious and fatal disease of Muscovy ducklings. The infectious clone methodology is a valuable tool to study the pathogenic mechanisms of viruses, but no infectious clone of MDPV is yet available. In this study, a plasmid clone containing the full-length genome of MDPV was constructed using the TA cloning methodology. This MDPV clone was found to be infectious after transfection of primary Muscovy duck embryo fibroblast cells and passage in embryonated Muscovy duck eggs. Site-directed mutagenesis showed that the K75N mutation in the VP1 protein of MDPV resulted in the partial attenuation of the virus. The availability of an MDPV infectious clone can facilitate investigation of the pathogenic mechanisms of MDPV and development of vaccines against diseases caused by MDPV.

Quantum cloning machines and their implementation in physical systems

International Nuclear Information System (INIS)

Wu Tao; Ye Liu; Fang Bao-Long

2013-01-01

We review the basic theory of approximate quantum cloning for discrete variables and some schemes for implementing quantum cloning machines. Several types of approximate quantum clones and their expansive quantum clones are introduced. As for the implementation of quantum cloning machines, we review some design methods and recent experimental results. (topical review - quantum information)

Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L.

Science.gov (United States)

Li, Fupeng; Wu, Baoduo; Qin, Xiaowei; Yan, Lin; Hao, Chaoyun; Tan, Lehe; Lai, Jianxiong

2014-08-10

In this study, we performed cloning and expression analysis of six putative sucrose transporter genes, designated TcSUT1, TcSUT2, TcSUT3, TcSUT4, TcSUT5 and TcSUT6, from the cacao genotype 'TAS-R8'. The combination of cDNA and genomic DNA sequences revealed that the cacao SUT genes contained exon numbers ranging from 1 to 14. The average molecular mass of all six deduced proteins was approximately 56 kDa (range 52 to 66 kDa). All six proteins were predicted to exhibit typical features of sucrose transporters with 12 trans-membrane spanning domains. Phylogenetic analysis revealed that TcSUT2 and TcSUT4 belonged to Group 2 SUT and Group 4 SUT, respectively, and the other TcSUT proteins were belonging to Group 1 SUT. Real-time PCR was conducted to investigate the expression pattern of each member of the SUT family in cacao. Our experiment showed that TcSUT1 was expressed dominantly in pods and that, TcSUT3 and TcSUT4 were highly expressed in both pods and in bark with phloem. Within pods, TcSUT1 and TcSUT4 were expressed more in the seed coat and seed from the pod enlargement stage to the ripening stage. TcSUT5 expression sharply increased to its highest expression level in the seed coat during the ripening stage. Expression pattern analysis indicated that TcSUT genes may be associated with photoassimilate transport into developing seeds and may, therefore, have an impact on seed production. Copyright © 2014 Elsevier B.V. All rights reserved.

Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA

International Nuclear Information System (INIS)

Indik, Z.; Yeh, H.; Ornstein-goldstein, N.; Sheppard, P.; Anderson, N.; Rosenbloom, J.C.; Peltonen, L.; Rosenbloom, J.

1987-01-01

Poly(A) + RNA, isolated from a single 7-mo fetal human aorta, was used to synthesize cDNA by the RNase H method, and the cDNA was inserted into λgt10. Recombinant phage containing elastin sequences were identified by hybridization with cloned, exon-containing fragments of the human elastin gene. Three clones containing inserts of 3.3, 2.7, and 2.3 kilobases were selected for further analysis. Three overlapping clones containing 17.8 kilobases of the human elastin gene were also isolated from genomic libraries. Complete sequence analysis of the six clones demonstrated that: (i) the cDNA encompassed the entire translated portion of the mRNA encoding 786 amino acids, including several unusual hydrophilic amino acid sequences not previously identified in porcine tropoelastin, (ii) exons encoding either hydrophobic or crosslinking domains in the protein alternated in the gene, and (iii) a great abundance of Alu repetitive sequences occurred throughout the introns. The data also indicated substantial alternative splicing of the mRNA. These results suggest the potential for significant variation in the precise molecular structure of the elastic fiber in the human population

Molecular cloning and functional expression of Lewis type α1,3/α1,4-fucosyltransferase cDNAs from Mangifera indica L.

Science.gov (United States)

Okada, Takahiro; Ihara, Hideyuki; Ito, Ritsu; Ikeda, Yoshitaka

2017-12-01

In higher plants, complex type N-glycans contain characteristic carbohydrate moieties that are not found in mammals. In particular, the attachment of the Lewis a (Le a ) epitope is currently the only known outer chain elongation that is present in plant N-glycans. Such a modification is of great interest in terms of the biological function of complex type N-glycans in plant species. However, little is known regarding the exact molecular basis underlying their Le a expression. In the present study, we cloned two novel Lewis type fucosyltransferases (MiFUT13) from mango fruit, Mangifera indica L., heterologously expressed the proteins and structurally and functionally characterized them. Using an HPLC-based assay, we demonstrated that the recombinant MiFUT13 proteins mediate the α1,4-fucosylation of acceptor tetrasaccharides with a strict preference for type I-based structure to type II. The results and other findings suggest that MiFUT13s are involved in the biosynthesis of Le a containing glycoconjugates in mango fruits. Copyright © 2017 Elsevier Ltd. All rights reserved.

cDNA Clones with Rare and Recurrent Mutations Found in Cancers | Office of Cancer Genomics

Science.gov (United States)

The CTD2 Center at UT- MD Anderson Cancer Center has developed High-Throughput Mutagenesis and Molecular Barcoding (HiTMMoB)1,2 pipeline to construct mutant alleles open reading frame expression clones that are either recurrent or rare in cancers. These barcoded genes can be used for context-specific functional validation, detection of novel biomarkers (pathway activation) and targets (drug sensitivity).

Why Clone?

Science.gov (United States)

... than expected. Could we really clone dinosaurs? In theory? Yes. You would need: A well-preserved source ... it raises a number of ethical, legal, and social challenges that need to be considered. The vast ...

Molecular cross-talk between sponge host and associated microbes

Digital Repository Service at National Institute of Oceanography (India)

Wang, X.; Brandt, D.; Thakur, N.L.; Wiens, M.; Batel, R.; Schroder, H.C.; Muller, W.E.G.

cited in modern textbooks, e.g. in Pechenik (2000), in which sponges are assumed to be composed of functionally independent cells. The introduction of molecular cloning of genes, coding for “informative” proteins increased in a rapid, self...). The first report in line with this new strategy was on the cloning of a galectin from the demosponge Geodia cydonium (Pfeifer et al., 1993; Hirabayashi and Kasai, 1993). This protein is known to be crucially important for cell-cell interactions (Müller et...

Therapeutic cloning in the mouse

Science.gov (United States)

Mombaerts, Peter

2003-01-01

Nuclear transfer technology can be applied to produce autologous differentiated cells for therapeutic purposes, a concept termed therapeutic cloning. Countless articles have been published on the ethics and politics of human therapeutic cloning, reflecting the high expectations from this new opportunity for rejuvenation of the aging or diseased body. Yet the research literature on therapeutic cloning, strictly speaking, is comprised of only four articles, all in the mouse. The efficiency of derivation of embryonic stem cell lines via nuclear transfer is remarkably consistent among these reports. However, the efficiency is so low that, in its present form, the concept is unlikely to become widespread in clinical practice. PMID:12949262

Leptospira borgpetersenii hybrid leucine-rich repeat protein: Cloning and expression, immunogenic identification and molecular docking evaluation.

Science.gov (United States)

Sritrakul, Tepyuda; Nitipan, Supachai; Wajjwalku, Worawidh; La-Ard, Anchalee; Suphatpahirapol, Chattip; Petkarnjanapong, Wimol; Ongphiphadhanakul, Boonsong; Prapong, Siriwan

2017-11-01

Leptospirosis is an important zoonotic disease, and the major outbreak of this disease in Thailand in 1999 was due largely to the Leptospira borgpetersenii serovar Sejroe. Identification of the leucine-rich repeat (LRR) LBJ_2271 protein containing immunogenic epitopes and the discovery of the LBJ_2271 ortholog in Leptospira serovar Sejroe, KU_Sej_R21_2271, led to further studies of the antigenic immune properties of KU_Sej_LRR_2271. The recombinant hybrid (rh) protein was created and expressed from a hybrid PCR fragment of KU_Sej_R21_2271 fused with DNA encoding the LBJ_2271 signal sequence for targeting protein as a membrane-anchoring protein. The fusion DNA was cloned into pET160/GW/D-TOPO® to form the pET160_hKU_R21_2271 plasmid. The plasmid was used to express the rhKU_Sej_LRR_2271 protein in Escherichia coli BL21 Star™ (DE3). The expressed protein was immunologically detected by Western blotting and immunoreactivity detection with hyperimmune sera, T cell epitope prediction by HLA allele and epitope peptide binding affinity, and potential T cell reactivity analysis. The immunogenic epitopes of the protein were evaluated and verified by HLA allele and epitope peptide complex structure molecular docking. Among fourteen best allele epitopes of this protein, binding affinity values of 12 allele epitopes remained unchanged compared to LBJ_2271. Two epitopes for alleles HLA-A0202 and -A0301 had higher IC 50 values, while T cell reactivity values of these peptides were better than values from LBJ_2271 epitopes. Eight of twelve epitope peptides had positive T-cell reactivity scores. Although the molecular docking of two epitopes, 3FPLLKEFLV11/47FPLLKEFLV55 and 50KLSTVPEGV58, into an HLA-A0202 model revealed a good fit in the docked structures, 50KLSTVPEGV58 and 94KLSTVPEEV102 are still considered as the proteins' best epitopes for allele HLA-A0202. The results of this study showed that rhKU_Sej_LRR_2271 protein contained natural immunological properties that should

Molecular cloning and characterization of a new peroxidase gene ...

African Journals Online (AJOL)

length cDNA of O.violaceus peroxidase gene (OvRCI, GenBank. Acc. No. AY428037) was 1220 bp and contained an 1128 bp open reading frame encoding a protein of 375 amino acids. Homology analysis and molecular modeling revealed that ...

Cloning Mice and Men: Prohibiting the Use of iPS Cells for Human Reproductive Cloning

OpenAIRE

Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

2010-01-01

The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation.

Human cloning: category, dignity, and the role of bioethics.

Science.gov (United States)

Shuster, Evelyne

2003-10-01

Human cloning has been simultaneously a running joke for massive worldwide publicity of fringe groups like the Raelians, and the core issue of an international movement at the United Nations in support of a treaty to ban the use of cloning techniques to produce a child (so called reproductive cloning). Yet, even though debates on human cloning have greatly increased since the birth of Dolly, the clone sheep, in 1997, we continue to wonder whether cloning is after all any different from other methods of medically assisted reproduction, and what exactly makes cloning an 'affront to the dignity of humans.' Categories we adopt matter mightily as they inform but can also misinform and lead to mistaken and unproductive decisions. And thus bioethicists have a responsibility to ensure that the proper categories are used in the cloning debates and denounce those who try to win the ethical debate through well-crafted labels rather than well-reasoned argumentations. But it is as important for bioethicists to take a position on broad issues such as human cloning and species altering interventions. One 'natural question' would be, for example, should there be an international treaty to ban human reproductive cloning?

[Cloning and law in Hungary].

Science.gov (United States)

Julesz, Máté

2015-03-01

Reproductive human cloning is prohibited in Hungary, as in many other countries. Therapeutic human cloning is not prohibited, just like in many other countries. Stem cell therapy is also allowed. Article III, paragraph (3) of the Hungarian basic law (constitution) strictly forbids total human cloning. Article 1 of the Additional Protocol to the Oviedo Convention, on the Prohibition of Cloning Human Beings (1998) stipulates that any intervention seeking to create a human being genetically identical to another human being, whether living or dead, is prohibited. In Hungary, according to Article 174 of the Criminal Code, total human cloning constitutes a crime. Article 180, paragraph (3) of the Hungarian Act on Health declares that embryos shall not be brought about for research purposes; research shall be conducted only on embryos brought about for reproductive purposes when this is authorized by the persons entitled to decide upon its disposal, or when the embryo is damaged. Article 180, paragraph (5) of the Hungarian Act on Health stipulates that multiple individuals who genetically conform to one another shall not be brought about. According to Article 181, paragraph (1) of the Hungarian Act on Health, an embryo used for research shall be kept alive for not longer than 14 days, not counting the time it was frozen for storage and the time period of research.

Emotional reactions to human reproductive cloning.

Science.gov (United States)

May, Joshua

2016-01-01

Extant surveys of people's attitudes towards human reproductive cloning focus on moral judgements alone, not emotional reactions or sentiments. This is especially important given that some (especially Leon Kass) have argued against such cloning on the ground that it engenders widespread negative emotions, like disgust, that provide a moral guide. To provide some data on emotional reactions to human cloning, with a focus on repugnance, given its prominence in the literature. This brief mixed-method study measures the self-reported attitudes and emotions (positive or negative) towards cloning from a sample of participants in the USA. Most participants condemned cloning as immoral and said it should be illegal. The most commonly reported positive sentiment was by far interest/curiosity. Negative emotions were much more varied, but anxiety was the most common. Only about a third of participants selected disgust or repugnance as something they felt, and an even smaller portion had this emotion come to mind prior to seeing a list of options. Participants felt primarily interested and anxious about human reproductive cloning. They did not primarily feel disgust or repugnance. This provides initial empirical evidence that such a reaction is not appropriately widespread. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Cloning and expression analysis of a novel ammonium transporter gene from eichhornia

International Nuclear Information System (INIS)

Li, Y.; Yan, G.; Zheng, L.

2014-01-01

In order to explore the molecular mechanism for Eichhornia crassipes to transport ammonium from outside, we cloned a novel ammonium transporter (EcAMT) gene from E. crassipes and identified its function by using yeast complementation experiment. The full-length cDNA of EcAMT contains a 1506 nucletide-long open reading frame which encodes a protein of 501 amino acids. Bioinformatics analysis predicted that EcAMT had 8 transmembrane regions. The expressions of EcAMT gene under three different nitrogen conditions were evaluated by quantitative reverse transcriptase PCR (qRT-PCR) and the results showed that the expression of EcAMT gene was up-regulated under nitrogen starvation. Our study results revealed some molecular mechanism of E. crassipes to absorb the ammonium in eutrophic water. (author)

Cloning of low dose radiation induced gene RIG1 by RACE based on non-cloned cDNA library

International Nuclear Information System (INIS)

Luo Ying; Sui Jianli; Tie Yi; Zhang Yuanping; Zhou Pingkun; Sun Zhixian

2001-01-01

Objective: To obtain full-length cDNA of radiation induced new gene RIG1 based on its EST fragment. Methods: Based on non-cloned cDNA library, enhanced nested RACE PCR and biotin-avidin labelled probe for magnetic bead purification was used to obtain full-length cDNA of RIG1. Results: About 1 kb of 3' end of RIG1 gene was successfully cloned by this set of methods and cloning of RIG1 5' end is proceeding well. Conclusion: The result is consistent with the design of experiment. This set of protocol is useful for cloning of full-length gene based on EST fragment

Quantitative discrimination of Aggregatibacter actinomycetemcomitans highly leukotoxic JP2 clone from non-JP2 clones in diagnosis of aggressive periodontitis

Directory of Open Access Journals (Sweden)

Yoshida Akihiro

2012-10-01

Full Text Available Abstract Background Aggregatibacter actinomycetemcomitans is the etiological agent of periodontitis, and there is a strong association between clone JP2 and aggressive periodontitis in adolescents of African descent. The JP2 clone has an approximately 530-bp deletion (∆530 in the promoter region of the lkt/ltx gene, which encodes leukotoxin, and this clone has high leukotoxic activity. Therefore, this clone is very important in aggressive periodontitis. To diagnose this disease, culture methods and conventional PCR techniques are used. However, quantitative detection based on qPCR for the JP2 clone has not been developed due to genetic difficulties. In this study, we developed a qPCR-based quantification method specific to the JP2 clone. Methods Based on our analysis of the DNA sequence of the lkt/ltx gene and its flanking region, we designed a reverse primer specific for the ∆530 deletion border sequence and developed a JP2-specific PCR-based quantification method using this primer. We also analyzed the DNA sequence of the ∆530 locus and found it to be highly conserved (97–100% among 17 non-JP2 strains. Using the ∆530 locus, we designed a qPCR primer–probe set specific to non-JP2 clones. Next, we determined the numbers of JP2 and non-JP2 clone cells in the periodontal pockets of patients with aggressive periodontitis. Results The JP2-specific primers specifically amplified the genomic DNA of the A. actinomycetemcomitans JP2 clone and did not react with other bacterial DNA, whereas the non-JP2 specific primers reacted only with A. actinomycetemcomitans non-JP2 clones. Samples from the 88 periodontal sites in the 11 patients with aggressive periodontitis were analyzed. The bacterial cell numbers in 88 periodontal sites ranged from 0 to 4.8 × 108 (mean 1.28 × 107 for JP2 clones and from 0 to 1.6 × 106 for non-JP2 clones (mean 1.84 × 105. There were significant differences in the JP2 cell number between a clinical attachment level

Molecular cloning and transcriptional activity of a new Petunia calreticulin gene involved in pistil transmitting tract maturation, progamic phase, and double fertilization.

Science.gov (United States)

Lenartowski, Robert; Suwińska, Anna; Prusińska, Justyna; Gumowski, Krzysztof; Lenartowska, Marta

2014-02-01

Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca²⁺-binding protein in multicellular eukaryotes. As an endoplasmic reticulum-resident protein, CRT plays a key role in many cellular processes including Ca²⁺ storage and release, protein synthesis, and molecular chaperoning in both animals and plants. CRT has long been suggested to play a role in plant sexual reproduction. To begin to address this possibility, we cloned and characterized the full-length cDNA of a new CRT gene (PhCRT) from Petunia. The deduced amino acid sequence of PhCRT shares homology with other known plant CRTs, and phylogenetic analysis indicates that the PhCRT cDNA clone belongs to the CRT1/CRT2 subclass. Northern blot analysis and fluorescent in situ hybridization were used to assess PhCRT gene expression in different parts of the pistil before pollination, during subsequent stages of the progamic phase, and at fertilization. The highest level of PhCRT mRNA was detected in the stigma-style part of the unpollinated pistil 1 day before anthesis and during the early stage of the progamic phase, when pollen is germinated and tubes outgrow on the stigma. In the ovary, PhCRT mRNA was most abundant after pollination and reached maximum at the late stage of the progamic phase, when pollen tubes grow into the ovules and fertilization occurs. PhCRT mRNA transcripts were seen to accumulate predominantly in transmitting tract cells of maturing and receptive stigma, in germinated pollen/growing tubes, and at the micropylar region of the ovule, where the female gametophyte is located. From these results, we suggest that PhCRT gene expression is up-regulated during secretory activity of the pistil transmitting tract cells, pollen germination and outgrowth of the tubes, and then during gamete fusion and early embryogenesis.

Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites*

OpenAIRE

Zhao, Ya-e; Wang, Zheng-hang; Xu, Yang; Xu, Ji-ru; Liu, Wen-yan; Wei, Meng; Wang, Chu-ying

2012-01-01

To our knowledge, few reports on Demodex studied at the molecular level are available at present. In this study our group, for the first time, cloned, sequenced and analyzed the chitin synthase (CHS) gene fragments of Demodex folliculorum, Demodex brevis, and Demodex canis (three isolates from each species) from Xi’an China, by designing specific primers based on the only partial sequence of the CHS gene of D. canis from Japan, retrieved from GenBank. Results show that amplification was succe...

Performance of new Hevea clones from IAC 400 series Perfomance de novos clones de Hevea da série IAC 400

Directory of Open Access Journals (Sweden)

Paulo de Souza Gonçalves

2007-06-01

Full Text Available The Hevea breeding program of Instituto Agronômico de Campinas (IAC has completed clonal evaluation on the following series: IAC 100, IAC 200 and IAC 300. The performance of 22 clones of Hevea brasiliensis (Willd. ex Adr. de Juss. Muell.-Arg., evolved at IAC, over a period of eleven years was evaluated in the Western Central part of the São Paulo State, Brazil. Among these 22 new clones, six were intraspecific hybrid clones (IAC 400, IAC 404, IAC 405, IAC 406, IAC 410, IAC 412 and the remaining are primary those resulted from selected ortets within half-sib progenies. An old popular clone RRIM 600, of Malaysian origin, was used as the control. The trial was laid out in a randomized block design with three replications. Yield performance over a period of four years, mean girth at the 11th year, girth increment before tapping and on tapping, thermal property of natural rubber produced, bark thickness, number of latex vessel rows in seven year virgin bark, percentage incidence of tapping panel dryness, wind damage and diseases like leaf and panel anthracnose have been observed. Sixty one percent of the clones were superior in relation to the control for yield. The clone IAC 400 recorded the highest yield (97.40 g tree-1 tap-1 over four years of tapping, followed by IAC 411 (78.87 tree-1 tap-1, whereas the control clone RRIM 600 recorded 50.86 g tree-1 tap-1. All selected clones were vigorous in growth. Girth increment of these clones was average to above average. Except for IAC 423, other clones had thick virgin bark at opening ranging from 4.84 mm for IAC 401 to 6.38 mm for IAC 416. The natural rubbers from IAC clones have shown good thermal stability up to 300ºC and no differences in the thermal behavior among rubber from clones of the IAC series and the clone RRIM 600 were found in inert atmosphere.O programa de melhoramento de Hevea do Instituto Agronômico de Campinas (IAC completou a avaliação dos clones da série IAC 100, IAC 200 e IAC

Molecular cloning and characterization of preproorexin in winter skate (Leucoraja ocellata).

Science.gov (United States)

MacDonald, Erin E; Volkoff, Hélène

2010-12-01

A 815 base pairs (bp) cDNA encoding for preproorexin (preproOX) was cloned in winter skate, a cartilaginous fish. Winter skate preproOX is 159 amino acids (aa) long and contains a 34 aa orexin A and 28 aa orexin B. The amino acid sequence of winter skate preproOX is more similar to tetrapod preproOXs (36-40% identity) than teleost preproOXs (23-33% identity). Whereas orexin B appears relatively well conserved among vertebrates, orexin A displays more variability, in particular due to an "insertion sequence" that is present in teleost fish, but not in skate and tetrapods. RT-PCR studies show that preproOX mRNA has a widespread distribution within the brain and is present in several peripheral tissues, including gastrointestinal tract, heart and testes. Fasting induced increases in preproOX expression in the hypothalamus, suggesting that orexin might play a role in the regulation of food intake in winter skate. Copyright © 2010 Elsevier Inc. All rights reserved.

Molecular cloning and expression in mammalian cells of ricin B chain

International Nuclear Information System (INIS)

Chang, M.

1987-01-01

In these studies, the cDNA encoding the B chain of ricin has been cloned and expressed in monkey kidney COS-M6 cells. The recombinant B chain was detected by labeling the transfected cells with 35 S-methionine and 35 S-cysteine and demonstrating secretion of a protein with a Mr of 30-32,000 which was not present in the medium of mock-transfected COS-M6 cells. This protein was specifically immunoprecipitated by an anti-ricin or anti-B chain antibody. The amount of recombinant B chain secreted by the COS-M6 cells was determined by radioimmunoassay to be 1-10 ng/ml of media. Virtually all the recombinant B chain formed active ricin when mixed with native A chain; it could also bind as effectively as native B chain to the galactose-containing glycoprotein, asialofetuin. These results indicate that the vast majority of recombinant B chains secreted into the medium of the COS-M6 cells retain biological function

Language and values in the human cloning debate: a web-based survey of scientists and Christian fundamentalist pastors.

Science.gov (United States)

Weasel, Lisa H; Jensen, Eric

2005-04-01

Over the last seven years, a major debate has arisen over whether human cloning should remain legal in the United States. Given that this may be the 'first real global and simultaneous news story on biotechnology' (Einsiedel et al., 2002, p.313), nations around the world have struggled with the implications of this newly viable scientific technology, which is often also referred to as somatic cell nuclear transfer. Since the successful cloning of Dolly the sheep in 1997, and with increasing media attention paid to the likelihood of a successful human reproductive clone coupled with research suggesting the medical potential of therapeutic cloning in humans, members of the scientific community and Christian fundamentalist leaders have become increasingly vocal in the debate over U.S. policy decisions regarding human cloning (Wilmut, 2000). Yet despite a surfeit of public opinion polls and widespread opining in the news media on the topic of human cloning, there have been no empirical studies comparing the views of scientists and Christian fundamentalists in this debate (see Evans, 2002a for a recent study of opinion polls assessing religion and attitudes toward cloning). In order to further investigate the values that underlie scientists' and Christian fundamentalist leader's understanding of human cloning, as well as their differential use of language in communicating about this issue, we conducted an open-ended, exploratory survey of practicing scientists in the field of molecular biology and Christian fundamentalist pastors. We then analyzed the responses from this survey using qualitative discourse analysis. While this was not necessarily a representative sample (in quantitative terms, see Gaskell & Bauer, 2000) of each of the groups and the response rate was limited, this approach was informative in identifying both commonalities between the two groups, such as a focus on ethical concerns about reproductive cloning and the use of scientific terminology, as well

Molecular cloning of a recA-like gene from the cyanobacterium Anabaena variabilis

International Nuclear Information System (INIS)

Owttrim, G.W.; Coleman, J.R.

1987-01-01

A recA-like gene isolated from the cyanobacterium Anabaena variabilis was cloned and partially characterized. When introduced into Escherichia coli recA mutants, the 7.5-kilobase-pair plasmid-borne DNA insert restored resistance to methyl methanesulfonate and UV irradiation, as well as recombination proficiency when measured by Hfr-mediated conjugation. The cyanobacterial recA gene restored spontaneous but not mitomycin C-induced prophage production. Restriction analysis and subcloning yielded a 1.5-kilobase-pair Sau3A fragment which also restored methylmethane sulfonate resistance and coded for a 38- to 40-kilodalton polypeptide when expressed in an in vitro transcription-translation system

Optimal cloning of mixed Gaussian states

International Nuclear Information System (INIS)

Guta, Madalin; Matsumoto, Keiji

2006-01-01

We construct the optimal one to two cloning transformation for the family of displaced thermal equilibrium states of a harmonic oscillator, with a fixed and known temperature. The transformation is Gaussian and it is optimal with respect to the figure of merit based on the joint output state and norm distance. The proof of the result is based on the equivalence between the optimal cloning problem and that of optimal amplification of Gaussian states which is then reduced to an optimization problem for diagonal states of a quantum oscillator. A key concept in finding the optimum is that of stochastic ordering which plays a similar role in the purely classical problem of Gaussian cloning. The result is then extended to the case of n to m cloning of mixed Gaussian states

Molecular cloning and expression of heteromeric ACCase subunit genes from Jatropha curcas.

Science.gov (United States)

Gu, Keyu; Chiam, Huihui; Tian, Dongsheng; Yin, Zhongchao

2011-04-01

Acetyl-CoA carboxylase (ACCase) catalyzes the biotin-dependent carboxylation of acetyl-CoA to produce malonyl-CoA, which is the essential first step in the biosynthesis of long-chain fatty acids. ACCase exists as a multi-subunit enzyme in most prokaryotes and the chloroplasts of most plants and algae, while it is present as a multi-domain enzyme in the endoplasmic reticulum of most eukaryotes. The heteromeric ACCase of higher plants consists of four subunits: an α-subunit of carboxyltransferase (α-CT, encoded by accA gene), a biotin carboxyl carrier protein (BCCP, encoded by accB gene), a biotin carboxylase (BC, encoded by accC gene) and a β-subunit of carboxyltransferase (β-CT, encoded by accD gene). In this study, we cloned and characterized the genes accA, accB1, accC and accD that encode the subunits of heteromeric ACCase in Jatropha (Jatropha curcas), a potential biofuel plant. The full-length cDNAs of the four subunit genes were isolated from a Jatropha cDNA library and by using 5' RACE, whereas the genomic clones were obtained from a Jatropha BAC library. They encode a 771 amino acid (aa) α-CT, a 286-aa BCCP1, a 537-aa BC and a 494-aa β-CT, respectively. The single-copy accA, accB1 and accC genes are nuclear genes, while the accD gene is located in chloroplast genome. Jatropha α-CT, BCCP1, BC and β-CT show high identity to their homologues in other higher plants at amino acid level and contain all conserved domains for ACCase activity. The accA, accB1, accC and accD genes are temporally and spatially expressed in the leaves and endosperm of Jatropha plants, which are regulated by plant development and environmental factors. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Molecular cloning, characterization and expression of the caffeic acid O-methyltransferase (COMT) ortholog from kenaf (Hibiscus cannabinus)

Science.gov (United States)

We cloned the full-length of the gene putatively encoding caffeic acid O-methyltransferase (COMT) from kenaf (Hibiscus cannabinus L.) using degenerate primers and the RACE (rapid amplification of cDNA ends) method. Kenaf is an herbaceous and rapidly growing dicotyledonous plant with great potential ...

[Telomere lengthening by trichostatin A treatment in cloned pigs].

Science.gov (United States)

Xie, Bing-Teng; Ji, Guang-Zhen; Kong, Qing-Ran; Mao, Jian; Shi, Yong-Qian; Liu, Shi-Chao; Wu, Mei-Ling; Wang, Juan; Liu, Lin; Liu, Zhong-Hua

2012-12-01

Telomeres are repeated GC rich sequences at the end of chromosomes, and shorten with each cell division due to DNA end replication problem. Previously, reprogrammed somatic cells of cloned animals display variable telomere elongation. However, it was reported that the cloned animals including Dolly do not reset telomeres and show premature aging. In this study, we investigated telomere function in cloned or transgenic cloned pigs, including the cloned Northeast Min pigs, eGFP, Mx, and PGC1α transgenic cloned pigs, and found that the telomere lengths of cloned pigs were significantly shorter than the nuclear donor adult fibroblasts and age-matched noncloned pigs (Pstage for 24 h. Consistent with previous reports, the developmental rate of SCNT embryos to the blastocyst stage was significantly increased compared with those of the control group (16.35% vs. 27.09%, 21.60% vs. 34.90%, Plengthen the telomere lengths of cloned pigs.

Trichostatin A (TSA) improves the development of rabbit-rabbit intraspecies cloned embryos, but not rabbit-human interspecies cloned embryos.

Science.gov (United States)

Shi, Li-Hong; Miao, Yi-Liang; Ouyang, Ying-Chun; Huang, Jun-Cheng; Lei, Zi-Li; Yang, Ji-Wen; Han, Zhi-Ming; Song, Xiang-Fen; Sun, Qing-Yuan; Chen, Da-Yuan

2008-03-01

The interspecies somatic cell nuclear transfer (iSCNT) technique for therapeutic cloning gives great promise for treatment of many human diseases. However, the incomplete nuclear reprogramming and the low blastocyst rate of iSCNT are still big problems. Herein, we observed the effect of TSA on the development of rabbit-rabbit intraspecies and rabbit-human interspecies cloned embryos. After treatment with TSA for 6 hr during activation, we found that the blastocyst rate of rabbit-rabbit cloned embryos was more than two times higher than that of untreated embryos; however, the blastocyst rate of TSA-treated rabbit-human interspecies cloned embryos decreased. We also found evident time-dependent histone deacetylation-reacetylation changes in rabbit-rabbit cloned embryos, but not in rabbit-human cloned embryos from fusion to 6 hr after activation. Our results suggest that TSA-treatment does not improve blastocyst development of rabbit-human iSCNT embryos and that abnormal histone deacetylation-reacetylation changes in iSCNT embryos may account for their poor blastocyst development. (c) 2008 Wiley-Liss, Inc.

Functional markers based molecular characterization and cloning of resistance gene analogs encoding NBS-LRR disease resistance proteins in finger millet (Eleusine coracana).

Science.gov (United States)

Panwar, Preety; Jha, Anand Kumar; Pandey, P K; Gupta, Arun K; Kumar, Anil

2011-06-01

Magnaporthe grisea, the blast fungus is one of the main pathological threats to finger millet crop worldwide. A systematic search for the blast resistance gene analogs was carried out, using functional molecular markers. Three-fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants encodes nucleotide binding site (NBS) leucine-rich repeat (LRR) proteins. NBS-LRR homologs have only been isolated on a limited scale from Eleusine coracana. Genomic DNA sequences sharing homology with NBS region of resistance gene analogs were isolated and characterized from resistant genotypes of finger millet using PCR based approach with primers designed from conserved regions of NBS domain. Attempts were made to identify molecular markers linked to the resistance gene and to differentiate the resistant bulk from the susceptible bulk. A total of 9 NBS-LRR and 11 EST-SSR markers generated 75.6 and 73.5% polymorphism respectively amongst 73 finger millet genotypes. NBS-5, NBS-9, NBS-3 and EST-SSR-04 markers showed a clear polymorphism which differentiated resistant genotypes from susceptible genotypes. By comparing the banding pattern of different resistant and susceptible genotypes, five DNA amplifications of NBS and EST-SSR primers (NBS-05(504,) NBS-09(711), NBS-07(688), NBS-03(509) and EST-SSR-04(241)) were identified as markers for the blast resistance in resistant genotypes. Principal coordinate plot and UPGMA analysis formed similar groups of the genotypes and placed most of the resistant genotypes together showing a high level of genetic relatedness and the susceptible genotypes were placed in different groups on the basis of differential disease score. Our results provided a clue for the cloning of finger millet blast resistance gene analogs which not only facilitate the process of plant breeding but also molecular characterization of blast resistance gene analogs from Eleusine coracana.

Inverse fusion PCR cloning.

Directory of Open Access Journals (Sweden)

Markus Spiliotis

Full Text Available Inverse fusion PCR cloning (IFPC is an easy, PCR based three-step cloning method that allows the seamless and directional insertion of PCR products into virtually all plasmids, this with a free choice of the insertion site. The PCR-derived inserts contain a vector-complementary 5'-end that allows a fusion with the vector by an overlap extension PCR, and the resulting amplified insert-vector fusions are then circularized by ligation prior transformation. A minimal amount of starting material is needed and experimental steps are reduced. Untreated circular plasmid, or alternatively bacteria containing the plasmid, can be used as templates for the insertion, and clean-up of the insert fragment is not urgently required. The whole cloning procedure can be performed within a minimal hands-on time and results in the generation of hundreds to ten-thousands of positive colonies, with a minimal background.

Combinations of probabilistic and approximate quantum cloning and deleting

International Nuclear Information System (INIS)

Qiu Daowen

2002-01-01

We first construct a probabilistic and approximate quantum cloning machine (PACM) and then clarify the relation between the PACM and other cloning machines. After that, we estimate the global fidelity of the approximate cloning that improves the previous estimation for the deterministic cloning machine; and also derive a bound on the success probability of producing perfect multiple clones. Afterwards, we further establish a more generalized probabilistic and approximate cloning and deleting machine (PACDM) and discuss the connections of the PACDM to some of the existing quantum cloning and deleting machines. Finally the global fidelity and a bound on the success probability of the PACDM are obtained. Summarily, the quantum devices established in this paper improve and also greatly generalize some of the existing machines

Reproductive cloning in humans and therapeutic cloning in primates: is the ethical debate catching up with the recent scientific advances?

Science.gov (United States)

Camporesi, S; Bortolotti, L

2008-09-01

After years of failure, in November 2007 primate embryonic stem cells were derived by somatic cellular nuclear transfer, also known as therapeutic cloning. The first embryo transfer for human reproductive cloning purposes was also attempted in 2006, albeit with negative results. These two events force us to think carefully about the possibility of human cloning which is now much closer to becoming a reality. In this paper we tackle this issue from two sides, first summarising what scientists have achieved so far, then discussing some of the ethical arguments in favour and against human cloning which are debated in the context of policy making and public consultation. Therapeutic cloning as a means to improve and save lives has uncontroversial moral value. As to human reproductive cloning, we consider and assess some common objections and failing to see them as conclusive. We do recognise, though, that there will be problems at the level of policy and regulation that might either impair the implementation of human reproductive cloning or make its accessibility restricted in a way that could become difficult to justify on moral grounds. We suggest using the time still available before human reproductive cloning is attempted successfully to create policies and institutions that can offer clear directives on its legitimate applications on the basis of solid arguments, coherent moral principles, and extensive public consultation.

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

International Nuclear Information System (INIS)

Collomb, J.; Finance, C.; Alabouch, S.; Laporte, J.

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32 P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors)

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

Energy Technology Data Exchange (ETDEWEB)

Collomb, J; Finance, C; Alabouch, S [Lab. de Microbiologie Moleculaire, Faculte des Sciences Pharmaceutiques et Biologiques, Univ. de Nancy I, Nancy (France); Laporte, J [Station de Virologie et d' Immunologie Moleculaires, INRA, Jouy-en-Josas (France)

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabeled with [sup 32]P and enzymatic labeled through covalent linkage to peroxidase for chemiluminescence detection. The radioactive probe 174 detected as little as 1-3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in fecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors).

Meat and milk compositions of bovine clones

Science.gov (United States)

Tian, X. Cindy; Kubota, Chikara; Sakashita, Kunihito; Izaike, Yoshiaki; Okano, Ryoichi; Tabara, Norio; Curchoe, Carol; Jacob, Lavina; Zhang, Yuqin; Smith, Sadie; Bormann, Charles; Xu, Jie; Sato, Masumi; Andrew, Sheila; Yang, Xiangzhong

2005-01-01

The technology is now available for commercial cloning of farm animals for food production, but is the food safe for consumers? Here, we provide data on >100 parameters that compare the composition of meat and milk from beef and dairy cattle derived from cloning to those of genetic- and breed-matched control animals from conventional reproduction. The cloned animals and the comparators were managed under the same conditions and received the same diet. The composition of the meat and milk from the clones were largely not statistically different from those of matched comparators, and all parameters examined were within the normal industry standards or previously reported values. The data generated from our match-controlled experiments provide science-based information desired by regulatory agencies to address public concerns about the safety of meat and milk from somatic animal clones. PMID:15829585

High-dimensional quantum cloning and applications to quantum hacking.

Science.gov (United States)

Bouchard, Frédéric; Fickler, Robert; Boyd, Robert W; Karimi, Ebrahim

2017-02-01

Attempts at cloning a quantum system result in the introduction of imperfections in the state of the copies. This is a consequence of the no-cloning theorem, which is a fundamental law of quantum physics and the backbone of security for quantum communications. Although perfect copies are prohibited, a quantum state may be copied with maximal accuracy via various optimal cloning schemes. Optimal quantum cloning, which lies at the border of the physical limit imposed by the no-signaling theorem and the Heisenberg uncertainty principle, has been experimentally realized for low-dimensional photonic states. However, an increase in the dimensionality of quantum systems is greatly beneficial to quantum computation and communication protocols. Nonetheless, no experimental demonstration of optimal cloning machines has hitherto been shown for high-dimensional quantum systems. We perform optimal cloning of high-dimensional photonic states by means of the symmetrization method. We show the universality of our technique by conducting cloning of numerous arbitrary input states and fully characterize our cloning machine by performing quantum state tomography on cloned photons. In addition, a cloning attack on a Bennett and Brassard (BB84) quantum key distribution protocol is experimentally demonstrated to reveal the robustness of high-dimensional states in quantum cryptography.

Molecular cloning and expression of five glutathione S-transferase (GST) genes from Banana (Musa acuminata L. AAA group, cv. Cavendish).

Science.gov (United States)

Wang, Zhuo; Huang, Suzhen; Jia, Caihong; Liu, Juhua; Zhang, Jianbin; Xu, Biyu; Jin, Zhiqiang

2013-09-01

Three tau class MaGSTs responded to abiotic stress, MaGSTF1 and MaGSTL1 responded to signaling molecules, they may play an important role in the growth of banana plantlet. Glutathione S-transferases (GST) are multifunctional detoxification enzymes that participate in a variety of cellular processes, including stress responses. In this study, we report the molecular characteristics of five GST genes (MaGSTU1, MaGSTU2, MaGSTU3, MaGSTF1 and MaGSTL1) cloned from banana (Musa acuminate L. AAA group, cv. Cavendish) using a RACE-PCR-based strategy. The predicted molecular masses of these GSTs range from 23.4 to 27.7 kDa and their pIs are acidic. At the amino acid level, they share high sequence similarity with GSTs in the banana DH-Pahang (AA group) genome. Phylogenetic analysis showed that the deduced amino acid sequences of MaGSTs also have high similarity to GSTs of other plant species. Expression analysis by semi-quantitative RT-PCR revealed that these genes are differentially expressed in various tissues. In addition, their expression is regulated by various stress conditions, including exposure to signaling molecules, cold, salinity, drought and Fusarium oxysporum f specialis(f. Sp) cubense Tropical Race 4 (Foc TR4) infection. The expression of the tau class MaGSTs (MaGSTU1, MaGSTU2 and MaGSTU3) mainly responded to cold, salinity and drought while MaGSTF1 and MaGSTL1 expressions were upregulated by signaling molecules. Our findings suggest that MaGSTs play a key role in both development and abiotic stress responses.

Molecular cloning, structural analysis and expression of a zinc ...

African Journals Online (AJOL)

The results of prokaryotic expression of ZnBP and overexpression of the ZnBP gene in A. thaliana improve our understanding of the function of this gene. Future studies should investigate the molecular mechanisms involved in gland morphogenesis in cotton. Key words: Gossypium hirsutum, pigment gland, zinc binding ...

Simultaneous cloning and expression of two cellulase genes from Bacillus subtilis newly isolated from Golden Takin (Budorcas taxicolor Bedfordi)

International Nuclear Information System (INIS)

Li, Wang; Huan, Xiajuan; Zhou, Ying; Ma, Qingyi; Chen, Yulin

2009-01-01

A bacterial strain with high cellulase activity was isolated of feces sample of Golden Takin (Budorcas taxicolor Bedfordi). The bacterium was classified and designated Bacillus subtilis LN by morphological and 16SrDNA gene sequence analysis. Two putative cellulase genes, CelL15 and CelL73, were simultaneously cloned from the isolated strain by PCR. The putative gene CelL15 consisted of an open reading frame (ORF) of 1470 nucleotides and encoded a protein of 490 amino acids with a molecular weight of 54 kDa. The CelL73 gene consisted of an open reading frame (ORF) of 741 nucleotides and encoded a protein of 247 amino acids with a molecular weight of 27 kDa. Both genes were purified and cloned into pET-28a for expression in Escherichia coli BL21 (DE3). The ability of E. coli to degrade cellulose was enhanced when the two recombinants were cultured together.

Willow yield is highly dependent on clone and site

DEFF Research Database (Denmark)

Ugilt Larsen, Søren; Jørgensen, Uffe; Lærke, Poul Erik

2014-01-01

Use of high-yielding genotypes is one of the means to achieve high yield and profitability in willow (Salix spp.) short rotation coppice. This study investigated the performance of eight willow clones (Inger, Klara, Linnea, Resolution, Stina, Terra Nova, Tora, Tordis) on five Danish sites......, differing considerably in soil type, climatic conditions and management. Compared to the best clone, the yield was up to 36 % lower for other clones across sites and up to 51 % lower within sites. Tordis was superior to other clones with dry matter yields between 5.2 and 10.2 Mg ha−1 year−1 during the first...... 3-year harvest rotation, and it consistently ranked as the highest yielding clone on four of the five sites and not significantly lower than the highest yielding clone on the fifth site. The ranking of the other clones was more dependent on site with significant interaction between clone and site...

Cloning mice and men: prohibiting the use of iPS cells for human reproductive cloning.

Science.gov (United States)

Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

2010-01-08

The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation. Copyright 2010 Elsevier Inc. All rights reserved.

Molecular Characterization of Tomato Yellow Leaf Curl Virus in Korea and the Construction of an Infectious Clone

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Bong Choon Lee

2015-06-01

Full Text Available Several tomato production regions in Korea were surveyed for tomato yellow leaf curl disease (TYLCD. Tomato leaf samples showing TYLCD-like symptoms were collected from Tongyeong (To, Geoje (Gi, and Gimhae (Gh cities of the southern part of Korea. Tomato yellow leaf curl virus (TYLCV was detected and the full-length genomes of the isolates were sequenced. The TYLCV isolates found in Korea shared high sequence identity (> 99% with TYLCV-IL [JR:Omu:Ng] (AB110217. Phylogenetic relationship analysis revealed that they formed two groups (with little genetic variability, and the To, Gj, and Gh isolates belonged to the TYLCV-IL group. An infectious clone of TYLCV-To (JQ013089 was constructed and agroinoculated into Nicotiana benthamiana, Nicotiana tabacum var. Xanthi, Petunia hybrida, Capsicum annuum, and Lycopersicon esculentum cv. Hausumomotaro. Agroinfection with a dimeric infectious clone of TYLCV-To induced severe leaf curling and stunting symptoms in these plants, excluding C. annuum. Tomato plants then developed typical yellow leaf curl symptoms.

Endangered wolves cloned from adult somatic cells.

Science.gov (United States)

Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Yuda, Fibrianto; Kim, Hye Jin; Hwang, Woo Suk; Hossein, Mohammad Shamim; Kim, Joung Joo; Shin, Nam Shik; Kang, Sung Keun; Lee, Byeong Chun

2007-01-01

Over the world, canine species, including the gray wolf, have been gradually endangered or extinct. Many efforts have been made to recover and conserve these canids. The aim of this study was to produce the endangered gray wolf with somatic cell nuclear transfer (SCNT) for conservation. Adult ear fibroblasts from a female gray wolf (Canis lupus) were isolated and cultured in vitro as donor cells. Because of limitations in obtaining gray wolf matured oocytes, in vivo matured canine oocytes obtained by flushing the oviducts from the isthmus to the infundibulum were used. After removing the cumulus cells, the oocyte was enucleated, microinjected, fused with a donor cell, and activated. The reconstructed cloned wolf embryos were transferred into the oviducts of the naturally synchronized surrogate mothers. Two pregnancies were detected by ultrasonography at 23 days of gestation in recipient dogs. In each surrogate dog, two fetal sacs were confirmed by early pregnancy diagnosis at 23 days, but only two cloned wolves were delivered. The first cloned wolf was delivered by cesarean section on October 18, 2005, 60 days after embryo transfer. The second cloned wolf was delivered on October 26, 2005, at 61 days postembryo transfer. Microsatellite analysis was performed with genomic DNA from the donor wolf, the two cloned wolves, and the two surrogate female recipients to confirm the genetic identity of the cloned wolves. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically identical to the donor wolf. In conclusion, we demonstrated live birth of two cloned gray wolves by nuclear transfer of wolf somatic cells into enucleated canine oocyte, indicating that SCNT is a practical approach for conserving endangered canids.

Clone tag detection in distributed RFID systems

Science.gov (United States)

Kamaludin, Hazalila; Mahdin, Hairulnizam

2018-01-01

Although Radio Frequency Identification (RFID) is poised to displace barcodes, security vulnerabilities pose serious challenges for global adoption of the RFID technology. Specifically, RFID tags are prone to basic cloning and counterfeiting security attacks. A successful cloning of the RFID tags in many commercial applications can lead to many serious problems such as financial losses, brand damage, safety and health of the public. With many industries such as pharmaceutical and businesses deploying RFID technology with a variety of products, it is important to tackle RFID tag cloning problem and improve the resistance of the RFID systems. To this end, we propose an approach for detecting cloned RFID tags in RFID systems with high detection accuracy and minimal overhead thus overcoming practical challenges in existing approaches. The proposed approach is based on consistency of dual hash collisions and modified count-min sketch vector. We evaluated the proposed approach through extensive experiments and compared it with existing baseline approaches in terms of execution time and detection accuracy under varying RFID tag cloning ratio. The results of the experiments show that the proposed approach outperforms the baseline approaches in cloned RFID tag detection accuracy. PMID:29565982

Clone tag detection in distributed RFID systems.

Science.gov (United States)

Kamaludin, Hazalila; Mahdin, Hairulnizam; Abawajy, Jemal H

2018-01-01

Although Radio Frequency Identification (RFID) is poised to displace barcodes, security vulnerabilities pose serious challenges for global adoption of the RFID technology. Specifically, RFID tags are prone to basic cloning and counterfeiting security attacks. A successful cloning of the RFID tags in many commercial applications can lead to many serious problems such as financial losses, brand damage, safety and health of the public. With many industries such as pharmaceutical and businesses deploying RFID technology with a variety of products, it is important to tackle RFID tag cloning problem and improve the resistance of the RFID systems. To this end, we propose an approach for detecting cloned RFID tags in RFID systems with high detection accuracy and minimal overhead thus overcoming practical challenges in existing approaches. The proposed approach is based on consistency of dual hash collisions and modified count-min sketch vector. We evaluated the proposed approach through extensive experiments and compared it with existing baseline approaches in terms of execution time and detection accuracy under varying RFID tag cloning ratio. The results of the experiments show that the proposed approach outperforms the baseline approaches in cloned RFID tag detection accuracy.

Sources of Blood Meals of Sylvatic Triatoma guasayana near Zurima, Bolivia, Assayed with qPCR and 12S Cloning

Science.gov (United States)

Lucero, David E.; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W.; Peña, Reynaldo; Morrissey, Leslie A.; Rizzo, Donna M.; Stevens, Lori

2014-01-01

Background In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. Methodology/Principal Findings We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). Conclusions/Significance We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors. PMID:25474154

Sources of blood meals of sylvatic Triatoma guasayana near Zurima, Bolivia, assayed with qPCR and 12S cloning.

Directory of Open Access Journals (Sweden)

David E Lucero

2014-12-01

Full Text Available In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps. Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia.We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens, five for chicken (Gallus gallus and unicolored blackbird (Agelasticus cyanopus, and one for opossum (Monodelphis domestica. Using the qPCR assay we detected chicken (13 vectors, and human (14 vectors blood meals as well as an additional blood meal source, Canis sp. (4 vectors.We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

[Human cloning in Muslim and Arab law].

Science.gov (United States)

Aldeeb Abu-Sahlieh, Sami A

2009-01-01

Cloning is a modern medical procedure that Muslim religious authorities treat en resorting to the general principles established by classical Muslim law based on the Koran and the Sunnah of Muhhamad as the messenger of God. In this regard, human beings are not capable of deciding what is or what is not lawful without resorting to divine norms. Cloning clashes with several principles. Firstly, the principle of the respect for life in relation to surpernumeraries, but Muslim authors are not in unanimous agreement on the determination of the moment at which life begins. Secondly, is the respect of progeny: cloning could only take place between a married couple. But even if these two principles are respected, cloning poses two major problems: the diversity of species expounded by the Koran and the Sunnah and a lack of interest. Which explains the quasi-unanimous opposition of Muslim writings regarding cloning.

Fusarium diversity in soil using a specific molecular approach and a cultural approach.

Science.gov (United States)

Edel-Hermann, Véronique; Gautheron, Nadine; Mounier, Arnaud; Steinberg, Christian

2015-04-01

Fusarium species are ubiquitous in soil. They cause plant and human diseases and can produce mycotoxins. Surveys of Fusarium species diversity in environmental samples usually rely on laborious culture-based methods. In the present study, we have developed a molecular method to analyze Fusarium diversity directly from soil DNA. We designed primers targeting the translation elongation factor 1-alpha (EF-1α) gene and demonstrated their specificity toward Fusarium using a large collection of fungi. We used the specific primers to construct a clone library from three contrasting soils. Sequence analysis confirmed the specificity of the assay, with 750 clones identified as Fusarium and distributed among eight species or species complexes. The Fusarium oxysporum species complex (FOSC) was the most abundant one in the three soils, followed by the Fusarium solani species complex (FSSC). We then compared our molecular approach results with those obtained by isolating Fusarium colonies on two culture media and identifying species by sequencing part of the EF-1α gene. The 750 isolates were distributed into eight species or species complexes, with the same dominant species as with the cloning method. Sequence diversity was much higher in the clone library than in the isolate collection. The molecular approach proved to be a valuable tool to assess Fusarium diversity in environmental samples. Combined with high throughput sequencing, it will allow for in-depth analysis of large numbers of samples. Published by Elsevier B.V.

Molecular cloning, structural analysis and mass spectrometric identification of native dioscorins of various yam species.

Science.gov (United States)

Tsai, Wei-Yi; Jheng, Yi-Jyun; Chen, Kuo-Hsuan; Lin, Kuo-Wei; Ho, Yen-Pen; Yang, Ching-Chi; Lin, Kuo-Chih

2013-03-15

Dioscorins are the major storage proteins of yam tubers. However, the molecular nature of their heterogeneity in tubers has not been fully elucidated. In this study the authors isolated the dioscorin gene families of Dioscorea japonica and Dioscorea pseudojaponica, performed matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and elucidated which dioscorin isoforms are the major constituents in tubers. The dioscorin gene families of D. japonica (Dj-dioA1-Dj-dioA4, Dj-dioB1 and Dj-dioB2) and D. pseudojaponica (Dp-dioA1-Dp-dioA5 and Dp-dioB1) were cloned from cDNA libraries of yam tubers. The dioscorins isolated from Dioscorea alata (Da-dioscorins), D. japonica (Dj-dioscorins) and D. pseudojaponica (Dp-dioscorins) were mainly monomers, with a few dimers. The monomers contained one intramolecular disulfide bond (Cys(28)-Cys(187)) and belonged to Class A dioscorins with two cysteine residues. The dimers consisted of Class B dioscorins with one intermolecular disulfide bond (Cys(40)-Cys(40)). Results of MALDI-TOF-MS revealed that the Da-dioscorins were mainly encoded by Da-dioA2, Da-dioA3 and Da-dioA4. The majority of the Dj-dioscorins were encoded by Dj-dioA1, Dj-dioA2, Dj-dioA3 and Dj-dioB2. The Dp-dioscorins mainly comprised proteins encoded by Dp-dioA1, Dp-dioA3, Dp-dioA4, Dp-dioB1 and Dp-dioB2. Determination of the constituents of dioscorin isoforms in yam tubers provides a basis for future studies of their physiological and biomedical functions. © 2012 Society of Chemical Industry.

Telomeres and the ethics of human cloning.

Science.gov (United States)

Allhoff, Fritz

2004-01-01

In search of a potential problem with cloning, I investigate the phenomenon of telomere shortening which is caused by cell replication; clones created from somatic cells will have shortened telomeres and therefore reach a state of senescence more rapidly. While genetic intervention might fix this problem at some point in the future, I ask whether, absent technological advances, this biological phenomenon undermines the moral permissibility of cloning.

Gaussian cloning of coherent states with known phases

International Nuclear Information System (INIS)

Alexanian, Moorad

2006-01-01

The fidelity for cloning coherent states is improved over that provided by optimal Gaussian and non-Gaussian cloners for the subset of coherent states that are prepared with known phases. Gaussian quantum cloning duplicates all coherent states with an optimal fidelity of 2/3. Non-Gaussian cloners give optimal single-clone fidelity for a symmetric 1-to-2 cloner of 0.6826. Coherent states that have known phases can be cloned with a fidelity of 4/5. The latter is realized by a combination of two beam splitters and a four-wave mixer operated in the nonlinear regime, all of which are realized by interaction Hamiltonians that are quadratic in the photon operators. Therefore, the known Gaussian devices for cloning coherent states are extended when cloning coherent states with known phases by considering a nonbalanced beam splitter at the input side of the amplifier

Molecular cloning and biochemical characterization of an α-amylase family from Aspergillus niger

Directory of Open Access Journals (Sweden)

Junying Wang

2018-03-01

Full Text Available Background: α-Amylase is widely used in the starch processing, food and paper industries, hydrolyzing starch, glycogen and other polysaccharides into glucose, maltose and oligosaccharides. An α-amylase gene family from Aspergillus niger CBS513.88 encode eight putative α-amylases. The differences and similarities, biochemical properties and functional diversity among these eight α-amylases remain unknown. Results: The eight genes were cloned and expressed in Pichia pastoris GS115 by shaking-flask fermentation under the induction of methanol. The sequence alignment, biochemical characterizations and product analysis of starch hydrolysis by these α-amylases were investigated. It is found that the eight α-amylases belonged to three different groups with the typical structure of fungal α-amylase. They exhibited maximal activities at 30–40°C except AmyG and were all stable at acidic pH. Ca2+ and EDTA had no effects on the activities of α-amylases except AmyF and AmyH, indicating that the six amylases were Ca2+ independent. Two novel α-amylases of AmyE and AmyF were found. AmyE hydrolyzed starch into maltose, maltotriose and a small amount of glucose, while AmyF hydrolyzed starch into mainly glucose. The excellent physical and chemical properties including high acidic stability, Ca2+-independent and high maltotriose-forming capacity make AmyE suitable in food and sugar syrup industries. Conclusions: This study illustrates that a gene family can encode multiple enzymes members having remarkable differences in biochemical properties. It provides not only new insights into evolution and functional divergence among different members of an α-amylase family, but the development of new enzymes for industrial application. Keywords: Biochemical properties, Food industry, Fungal α-amylase, Glycosyl hydrolase family, Glycosyl hydrolase family, Industrial application, Paper industry, Recombinant Pichia pastoris, Starch processing, α-amylase cloning

[The discrete horror of cloning].

Science.gov (United States)

Guibourg, Ricardo A

2009-01-01

The author raises the topic of cloning after the decision of the Argentine government, which concerned for the "dignity of the human person", passed a decree of need and urgency, No. 200/97 (Annex), prohibiting cloning experiments with human beings. Therefore, considering that the topic is so terribly urgent and necessary, the author feels it is timely to consider it.

Technological Literacy and Human Cloning. Resources in Technology.

Science.gov (United States)

Baird, Steven L.

2002-01-01

Discusses how technology educators can deal with advances in human genetics, specifically, cloning. Includes a definition and history of cloning, discusses its benefits, and looks at social concerns and arguments for and against human cloning. Includes classroom activities and websites. (Contains 10 references.) (JOW)

Defended to the Nines: 25 Years of Resistance Gene Cloning Identifies Nine Mechanisms for R Protein Function[OPEN

Science.gov (United States)

2018-01-01

Plants have many, highly variable resistance (R) gene loci, which provide resistance to a variety of pathogens. The first R gene to be cloned, maize (Zea mays) Hm1, was published over 25 years ago, and since then, many different R genes have been identified and isolated. The encoded proteins have provided clues to the diverse molecular mechanisms underlying immunity. Here, we present a meta-analysis of 314 cloned R genes. The majority of R genes encode cell surface or intracellular receptors, and we distinguish nine molecular mechanisms by which R proteins can elevate or trigger disease resistance: direct (1) or indirect (2) perception of pathogen-derived molecules on the cell surface by receptor-like proteins and receptor-like kinases; direct (3) or indirect (4) intracellular detection of pathogen-derived molecules by nucleotide binding, leucine-rich repeat receptors, or detection through integrated domains (5); perception of transcription activator-like effectors through activation of executor genes (6); and active (7), passive (8), or host reprogramming-mediated (9) loss of susceptibility. Although the molecular mechanisms underlying the functions of R genes are only understood for a small proportion of known R genes, a clearer understanding of mechanisms is emerging and will be crucial for rational engineering and deployment of novel R genes. PMID:29382771

[Methicillin-sensitive Staphylococcus aureus isolates related to USA300 clone: Origin of community-genotype MRSA in Colombia?].

Science.gov (United States)

Escobar-Pérez, Javier Antonio; Castro, Betsy Esperanza; Márquez-Ortiz, Ricaurte Alejandro; Gaines, Sebastián; Chavarro, Bibiana; Moreno, Jaime; Leal, Aura Lucía; Vanegas, Natasha

2014-04-01

USA300 is a genetic lineage found both in methicillin-resistant (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) isolates. In Colombia, hospital and community MRSA infections are caused by a USA300-related community genotype MRSA (CG-MRSA) clone. The genetic origin of this clone is unknown yet. To identify and characterize methicillin-resistant (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates in order to improve the information about the origin of the CG-MRSA isolates in Colombia. USA300-related MSSA isolates were detected and characterized from a study of 184 S. aureus isolates (90 MRSA and 94 MSSA) recovered from infections. The genetic relatedness of the isolates was established by means of pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and protein A gene typification ( spa typing). Among 184 isolates, 27 (14.7%) showed molecular characteristics and genetic relationship with the USA300 clone, of which 18 were MRSA and nine were MSSA. All USA300-related MRSA harbored Staphylococcal cassette chromosome mec (SCC mec ) IVc (3.1.2). In the MSSA isolates, SCC mec remnants or att B duplicate sites were not detected. In Colombia, the CG-MRSA isolates probably originated in the dissemination of an USA300-related MSSA clone which later acquired SCC mec IVc.

Molecular cloning and characterization of the porcine prostaglandin transporter (SLCO2A1: evaluation of its role in F4 mediated neonatal diarrhoea

Directory of Open Access Journals (Sweden)

Cox Eric

2009-10-01

Full Text Available Abstract Background Because prostaglandins are involved in many (pathophysiological processes, SLCO2A1 was already characterized in several species in an attempt to unravel specific processes/deficiencies. Here, we describe the molecular cloning and characterization of the porcine ortholog in order to evaluate its possible involvement in F4 enterotoxigenic E. coli mediated neonatal diarrhoea, based on a positional candidate gene approach study. Results Porcine SLCO2A1 is organized in 14 exons, containing an open reading frame of 1935 bp, encoding a 12-transmembrane organic anion cell surface transporter of 644 aa. The -388 to -5 upstream region comprises a (CpG48 island containing a number of conserved promoter elements, including a TATA box. A potential alternative promoter region was found in the conserved -973 to -700 upstream region. No consensus polyadenylation signal was discovered in the 3' UTR. Repeat sequences were found in 15% of all the non coding sequences. As expected for a multifunctional protein, a wide tissue distribution was observed. mRNA expression was found in the adrenal gland, bladder, caecum, colon (centripetal coil/centrifugal coil, diaphragm, duodenum, gallbladder, heart, ileum, jejunum, kidney, liver, longissimus dorsi muscle, lung, lymph node, mesenterium, rectum, spleen, stomach, tongue and ureter, but not in the aorta, oesophagus and pancreas. The promoter region and the exons (including the splice sites of SLCO2A1 were resequenced in 5 F4ab/ac receptor positive and 5 F4ab/ac receptor negative pigs. Two silent and 2 missense (both S → L at position 360 and 633 mutations were found, but none was associated with the F4ab/ac receptor phenotype. In addition, no phenotype associated differential mRNA expression or alternative/abberant splicing/polyadenylation was found in the jejunum. Conclusion The molecular cloning and characterization of porcine SLCO2A1 not only contributes to the already existing knowledge about the

Use of electroporation for high-molecular-weight DNA-mediated gene transfer.

Science.gov (United States)

Jastreboff, M M; Ito, E; Bertino, J R; Narayanan, R

1987-08-01

Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.

Molecular cloning and expression analysis of three omega-6 desaturase genes from purslane (Portulaca oleracea L.).

Science.gov (United States)

Teixeira, M C; Coelho, N; Olsson, M E; Brodelius, P E; Carvalho, I S; Brodelius, M

2009-07-01

Two full-length cDNA clones of PoleFAD2 and one full-length cDNA clone of PoleFAD6, encoding omega-6 fatty acid desaturases, the key enzymes for the conversion of oleic into linoleic acid, were isolated from purslane (Portulaca oleracea L.) leaves and seeds. The deduced amino acid sequence of both isoforms of PoleFAD2 showed higher similarities to other microsomal omega-6 desaturases then to PoleFAD6 or other plastidial orthologues, and vice versa. Expression analysis by RT-PCR showed that all genes are expressed in all tissues of purslane tested, but higher levels of mRNA accumulation were detected in reproductive organs and cells that proliferate rapidly or store lipids. Wounding affected the levels of mRNA accumulation of both, FAD2 and FAD6 genes in purslane leaves, while chilling stress affected only FAD2 transcript level. The expression patterns observed reflect the discrete roles of these genes in membrane synthesis for cell division, thylakoid development, and lipid storage or in the biosynthetic pathway for the production of signaling molecules that influence plant development or defense.

Genetic superiority of exotic clones over indigenous clones for quantitative and qualitative traits

International Nuclear Information System (INIS)

Khan, I.A.; Khatri, A.; Ahmad, M.; Siddiqui, N.A.; Dahar, M.H.; Khanzada, M.H.; Nizamani, G.S.

1997-01-01

Seventeen exotic sugar cane clones along with two local checks (BL4 and L116) were planted for three consecutive years (1989-90 to 1991-92) and evaluated for cane yield, yield components (plant height, cane girth, stalks per stool, stool weight), fibre, sucrose and sugar yield. Two exotic clones AEC82-1026 and AEC86-329 proved to be significantly (p< 0.05) superior in cane yield (130.62 and 114.87 t/ha respectively) and sugar yield 18.10 and 19.33 t/ha respectively) to both checks, cane and sugar yield of BL4 were 100.73 and 12.69 t/ha and that of L116 were 74.19 11.03 t/ha respectively. Cane and sugar yields were positively (P<0.01) correlated with plant height, cane girth and weight per stool. These promising clones would be subjected to extensive studies for cane yield in different parts of Sindh province. (author)

Molecular Cloning and Expression Analysis of a Hexokinase Gene, MdHXK1 in Apple

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Jin Zhao

2016-03-01

Full Text Available A hexokinase gene named MdHXK1 (MDP0000309677 was cloned from ‘Gala’ apple (Malus × domestica Borkh.. Sequence analysis showed that the MdHXK1 gene was 1 497 bp long and encoded 499 amino acids. The predicted molecular mass of this protein was 54.05 kD, and the pI was 5.76. A phylogenetic tree indicated apple MdHXK1 exhibited the highest sequence similarity to Pyrus bretschneideri PbHXK1. Analysis of the functional domain showed that the MdHXK1 protein included two conserved kinase domains. The prediction of subcellular localization suggested that the MdHXK1 protein was mainly localized in the cytoplasm. There was an indication that MdHXK1 existed as one copy in the apple genome by Southern blotting. Silico analysis suggested that the promoter sequence contained several typical cis-acting elements, including defense, sugar signaling and phytohormone responsive elements. Quantitative real-time PCR analysis demonstrated that the MdHXK1 gene was mainly expressed in stem and flower tissues. During the development of apple fruits, the expression of the MdHXK1 gene initially increased and then decreased. The changes on Glc phosphorylation relative activity and glucose concentration showed the same trend. In addition, the expression of this gene was induced by salt stress, low temperature, and abscisic acid (ABA. Finally, we obtained and purified the fused MdHXK1 protein by recombinant prokaryotic expression. Studies have demonstrated that MdHXK1 may participate in sugar metabolism in apple fruits. Enzyme encoded by MdHXK1 is a key factor in the mediation of sugar accumulation. Recently, researchers on hexokinase at home and abroad mainly focused on model plants, such as Arabidopsis, tobacco and rice, but orchard fruit like apple were underresearched. Our research established the foundation for the further study of the functions of MdHXK1.

Cloning, purification and crystallization of Thermus thermophilus proline dehydrogenase

International Nuclear Information System (INIS)

White, Tommi A.; Tanner, John J.

2005-01-01

Cloning, purification and crystallization of T. thermophilus proline dehydrogenase is reported. The detergent n-octyl β-d-glucopyranoside was used to reduce polydispersity, which enabled crystallization. Nature recycles l-proline by converting it to l-glutamate. This four-electron oxidation process is catalyzed by the two enzymes: proline dehydrogenase (PRODH) and Δ 1 -pyrroline-5-carboxylate dehydrogenase. This note reports the cloning, purification and crystallization of Thermus thermophilus PRODH, which is the prototype of a newly discovered superfamily of bacterial monofunctional PRODHs. The results presented here include production of a monodisperse protein solution through use of the detergent n-octyl β-d-glucopyranoside and the growth of native crystals that diffracted to 2.3 Å resolution at Advanced Light Source beamline 4.2.2. The space group is P2 1 2 1 2 1 , with unit-cell parameters a = 82.2, b = 89.6, c = 94.3 Å. The asymmetric unit is predicted to contain two protein molecules and 46% solvent. Molecular-replacement trials using a fragment of the PRODH domain of the multifunctional Escherichia coli PutA protein as the search model (24% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of T. thermophilus PRODH will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative

Preservation and Reproduction of Microminipigs by Cloning Technology.

Science.gov (United States)

Enya, Satoko; Kawarasaki, Tatsuo; Otake, Masayoshi; Kangawa, Akihisa; Uenishi, Hirohide; Mikawa, Satoshi; Nishimura, Takashi; Kuwahawa, Yasushi; Shibata, Masatoshi

Microminipigs have been maintained in small populations of closed colonies, involving risks of inbreeding depression and genetic drift. In order to avoid these risks, we assessed the applicability of cloning technology. Male and female clones were produced from a stock of cryopreserved somatic cells, obtaining offspring by means of natural mating. Phenotypic and genotypic characteristics of original microminipigs, clones and their offspring were analyzed and recorded. Clones presented characteristics similar to those of the cell-stock data. Although the body weight of clones tended to be heavier than that of the cell-stock data, body weights of their offspring were similar to those of previous reports. Thus, cloned microminipigs have the potential to be a valuable genetic resource for reproduction and breeding. Our proposed methodology might be useful to provide a large number of animals with adequate quality from a limited population with sufficient genetic diversity. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

[Product safety analysis of somatic cell cloned bovine].

Science.gov (United States)

Hua, Song; Lan, Jie; Song, Yongli; Lu, Chenglong; Zhang, Yong

2010-05-01

Somatic cell cloning (nuclear transfer) is a technique through which the nucleus (DNA) of a somatic cell is transferred into an enucleated oocyte for the generation of a new individual, genetically identical to the somatic cell donor. It could be applied for the enhancement of reproduction rate and the improvement of food products involving quality, yield and nutrition. In recent years, the United States, Japan and Europe as well as other countries announced that meat and milk products made from cloned cattle are safe for human consumption. Yet, cloned animals are faced with a wide range of health problems, with a high death rate and a high incidence of disease. The precise causal mechanisms for the low efficiency of cloning remain unclear. Is it safe that any products from cloned animals were allowed into the food supply? This review focuses on the security of meat, milk and products from cloned cattle based on the available data.

Generating West Nile Virus from an Infectious Clone.

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Vandergaast, Rianna; Fredericksen, Brenda L

2016-01-01

WNV infectious clones are valuable tools for elucidating WNV biology. Nevertheless, relatively few infectious WNV clones have been generated because their construction is hampered by the instability of flaviviral genomes. More recently, advances in cloning techniques as well as the development of several two-plasmid WNV infectious clone systems have facilitated the generation of WNV infectious clones. Here we described a protocol for recovering WNV from a two-plasmid system. In this approach, large quantities of these constructs are digested with restriction enzymes to produce complementary restriction sites at the 3' end of the upstream fragment and the 5' end of the downstream fragment. These fragments are then annealed to produce linear template for in vitro transcription to synthesize infectious RNA. The resulting RNA is transfected into cells and after several days WNV is recovered in the culture supernatant. This method can be used to generate virus from infectious clones encoding high- and low-pathogenicity strains of WNV, as well as chimeric virues.

Heterosis and combining ability: a diallel cross of three geographically isolated populations of Pacific abalone Haliotis discus hannai Ino

Science.gov (United States)

Deng, Yuewen; Liu, Xiao; Zhang, Guofan; Wu, Fucun

2010-11-01

We conducted a complete diallel cross among three geographically isolated populations of Pacific abalone Haliotis discus hannai Ino to determine the heterosis and the combining ability of growth traits at the spat stage. The three populations were collected from Qingdao (Q) and Dalian (D) in China, and Miyagi (M) in Japan. We measured the shell length, shell width, and total weight. The magnitude of the general combining ability (GCA) variance was more pronounced than the specific combining ability (SCA) variance, which is evidenced by both the ratio of the genetic component in total variation and the GCA/SCA values. The component variances of GCA and SCA were significant for all three traits ( P<0.05), indicating the importance of additive and non-additive genetic effects in determining the expression of these traits. The reciprocal maternal effects (RE) were also significant for these traits ( P<0.05). Our results suggest that population D was the best general combiner in breeding programs to improve growth traits. The DM cross had the highest heterosis values for all three traits.

In vivo elimination of parental clones in general and site-directed mutagenesis.

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Holland, Erika G; Acca, Felicity E; Belanger, Kristina M; Bylo, Mary E; Kay, Brian K; Weiner, Michael P; Kiss, Margaret M

2015-02-01

The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5'-CCGCGG-3' and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 10(7) from a single transformation and with greater than 90% recombinant clones. Copyright © 2014 Elsevier B.V. All rights reserved.

Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.

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Tóth, Eszter; Huszár, Krisztina; Bencsura, Petra; Kulcsár, Péter István; Vodicska, Barbara; Nyeste, Antal; Welker, Zsombor; Tóth, Szilvia; Welker, Ervin

2014-01-01

The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease--a Body Double of the Type IIP enzyme--is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program (http://group.szbk.u-szeged.hu/welkergr/body_double/index.html) for the selection of suitable Body Double enzymes and the design of the appropriate primers.

Cloning, expression and purification of d-tagatose 3-epimerase gene from Escherichia coli JM109.

Science.gov (United States)

He, Xiaoliang; Zhou, Xiaohui; Yang, Zi; Xu, Le; Yu, Yuxiu; Jia, Lingling; Li, Guoqing

2015-10-01

An unknown d-tagatose 3-epimerase (DTE) containing a IoIE domain was identified and cloned from Escherichia coli. This gene was subcloned into the prokaryotic expression vector pET-15b, and induced by IPTG in E. coli BL21 expression system. Through His-select gel column purification and fast-protein liquid chromatography, highly purified and stable DTE protein was produced. The molecular weight of the DTE protein was estimated to be 29.8kDa. The latest 83 DTE sequences from public database were selected and analyzed by molecular clustering, multi-sequence alignment. DTEs were roughly divided into five categories. Copyright © 2015 Elsevier Inc. All rights reserved.

Cloning and molecular characterization of the salt-regulated jojoba ScRab cDNA encoding a small GTP-binding protein.