Incidence of abnormal offspring from cloning and other assisted reproductive technologies.

Science.gov (United States)

Hill, Jonathan R

2014-02-01

In animals produced by assisted reproductive technologies, two abnormal phenotypes have been characterized. Large offspring syndrome (LOS) occurs in offspring derived from in vitro cultured embryos, and the abnormal clone phenotype includes placental and fetal changes. LOS is readily apparent in ruminants, where a large calf or lamb derived from in vitro embryo production or cloning may weigh up to twice the expected body weight. The incidence of LOS varies widely between species. When similar embryo culture conditions are applied to nonruminant species, LOS either is not as dramatic or may even be unapparent. Coculture with serum and somatic cells was identified in the 1990s as a risk factor for abnormal development of ruminant pregnancies. Animals cloned from somatic cells may display a combination of fetal and placental abnormalities that are manifested at different stages of pregnancy and postnatally. In highly interventional technologies, such as nuclear transfer (cloning), the incidence of abnormal offspring continues to be a limiting factor to broader application of the technique. This review details the breadth of phenotypes found in nonviable pregnancies, together with the phenotypes of animals that survive the transition to extrauterine life. The focus is on animals produced using in vitro embryo culture and nuclear transfer in comparison to naturally occurring phenotypes.

Transformation and Stability of Cloned Polysaccharidase Genes in Gram-Positive Bacteria

OpenAIRE

EKİNCİ, Mehmet Sait

2014-01-01

Polysaccharidase genes from rumen bacteria were transferred to and expressed in ruminal and non-ruminal Gram-positive bacteria. The transformation efficiency and genetic stability of polysaccharidase genes in bacteria from different habitats were investigated. PCR amplification of cloned polysaccharidase genes from Escherichia coli, Lactococcus lactis, Enterococcus faecalis, Streptococcus sanguis and S. bovis strain 26 showed that rearrangement of plasmid and the gene fragment did not occur. ...

[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.

Tumor progression: analysis of the instability of the metastatic phenotype, sensitivity to radiation and chemotherapy

International Nuclear Information System (INIS)

Welch, D.R.

1984-01-01

The major complications for tumor therapy are 1) tumor spread (metastasis); 2) the mixed nature of tumors (heterogeneity); and 3) the capacity of tumors to evolve (progress). To study these tumor characteristics, the rat 13762NF mammary adenocarcinoma was cloned and studied for metastatic properties and sensitivities to therapy (chemotherapy, radiation and hyperthermia). The cell clones were heterogeneous and no correlation between metastatic potential and therapeutic sensitivities was observed. Further, these phenotypes were unstable during pasage in vitro; yet, the changes were clone dependent and reproducible using different cryoprotected cell stocks. To understand the phenotypic instability, subclones were isolated from low and high passage cell clones. The results demonstrated that 1) tumor cells are heterogeneous for multiple phenotypes; 2) tumor cells are unstable for multiple phenotypes; 3) the magnitude, direction and time of occurrence of phenotypic drift is clone dependent; 4) the sensitivity of cell clones to ionizing radiation (γ or heat) and chemotherapy agents is independent of their metastatic potential; 5) shifts in metastatic potential and sensitivity to therapy may occur simultaneously but are not linked; and 6) tumor cells independently diverge to form several subpopulations with unique phenotypic profiles

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.

Heterogeneity in induced thermal resistance of rat tumor cell clones

International Nuclear Information System (INIS)

Tomasovic, S.P.; Rosenblatt, P.L.; Heitzman, D.

1983-01-01

Four 13762NF rat mammary adenocarcinoma clones were examined for their survival response to heating under conditions that induced transient thermal resistance (thermotolerance). Clones MTC and MTF7 were isolated from the subcutaneous locally growing tumor, whereas clones MTLn2 and MTLn3 were derived from spontaneous lung metastases. There was heterogeneity among these clones in thermotolerance induced by either fractionated 45 0 C or continuous 42 0 C heating, but the order of sensitivity was not necessarily the same. The clones developed thermal resistance at different rates and to different degrees within the same time intervals. There was heterogeneity between clones isolated from within either the primary site or metastatic lesions. However, clones derived from metastatic foci did not intrinsically acquire more or less thermotolerance to fractionated 45 0 C or continuous 42 0 C heating than did clones from the primary tumor. Further, there was no apparent relationship between any phenotypic properties that conferred more or less thermotolerance in vitro and any phenotypic properties that conferred enhanced metastatic success of these same clones by spontaneous (subcutaneous) or experimental (intravenous) routes in vivo. These tumor clones also differ in their karyotype, metastatic potential, cell surface features, sensitivity to x-irradiation and drugs, and ability to repair sublethal radiation damage. These results provide further credence to the concept that inherent heterogeneity within tumors may be as important in therapeutic success as other known modifiers of outcome such as site and treatment heterogeneity

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.

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.

Complementation of the UV-sensitive phenotype of a xeroderma pigmentosum human cell line by transfection with a cDNA clone library

International Nuclear Information System (INIS)

Teitz, T.; Naiman, T.; Avissar, S.S.; Bar, S.; Okayama, H.; Canaani, D.

1987-01-01

In previous work, a xeroderma pigmentosum cell line belonging to complementation group C was established by transformation with origin-defective simian virus 40. We now report the complementation of the UV sensitivity of this cell line by gene transfer. A human cDNA clone library constructed in a mammalian expression vector, and itself incorporated in a lambda phage vector, was introduced into the cells as a calcium phosphate precipitate. Following selection to G418 resistance, provided by the neo gene of the vector, transformants were selected for UV resistance. Twenty-one cell clones were obtained with UV-resistance levels typical of normal human fibroblasts. All transformants contained vector DNA sequences in their nuclei. Upon further propagation in the absence of selection for G418 resistance, about half of the primary transformants remained UV-resistant. Secondary transformants were generated by transfection with a partial digest of total chromosomal DNA from one of these stable transformants. This resulted in 15 G418-resistant clones, 2 of which exhibited a UV-resistant phenotype. The other primary clones lost UV resistance rapidly when subcultured in the absence of G418. Importantly, several retained UV resistance under G418 selection pressure. The acquisition of UV resistance by secondary transformants derived by transfection of DNA from a stable primary transformant, and the linkage between G418 and UV resistances in the unstable primary transformants, strongly suggests that the transformants acquired UV resistance through DNA-mediated gene transfer and not by reversion

DNA Methylation in Peripheral Blood Cells of Pigs Cloned by Somatic Cell Nuclear Transfer

DEFF Research Database (Denmark)

Gao, Fei; Li, Shengting; Lin, Lin

2011-01-01

To date, the genome-wide DNA methylation status of cloned pigs has not been investigated. Due to the relatively low success rate of pig cloning by somatic cell nuclear transfer, a better understanding of the epigenetic reprogramming and the global methylation patterns associated with development...... in cloned pigs is required. In this study we applied methylation-specific digital karyotyping tag sequencing by Solexa technology and investigated the genome-wide DNA methylation profiles of peripheral blood cells in cloned pigs with normal phenotypes in comparison with their naturally bred controls....... In the result, we found that globally there was no significant difference of DNA methylation patterns between the two groups. Locus-specifically, some genes involved in embryonic development presented a generally increased level of methylation. Our findings suggest that in cloned pigs with normal phenotypes...

Chitinase determinants of Vibrio vulnificus: gene cloning and applications of a chitinase probe

International Nuclear Information System (INIS)

Wortman, A.T.; Somerville, C.C.; Colwell, R.R.

1986-01-01

To initiate study of the genetic control of chitinolytic activity in vibrios, the chitobiase gene was isolated by cloning chromosomal DNA prepared from Vibrio vulnificus. Chimeric plasmids were constructed from Sau3A I partial digests of chromosomal DNA by ligating 5 to 15-kilobase fragments into the BamHI site, i.e., in the Tc/sup r/ gene, of pBR322 (Am/sup r/Tc/sup r/). The resulting plasmids were transformed into Escherichia coli DH1. Chitobiase activity of the insert-bearing clones was detected by using a chromogenic substrate, p-nitrophenyl-N-acetyl-β,D-glucosaminide, and confirmed by the appearance of a fluorescent end product from the hydrolysis of 4-methylumbelliferyl-β,D-N-N'-diacetylchitiobiose. Endochitinase activity was demonstrated by liberation of water-soluble products produced by the degradation of [ 3 H]chitin. Transformation of E. coli Y10R (lacY) with plasmids from chitinase-positive clones restored the lactose-positive phenotype, suggesting the presence of a permease associated with chitinase activity. Physical mapping of plasmids containing the chitinase determinants indicate that transcription of these genes in E. coli may be initiated at a V. vulnificus promoter

Expressão fenotípica de clones de seringueira na região noroeste do estado de São Paulo Phenotypic expression of rubber tree clones in the northwestern region of São Paulo state

Directory of Open Access Journals (Sweden)

Paulo de Souza Gonçalves

2006-01-01

Full Text Available O desenvolvimento de novos clones de seringueira [Hevea brasiliensis (Willd. ex Adr. de Juss. Muell.-Arg.] com alto potencial de produção aliado a outros caracteres secundários desejáveis é de fundamental importância para uma heveicultura sustentável e competitiva. O objetivo deste trabalho foi avaliar a expressão fenotípica de caracteres superiores em 17 clones de seringueira, tendo em vista a escolha dos mais promissores. Em campo, o experimento obedeceu ao delineamento de blocos ao acaso com três repetições e parcelas lineares de seis plantas. Pelos resultados, verificou-se que o clone IAC 40 foi o mais produtivo, com média de 2.316 kg de borracha seca ha-1 ano-1 no período de seis anos, seguido pelo clone IAC 300 (1.921 kg, enquanto o clone-testemunha, RRIM 600 produziu 1.493 kg. Observou-se na maior parte dos clones, crescimento superior em relação à testemunha. A porcentagem de plantas aptas à sangria variou de 40% (IAC 329 a 100% (IAC 327. Exceto nos clones IAC 56, IAC 331 e IAN 3156 com 7,21 mm, 7,18 mm e 6,40 mm respectivamente, em todos os demais notou-se espessura de casca virgem inferior ao clone RRIM 600 (6,38 mm. Com exceção do IAN 3156, os demais clones tiveram baixa incidência de secamento de painel. O bom desempenho de todos os clones IAC e amazônicos (IAN, Fx e RO permite que sejam recomendados para plantio em pequena escala, ao tempo em que serão avaliados para futura recomendação em grande escala envolvendo diferentes ambientes do Estado de São Paulo.The development of new clones with high production combined to other desirable secondary characters is fundamental for a sustainable and competitive rubber tree cultivation. The objective of this study was to evaluate, during a period of 13 years, the phenotypic expression of superior characters of 17 clones of rubber tree grown in the plateau region of São Paulo State, Brazil. The treatments were arranged in a randomized block design with three

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.

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.

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.

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

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

Liquid Chromatography–Mass Spectrometry Based Metabolomics Study of Cloned versus Normal Pigs Fed Either Restricted or Ad Libitum High-Energy Diets

DEFF Research Database (Denmark)

Christensen, Kirstine Lykke; Hedemann, Mette Skou; Jørgensen, Henry

2012-01-01

Genetically identical cloned pigs should in principle eliminate biological variation and provide more pronounced effects when subjected to, e.g., dietary interventions, but little is known about how phenotype and phenotypic variation is affected by cloning. Therefore, an investigation...... of the metabolome of cloned pigs compared to normal control pigs was performed to elucidate the variation and possible differences in the metabolic phenotypes during a dietary intervention. A total of 19 control pigs and 17 cloned pigs were given the same high-energy dense diet either ad libitum or in a restricted...... manner (60% of ad libitum) for 6 months, and plasma was subjected to liquid chromatography–mass spectrometry nontargeted metabolomics and biochemical analyses. Low systemic levels of IGF-1 could indicate altered growth conditions and energy metabolism in cloned pigs. In response to ad libitum feeding...

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.

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.

Disease-causing mitochondrial heteroplasmy segregated within induced pluripotent stem cell clones derived from a patient with MELAS.

Science.gov (United States)

Folmes, Clifford D L; Martinez-Fernandez, Almudena; Perales-Clemente, Ester; Li, Xing; McDonald, Amber; Oglesbee, Devin; Hrstka, Sybil C; Perez-Terzic, Carmen; Terzic, Andre; Nelson, Timothy J

2013-07-01

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual

Intraclonal genome diversity of Pseudomonas aeruginosa clones CHA and TB

Science.gov (United States)

2013-01-01

Background Adaptation of Pseudomonas aeruginosa to different living conditions is accompanied by microevolution resulting in genomic diversity between strains of the same clonal lineage. In order to detect the impact of colonized habitats on P. aeruginosa microevolution we determined the genomic diversity between the highly virulent cystic fibrosis (CF) isolate CHA and two temporally and geographically unrelated clonal variants. The outcome was compared with the intraclonal genome diversity between three more closely related isolates of another clonal complex. Results The three clone CHA isolates differed in their core genome in several dozen strain specific nucleotide exchanges and small deletions from each other. Loss of function mutations and non-conservative amino acid replacements affected several habitat- and lifestyle-associated traits, for example, the key regulator GacS of the switch between acute and chronic disease phenotypes was disrupted in strain CHA. Intraclonal genome diversity manifested in an individual composition of the respective accessory genome whereby the highest number of accessory DNA elements was observed for isolate PT22 from a polluted aquatic habitat. Little intraclonal diversity was observed between three spatiotemporally related outbreak isolates of clone TB. Although phenotypically different, only a few individual SNPs and deletions were detected in the clone TB isolates. Their accessory genome mainly differed in prophage-like DNA elements taken up by one of the strains. Conclusions The higher geographical and temporal distance of the clone CHA isolates was associated with an increased intraclonal genome diversity compared to the more closely related clone TB isolates derived from a common source demonstrating the impact of habitat adaptation on the microevolution of P. aeruginosa. However, even short-term habitat differentiation can cause major phenotypic diversification driven by single genomic variation events and uptake of phage

Identification and cloning of a prethymic precursor T lymphocyte from a population of common acute lymphoblastic leukemia antigen (CALLA)-positive fetal bone marrow cells

DEFF Research Database (Denmark)

Hokland, P; Hokland, M; Daley, J

1987-01-01

We have cloned common acute lymphoblastic leukemia (CALLA)-positive cells from human fetal bone marrow containing less than 1 in 10,000 E-RFC in round-bottomed microtiter wells (one cell per well) using the autocloning unit of an EPICS-V cell sorter. Expansion of such cells (with IL-2 and heavily...... irradiated autologous thymocytes as feeder cells) resulted in growth in 6-14% of the wells (mean, 11%) with cells with mature T lymphocyte phenotype. Two-color fluorescence analysis of outgrowing cultures furthermore ascertained that these cells had differentiated through a phase of simultaneous expression...... of T4 and T8 antigens and at the same time expression of the thymocyte-associated T6 antigens. Thus, given the fact that 10-20% of T cell acute lymphoblastic leukemia (T-ALLs) are CALLA+, we have been able to identify a human prethymic T lymphocyte population that might be the normal counterpart...

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.

Variation in biological properties of cauliflower mosaic virus clones.

Science.gov (United States)

al-Kaff, N; Covey, S N

1994-11-01

Infectious clones were prepared from virion DNA of three cauliflower mosaic virus (CaMV) isolates, 11/3, Xinjiang (XJ), and Aust, to investigate pathogenic variation in virus populations. Of 10 infectious clones obtained for isolate 11/3, four pathotypes were identified, each producing symptoms in turnip that differed from those of the 11/3 wild-type. Virus from two clonal groups of 11/3 was transmissible by aphids whereas that from two others was not. Of the five infectious clones obtained from isolate XJ, two groups were identified, one of which differed symptomatically from the wild-type. Only one infectious clone was obtained from isolate Aust and this had properties similar to the wild-type. Restriction enzyme polymorphisms were found in some clonal groups and these correlated with symptoms. Other groups with different pathogenic properties could not be distinguished apart by restriction site polymorphisms. Further variation was observed in the nucleotide sequences of gene II (coding for aphid transmission factor) from these viruses as compared with other CaMV isolates. In the aphid non-transmissible clones of isolate 11/3, one had a Gly to Arg mutation in gene II similar to that of other non-deleted non-transmissible CaMV isolates. The second had a 322 bp deletion at the site of a small direct repeat similar to that of isolate CM4-184 although occurring in a different position. The gene II deletion of isolate 11/3 produced a frame-shift that separated genes II and III by 60 bp. Most CaMV clones studied remained biologically stable producing similar symptoms during subsequent passages. However, one clone (11/3-7) produced two new biotypes during its first passage suggesting that it was relatively unstable. Our results show that wild-type populations of CaMV contain a range of infectious genome variants with contrasting biological properties and differing stability. We suggest that a variety of significant viral phenotypic changes can occur during each

O-Linked β-N-Acetylglucosaminylation (O-GlcNAcylation) in Primary and Metastatic Colorectal Cancer Clones and Effect of N-Acetyl-β-d-glucosaminidase Silencing on Cell Phenotype and Transcriptome*

Science.gov (United States)

Yehezkel, Galit; Cohen, Liz; Kliger, Adi; Manor, Esther; Khalaila, Isam

2012-01-01

O-Linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is a regulatory post-translational modification occurring on the serine or threonine residues of nucleocytoplasmic proteins. O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), which are responsible for O-GlcNAc addition and removal, respectively. Although O-GlcNAcylation was found to play a significant role in several pathologies such as type II diabetes and neurodegenerative diseases, the role of O-GlcNAcylation in the etiology and progression of cancer remains vague. Here, we followed O-GlcNAcylation and its catalytic machinery in metastatic clones of human colorectal cancer and the effect of OGA knockdown on cellular phenotype and on the transcriptome. The colorectal cancer SW620 metastatic clone exhibited increased O-GlcNAcylation and decreased OGA expression compared with its primary clone, SW480. O-GlcNAcylation elevation in SW620 cells, through RNA interference of OGA, resulted in phenotypic alterations that included acquisition of a fibroblast-like morphology, which coincides with epithelial metastatic progression and growth retardation. Microarray analysis revealed that OGA silencing altered the expression of about 1300 genes, mostly involved in cell movement and growth, and specifically affected metabolic pathways of lipids and carbohydrates. These findings support the involvement of O-GlcNAcylation in various aspects of tumor cell physiology and suggest that this modification may serve as a link between metabolic changes and cancer. PMID:22730328

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.

PAF-receptor is preferentially expressed in a distinct synthetic phenotype of smooth muscle cells cloned from human internal thoracic artery: Functional implications in cell migration

International Nuclear Information System (INIS)

Stengel, Dominique; O'Neil, Caroline; Brocheriou, Isabelle; Karabina, Sonia-Athina; Durand, Herve; Caplice, Noel M.; Pickering, J. Geoffrey; Ninio, Ewa

2006-01-01

Platelet-activating-Factor (PAF) and its structural analogues formed upon low density lipoprotein oxidation are involved in atherosclerotic plaque formation and may signal through PAF-receptor (PAF-R) expressed in human macrophages and in certain smooth muscle cells (SMCs) in the media, but rarely in the intima of human plaques. Our aim was to determine which SMC phenotype expresses PAF-R and whether this receptor is functional in cell migration. Circulating SMC progenitors and two phenotypically distinct clones of proliferative, epithelioid phenotype vs contractile, spindle-shaped SMCs from the media of adult internal thoracic artery were studied for the presence of PAF-receptor (PAF-R). The levels of specific mRNA were obtained by reverse transcription/real-time PCR, the protein expression was deduced from immunohistochemistry staining, and the functional transmigration assay was performed by Boyden chamber-type chemotaxis assay. Only SMCs of spindle-shape and synthetic phenotype expressed both mRNA and PAF-R protein and in the functional test migrated at low concentrations of PAF. Two unrelated, specific PAF-R antagonists inhibited PAF-induced migration, but did not modify the migration initiated by PDGF. The presence of functional PAF-R in arterial spindle-shaped SMCs of synthetic phenotype may be important for their migration from the media into the intima and atherosclerotic plaques formation

Arylesterase Phenotype-Specific Positive Association Between Arylesterase Activity and Cholinesterase Specific Activity in Human Serum

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Yutaka Aoki

2014-01-01

Full Text Available Context: Cholinesterase (ChE specific activity is the ratio of ChE activity to ChE mass and, as a biomarker of exposure to cholinesterase inhibitors, has a potential advantage over simple ChE activity. Objective: To examine the association of several potential correlates (serum arylesterase/paraoxonase activity, serum albumin, sex, age, month of blood collection, and smoking with plasma ChE specific activity. Methods: We analyzed data from 195 cancer-free controls from a nested case-control study, accounting for potential confounding. Results: Arylesterase activity had an independent, statistically significant positive association with ChE specific activity, and its magnitude was the greatest for the arylesterase phenotype corresponding to the QQ PON1192 genotype followed by phenotypes corresponding to QR and RR genotypes. Serum albumin was positively associated with ChE specific activity. Conclusions: Plasma arylesterase activity was positively associated with plasma ChE specific activity. This observation is consistent with protection conferred by a metabolic phenotype resulting in reduced internal dose.

Stochasticity or the fatal `imperfection' of cloning

Indian Academy of Sciences (India)

2005-01-07

Jan 7, 2005 ... The concept of clone is analysed with the aim of exploring the limits to which a phenotype can be said to be determined geneticaly. First of all, mutations that result from the replication, topological manipulation or lesion of DNA introduce a source of heritable variation in an otherwise identical genetic ...

Learning, memory and exploratory similarities in genetically identical cloned dogs.

Science.gov (United States)

Shin, Chi Won; Kim, Geon A; Park, Won Jun; Park, Kwan Yong; Jeon, Jeong Min; Oh, Hyun Ju; Kim, Min Jung; Lee, Byeong Chun

2016-12-30

Somatic cell nuclear transfer allows generation of genetically identical animals using donor cells derived from animals with particular traits. To date, few studies have investigated whether or not these cloned dogs will show identical behavior patterns. To address this question, learning, memory and exploratory patterns were examined using six cloned dogs with identical nuclear genomes. The variance of total incorrect choice number in the Y-maze test among cloned dogs was significantly lower than that of the control dogs. There was also a significant decrease in variance in the level of exploratory activity in the open fields test compared to age-matched control dogs. These results indicate that cloned dogs show similar cognitive and exploratory patterns, suggesting that these behavioral phenotypes are related to the genotypes of the individuals.

Establishment and Analysis of Cancer Stem-Like and Non-Cancer Stem-Like Clone Cells from the Human Colon Cancer Cell Line SW480.

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Akari Takaya

Full Text Available Human cancer stem-like cells (CSCs/cancer-initiating cells (CICs can be isolated as side population (SP cells, aldehyde dehydrogenase high (ALDHhigh cells or cell surface marker-positive cells including CD44+ cells and CD133+ cells. CSCs/CICs and non-CSCs/CICs are unstable in in vitro culture, and CSCs/CICs can differentiate into non-CSCs/CICs and some non-CSCs/CICs can dedifferentiate into CSCs/CICs. Therefore, experiments using a large amount of CSCs/CICs are technically very difficult. In this study, we isolated single cell clones from SP cells and main population (MP cells derived from the human colon cancer cell line SW480. SP analysis revealed that SP clone cells had relatively high percentages of SP cells, whereas MP clone cells showed very few SP cells, and the phenotypes were sustainable for more than 2 months of in vitro culture. Xenograft transplantation revealed that SP clone cells have higher tumor-initiating ability than that of MP clone cells and SP clone cell showed higher chemo-resistance compared with MP clone cells. These results indicate that SP clone cells derived from SW480 cells are enriched with CSCs/CICs, whereas MP clone cells are pure non-CSCs/CICs. SP clone cells and MP clone cells are a very stable in vitro CSC/CIC-enriched and non-CSC/CIC model for further analysis.

Establishment and Analysis of Cancer Stem-Like and Non-Cancer Stem-Like Clone Cells from the Human Colon Cancer Cell Line SW480.

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Takaya, Akari; Hirohashi, Yoshihiko; Murai, Aiko; Morita, Rena; Saijo, Hiroshi; Yamamoto, Eri; Kubo, Terufumi; Nakatsugawa, Munehide; Kanaseki, Takayuki; Tsukahara, Tomohide; Tamura, Yasuaki; Takemasa, Ichiro; Kondo, Toru; Sato, Noriyuki; Torigoe, Toshihiko

2016-01-01

Human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be isolated as side population (SP) cells, aldehyde dehydrogenase high (ALDHhigh) cells or cell surface marker-positive cells including CD44+ cells and CD133+ cells. CSCs/CICs and non-CSCs/CICs are unstable in in vitro culture, and CSCs/CICs can differentiate into non-CSCs/CICs and some non-CSCs/CICs can dedifferentiate into CSCs/CICs. Therefore, experiments using a large amount of CSCs/CICs are technically very difficult. In this study, we isolated single cell clones from SP cells and main population (MP) cells derived from the human colon cancer cell line SW480. SP analysis revealed that SP clone cells had relatively high percentages of SP cells, whereas MP clone cells showed very few SP cells, and the phenotypes were sustainable for more than 2 months of in vitro culture. Xenograft transplantation revealed that SP clone cells have higher tumor-initiating ability than that of MP clone cells and SP clone cell showed higher chemo-resistance compared with MP clone cells. These results indicate that SP clone cells derived from SW480 cells are enriched with CSCs/CICs, whereas MP clone cells are pure non-CSCs/CICs. SP clone cells and MP clone cells are a very stable in vitro CSC/CIC-enriched and non-CSC/CIC model for further analysis.

Hybrid clone cells derived from human breast epithelial cells and human breast cancer cells exhibit properties of cancer stem/initiating cells.

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Gauck, Daria; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S; Dittmar, Thomas

2017-08-02

The biological phenomenon of cell fusion has been associated with cancer progression since it was determined that normal cell × tumor cell fusion-derived hybrid cells could exhibit novel properties, such as enhanced metastatogenic capacity or increased drug resistance, and even as a mechanism that could give rise to cancer stem/initiating cells (CS/ICs). CS/ICs have been proposed as cancer cells that exhibit stem cell properties, including the ability to (re)initiate tumor growth. Five M13HS hybrid clone cells, which originated from spontaneous cell fusion events between M13SV1-EGFP-Neo human breast epithelial cells and HS578T-Hyg human breast cancer cells, and their parental cells were analyzed for expression of stemness and EMT-related marker proteins by Western blot analysis and confocal laser scanning microscopy. The frequency of ALDH1-positive cells was determined by flow cytometry using AldeRed fluorescent dye. Concurrently, the cells' colony forming capabilities as well as the cells' abilities to form mammospheres were investigated. The migratory activity of the cells was analyzed using a 3D collagen matrix migration assay. M13HS hybrid clone cells co-expressed SOX9, SLUG, CK8 and CK14, which were differently expressed in parental cells. A variation in the ALDH1-positive putative stem cell population was observed among the five hybrids ranging from 1.44% (M13HS-7) to 13.68% (M13HS-2). In comparison to the parental cells, all five hybrid clone cells possessed increased but also unique colony formation and mammosphere formation capabilities. M13HS-4 hybrid clone cells exhibited the highest colony formation capacity and second highest mammosphere formation capacity of all hybrids, whereby the mean diameter of the mammospheres was comparable to the parental cells. In contrast, the largest mammospheres originated from the M13HS-2 hybrid clone cells, whereas these cells' mammosphere formation capacity was comparable to the parental breast cancer cells. All M13HS

Cloning, sequencing, disruption and phenotypic analysis of uvsC, an Aspergillus nidulans homologue of yeast RAD51.

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van Heemst, D; Swart, K; Holub, E F; van Dijk, R; Offenberg, H H; Goosen, T; van den Broek, H W; Heyting, C

1997-05-01

We have cloned the uvsC gene of Aspergillus nidulans by complementation of the A. nidulans uvsC114 mutant. The predicted protein UVSC shows 67.4% sequence identity to the Saccharomyces cerevisiae Rad51 protein and 27.4% sequence identity to the Escherichia coli RecA protein. Transcription of uvsC is induced by methyl-methane sulphonate (MMS), as is transcription of RAD51 of yeast. Similar levels of uvsC transcription were observed after MMS induction in a uvsC+ strain and the uvsC114 mutant. The coding sequence of the uvsC114 allele has a deletion of 6 bp, which results in deletion of two amino acids and replacement of one amino acid in the translation product. In order to gain more insight into the biological function of the uvsC gene, a uvsC null mutant was constructed, in which the entire uvsC coding sequence was replaced by a selectable marker gene. Meiotic and mitotic phenotypes of a uvsC+ strain, the uvsC114 mutant and the uvsC null mutant were compared. The uvsC null mutant was more sensitive to both UV and MMS than the uvsC114 mutant. The uvsC114 mutant arrested in meiotic prophase-I. The uvsC null mutant arrested at an earlier stage, before the onset of meiosis. One possible interpretation of these meiotic phenotypes is that the A. nidulans homologue of Rad51 of yeast has a role both in the specialized processes preceding meiosis and in meiotic prophase I.

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

Human T cell leukemia/lymphoma virus type I infection of a CD4+ proliferative/cytotoxic T cell clone progresses in at least two distinct phases based on changes in function and phenotype of the infected cells

NARCIS (Netherlands)

Yssel, H.; de Waal Malefyt, R.; Duc Dodon, M. D.; Blanchard, D.; Gazzolo, L.; de Vries, J. E.; Spits, H.

1989-01-01

The effect of human T cell leukemia/lymphoma virus type I (HTLV-I) infection on the function and the phenotype of a human proliferating/cytotoxic T cell clone, specific for tetanus toxin, was investigated. During the period after infection, two distinct phases were observed, based on growth

Analysis of IgG4-positive clones in affected organs of IgG4-related disease.

Science.gov (United States)

Kakuchi, Yasushi; Yamada, Kazunori; Ito, Kiyoaki; Hara, Satoshi; Fujii, Hiroshi; Yamagishi, Masakazu; Kawano, Mitsuhiro

2016-11-01

We investigated class switch reaction (CSR) in affected organs and evaluated whether the same or genetically related clones exist in IgG4-RD. We studied three patients with IgG4-RD. Total cellular RNA was extracted from salivary glands and peripheral blood and lung tissue. Activation-induced cytidine deaminase (AID) and immunoglobulin heavy chain third complementarity determining region (IgVH-CDR3) of IgM and IgG4 were detected by reverse transcription polymerase chain reaction (RT-PCR). We analyzed the clonal relationship of infiltrating IgG4-positive cells, as compared with IgM. We determined the existence of common clones among organs and patients. AID was expressed in salivary glands of all patients and lung tissue in one. Closely related IgVH-CDR3 sequences in infiltrating IgG4-positive cells were detected in salivary glands and lung tissue. Identical IgVH-CDR3 sequence between IgM and IgG4 in salivary glands was detected in one patient, indicating CSR in salivary glands. Identical IgVH-CDR3 sequences of IgG4-positive cells were detected between salivary glands and peripheral blood in two patients. Four identical sequences of IgVH-CDR3 existed between patients. Interestingly, one of the four sequences was detected in all patients. Our results demonstrate the existence of common antigen(s) shared by patients with IgG4-RD.

Phenotypic and genotypic characterization of vancomycin-resistant Enterococcus faecium clinical isolates from two hospitals in Mexico: First detection of VanB phenotype-vanA genotype.

Science.gov (United States)

Bocanegra-Ibarias, Paola; Flores-Treviño, Samantha; Camacho-Ortiz, Adrián; Morfin-Otero, Rayo; Villarreal-Treviño, Licet; Llaca-Díaz, Jorge; Martínez-Landeros, Erik Alan; Rodríguez-Noriega, Eduardo; Calzada-Güereca, Andrés; Maldonado-Garza, Héctor Jesús; Garza-González, Elvira

2016-01-01

Enterococcus faecium has emerged as a multidrug-resistant nosocomial pathogen involved in outbreaks worldwide. Our aim was to determine the antimicrobial susceptibility, biofilm production, and clonal relatedness of vancomycin-resistant E. faecium (VREF) clinical isolates from two hospitals in Mexico. Consecutive clinical isolates (n=56) were collected in two tertiary care hospitals in Mexico from 2011 to 2014. VREF isolates were characterized by phenotypic and molecular methods including pulsed-field gel electrophoresis (PFGE). VREF isolates were highly resistant to vancomycin, erythromycin, norfloxacin, high-level streptomycin, and teicoplanin, and showed lower resistance to tetracycline, nitrofurantoin and quinupristin-dalfopristin. None of the isolates were resistant to linezolid. The vanA gene was detected in all isolates. Two VanB phenotype-vanA genotype isolates, highly resistant to vancomycin and susceptible to teicoplanin, were detected. Furthermore, 17.9% of the isolates were classified as biofilm producers, and the espfm gene was found in 98.2% of the isolates. A total of 37 distinct PFGE patterns and 6 clones (25% of the isolates as clone A, 5.4% as clone B, and 3.6% each as clone C, D, E, and F) were detected. Clone A was detected in 5 different wards of the same hospital during 14 months of surveillance. The high resistance to most antimicrobial agents and the moderate cross-transmission of VREF detected accentuates the need for continuous surveillance of E. faecium in the hospital setting. This is also the first reported incidence of the E. faecium VanB phenotype-vanA genotype in the Americas. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Farmer participatory evaluation of eight elite clones of cocoyam ...

African Journals Online (AJOL)

Eight elite clones of cocoyam were evaluated for high yield, tolerance to major diseases and pests and culinary properties. Phenotypic attributes evaluated at peak vegetative phase 24 weeks after planting (24 WAP) were plant height, number of leaves and leaf area. At harvest, 12 months after planting (12 MAP), variables ...

Map-based cloning and expression analysis of BMR-6 in sorghum

Indian Academy of Sciences (India)

CAD), using a map-based cloning approach. Genetic complementation confirmed that CAD is responsible for the BMR-6 phenotype. BMR-6 gene was expressed in all tested sorghum tissues, with the highest being in midrib and stem. Transient ...

Establishment and characterization of Epstein-Barr virus-specific human CD4+ T lymphocyte clones

International Nuclear Information System (INIS)

Honda, S.; Okuno, K.; Yasutomi, M.; Takasaki, T.; Kurane, I.

1998-01-01

We developed a simple method for establishing Epstein-Barr virus (EBV)-specific, human CD4+ T cell clones. The method originates from our experience that the regression of cell growth in in vitro EBV transformation of B cells occurs when round lymphoid cells appear in the culture. Peripheral blood mononuclear cells were cultured with EBV; and IL-2 (20 U/ml) was added to the culture on day 17 after the virus addition. The phenotype of the growing cells was CD3+ , CD4+ , and CD8-. The cells were cytotoxic for autologous lymphoblastoid B cell line (LCL) and EBV-super-infected autologous LCL. The cytotoxic T lymphocytes (CTLs) were confirmed to be CD4+ T cells but not CD8+ T cells in the culture. CTL clones were established by a limiting dilution method. All the CTL clones had the phenotype of CD3+ , CD4+ and CD8-, and proliferated in response to autologous LCL. They produced interferon (IFN)-gamma, interleukin 2 (IL-2) and tumour necrosis factor (TNF)-beta but not IL-4. All but one clone responded to both autologous, EBV-super-infected and non-super-infected LCLs. Proliferative and cytotoxic responses to allogeneic LCLs were heterogeneous. These results suggest that this method induces heterogeneous, EBV-specific CD4+ CTL clones and is useful for analysis of CD4+ T cells in EBV infections. (authors)

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/

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...

Clonal stability of latex yield in eleven clones of Hevea brasiliensis Muell. Arg.

Directory of Open Access Journals (Sweden)

K.O. Omokhafe

2003-01-01

Full Text Available Eleven Hevea brasiliensis clones were evaluated for clonal stability of latex yield. A randomized complete block design was used with four replicates, two locations, seven years and three periods per year. Stability analysis was based on clone x year and clone x year x location interactions. Five stability parameters viz environmental variance, shukla's stability variance, regression of clonal latex yield on environmental index, variance due to regression and variance due to deviation from regression were applied. There was significant clone x environment effect at the two levels of interaction. Among the eleven clones, C 162 was outstanding for clonal stability and it can serve as donor parent for stability alleles. Three clones (C 76, C 150 and C 154 were also stable. The four stable clones (C 76, C 150, C 154 and C 162 are suitable for broad-spectrum recommendation for latex yield. Five clones (C 83, C 143, C 163, C 202 and RRIM 600 will require environment-specific recommendation because of their unstable phenotype. The stability feature of two clones (C 145 and C 159 was not clear and this will be investigated in subsequent studies.

Persistence of collagen type II-specific T-cell clones in the synovial membrane of a patient with rheumatoid arthritis

International Nuclear Information System (INIS)

Londei, M.; Savill, C.M.; Verhoef, A.; Brennan, F.; Leech, Z.A.; Feldmann, M.; Duance, V.; Maini, R.N.

1989-01-01

Rheumatoid arthritis is an autoimmune disease characterized by T-cell infiltration of the synovium of joints. Analysis of the phenotype and antigen specificity of the infiltrating cells may thus provide insight into the pathogenesis of rheumatoid arthritis. T cells were cloned with interleukin 2, a procedure that selects for in vivo-activated cells. All clones had the CD4 CDW29 phenotype. Their antigen specificity was tested by using a panel of candidate joint autoantigens. Four of 17 reacted against autologous blood mononuclear cells. Two clones proliferated in response to collagen type II. After 21 months, another set of clones was derived from synovial tissue of the same joint. One of eight clones tested showed a strong proliferative response against collagen type II. The uncloned synovial T cells of a third operation from another joint also responded to collagen type II. The persistence of collagen type II-specific T cells in active rheumatoid joints over a period of 3 years suggests that collagen type II could be one of the autoantigens involved in perpetuating the inflammatory process in rheumatoid arthritis

Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer.

Science.gov (United States)

Lanza, R P; Cibelli, J B; Diaz, F; Moraes, C T; Farin, P W; Farin, C E; Hammer, C J; West, M D; Damiani, P

2000-01-01

Approximately 100 species become extinct a day. Despite increasing interest in using cloning to rescue endangered species, successful interspecies nuclear transfer has not been previously described, and only a few reports of in vitro embryo formation exist. Here we show that interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic and phenotypic development through implantation and the late stages of fetal growth. Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction, (Species Survival Plan cloned animals was gaurus in origin. The gaur nuclei were shown to direct normal fetal development, with differentiation into complex tissue and organs, even though the mitochondrial DNA (mtDNA) within all the tissue types evaluated was derived exclusively from the recipient bovine oocytes. These results suggest that somatic cell cloning methods could be used to restore endangered, or even extinct, species and populations.

Variation in gene expression within clones of the earthworm Dendrobaena octaedra.

Directory of Open Access Journals (Sweden)

Marina Mustonen

Full Text Available Gene expression is highly plastic, which can help organisms to both acclimate and adapt to changing environments. Possible variation in gene expression among individuals with the same genotype (among clones is not widely considered, even though it could impact the results of studies that focus on gene expression phenotypes, for example studies using clonal lines. We examined the extent of within and between clone variation in gene expression in the earthworm Dendrobaena octaedra, which reproduces through apomictic parthenogenesis. Five microsatellite markers were developed and used to confirm that offspring are genetic clones of their parent. After that, expression of 12 genes was measured from five individuals each from six clonal lines after exposure to copper contaminated soil. Variation in gene expression was higher over all genotypes than within genotypes, as initially assumed. A subset of the genes was also examined in the offspring of exposed individuals in two of the clonal lines. In this case, variation in gene expression within genotypes was as high as that observed over all genotypes. One gene in particular (chymotrypsin inhibitor also showed significant differences in the expression levels among genetically identical individuals. Gene expression can vary considerably, and the extent of variation may depend on the genotypes and genes studied. Ensuring a large sample, with many different genotypes, is critical in studies comparing gene expression phenotypes. Researchers should be especially cautious inferring gene expression phenotypes when using only a single clonal or inbred line, since the results might be specific to only certain genotypes.

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.

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.

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.

Detection of paroxysmal nocturnal hemoglobinuria clones in patients with myelodysplastic syndromes and related bone marrow diseases, with emphasis on diagnostic pitfalls and caveats.

Science.gov (United States)

Wang, Sa A; Pozdnyakova, Olga; Jorgensen, Jeffrey L; Medeiros, L Jeffrey; Stachurski, Dariusz; Anderson, Mary; Raza, Azra; Woda, Bruce A

2009-01-01

The presence of paroxysmal nocturnal hemoglobinuria clones in the setting of aplastic anemia or myelodysplastic syndrome has been shown to have prognostic and therapeutic implications. However, the status of paroxysmal nocturnal hemoglobinuria clones in various categories of myelodysplastic syndrome and in other bone marrow disorders is not well-studied. By using multiparameter flow cytometry immunophenotypic analysis with antibodies specific for four glycosylphosphatidylinositol-anchored proteins (CD55, CD59, CD16, CD66b) and performing an aerolysin lysis confirmatory test in representative cases, we assessed the paroxysmal nocturnal hemoglobinuria-phenotype granulocytes in 110 patients with myelodysplastic syndrome, 15 with myelodysplastic/myeloproliferative disease, 5 with idiopathic myelofibrosis and 6 with acute myeloid leukemia. Paroxysmal nocturnal hemoglobinuria-phenotype granulocytes were detected in nine patients with low grade myelodysplastic syndrome who showed clinicopathological features of bone marrow failure, similar to aplastic anemia. All paroxysmal nocturnal hemoglobinuria-positive cases demonstrated loss of the four glycosylphosphatidylinositol-anchored proteins, with CD16(-)CD66b(-) clones being larger than those of CD55(-)CD59(-) (p<0.05). Altered glycosylphosphatidylinositol-anchored protein expression secondary to granulocytic hypogranulation, immaturity, and/or immunophenotypic abnormalities was present in a substantial number of cases and diagnostically challenging. These results show that routine screening for paroxysmal nocturnal hemoglobinuria clones in patients with an intrinsic bone marrow disease who show no clinical evidence of hemolysis has an appreciable yield in patients with low grade myelodysplastic syndromes. The recognition of diagnostic caveats and pitfalls associated with the underlying intrinsic bone marrow disease is essential in interpreting paroxysmal nocturnal hemoglobinuria testing correctly. In our experience, the CD

Proteins interacting with cloning scars: a source of false positive protein-protein interactions.

Science.gov (United States)

Banks, Charles A S; Boanca, Gina; Lee, Zachary T; Florens, Laurence; Washburn, Michael P

2015-02-23

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine "cloning scar" present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected.

Yield Evaluation of Nutrient-rich Potato Clones in High Hill of Nepal

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Binod Prasad Luitel

2017-05-01

Full Text Available A study was conducted to evaluate the yield of nutrient-rich potato clones in high-hill districts: Dolakha and Jumla of Nepal during the years 2013 and 2014, respectively. Fourteen potato clones were tested as on-station and on-farm experiments at both districts, and those fourteen clones were compared to ‘Lady Rosita’ and ‘Jumli Local’ respectively as the check varieties in the first year experiment, 2013. Eight promising clones were selected from the first year experiment, and were evaluated and compared with same local varieties in the consecutive year, 2014. Two clones namely; CIP 395112.32 (19.3 tha-1 and CIP 393073.179 (17.8 tha-1 exhibited superior marketable tuber yield than that of ‘Lady Rosita’(14.2 tha-1 in Dolakha and five CIP clones namely; 395112.32 (25.5 tha-1, 393073.179 (22.5 tha-1, 394611.112 (20.9 tha-1, 390478.9 (19.9 tha-1 and 395017.229 (17.0 tha-1 showed higher marketable tuber yield than ‘Jumli Local’(14.5 tha-1. Based on two years’ phenotypic and tuber yield result, clones CIP 395112.32 and CIP 393073.179 are recommended to potato growers at high hills of Nepal for commercial cultivation.

Genetic and phenotypic characteristics of importance for clonal success and diversity in Salmonella

DEFF Research Database (Denmark)

Müller, Karoline

dominance of certain clones. These epidemically successful clones are often resistant to antibiotics and associated with severe human illness. They pose a major threat to public health and lead to heavy economic losses. So far, little is known about the environmental and bacterial factors leading...... to the emergence of successful clones. However, resistance to multiple antimicrobial drugs and quinolones seems to contribute to the epidemic success of Salmonella as it is associated with an increased severity of illness and epidemicity. In order to predict and prevent future outbreaks and epidemics, research...... should focus on the evolutionary mechanisms of emerging success clones. The ability to spread in different food production sectors and to cause human disease seems critical for a clone to become successful. The aim of this PhD study was to identify common phenotypic and genetic traits of success clones...

A hybrid approach identifies metabolic signatures of high-producers for chinese hamster ovary clone selection and process optimization.

Science.gov (United States)

Popp, Oliver; Müller, Dirk; Didzus, Katharina; Paul, Wolfgang; Lipsmeier, Florian; Kirchner, Florian; Niklas, Jens; Mauch, Klaus; Beaucamp, Nicola

2016-09-01

In-depth characterization of high-producer cell lines and bioprocesses is vital to ensure robust and consistent production of recombinant therapeutic proteins in high quantity and quality for clinical applications. This requires applying appropriate methods during bioprocess development to enable meaningful characterization of CHO clones and processes. Here, we present a novel hybrid approach for supporting comprehensive characterization of metabolic clone performance. The approach combines metabolite profiling with multivariate data analysis and fluxomics to enable a data-driven mechanistic analysis of key metabolic traits associated with desired cell phenotypes. We applied the methodology to quantify and compare metabolic performance in a set of 10 recombinant CHO-K1 producer clones and a host cell line. The comprehensive characterization enabled us to derive an extended set of clone performance criteria that not only captured growth and product formation, but also incorporated information on intracellular clone physiology and on metabolic changes during the process. These criteria served to establish a quantitative clone ranking and allowed us to identify metabolic differences between high-producing CHO-K1 clones yielding comparably high product titers. Through multivariate data analysis of the combined metabolite and flux data we uncovered common metabolic traits characteristic of high-producer clones in the screening setup. This included high intracellular rates of glutamine synthesis, low cysteine uptake, reduced excretion of aspartate and glutamate, and low intracellular degradation rates of branched-chain amino acids and of histidine. Finally, the above approach was integrated into a workflow that enables standardized high-content selection of CHO producer clones in a high-throughput fashion. In conclusion, the combination of quantitative metabolite profiling, multivariate data analysis, and mechanistic network model simulations can identify metabolic

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 ...

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.

Generation of Infectious Poliovirus with Altered Genetic Information from Cloned cDNA.

Science.gov (United States)

Bujaki, Erika

2016-01-01

The effect of specific genetic alterations on virus biology and phenotype can be studied by a great number of available assays. The following method describes the basic protocol to generate infectious poliovirus with altered genetic information from cloned cDNA in cultured cells.The example explained here involves generation of a recombinant poliovirus genome by simply replacing a portion of the 5' noncoding region with a synthetic gene by restriction cloning. The vector containing the full length poliovirus genome and the insert DNA with the known mutation(s) are cleaved for directional cloning, then ligated and transformed into competent bacteria. The recombinant plasmid DNA is then propagated in bacteria and transcribed to RNA in vitro before RNA transfection of cultured cells is performed. Finally, viral particles are recovered from the cell culture.

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.

Cloning of regions required for contact hemolysis and entry into LLC-MK2 cells from Shigella sonnei form I plasmid: virF is a positive regulator gene for these phenotypes.

OpenAIRE

Kato, J; Ito, K; Nakamura, A; Watanabe, H

1989-01-01

Two distinct regions required for both contact hemolysis and entry into LLC-MK2 cells were cloned into Escherichia coli from the Shigella sonnei form I plasmid, pSS120. The first region was cloned into an E. coli HB101 strain containing noninvasive Tn1 insertion mutants of the form I plasmid, and expression of ipa (invasion plasmid antigen) gene products was restored. The plasmid carrying the first region was then transformed into E. coli lacking the form I plasmid, and additional DNA fragmen...

Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattle.

Science.gov (United States)

Mesquita, Fernando S; Machado, Sergio A; Drnevich, Jenny; Borowicz, Pawel; Wang, Zhongde; Nowak, Romana A

2013-01-30

Poor success rates in somatic cell cloning are often attributed to abnormal early embryonic development as well as late abnormal fetal growth and placental development. Although promising results have been reported following chromatin transfer (CT), a novel cloning method that includes the remodeling of the donor nuclei in vitro prior to their transfer into enucleated oocytes, animals cloned by CT show placental abnormalities similar to those observed following conventional nuclear transfer. We hypothesized that the placental gene expression pattern from cloned fetuses was ontologically related to the frequently observed placental phenotype. The aim of the present study was to compare global gene expression by microarray analysis of Day 44-47 cattle placentas derived from CT cloned fetuses with those derived from in vitro fertilization (i.e. control), and confirm the altered mRNA and protein expression of selected molecules by qRT-PCR and immunohistochemistry, respectively. The differentially expressed genes identified in the present study are known to be involved in a range of activities associated with cell adhesion, cell cycle control, intracellular transport and proteolysis. Specifically, an imprinted gene, involved with cell proliferation and placentomegaly in humans (CDKN1C) and a peptidase that serves as a marker for non-invasive trophoblast cells in human placentas (DPP4), had mRNA and protein altered in CT placentas. It was concluded that the altered pattern of gene expression observed in CT samples may contribute to the abnormal placental development phenotypes commonly identified in cloned offspring, and that expression of imprinted as well as trophoblast invasiveness-related genes is altered in cattle cloned by CT. Copyright © 2012 Elsevier B.V. All rights reserved.

A lesion mimic phenotype in tomato obtained by isolating and silencing an Lls1 homologue

NARCIS (Netherlands)

Spassieva, S; Hille, J

Lesion mimic phenotypes serve as a tool to study the regulation of cell death in plants. In order to obtain a tomato lesion mimic phenotype, we used the conservation of the lethal leaf spot 1 (Lls1) genes between plant species. The tomato Lls1 homologue was cloned, sequenced and analyzed. It showed

Bacterial Artificial Chromosome Clones of Viruses Comprising the Towne Cytomegalovirus Vaccine

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Xiaohong Cui

2012-01-01

Full Text Available Bacterial artificial chromosome (BAC clones have proven invaluable for genetic manipulation of herpesvirus genomes. BAC cloning can also be useful for capturing representative genomes that comprise a viral stock or mixture. The Towne live attenuated cytomegalovirus vaccine was developed in the 1970s by serial passage in cultured fibroblasts. Although its safety, immunogenicity, and efficacy have been evaluated in nearly a thousand human subjects, the vaccine itself has been little studied. Instead, genetic composition and in vitro growth properties have been inferred from studies of laboratory stocks that may not always accurately represent the viruses that comprise the vaccine. Here we describe the use of BAC cloning to define the genotypic and phenotypic properties of viruses from the Towne vaccine. Given the extensive safety history of the Towne vaccine, these BACs provide a logical starting point for the development of next-generation rationally engineered cytomegalovirus vaccines.

Bacterial artificial chromosome clones of viruses comprising the towne cytomegalovirus vaccine.

Science.gov (United States)

Cui, Xiaohong; Adler, Stuart P; Davison, Andrew J; Smith, Larry; Habib, El-Sayed E; McVoy, Michael A

2012-01-01

Bacterial artificial chromosome (BAC) clones have proven invaluable for genetic manipulation of herpesvirus genomes. BAC cloning can also be useful for capturing representative genomes that comprise a viral stock or mixture. The Towne live attenuated cytomegalovirus vaccine was developed in the 1970s by serial passage in cultured fibroblasts. Although its safety, immunogenicity, and efficacy have been evaluated in nearly a thousand human subjects, the vaccine itself has been little studied. Instead, genetic composition and in vitro growth properties have been inferred from studies of laboratory stocks that may not always accurately represent the viruses that comprise the vaccine. Here we describe the use of BAC cloning to define the genotypic and phenotypic properties of viruses from the Towne vaccine. Given the extensive safety history of the Towne vaccine, these BACs provide a logical starting point for the development of next-generation rationally engineered cytomegalovirus vaccines.

Realizing directional cloning using sticky ends produced by 3′-5 ...

Indian Academy of Sciences (India)

http://www.ias.ac.in/jbiosci. J. Biosci. 38(5), December 2013, 857–866, © Indian Academy of Sciences. Supplementary material. Supplementary figure 1. Sequencing of PCR positive clones. (A) Forward insertion of non-directional cloning. (B) Reverse insertion of non-directional cloning. (C) Forward insertion of directional ...

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?

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)

Cloning of Salmonella typhimurium DNA encoding mutagenic DNA repair

International Nuclear Information System (INIS)

Thomas, S.M.; Sedgwick, S.G.

1989-01-01

Mutagenic DNA repair in Escherichia coli is encoded by the umuDC operon. Salmonella typhimurium DNA which has homology with E. coli umuC and is able to complement E. coli umuC122::Tn5 and umuC36 mutations has been cloned. Complementation of umuD44 mutants and hybridization with E. coli umuD also occurred, but these activities were much weaker than with umuC. Restriction enzyme mapping indicated that the composition of the cloned fragment is different from the E. coli umuDC operon. Therefore, a umu-like function of S. typhimurium has been found; the phenotype of this function is weaker than that of its E. coli counterpart, which is consistent with the weak mutagenic response of S. typhimurium to UV compared with the response in E. coli

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

Optimal multicopy asymmetric Gaussian cloning of coherent states

International Nuclear Information System (INIS)

Fiurasek, Jaromir; Cerf, Nicolas J.

2007-01-01

We investigate the asymmetric Gaussian cloning of coherent states which produces M copies from N input replicas in such a way that the fidelity of each copy may be different. We show that the optimal asymmetric Gaussian cloning can be performed with a single phase-insensitive amplifier and an array of beam splitters. We obtain a simple analytical expression characterizing the set of optimal asymmetric Gaussian cloning machines and prove the optimality of these cloners using the formalism of Gaussian completely positive maps and semidefinite programming techniques. We also present an alternative implementation of the asymmetric cloning machine where the phase-insensitive amplifier is replaced with a beam splitter, heterodyne detector, and feedforward

Optimal multicopy asymmetric Gaussian cloning of coherent states

Science.gov (United States)

Fiurášek, Jaromír; Cerf, Nicolas J.

2007-05-01

We investigate the asymmetric Gaussian cloning of coherent states which produces M copies from N input replicas in such a way that the fidelity of each copy may be different. We show that the optimal asymmetric Gaussian cloning can be performed with a single phase-insensitive amplifier and an array of beam splitters. We obtain a simple analytical expression characterizing the set of optimal asymmetric Gaussian cloning machines and prove the optimality of these cloners using the formalism of Gaussian completely positive maps and semidefinite programming techniques. We also present an alternative implementation of the asymmetric cloning machine where the phase-insensitive amplifier is replaced with a beam splitter, heterodyne detector, and feedforward.

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

significantly higher birth rate of healthy clones (0.5009% vs. 0.3362% and 0.2433%) than that resulting from P,D,L,Y-AFBs and P,D,L,Y-FFBs. This suggests that using LW-AFBs as donor cells results in a higher cloning efficiency in pigs, compared with the other two donor fibroblast cell types. The birth rate of healthy clones was significantly improved when the number of transferred cloned embryos was increased from 150-199 to 200-450 per recipient. However, increase of the number of transferred embryos from 200-249 to 250-450 per surrogate did not change the birth rate of healthy clones. This suggests that transfer of excessive (250-450) cloned embryos to an individual surrogate is not necessary for increasing the cloning efficiency in pigs, and the relatively optimal number of reconstructed embryos transferred to individual recipient is 200-249. Furthermore, our results indicated that the numbers of total born clones, clones born alive, and clones born healthy per litter have a significantly high positive correlation with each other. The present study provides useful information for improving SCNT efficiency in pigs.

Major histocompatibility complex-unrestricted cytolytic activity of human T cells: analysis of precursor frequency and effector phenotype

International Nuclear Information System (INIS)

Patel, S.S.; Thiele, D.L.; Lipsky, P.E.

1987-01-01

The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. All of the 198 clones generated by this method were T cells (CD2 + , CD3 + , CD4 + or CD2 + , CD3 + , CD8 + ) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis, measured by 51 Cr release, was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur of K562 was not mediated by a soluble factor secreted by the clones. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD 16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype

Early selection of elite clones of an ornamental bromeliad in vitro Seleção precoce in vitro de clones elite de uma bromélia ornamental

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Candida Elisa Manfio

2010-07-01

Full Text Available Orthophytum grossiorum is a typical bromeliad from Atlantic forestry threatened of extinction. The objectives of this research were to select O. grossiorum clones with ornamental values easy to propagate in vitro, and establish in vitro propagation protocols for these clones. The project was developed in three steps: germination and in vitro selection of seedlings responsive to BAP (6-benzylaminopurine, selection of clones with ornamental values, and establishment of protocol for in vitro propagation of the selected clones. In the first step only 18.33% of plantlets germinated in vitro were responsive to BAP. These plantlets were selected and replicated in vitro several times, each replicated plantlet constituting a clone. In the second step these clones were established ex vitro and surveyed for ornamental attributes. Five out of 11 clones were selected in this step. These clones presented distinct phenotypic traits and were considered of high ornamental quality. In the third step a protocol for in vitro propagation was developed for each selected clone.Orthophytum grossiorum é uma bromélia ameaçada de extinção típica de Mata Atlântica. Os objetivos deste trabalho foram selecionar clones de O. grossiorum com potencial ornamental e de fácil propagação in vitro e estabelecer protocolo de propagação in vitro para esses clones. O trabalho foi desenvolvido em três etapas: germinação e em seleção in vitro de plântulas responsivas a BAP (6-benzylaminopurine, seleção de clones com valores ornamentais e estabelecimento de protocolo para propagação in vitro dos clones selecionados. Na primeira etapa, foi observado que apenas 18.33% das plântulas germinadas in vitro eram responsivas a BAP. Essas plântulas foram selecionadas e reproduzidas em in vitro, e cada plântula selecionada e reproduzida constituiu um clone. Na segunda etapa, esses clones foram estabelecidos ex vitro e selecionados em relação aos atributos ornamentais

Assessment of mitochondrial functions in Daphnia pulex clones using high-resolution respirometry.

Science.gov (United States)

Kake-Guena, Sandrine A; Touisse, Kamal; Vergilino, Roland; Dufresne, France; Blier, Pierre U; Lemieux, Hélène

2015-06-01

The objectives of our study were to adapt a method to measure mitochondrial function in intact mitochondria from the small crustacean Daphnia pulex and to validate if this method was sensitive enough to characterize mitochondrial metabolism in clones of the pulex complex differing in ploidy levels, mitochondrial DNA haplotypes, and geographic origins. Daphnia clones belonging to the Daphnia pulex complex represent a powerful model to delineate the link between mitochondrial DNA evolution and mitochondrial phenotypes, as single genotypes with divergent mtDNA can be grown under various experimental conditions. Our study included two diploid clones from temperate environments and two triploid clones from subarctic environments. The whole animal permeabilization and measurement of respiration with high-resolution respirometry enabled the measurement of the functional capacity of specific mitochondrial complexes in four clones. When expressing the activity as ratios, our method detected significant interclonal variations. In the triploid subarctic clone from Kuujjurapik, a higher proportion of the maximal physiological oxidative phosphorylation (OXPHOS) capacity of mitochondria was supported by complex II, and a lower proportion by complex I. The triploid subarctic clone from Churchill (Manitoba) showed the lowest proportion of the maximal OXPHOS supported by complex II. Additional studies are required to determine if these differences in mitochondrial functions are related to differences in mitochondrial haplotypes or ploidy level and if they might be associated with fitness divergences and therefore selective value. © 2015 Wiley Periodicals, Inc.

Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

Energy Technology Data Exchange (ETDEWEB)

Henderson, R.F.; Waide, J.J.; Lechner, J.F.

1995-12-01

A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

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.

Experimental alternatives for evaluation of progenies and clones in eucalyptus breeding programs

Directory of Open Access Journals (Sweden)

Souza Elaine Aparecida de

2003-01-01

Full Text Available The feasibility of using augmented block designs and spatial analysis methods for early stage selection in eucalyptus breeding programs was tested. A total of 113 half-sib progenies of Eucalyptus urophylla and eight clones were evaluated in an 11 x 11 triple lattice experiment at two locations: Posto da Mata (Bahia, Brazil and São Mateus (Minas Gerais, Brazil. Four checks were randomly allocated within each block. Plots consisted of 15 m long rows containing 6 plants spaced 3 m apart. The girth at breast height (cm/plant was evaluated at 19 and 26 months of age. Variance analyses were performed according to the following methods: lattice design, randomized complete block design, augmented block design, Papadakis method, moving means method, and check plots. Comparisons among different methods were based on the magnitude of experimental errors and precision of the estimates of genetic and phenotypic parameters. General results indicated that augmented block design is useful to evaluate progenies and clones in early selection in eucalyptus breeding programs using moderate and low selection intensities. However, this design is not suitable for estimating genetic and phenotypic parameters due to its low precision. Check plots, nearest neighbour, Papadakis (1937, and moving means methods were efficient in removing the heterogeneity within blocks. These efficiencies were compared to that in lattice analysis for estimation of genetic and phenotypic parameters.

Innate immune responses to obesity in cloned and wild-type domestic pig

DEFF Research Database (Denmark)

Højbøge, Tina Rødgaard; Skovgaard, Kerstin; Stagsted, Jan

as a refined pig model for obesity-induced innate host responses by reducing pig-to-pig biological variation compared to wild-type (WT) pigs (n=19). Pigs were fed ad libitum with a high fat/high sucrose diet to induce obesity or kept lean on a restricted diet (60% of ad libitum intake) beginning at three...... months of age. mRNA expression levels were determined for 39 innate immune factors on a high-throughput qPCR system in samples from liver, abdominal fat, mesenteric fat and subcutaneous fat. Previous findings have suggested that cloning may affect certain phenotypic traits of pigs including basic...... concentrations and responsiveness of components of the innate immune system. Terminal body weights at 7½ - 9½ months of age were significantly higher for both (WT and cloned) obese groups compared to the lean groups. However, obese WT pigs weighed significantly more than obese cloned pigs (P

Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

Directory of Open Access Journals (Sweden)

Sandra Arenhart

Full Text Available Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3. The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

Establishment and characterization of canine parvovirus-specific murine CD4+ T cell clones and their use for the delineation of T cell epitopes.

NARCIS (Netherlands)

G.F. Rimmelzwaan (Guus); R.W.J. van der Heijden (Roger); E.J. Tijhaar (Edwin); M.C.M. Poelen (Martien); J. Carlson; A.D.M.E. Osterhaus (Albert); F.G.C.M. Uytdehaag (Fons)

1990-01-01

textabstractCanine parvovirus (CPV)-specific T cell clones were generated by culturing lymph node cells from CPV-immunized BALB/c mice at limiting dilutions in the presence of CPV antigen and interleukin-2 (IL-2). All isolated T cell clones exhibited the cell surface phenotype Thy1+, CD4+, CD8- and

Public perceptions of animal cloning

DEFF Research Database (Denmark)

Jelsøe, Erling; Vincentsen, Ulla; Andersen, Ida-Elisabeth

What was from the outset meant to be a survey testing predefined categories of ethical positions related to new biotechnologies with animal cloning as an example was subsequently developed into a process of broader involvement of groups of citizens in the issue. The survey was conducted at meetings...... in four different cities in Denmark. The participants were introduced to animal cloning and after that they filled out the questionnaire. Finally, the issue was discussed in focus groups. The process as a whole was run in a dialogue oriented way. Through the information they received in combination...... with reflecting on the survey questions the participants were well prepared for discussions in the focus groups. This approach made it possible, on the one hand to get a measure of the citizen's perceptions of the ethical aspects of animal cloning, but also to go deeper into their own thoughts of the issue...

Analysis of polymorphisms in milk proteins from cloned and sexually reproduced goats.

Science.gov (United States)

Xing, H; Shao, B; Gu, Y Y; Yuan, Y G; Zhang, T; Zang, J; Cheng, Y

2015-12-08

This study evaluates the relationship between the genotype and milk protein components in goats. Milk samples were collected from cloned goats and normal white goats during different postpartum (or abortion) phases. Two cloned goats, originated from the same somatic line of goat mammary gland epithelial cells, and three sexually reproduced normal white goats with no genetic relationships were used as the control. The goats were phylogenetically analyzed by polymerase chain reaction-restriction fragment length polymorphism. The milk protein components were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results indicated that despite the genetic fingerprints being identical, the milk protein composition differed between the two cloned goats. The casein content of cloned goat C-50 was significantly higher than that of cloned goat C-4. Conversely, although the genetic fingerprints of the normal white goats N-1, N-2, and N-3 were not identical, the milk protein profiles did not differ significantly in their milk samples (obtained on postpartum day 15, 20, 25, 30, and 150). These results indicated an association between milk protein phenotypes and genetic polymorphisms, epigenetic regulation, and/or non-chromosomal factors. This study extends the knowledge of goat milk protein polymorphisms, and provides new strategies for the breeding of high milk-yielding goats.

Probabilistic quantum cloning of a subset of linearly dependent states

Science.gov (United States)

Rui, Pinshu; Zhang, Wen; Liao, Yanlin; Zhang, Ziyun

2018-02-01

It is well known that a quantum state, secretly chosen from a certain set, can be probabilistically cloned with positive cloning efficiencies if and only if all the states in the set are linearly independent. In this paper, we focus on probabilistic quantum cloning of a subset of linearly dependent states. We show that a linearly-independent subset of linearly-dependent quantum states {| Ψ 1⟩,| Ψ 2⟩,…,| Ψ n ⟩} can be probabilistically cloned if and only if any state in the subset cannot be expressed as a linear superposition of the other states in the set {| Ψ 1⟩,| Ψ 2⟩,…,| Ψ n ⟩}. The optimal cloning efficiencies are also investigated.

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.

Rabies virus-specific human T cell clones provide help for an in vitro antibody response against neutralizing antibody-inducing determinants of the viral glycoprotein.

NARCIS (Netherlands)

H. Bunschoten; R.J. Klapmuts; I.J.Th.M. Claassen (Ivo); S.D. Reijneveld; A.D.M.E. Osterhaus (Albert); F.G.C.M. Uytdehaag (Fons)

1989-01-01

textabstractHuman T cell clones were prepared from peripheral blood mononuclear cells from a vaccinated human donor and kept in culture in the presence of rabies virus antigen and growth factors. Phenotypic analysis of the T cell clones revealed expression of the CD3 and CD4 cell surface markers,

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

Cloning humans? Current science, current views, and a perspective from Christianity.

Science.gov (United States)

Brun, Rudolf B

2002-01-01

Therapeutic cloning is urgent and should be vigorously supported. To successfully argue for this position, the distinction between a human embryo and a human nuclear transplant may be helpful. Even if current technical difficulties should be solved, global legislation should prohibit cloning for the purpose of fabricating babies. This position originates from a view on human nature in general and from a Christian perspective in particular.

X-linked hypophosphatemia. A phenotype in search of a cause.

Science.gov (United States)

Tenenhouse, H S; Scriver, C R

1992-05-01

XLH is an important disease, it is the subject of several classic articles in the medical sciences (Scriver et al., 1991), and it has been an important stimulus to study renal hypophosphatemias and how they are involved in rickets and osteomalacia (Scriver, 1974; Scriver and Tenenhouse, 1991). Renal transport is the major determinant of phosphate homeostasis in mammals and it is unlikely that this important biochemical parameter would have been left by evolution to a single renal transport system. Together physiologists and geneticists found that the mammalian kidney has several gene products dedicated to phosphate transport. That has implications for biochemists in search of a membrane protein to clone and explain XLH, for example. Let us suppose the transporter affected in XLH is cloned. Will it be the product of the XLH (or Hyp or Gy) locus? One will not know until the transporter gene is mapped. There is no question of the X-chromosome locus product being protein kinase C for example, since it maps to autosomes. But where does one start in the search for the X-chromosome locus? With the elusive putative diffusible factor or with the transporter, or perhaps with an enzyme in vitamin D hormone metabolism? Which goes to say that it is necessary to know the phenotype to arrive at the right locus. Or is it? Sufficient physical mapping of region Xp22.31-p21.3 will eventually lead to positional cloning of the Hyp gene. What will it be?(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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

Hydraulic and mechanical properties of young Norway spruce clones related to growth and wood structure

Science.gov (United States)

ROSNER, SABINE; KLEIN, ANDREA; MÜLLER, ULRICH; KARLSSON, BO

2011-01-01

Summary Stem segments of eight five-year-old Norway spruce (Picea abies (L.) Karst.) clones differing in growth characteristics were tested for maximum specific hydraulic conductivity (ks100), vulnerability to cavitation and behavior under mechanical stress. The vulnerability of the clones to cavitation was assessed by measuring the applied air pressure required to cause 12 and 50% loss of conductivity (Ψ12, Ψ50) and the percent loss of conductivity at 4 MPa applied air pressure (PLC4MPa). The bending strength and stiffness and the axial compression strength and stiffness of the same stem segments were measured to characterize wood mechanical properties. Growth ring width, wood density, latewood percentage, lumen diameter, cell wall thickness, tracheid length and pit dimensions of earlywood cells, spiral grain and microfibril angles were examined to identify structure–function relationships. High ks100 was strongly and positively related to spiral grain angle, which corresponded positively to tracheid length and pit dimensions. Spiral grain may reduce flow resistance of the bordered pits of the first earlywood tracheids, which are characterized by rounded tips and an equal distribution of pits along the entire length. Wood density was unrelated to hydraulic vulnerability parameters. Traits associated with higher hydraulic vulnerability were long tracheids, high latewood percentage and thick earlywood cell walls. The positive relationship between earlywood cell wall thickness and vulnerability to cavitation suggest that air seeding through the margo of bordered pits may occur in earlywood. There was a positive phenotypic and genotypic relationship between ks100 and PLC4MPa, and both parameters were positively related to tree growth rate. Variability in mechanical properties depended mostly on wood density, but also on the amount of compression wood. Accordingly, hydraulic conductivity and mechanical strength or stiffness showed no tradeoff. PMID:17472942

Toward understanding of the role of reversibility of phenotypic switching in the evolution of resistance to therapy

Science.gov (United States)

Horvath, D.; Brutovsky, B.

2018-06-01

Reversibility of state transitions is intensively studied topic in many scientific disciplines over many years. In cell biology, it plays an important role in epigenetic variation of phenotypes, known as phenotypic plasticity. More interestingly, the cell state reversibility is probably crucial in the adaptation of population phenotypic heterogeneity to environmental fluctuations by evolving bet-hedging strategy, which might confer to cancer cells resistance to therapy. In this article, we propose a formalization of the evolution of highly reversible states in the environments of periodic variability. Two interrelated models of heterogeneous cell populations are proposed and their behavior is studied. The first model captures selection dynamics of the cell clones for the respective levels of phenotypic reversibility. The second model focuses on the interplay between reversibility and drug resistance in the particular case of cancer. Overall, our results show that the threshold dependencies are emergent features of the investigated model with eventual therapeutic relevance. Presented examples demonstrate importance of taking into account cell to cell heterogeneity within a system of clones with different reversibility quantified by appropriately chosen genetic and epigenetic entropy measures.

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.

Development of high-throughput phenotyping of metagenomic clones from the human gut microbiome for modulation of eukaryotic cell growth.

Science.gov (United States)

Gloux, Karine; Leclerc, Marion; Iliozer, Harout; L'Haridon, René; Manichanh, Chaysavanh; Corthier, Gérard; Nalin, Renaud; Blottière, Hervé M; Doré, Joël

2007-06-01

Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.

GenMapDB: a database of mapped human BAC clones

OpenAIRE

Morley, Michael; Arcaro, Melissa; Burdick, Joshua; Yonescu, Raluca; Reid, Thomas; Kirsch, Ilan R.; Cheung, Vivian G.

2001-01-01

GenMapDB (http://genomics.med.upenn.edu/genmapdb) is a repository of human bacterial artificial chromosome (BAC) clones mapped by our laboratory to sequence-tagged site markers. Currently, GenMapDB contains over 3000 mapped clones that span 19 chromosomes, chromosomes 2, 4, 5, 9–22, X and Y. This database provides positional information about human BAC clones from the RPCI-11 human male BAC library. It also contains restriction fragment analysis data and end sequen...

Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.

Science.gov (United States)

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.

Establishment of primary bovine intestinal epithelial cell culture and clone method.

Science.gov (United States)

Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

2017-01-01

The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

Development of new USER-based cloning vectors for multiple genes expression in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Kildegaard, Kanchana Rueksomtawin; Jensen, Niels Bjerg; Maury, Jerome

2013-01-01

auxotrophic and dominant markers for convenience of use. Our vector set also contains both integrating and multicopy vectors for stability of protein expression and high expression level. We will make the new vector system available to the yeast community and provide a comprehensive protocol for cloning...... the production strain with the proper phenotype and product yield. However, the sequential number of metabolic engineering is time-consuming. Furthermore, the number of available selectable markers is also limiting the number of genetic modifications. To overcome these limitations, we have developed a new set...... of shuttle vectors for convenience of use for high-throughput cloning and selectable marker recycling. The new USER-based cloning vectors consist of a unique USER site and a CRE-loxP-mediated marker recycling system. The USER site allows insertion of genes of interest along with a bidirectional promoter...

Cloning and properties of the Salmonella typhimurium tricarboxylate transport operon in Escherichia coli

International Nuclear Information System (INIS)

Widenhorn, K.A.; Boos, W.; Somers, J.M.; Kay, W.W.

1988-01-01

The tricarboxylate transport operon (tctI) was cloned in Escherichia coli as a 12-kilobase (kb) fragment from an EcoRI library of the Salmonella typhimurium chromosome in λgtWES. It was further subcloned as a 12-kb fragment into pACYC184 and as an 8-kb fragment into pBR322. By insertional mutagenesis mediated by λTn5, restriction mapping, and phenotypic testing, the tctI operon was localized to a 4.5-kb region. The tctC gene which encodes a periplasmic binding protein (C-protein) was located near the center of the insert. E. coli/tctI clones on either multicopy or single-copy vectors grew on the same tricarboxylates as S. typhimurium, although unusually long growth lags were observed. E. coli/tctI clones exhibited similar [ 14 C] fluorocitrate transport kinetics to those of S. typhimurium, whereas E. coli alone was virtually impermeable to [ 14 C] fluorocitrate. The periplasmic C proteins (C1 and C2 isoelectric forms) were produced in prodigious quantities from the cloned strains. Motile E. coli/tctI clones were not chemotactic toward citrate, whereas tctI deletion mutants of S. typhimurium were. Taken together, these observations indicate that tctI is not an operon involved in chemotaxis

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.

Genotype-environment interaction and phenotypic stability for girth growth and rubber yield of Hevea clones in São Paulo State, Brazil

Directory of Open Access Journals (Sweden)

Gonçalves Paulo de Souza

2003-01-01

Full Text Available The best-yielding, best vigour and most stable Hevea clones are identified by growing clones in different environments. However, research on the stability in Hevea brasiliensis (Willd. Adr. ex Juss. Muell.-Arg. is scarce. The objectives of this work were to assess genotype-environment interaction and determine stable genotypes. Stability analysis were performed on results for girth growth and rubber yield of seven clones from five comparative trials conducted over 10 years (girth growth and four years (rubber yield in São Paulo State, Brazil. Stability was estimated using the Eberhart and Russell (1966 method. Year by location and location variability were the dominant sources of interactions. The stability analysis identified GT 1 and IAN 873 as the most stable clones for girth growth and rubber yield respectively since their regression coefficients were almost the unity (b = 1 and they had one of the lowest deviations from regressions (S2di. Their coefficient of determination (R² was as high as 89.5% and 89.8% confirming their stability. In contrast, clones such as PB 235, PR 261, and RRIM 701 for girth growth and clones such as GT 1 for rubber yield with regression coefficients greater than one were regarded as sensitive to environment changes.

Ovulation Statuses of Surrogate Gilts Are Associated with the Efficiency of Excellent Pig Cloning.

Science.gov (United States)

Huan, Yanjun; Hu, Kui; Xie, Bingteng; Shi, Yongqian; Wang, Feng; Zhou, Yang; Liu, Shichao; Huang, Bo; Zhu, Jiang; Liu, Zhongfeng; He, Yilong; Li, Jingyu; Kong, Qingran; Liu, Zhonghua

2015-01-01

Somatic cell nuclear transfer (SCNT) is an assisted reproductive technique that can produce multiple copies of excellent livestock. However, low cloning efficiency limits the application of SCNT. In this study, we systematically investigated the major influencing factors related to the overall cloning efficiency in pigs. Here, 13620 cloned embryos derived from excellent pigs were transferred into 79 surrogate gilts, and 119 live cloned piglets were eventually generated. During cloning, group of cloned embryos derived from excellent Landrace or Large white pigs presented no significant differences of cleavage and blastocyst rates, blastocyst cell numbers, surrogate pregnancy and delivery rates, average numbers of piglets born and alive and cloning efficiencies, and group of 101-150, 151-200 or 201-250 cloned embryos transferred per surrogate also displayed a similar developmental efficiency. When estrus stage of surrogate gilts was compared, group of embryo transfer on Day 2 of estrus showed significantly higher pregnancy rate, delivery rate, average number of piglets born, average alive piglet number or cloning efficiency than group on Day 1, Day 3, Day 4 or Day 5, respectively (Pcloning efficiency (Pcloning efficiency. And more, follicle puncture for preovulation, not transfer position shallowed for preovulation or deepened for postovulation, significantly improved the average number of piglets alive and cloning efficiency (Pcloning efficiency of excellent pigs, and follicle puncture, not transfer position change, improved cloning efficiency. This work would have important implications in preserving and breeding excellent livestock and improving the overall cloning efficiency.

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)

Control of the proportion of inner cells by asymmetric divisions and the ensuing resilience of cloned rabbit embryos

Science.gov (United States)

Duranthon, Véronique

2018-01-01

ABSTRACT Mammalian embryo cloning by nuclear transfer has a low success rate. This is hypothesized to correlate with a high variability of early developmental steps that segregate outer cells, which are fated to extra-embryonic tissues, from inner cells, which give rise to the embryo proper. Exploring the cell lineage of wild-type embryos and clones, imaged in toto until hatching, highlights the respective contributions of cell proliferation, death and asymmetric divisions to phenotypic variability. Preferential cell death of inner cells in clones, probably pertaining to the epigenetic plasticity of the transferred nucleus, is identified as a major difference with effects on the proportion of inner cell. In wild type and clones, similar patterns of outer cell asymmetric divisions are shown to be essential to the robust proportion of inner cells observed in wild type. Asymmetric inner cell division, which is not described in mice, is identified as a regulator of the proportion of inner cells and likely gives rise to resilient clones. PMID:29567671

[The status of human cloning in the international setting].

Science.gov (United States)

Rey del Castillo, Javier

2006-01-01

The General Assembly of the United Nations submitted a Declaration on Human Cloning in March 2005. The text of such Declaration was the result of a difficult and long process, taking more than three years. Being a Declaration instead of a Resolution, it has not legal capability in inforcing United Nations members to act according to its recommendations. This article begins with an explanation of several terms referred to cloning. Different countries' legislation on cloning is analyzed. Positions of the same countries at the Convention of the United Nations are as well analyzed. Comparing both countries' views shows that national legislation on cloning is independent and orientated by some countries' particular interests and biological and ethical views on these issues. Future developments on human cloning and its applications will be shared among all countries, both the ones currently allowing and supporting "therapeutic" cloning and the ones now banning it. In such case, it would be important to reach agreements on these issues at an international level. The article discusses possible legislative developments and offers some proposals to reach such agreements.

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…

Clonal structure of Trypanosoma cruzi Colombian strain (biodeme Type III: biological, isoenzymic and histopathological analysis of seven isolated clones

Directory of Open Access Journals (Sweden)

Camandaroba Edson Luiz Paes

2001-01-01

Full Text Available The clonal structure of the Colombian strain of Trypanosoma cruzi, biodeme Type III and zymodeme 1, was analyzed in order to characterize its populations and to establish its homogeneity or heterogeneity. Seven isolated clones presented the basic characteristics of Biodeme Type III, with the same patterns of parasitemic curves, tissue tropism to skeletal muscle and myocardium, high pathogenicity with extensive necrotic-inflammatory lesions from the 20th to 30th day of infection. The parental strain and its clones C1, C3, C4 and C6, determined the higher levels of parasitemia, 20 to 30 days of infection, with high mortality rate up to 30 days (79 to 100%; clones C2, C5 and C7 presented lower levels of parasitemia, with low mortality rates (7.6 to 23%. Isoenzymic patterns, characteristic of zymodeme 1, (Z1 were similar for the parental strain and its seven clones. Results point to a phenotypic homogeneity of the clones isolated from the Colombian strain and suggest the predominance of a principal clone, responsible for the biological behavior of the parental strain and clones.

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

Sequence of a cloned cDNA encoding human ribosomal protein S11

Energy Technology Data Exchange (ETDEWEB)

Lott, J B; Mackie, G A

1988-02-11

The authors have isolated a cloned cDNA that encodes human ribosomal protein (rp) S11 by screening a human fibroblast cDNA library with a labelled 204 bp DNA fragment encompassing residues 212-416 of pRS11, a rat rp Sll cDNA clone. The human rp S11 cloned cDNA consists of 15 residues of the 5' leader, the entire coding sequence and all 51 residues of the 3' untranslated region. The predicted amino acid sequence of 158 residues is identical to rat rpS11. The nucleotide sequence in the coding region differs, however, from that in rat in the first position in two codons and in the third position in 44 codons.

Cloning changes the response to obesity of innate immune factors in blood, liver, and adipose tissues in domestic pigs.

Science.gov (United States)

Rødgaard, Tina; Skovgaard, Kerstin; Stagsted, Jan; Heegaard, Peter M H

2013-06-01

The objective of this study was to evaluate the usefulness of cloned pigs as porcine obesity models reflecting obesity-associated changes in innate immune factor gene expression profiles. Liver and adipose tissue expression of 43 innate immune genes as well as serum concentrations of six immune factors were analyzed in lean and diet-induced obese cloned domestic pigs and compared to normal domestic pigs (obese and lean). The number of genes affected by obesity was lower in cloned animals than in control animals. All genes affected by obesity in adipose tissues of clones were downregulated; both upregulation and downregulation were observed in the controls. Cloning resulted in a less differentiated adipose tissue expression pattern. Finally, the serum concentrations of two acute-phase proteins (APPs), haptoglobin (HP) and orosomucoid (ORM), were increased in obese clones as compared to obese controls as well as lean clones and controls. Generally, the variation in phenotype between individual pigs was not reduced in cloned siblings as compared to normal siblings. Therefore, we conclude that cloning limits both the number of genes responding to obesity as well as the degree of tissue-differentiated gene expression, concomitantly with an increase in APP serum concentrations only seen in cloned, obese pigs. This may suggest that the APP response seen in obese, cloned pigs is a consequence of the characteristic skewed gene response to obesity in cloned pigs, as described in this work. This should be taken into consideration when using cloned animals as models for innate responses to obesity.

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

Origin and characteristics of Pseudomonas aeruginosa PAO1 clones surviving after the induction of transposable prophages

Energy Technology Data Exchange (ETDEWEB)

Krylov, V.N.; Solov`era, T.I.; Burkal`tseva, M.V. [State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow (Russian Federation)] [and others

1995-08-01

Various mutations cancelling the lethal effect of phage lytic development and simultaneous phenotypic modifications were found in rare clones surviving after incubation at 42{degrees}C of Pseudomonas aeruginosa (D3112 cts15), lysogenic for thermoinducible mutant cts15 of the transposable prophage (TP) D3112. All mutations arose prior to thermal induction. Temperature induction of other bacteriophages (nontransposable) did not lead to selection of bacterial morphological mutants. Therefore, it was concluded that mutagenesis occurred upon the partial (reversible) TP derepression accompanied by coupled replication-transposition of TP, the latter being the direct cause of the mutator effect. Isolation of the P. aeruginosa PAO1 mutant R10 (this mutant is resistant to infection with TP at 42{degrees}C) allowed the proper selection and examination of numerous survivors. Comparison of their types derived from lysogens with different prophage location indicated that the number of secondary sites where TP integration is possible without the loss of cell viability is limited. Several transposition events occurred in the history of some survivors (during a repeated or single derepression event). Type D clones, which produce small colonies, are of special interest, because mechanisms underlying the survival of such clones are extremely diverse, and their phenotypes indicate the possibility of stable chromosomal rearrangements in the genome of P. aeruginosa. 16 refs., 2 figs., 2 tabs.

Quantitative Seq-LGS: Genome-Wide Identification of Genetic Drivers of Multiple Phenotypes in Malaria Parasites

KAUST Repository

Abkallo, Hussein M.

2016-10-01

Identifying the genetic determinants of phenotypes that impact on disease severity is of fundamental importance for the design of new interventions against malaria. Traditionally, such discovery has relied on labor-intensive approaches that require significant investments of time and resources. By combining Linkage Group Selection (LGS), quantitative whole genome population sequencing and a novel mathematical modeling approach (qSeq-LGS), we simultaneously identified multiple genes underlying two distinct phenotypes, identifying novel alleles for growth rate and strain specific immunity (SSI), while removing the need for traditionally required steps such as cloning, individual progeny phenotyping and marker generation. The detection of novel variants, verified by experimental phenotyping methods, demonstrates the remarkable potential of this approach for the identification of genes controlling selectable phenotypes in malaria and other apicomplexan parasites for which experimental genetic crosses are amenable.

Variations and voids: the regulation of human cloning around the world.

Science.gov (United States)

Pattinson, Shaun D; Caulfield, Timothy

2004-12-13

No two countries have adopted identical regulatory measures on cloning. Understanding the complexity of these regulatory variations is essential. It highlights the challenges associated with the regulation of a controversial and rapidly evolving area of science and sheds light on a regulatory framework that can accommodate this reality. Using the most reliable information available, we have performed a survey of the regulatory position of thirty countries around the world regarding the creation and use of cloned embryos (see Table 1). We have relied on original and translated legislation, as well as published sources and personal communications. We have examined the regulation of both reproductive cloning (RC) and non-reproductive cloning (NRC). While most of the countries studied have enacted national legislation, the absence of legislation in seven of these countries should not be equated with the absence of regulation. Senator Morin was not correct in stating that the majority of recent legislation bans both RC and NRC. Recent regulatory moves are united only with regard to the banning of RC. While NRC is not permitted in seventeen of the countries examined, it could be permitted in up to thirteen countries. There is little consensus on the various approaches to cloning laws and policies, and the regulatory position in many countries remains uncertain.

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

What justifies the United States ban on federal funding for nonreproductive cloning?

Science.gov (United States)

Cunningham, Thomas V

2013-11-01

This paper explores how current United States policies for funding nonreproductive cloning are justified and argues against that justification. I show that a common conceptual framework underlies the national prohibition on the use of public funds for cloning research, which I call the simple argument. This argument rests on two premises: that research harming human embryos is unethical and that embryos produced via fertilization are identical to those produced via cloning. In response to the simple argument, I challenge the latter premise. I demonstrate there are important ontological differences between human embryos (produced via fertilization) and clone embryos (produced via cloning). After considering the implications my argument has for the morality of publicly funding cloning for potential therapeutic purposes and potential responses to my position, I conclude that such funding is not only ethically permissible, but also humane national policy.

Ovulation Statuses of Surrogate Gilts Are Associated with the Efficiency of Excellent Pig Cloning.

Directory of Open Access Journals (Sweden)

Yanjun Huan

Full Text Available Somatic cell nuclear transfer (SCNT is an assisted reproductive technique that can produce multiple copies of excellent livestock. However, low cloning efficiency limits the application of SCNT. In this study, we systematically investigated the major influencing factors related to the overall cloning efficiency in pigs. Here, 13620 cloned embryos derived from excellent pigs were transferred into 79 surrogate gilts, and 119 live cloned piglets were eventually generated. During cloning, group of cloned embryos derived from excellent Landrace or Large white pigs presented no significant differences of cleavage and blastocyst rates, blastocyst cell numbers, surrogate pregnancy and delivery rates, average numbers of piglets born and alive and cloning efficiencies, and group of 101-150, 151-200 or 201-250 cloned embryos transferred per surrogate also displayed a similar developmental efficiency. When estrus stage of surrogate gilts was compared, group of embryo transfer on Day 2 of estrus showed significantly higher pregnancy rate, delivery rate, average number of piglets born, average alive piglet number or cloning efficiency than group on Day 1, Day 3, Day 4 or Day 5, respectively (P<0.05. And, in comparison with the preovulation and postovulation groups, group of surrogate gilts during periovulation displayed a significantly higher overall cloning efficiency (P<0.05. Further investigation of surrogate estrus stage and ovulation status displayed that ovulation status was the real factor underlying estrus stage to determine the overall cloning efficiency. And more, follicle puncture for preovulation, not transfer position shallowed for preovulation or deepened for postovulation, significantly improved the average number of piglets alive and cloning efficiency (P<0.05. In conclusion, our results demonstrated that ovulation status of surrogate gilts was the fundamental factor determining the overall cloning efficiency of excellent pigs, and follicle

Public and Private Preferences for Animal Cloning Policies

OpenAIRE

Brooks, Kathleen R.; Lusk, Jayson L.

2012-01-01

Data on individuals’ private shopping choices are often used to draw conclusions about their desires for food policies. The purpose of this paper is to test this often-implicit assumption using data from a nationwide survey about animal cloning. We find that although individuals’ private choices indicate a strong desire to avoid meat and milk from cloned cattle, public choices predict that only 40.29% have a positive WTP for such a ban. The results suggest caution is necessary when inferr...

Evaluation of flooring produced from small diameters logs of Eucalyptus sp. clones

Directory of Open Access Journals (Sweden)

José Reinaldo Moreira da Silva

2010-12-01

Full Text Available This study evaluated two Eucalyptus clones, MN 249 and MN 89, for the flooring production using small diameters logs. It was considered the wood physical properties - NBR 7190/97 (ABNT, 1997 and simulation of the product in service (ASTM D 2394/83 with two thicknesses, 8 and 14 mm. The basic density of the clone 89 NM was the highest one (0,615 g/cm3. The contractions were more pronounced in clone NM 249, however, the anisotropy coefficient of this clone was small. In the simulation tests, the floor produced by clone MN 249 presented the lowest deformation rate. The floor of 8 mm, in addition to the differences between clones, there was significant interaction between the positions for the indentation test caused by loads applied in small areas. The deformations obtained for the floor with 14 mm thickness, produced with the MN clone 89, were higher than those found in the literature for the indentation load applied on a small area test. The clone MN 249 presented the best results in both thicknesses.

Identification of a subset of perpheral T-cell lymphoma, not otherwise specified, characterized by FOXP3-positive regulatory T-cell phenotype, HTLV-1 negativity and poor outcome

DEFF Research Database (Denmark)

Pedersen, Martin Bjerregård; Hamilton-Dutoit, Stephen Jacques; Bendix, Knud

2014-01-01

Identification of a subset of perpheral T-cell lymphoma, not otherwise specified, characterized by FOXP3-positive regulatory T-cell phenotype, HTLV-1 negativity and poor outcome.......Identification of a subset of perpheral T-cell lymphoma, not otherwise specified, characterized by FOXP3-positive regulatory T-cell phenotype, HTLV-1 negativity and poor outcome....

A NEW CELL CLONE DERIVED FROM TRICHOPLUSIA NI TN5B1-4 CELLS

Institute of Scientific and Technical Information of China (English)

Jian-xiaoTian; Chang-youLi; Gui-lingZheng; Guo-xunLi; PingWang; Granados

2004-01-01

The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B 1-4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins,however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B 1-4 cells. The growth characteristics,productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B 1-4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing β-galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B 1-4 cells.

Towards an understanding of British public attitudes concerning human cloning.

Science.gov (United States)

Shepherd, Richard; Barnett, Julie; Cooper, Helen; Coyle, Adrian; Moran-Ellis, Jo; Senior, Victoria; Walton, Chris

2007-07-01

The ability of scientists to apply cloning technology to humans has provoked public discussion and media coverage. The present paper reports on a series of studies examining public attitudes to human cloning in the UK, bringing together a range of quantitative and qualitative methods to address this question. These included a nationally representative survey, an experimental vignette study, focus groups and analyses of media coverage. Overall the research presents a complex picture of attitude to and constructions of human cloning. In all of the analyses, therapeutic cloning was viewed more favourably than reproductive cloning. However, while participants in the focus groups were generally negative about both forms of cloning, and this was also reflected in the media analyses, quantitative results showed more positive responses. In the quantitative research, therapeutic cloning was generally accepted when the benefits of such procedures were clear, and although reproductive cloning was less accepted there was still substantial support. Participants in the focus groups only differentiated between therapeutic and reproductive cloning after the issue of therapeutic cloning was explicitly raised; initially they saw cloning as being reproductive cloning and saw no real benefits. Attitudes were shown to be associated with underlying values associated with scientific progress rather than with age, gender or education, and although there were a few differences in the quantitative data based on religious affiliation, these tended to be small effects. Likewise in the focus groups there was little direct appeal to religion, but the main themes were 'interfering with nature' and the 'status of the embryo', with the latter being used more effectively to try to close down further discussion. In general there was a close correspondence between the media analysis and focus group responses, possibly demonstrating the importance of media as a resource, or that the media reflect

T cell clones which share T cell receptor epitopes differ in phenotype, function and specificity

NARCIS (Netherlands)

Yssel, H.; Blanchard, D.; Boylston, A.; de Vries, J. E.; Spits, H.

1986-01-01

Recently, we described a monoclonal antibody (3D6) that reacts with the T cell receptor (Ti) of the T leukemic cell line HPB-ALL and that cross-reacts with 2-10% of the T cells of normal healthy individuals. In this study we report the establishment of T cell clones that are 3D6+ but that differ in

Bengal Bay clone ST772-MRSA-V outbreak: conserved clone causes investigation challenges.

Science.gov (United States)

Blomfeldt, A; Larssen, K W; Moghen, A; Haugum, K; Steen, T W; Jørgensen, S B; Aamot, H V

2017-03-01

The Bengal Bay clone, ST772-MRSA-V, associated with multi-drug resistance, Panton-Valentine leukocidin (PVL) and skin and soft tissue infections, is emerging worldwide. In Norway, a country with low prevalence of meticillin-resistant Staphylococcus aureus (MRSA), increased occurrence of ST772-MRSA-V has also caused hospital outbreaks. The conserved nature of this clone challenged the outbreak investigations. To evaluate the usefulness of S. aureus protein A (spa) typing, multiple-locus variable number tandem repeat fingerprinting/analysis (MLVF/MLVA) and pulsed-field gel electrophoresis (PFGE) when investigating outbreaks with a conserved MRSA clone. A panel of 25 MRSA isolates collected in 2004-2014, consisting of six hospital outbreak isolates and 19 sporadic isolates, were analysed using spa typing, polymerase chain reaction detection of genes encoding PVL, MLVF/MLVA and PFGE. All isolates were ST772-MRSA-V-t657 and resistant to erythromycin, gentamicin and norfloxacin, and 88% were PVL positive. PFGE could not discriminate between the isolates (≥85% similarity). MLVF resolved five types [Simpson's index of diversity (SID)=0.56], MLVA resolved six types (SID=0.66), and both methods separated the hospital isolates into two defined outbreaks. MLVF/MLVA could not discriminate all epidemiologically unlinked cases and identical genotypes originated from a timespan of 10 years. MLVA was regarded as most suitable due to its higher discriminatory power and ability to provide unambiguous profiles. However, the Bengal Bay clone may require higher resolution methods for exact demarcation of outbreaks due to low diversity among isolates. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Preliminary assessment of the grading of Eucalyptus clones using carbon isotope discrimination

International Nuclear Information System (INIS)

Bond, W.J.; Stock, W.D.

1990-01-01

Stable carbon isotopes were analysed in leaf material of nine Eucalyptus clones grown in field trials in the eastern Transvaal. Carbon isotope ratios, measured as d 13 C, differed within tree canopies, between replicate trees and between clones. Values from both north and south canopy positions were correlated with tree height after 13 months. Unexplained variation in the correlation may be interpreted, theoretically, as an indication that some clones use less water for the same level of productivity. With further testing, the method may have promise for early screening of clones in genotype/environment interaction trials and in selecting water-efficient trees. 3 figs., 3 tabs., 7 refs

A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype

Science.gov (United States)

Woeste, K. E.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

2000-01-01

A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resulted in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a competitive inhibitor of ethylene binding. In contrast to the previously identified ran1-1 and ran1-2 alleles that are morphologically indistinguishable from wild-type plants, this ran1-3 allele results in a rosette-lethal phenotype. The predicted protein encoded by the RAN1 gene is similar to the Wilson and Menkes disease proteins and yeast Ccc2 protein, which are integral membrane cation-transporting P-type ATPases involved in copper trafficking. Genetic epistasis analysis indicated that RAN1 acts upstream of mutations in the ethylene receptor gene family. However, the rosette-lethal phenotype of ran1-3 was not suppressed by ethylene-insensitive mutants, suggesting that this mutation also affects a non-ethylene-dependent pathway regulating cell expansion. The phenotype of ran1-3 mutants is similar to loss-of-function ethylene receptor mutants, suggesting that RAN1 may be required to form functional ethylene receptors. Furthermore, these results suggest that copper is required not only for ethylene binding but also for the signaling function of the ethylene receptors.

Path analysis of the energy density of wood in eucalyptus clones.

Science.gov (United States)

Couto, A M; Teodoro, P E; Trugilho, P F

2017-03-16

Path analysis has been used for establishing selection criteria in genetic breeding programs for several crops. However, it has not been used in eucalyptus breeding programs yet. In the present study, we aimed to identify the wood technology traits that could be used as the criteria for direct and indirect selection of eucalyptus genotypes with high energy density of wood. Twenty-four eucalyptus clones were evaluated in a completely randomized design with five replications. The following traits were assessed: basic wood density, total extractives, lignin content, ash content, nitrogen content, carbon content, hydrogen content, sulfur content, oxygen content, higher calorific power, holocellulose, and energy density. After verifying the variability of all evaluated traits among the clones, a two-dimensional correlation network was used to determine the phenotypic patterns among them. The obtained coefficient of determination (0.94) presented a higher magnitude in relation to the effect of the residual variable, and it served as an excellent model for explaining the genetic effects related to the variations observed in the energy density of wood in all eucalyptus clones. However, for future studies, we recommend evaluating other traits, especially the morphological traits, because of the greater ease in their measurement. Selecting clones with high basic density is the most promising strategy for eucalyptus breeding programs that aim to increase the energy density of wood because of its high heritability and magnitude of the cause-and-effect relationship with this trait.

Evolution of the Pseudomonas aeruginosa mutational resistome in an international Cystic Fibrosis clone.

Science.gov (United States)

López-Causapé, Carla; Sommer, Lea Mette; Cabot, Gabriel; Rubio, Rosa; Ocampo-Sosa, Alain A; Johansen, Helle Krogh; Figuerola, Joan; Cantón, Rafael; Kidd, Timothy J; Molin, Soeren; Oliver, Antonio

2017-07-17

Emergence of epidemic clones and antibiotic resistance development compromises the management of Pseudomonas aeruginosa cystic fibrosis (CF) chronic respiratory infections. Whole genome sequencing (WGS) was used to decipher the phylogeny, interpatient dissemination, WGS mutator genotypes (mutome) and resistome of a widespread clone (CC274), in isolates from two highly-distant countries, Australia and Spain, covering an 18-year period. The coexistence of two divergent CC274 clonal lineages was revealed, but without evident geographical barrier; phylogenetic reconstructions and mutational resistome demonstrated the interpatient transmission of mutators. The extraordinary capacity of P. aeruginosa to develop resistance was evidenced by the emergence of mutations in >100 genes related to antibiotic resistance during the evolution of CC274, catalyzed by mutator phenotypes. While the presence of classical mutational resistance mechanisms was confirmed and correlated with resistance phenotypes, results also showed a major role of unexpected mutations. Among them, PBP3 mutations, shaping up β-lactam resistance, were noteworthy. A high selective pressure for mexZ mutations was evidenced, but we showed for the first time that high-level aminoglycoside resistance in CF is likely driven by mutations in fusA1/fusA2, coding for elongation factor G. Altogether, our results provide valuable information for understanding the evolution of the mutational resistome of CF P. aeruginosa.

Cloning of the relative genes of endocrine exophthalmos

International Nuclear Information System (INIS)

Zheng, JG

2004-01-01

respectively, and was labeled with digoxigenin (DIG.). The colony PCR products of the relative genes of endocrine exophthalmos were degenerated, dotted on the nylon membrane, and hybridized with the labeled probes to test the positive clone. The inserted fragments of positive clone was sequenced by a professional biotechnical group. Results: The intact and high-pure mRNA was obtained. The double chains of cDNA were synthesized by using the mRNA as a model. The Hae III products was less than 3 kb. The efficacies of subtraction suggested that the subtraction happened really and the experience was successful. The PCR products could be used to construct the suppressive subtractive library. Fourty eight white bacteria clonies selected randomly had the amplification products of 2 transformants with double bands, the others with single one. Through dot blots, the transformation which had hybridization signal with thyroid tissues of endocrine exophthalmos, and had no that with thyroid tissues without endocrine exophthalmos, was positive clones. The positive ratio was about 40%. The insert fragments in the positive clones were endocrine exophthalmos-related genes/ cDNA fragments. Conclusions: The endocrine exophthalmos-specific subtractive products obtained by suppression subtractive hybridization were cloned. After a high throughput screening, the endocrine exophthalmos -related genes or cDNA fragments were obtained. (authors)

Variations and voids: the regulation of human cloning around the world

Directory of Open Access Journals (Sweden)

Caulfield Timothy

2004-12-01

Full Text Available Abstract Background No two countries have adopted identical regulatory measures on cloning. Understanding the complexity of these regulatory variations is essential. It highlights the challenges associated with the regulation of a controversial and rapidly evolving area of science and sheds light on a regulatory framework that can accommodate this reality. Methods Using the most reliable information available, we have performed a survey of the regulatory position of thirty countries around the world regarding the creation and use of cloned embryos (see Table 1. We have relied on original and translated legislation, as well as published sources and personal communications. We have examined the regulation of both reproductive cloning (RC and non-reproductive cloning (NRC. Results While most of the countries studied have enacted national legislation, the absence of legislation in seven of these countries should not be equated with the absence of regulation. Senator Morin was not correct in stating that the majority of recent legislation bans both RC and NRC. Recent regulatory moves are united only with regard to the banning of RC. While NRC is not permitted in seventeen of the countries examined, it could be permitted in up to thirteen countries. Conclusions There is little consensus on the various approaches to cloning laws and policies, and the regulatory position in many countries remains uncertain.

Positional cloning identifies zebrafish one-eyed pinhead as a permissive EGF-related ligand required during gastrulation.

Science.gov (United States)

Zhang, J; Talbot, W S; Schier, A F

1998-01-23

The zebrafish one-eyed pinhead (oep) mutation disrupts embryonic development, resulting in cyclopia and defects in endoderm, prechordal plate, and ventral neuroectoderm formation. We report the molecular isolation of oep using a positional cloning approach. The oep gene encodes a novel EGF-related protein with similarity to the EGF-CFC proteins cripto, cryptic, and FRL-1. Wild-type oep protein contains a functional signal sequence and is membrane-associated. Following ubiquitous maternal and zygotic expression, highest levels of oep mRNA are found in the gastrula margin and in axial structures and forebrain. Widespread misexpression of both membrane-attached and secreted forms of oep rescues prechordal plate and forebrain development in mutant embryos but does not lead to the ectopic induction of these cell types in wild-type fish. These results establish an essential but permissive role for an EGF-related ligand during vertebrate gastrulation.

Two cloned β thalassemia genes are associated with amber mutations at codon 39

Science.gov (United States)

Pergolizzi, Robert; Spritz, Richard A.; Spence, Sally; Goossens, Michel; Kan, Yuet Wai; Bank, Arthur

1981-01-01

Two β globin genes from patients with the β+ thalassemia phenotype have been cloned and sequenced. A single nucleotide change from CAG to TAG (an amber mutation) at codon 39 is the only difference from normal in both genes analyzed. The results are consistent with the assumption that both patients are doubly heterozygous for β+ and β° thalassemia, and that we have isolated and analyzed the β° thalassemia gene. Images PMID:6278453

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.

Development and analysis of a tick-borne encephalitis virus infectious clone using a novel and rapid strategy.

Science.gov (United States)

Gritsun, T S; Gould, E A

1998-12-01

In less than 1 month we have constructed an infectious clone of attenuated tick-borne encephalitis virus (strain Vasilchenko) from 100 microl of unpurified virus suspension using long high fidelity PCR and a modified bacterial cloning system. Optimization of the 3' antisense primer concentration was essential to achieve PCR synthesis of an 11 kb cDNA copy of RNA from infectious virus. A novel system utilising two antisense primers, a 14-mer for reverse transcription and a 35-mer for long PCR, produced high yields of genomic length cDNA. Use of low copy number Able K cells and an incubation temperature of 28 degrees C increased the genetic stability of cloned cDNA. Clones containing 11 kb cDNA inserts produced colonies of reduced size, thus providing a positive selection system for full length clones. Sequencing of the infectious clone emphasised the improved fidelity of the method compared with conventional PCR and cloning methods. A simple and rapid strategy for genetic manipulation of the infectious clone is also described. These developments represent a significant advance in recombinant technology and should be applicable to positive stranded RNA viruses which cannot easily be purified or genetically manipulated.

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...

Positional RNA-Seq identifies candidate genes for phenotypic engineering of sexual traits

NARCIS (Netherlands)

Arbore, Roberto; Sekii, Kiyono; Beisel, Christian; Ladurner, Peter; Berezikov, Eugene; Schaerer, Lukas

2015-01-01

Introduction: RNA interference (RNAi) of trait-specific genes permits the manipulation of specific phenotypic traits ("phenotypic engineering") and thus represents a powerful tool to test trait function in evolutionary studies. The identification of suitable candidate genes, however, often relies on

CD11c-positive cells from brain, spleen, lung, and liver exhibit site-specific immune phenotypes and plastically adapt to new environments.

Science.gov (United States)

Immig, Kerstin; Gericke, Martin; Menzel, Franziska; Merz, Felicitas; Krueger, Martin; Schiefenhövel, Fridtjof; Lösche, Andreas; Jäger, Kathrin; Hanisch, Uwe-Karsten; Biber, Knut; Bechmann, Ingo

2015-04-01

The brain's immune privilege has been also attributed to the lack of dendritic cells (DC) within its parenchyma and the adjacent meninges, an assumption, which implies maintenance of antigens rather than their presentation in lymphoid organs. Using mice transcribing the green fluorescent protein under the promoter of the DC marker CD11c (itgax), we identified a juxtavascular population of cells expressing this DC marker and demonstrated their origin from bone marrow and local microglia. We now phenotypically compared this population with CD11c/CD45 double-positive cells from lung, liver, and spleen in healthy mice using seven-color flow cytometry. We identified unique, site-specific expression patterns of F4/80, CD80, CD86, CX3CR1, CCR2, FLT3, CD103, and MHC-II. Furthermore, we observed the two known CD45-positive populations (CD45(high) and CD45(int) ) in the brain, whereas liver, lung, and spleen exhibited a homogeneous CD45(high) population. CD11c-positive microglia lacked MHC-II expression and CD45(high) /CD11c-positive cells from the brain have a lower percentage of MHC-II-positive cells. To test whether phenotypical differences are fixed by origin or specifically develop due to environmental factors, we transplanted brain and spleen mononuclear cells on organotypic slice cultures from brain (OHSC) and spleen (OSSC). We demonstrate that adaption and ramification of MHC-II-positive splenocytes is paralleled by down-regulation of MHC-II, whereas brain-derived mononuclear cells neither ramified nor up-regulated MHC-II in OSSCs. Thus, brain-derived mononuclear cells maintain their MHC-II-negative phenotype within the environment of an immune organ. Intraparenchymal CD11c-positive cells share immunophenotypical characteristics of DCs from other organs but remain unique for their low MHC-II expression. © 2014 Wiley Periodicals, Inc.

Peripheral blood and intrathyroidal T cell clones from patients with thyroid autoimmune diseases.

Science.gov (United States)

Massart, C; Caroff, G; Maugendre, D; Genetet, N; Gibassier, J

1999-01-01

For a better understanding of the pathogenesis of thyroid autoimmune diseases, we have studied morphological and functional properties of T clones from peripheral blood lymphocytes (PBL) and from intrathyroidal lymphocytes (ITL) obtained from 3 patients with Graves' disease or 1 Hashimoto's thyroiditis. Investigations were carried out on clones cultured alone or cocultured with autologous thyrocytes. Clonage efficiency ranged from 30% to 33% for PBL and 10% to 36% for ITL. A predominance of CD4-positive clones was observed whatever the origin of the lymphocytes or the autoimmune pathology. Gamma interferon (IFN-gamma) was detected in the majority (17/19) of the clones tested. Intracytoplasmic interleukin (IL-4) was secreted in 7/19 clones and both cytokines were produced in 5/19 clones. In coculture a proliferative response and tumour necrosis factor (TNF-alpha) production were observed with 6 clones (4 from Graves thyrocytes and 2 from thyroiditis). No cytotoxic clone was derived from Graves or thyroiditis tissues. These data demonstrate that the large majority of T clones are principally CD4-T cells; all the clones secreted TNF-alpha and a large majority produced IFN-gamma. Only a few clones produced IL-4 alone or associated with IFN-gamma. Six T clones induced proliferative response and of TNF-alpha secretion in coculture. Further investigations must be performed on these antigen-reactive T clones to analyse their role in the pathogenesis of the human thyroid autoimmune diseases.

Anti-idiotypic antibodies against UV-induced tumor-specific CTL clones. Preparation in syngeneic combination

International Nuclear Information System (INIS)

Kuribayashi, K.; Tanaka, C.; Matsubayashi, Y.; Masuda, T.; Udono, H.; Abe, M.; Nakayama, E.; Shiku, H.

1988-01-01

In this study, we first established several CTL clones of (BALB/c x C57BL/6)F1 origin that were specific for either syngeneic UV female 1 or UV male 1 fibrosarcoma cell lines. All the CTL clones had Thy-1+ Lyt-2+ L3T4- phenotypes and showed Kd restriction when lysing the corresponding target cells. Sera obtained from syngeneic animals immunized with three CTL clones, 10B-5 for UV female 1, and CTL9 and CTL10 for UV male 1, showed specific inhibition of target cell lysis with the corresponding CTL clones. The inhibitory activities were found in sera of the majority of immunized animals. Because the inhibitory activity resides in protein A-binding fraction, mAb were produced by hybridizing spleen cells of hyperimmune animals. N1-56 was thus obtained from a mouse immunized with 10B-5 CTL clone reactive with UV female 1. N1-56 was clonotype specific, reacting with 10B-5 but not with other CTL lines or leukemia cell lines. No N1-56+ cells were detectable in thymocytes, lymph node cells, or spleen cells of either naive or UV female 1-immune CB6F1 mice. Immunoprecipitation showed that N1-56 reacts with 90,000 Mr molecules on 10B-5 CTL clone under nonreducing conditions and 45,000 Mr molecules under reducing conditions, indicating its reactivities with idiotypic determinants of TCR on the CTL clone. N1-56 inhibited lytic activity of 10B-5, but neither N1-56 nor alpha-10B-5 hyperimmune serum inhibited that of alpha-UV female 1 mixed lymphocyte tumor cell culture cells. N1-56 induced proliferation of 10B-5 without addition of Ag

Determining the Possible Etiology of Hospital-Acquired Pneumonia Using a Clone Library Analysis in Japan.

Science.gov (United States)

Yatera, Kazuhiro; Noguchi, Shingo; Yamasaki, Kei; Kawanami, Toshinori; Fukuda, Kazumasa; Naito, Keisuke; Akata, Kentaro; Kido, Takashi; Ishimoto, Hiroshi; Sakamoto, Noriho; Taniguchi, Hatsumi; Mukae, Hiroshi

2017-05-01

Obtaining precise etiological information regarding causative bacteria is important for the proper use of antimicrobials in hospital-acquired pneumonia (HAP), which is associated with a high rate of mortality. The aim of this study was to comparatively investigate the bacterial diversity in bronchoalveolar lavage fluid (BALF) in Japanese patients with HAP by the clone library method using the 16S rRNA gene. This study included Japanese patients with HAP who were treated at our hospital and referring hospitals. BALF specimens were obtained from pneumonia lesions identified on chest radiographs and/or computed tomography. Sputum specimens were also evaluated in patients with sputum production. Sixty-eight patients were ultimately enrolled. BALF cultivation revealed bacterial positivity in 53 of 68 (77.9%) patients, and Staphylococcus aureus (30.9%) was the most frequently isolated, followed by Pseudomonas aeruginosa (16.2%), and Escherichia coli (10.3%). In contrast, the clone library analysis identified the presence of some bacterial phenotype in 65 of 68 (95.6%) patients, and streptococci (16.2%), Corynebacterium species (11.8%), anaerobes (10.3%) were frequently detected as the predominant phylotypes. Both methods tended to detect S. aureus, Klebsiella pneumoniae, and E. coli in patients with late-onset pneumonia. In addition, the cases that phylotypes of S. aureus and P. aeruginosa were found to account for > 5% of the bacterial flora of each case were 42.9% and 72.7%, respectively. These results indicate that attention should be paid to the roles of gram-positive bacilli such as streptococci, Corynebacterium species and anaerobes, in addition to Gram-negative bacilli, in the pathogenesis of HAP.

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…

Microscopic Examination of Distribution and Phenotypic Properties of Phylogenetically Diverse Chloroflexaceae-Related Bacteria in Hot Spring Microbial Mats

DEFF Research Database (Denmark)

Nübel, U.; Bateson, Mary M.; Vandieken, V.

2002-01-01

We investigated the diversity, distribution, and phenotypes of uncultivated Chloroflexaceae-related bacteria in photosynthetic microbial mats of an alkaline hot spring (Mushroom Spring, Yellowstone National Park). By applying a directed PCR approach, molecular cloning, and sequence analysis of 16S...

Chromosomal mapping of canine-derived BAC clones to the red fox and American mink genomes.

Science.gov (United States)

Kukekova, Anna V; Vorobieva, Nadegda V; Beklemisheva, Violetta R; Johnson, Jennifer L; Temnykh, Svetlana V; Yudkin, Dmitry V; Trut, Lyudmila N; Andre, Catherine; Galibert, Francis; Aguirre, Gustavo D; Acland, Gregory M; Graphodatsky, Alexander S

2009-01-01

High-quality sequencing of the dog (Canis lupus familiaris) genome has enabled enormous progress in genetic mapping of canine phenotypic variation. The red fox (Vulpes vulpes), another canid species, also exhibits a wide range of variation in coat color, morphology, and behavior. Although the fox genome has not yet been sequenced, canine genomic resources have been used to construct a meiotic linkage map of the red fox genome and begin genetic mapping in foxes. However, a more detailed gene-specific comparative map between the dog and fox genomes is required to establish gene order within homologous regions of dog and fox chromosomes and to refine breakpoints between homologous chromosomes of the 2 species. In the current study, we tested whether canine-derived gene-containing bacterial artificial chromosome (BAC) clones can be routinely used to build a gene-specific map of the red fox genome. Forty canine BAC clones were mapped to the red fox genome by fluorescence in situ hybridization (FISH). Each clone was uniquely assigned to a single fox chromosome, and the locations of 38 clones agreed with cytogenetic predictions. These results clearly demonstrate the utility of FISH mapping for construction of a whole-genome gene-specific map of the red fox. The further possibility of using canine BAC clones to map genes in the American mink (Mustela vison) genome was also explored. Much lower success was obtained for this more distantly related farm-bred species, although a few BAC clones were mapped to the predicted chromosomal locations.

Nef does not contribute to replication differences between R5 pre-AIDS and AIDS HIV-1 clones from patient ACH142

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Rekosh David

2008-05-01

Full Text Available Abstract AIDS-associated, CCR5-tropic (R5 HIV-1 clones, isolated from a patient that never developed CXCR4-tropic HIV-1, replicate to a greater extent and cause greater cytopathic effects than R5 HIV-1 clones isolated before the onset of AIDS. Previously, we showed that HIV-1 Env substantially contributed to the enhanced replication of an AIDS clone. In order to determine if Nef makes a similar contribution, we cloned and phenotypically analyzed nef genes from a series of patient ACH142 derived R5 HIV-1 clones. The AIDS-associated Nef contains a series of residues found in Nef proteins from progressors 1. In contrast to other reports 123, this AIDS-associated Nef downmodulated MHC-I to a greater extent and CD4 less than pre-AIDS Nef proteins. Additionally, all Nef proteins enhanced infectivity similarly in a single round of replication. Combined with our previous study, these data show that evolution of the HIV-1 env gene, but not the nef gene, within patient ACH142 significantly contributed to the enhanced replication and cytopathic effects of the AIDS-associated R5 HIV-1 clone.

Biological characterization of clones derived from the edmonston strain of measles virus in comparison with schwarz and CAM-70 vaccine strains

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Maria Beatriz Junqueira Borges

1996-08-01

Full Text Available Four virus clones were derived from the Edmonston strain of measles virus by repeated plaque purification. These clones were compared with the vaccine strains Schwarz and CAM-70 in terms of biological activities including plaque formation, hemagglutination, hemolysis and replication in Vero cells and chick embryo fibroblasts (CEF. Two clones of intermediate plaque yielded mixed plaque populations on subcultivation whereas the other two, showing small and large plaque sizes, showed stable plaque phenotypes. The vaccine strains showed consistent homogeneous plaque populations. All the Edmonston clones showed agglutination of monkey erythrocytes in isotonic solution while both vaccine strains hemagglutinated only in the presence of high salt concentrations. Variation in the hemolytic activity was observed among the four clones but no hemolytic activity was detected for the vaccine virus strains. Vaccine strains replicated efficiently both in Vero cells and CEF. All four clones showed efficient replication in Vero cells but different replication profiles in CEF. Two of them replicated efficiently, one was of intermediate efficiency and the other showed no replication in CEF. Two of the clones showed characteristics similar to vaccine strains. One in terms of size and homogeneity of plaques, the other for a low hemolytic activity and both for the efficiency of propagation in CEF.

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...

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

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

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.

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

[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.

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)

MHC Intratumoral Heterogeneity May Predict Cancer Progression and Response to Immunotherapy

Directory of Open Access Journals (Sweden)

Irene Romero

2018-01-01

Full Text Available An individual tumor can present intratumoral phenotypic heterogeneity, containing tumor cells with different phenotypes that do not present irreversible genetic alterations. We have developed a mouse cancer model, named GR9, derived from a methylcholanthrene-induced fibrosarcoma that was adapted to tissue culture and cloned into different tumor cell lines. The clones showed diverse MHC-I phenotypes, ranging from highly positive to weakly positive MHC-I expression. These MHC-I alterations are due to reversible molecular mechanisms, because surface MHC-I could be recovered by IFN-γ treatment. Cell clones with high MHC-I expression demonstrated low local oncogenicity and high spontaneous metastatic capacity, whereas MHC-I-low clones showed high local oncogenicity and no spontaneous metastatic capacity. Although MHC-I-low clones did not metastasize, they produced MHC-I-positive dormant micrometastases controlled by the host immune system, i.e., in a state of immunodormancy. The metastatic capacity of each clone was directly correlated with the host T-cell subpopulations; thus, a strong decrease in cytotoxic and helper T lymphocytes was observed in mice with numerous metastases derived from MHC-I positive tumor clones but a strong increase was observed in those with dormant micrometastases. Immunotherapy was administered to the hosts after excision of the primary tumor, producing a recovery in their immune status and leading to the complete eradication of overt spontaneous metastases or their decrease. According to these findings, the combination of MHC-I surface expression in primary tumor and metastases with host T-cell subsets may be a decisive indicator of the clinical outcome and response to immunotherapy in metastatic disease, allowing the identification of responders to this approach.

Positional cloning of "Lisch-Like", a candidate modifier of susceptibility to type 2 diabetes in mice.

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Marija Dokmanovic-Chouinard

2008-07-01

Full Text Available In 404 Lep(ob/ob F2 progeny of a C57BL/6J (B6 x DBA/2J (DBA intercross, we mapped a DBA-related quantitative trait locus (QTL to distal Chr1 at 169.6 Mb, centered about D1Mit110, for diabetes-related phenotypes that included blood glucose, HbA1c, and pancreatic islet histology. The interval was refined to 1.8 Mb in a series of B6.DBA congenic/subcongenic lines also segregating for Lep(ob. The phenotypes of B6.DBA congenic mice include reduced beta-cell replication rates accompanied by reduced beta-cell mass, reduced insulin/glucose ratio in blood, reduced glucose tolerance, and persistent mild hypoinsulinemic hyperglycemia. Nucleotide sequence and expression analysis of 14 genes in this interval identified a predicted gene that we have designated "Lisch-like" (Ll as the most likely candidate. The gene spans 62.7 kb on Chr1qH2.3, encoding a 10-exon, 646-amino acid polypeptide, homologous to Lsr on Chr7qB1 and to Ildr1 on Chr16qB3. The largest isoform of Ll is predicted to be a transmembrane molecule with an immunoglobulin-like extracellular domain and a serine/threonine-rich intracellular domain that contains a 14-3-3 binding domain. Morpholino knockdown of the zebrafish paralog of Ll resulted in a generalized delay in endodermal development in the gut region and dispersion of insulin-positive cells. Mice segregating for an ENU-induced null allele of Ll have phenotypes comparable to the B.D congenic lines. The human ortholog, C1orf32, is in the middle of a 30-Mb region of Chr1q23-25 that has been repeatedly associated with type 2 diabetes.

Software-Supported USER Cloning Strategies for Site-Directed Mutagenesis and DNA Assembly

DEFF Research Database (Denmark)

Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen

2015-01-01

USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER...... cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein...... (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at ....

Galectin-7 Expression Potentiates HER-2-Positive Phenotype in Breast Cancer.

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Andrée-Anne Grosset

Full Text Available HER-2 positive tumors are among the most aggressive subtypes of breast cancer and are frequently associated with metastasis and poor outcome. As with other aggressive subtypes of breast cancer, these tumors are associated with abnormally high expression of galectin-7 (gal-7, which confers metastatic breast tumor cells with increased invasive behavior. Although previous studies in the rat model of breast tumorigenesis have shown that gal-7 is also increased in primary breast tumor, its contribution to the development of the primary breast tumors remains unclear. In the present work, we have used genetically-engineered gal-7-deficient mice to examine the role of gal-7 in the development of the mammary gland and of breast cancer. Using histological and immunohistological analysis of whole mammary glands at different stages of development, we detected no significant changes between normal and gal-7-deficient mice. To test the involvement of gal-7 in breast cancer, we next examined the effects of loss of gal-7 on mammary tumor development by crossing gal-7-deficient mice with the mammary tumor transgenic mouse strain FVB-Tg(MMTV-Erbb2NK1Mul/J. Finally, assessment of mice survival and tumor volume showed a delay of mammary tumor growth in the absence of systemic gal-7. These data suggest that gal-7 could potentiate the phenotype of HER-2 positive primary breast cancer.

Nucleotide sequence analysis of the recA gene and discrimination of the three isolates of urease-positive thermophilic Campylobacter (UPTC) isolated from seagulls (Larus spp.) in Northern Ireland.

Science.gov (United States)

Matsuda, M; Tai, K; Moore, J E; Millar, B C; Murayama, O

2004-01-01

Nucleotide sequencing after TA cloning of the amplicon of the almost-full length recA gene from three strains of UPTC (A1, A2, and A3) isolated from seagulls in Northern Ireland, the phenotypical and genotypical characteristics of which have been demonstrated to be indistinguishable, clarified nucleotide differences at three nucleotide positions among the three strains. In conclusion, the nucleotide sequences of the recA gene were found to discriminate among the three strains of UPTC, A1, A2, and A3, which are indistinguishable phenotypically and genotypically. Thus, the present study strongly suggests that nucleotide sequence data of the amplicon of a suitable gene or region could aid in discriminating among isolates of the UPTC group, which are indistinguishable phenotypically and genotypically. Copyright 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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

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

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

Comparison of the miRNA profiles in HPV-positive and HPV-negative tonsillar tumors and a model system of human keratinocyte clones

International Nuclear Information System (INIS)

Vojtechova, Zuzana; Sabol, Ivan; Salakova, Martina; Smahelova, Jana; Zavadil, Jiri; Turek, Lubomir; Grega, Marek; Klozar, Jan; Prochazka, Bohumir; Tachezy, Ruth

2016-01-01

Better insights into the molecular changes involved in virus-associated and -independent head and neck cancer may advance our knowledge of HNC carcinogenesis and identify critical disease biomarkers. Here we aimed to characterize the expression profiles in a matched set of well-characterized HPV-dependent and HPV-independent tonsillar tumors and equivalent immortalized keratinocyte clones to define potential and clinically relevant biomarkers of HNC of different etiology. Fresh frozen tonsillar cancer tissues were analyzed together with non-malignant tonsillar tissues and compared with cervical tumors and normal cervical tissues. Furthermore, relative miRNAs abundance levels of primary and immortalized human keratinocyte clones were evaluated. The global quantitation of miRNA gene abundance was performed using a TaqMan Low Density Array system. The confirmation of differentially expressed miRNAs was performed on a set of formalin-fixed paraffin-embedded tumor samples enriched for the tumor cell fraction by macrodissection. We defined 46 upregulated and 31 downregulated miRNAs characteristic for the HPV-positive tonsillar tumors and 42 upregulated miRNAs and 42 downregulated miRNAs characteristic for HPV-independent tumors. In comparison with the expression profiles in cervical tumors, we defined miR-141-3p, miR-15b-5p, miR-200a-3p, miR-302c-3p, and miR-9-5p as specific for HPV induced malignancies. MiR-335-5p, miR-579-3p, and miR-126-5p were shared by the expression profiles of HPV-positive tonsillar tumors and of the HPV immortalized keratinocyte clones, whereas miR-328-3p, miR-34c-3p, and miR-885-5p were shared by the miRNA profiles of HPV-negative tonsillar tumors and the HPV-negative keratinocytes. We identified the miRNAs characteristic for HPV-induced tumors and tonsillar tumors of different etiology, and the results were compared with those of the model system. Our report presents the basis for further investigations leading to the identification of

Human cloning laws, human dignity and the poverty of the policy making dialogue.

Science.gov (United States)

Caulfield, Timothy

2003-07-29

The regulation of human cloning continues to be a significant national and international policy issue. Despite years of intense academic and public debate, there is little clarity as to the philosophical foundations for many of the emerging policy choices. The notion of "human dignity" is commonly used to justify cloning laws. The basis for this justification is that reproductive human cloning necessarily infringes notions of human dignity. The author critiques one of the most commonly used ethical justifications for cloning laws - the idea that reproductive cloning necessarily infringes notions of human dignity. He points out that there is, in fact, little consensus on point and that the counter arguments are rarely reflected in formal policy. Rarely do domestic or international instruments provide an operational definition of human dignity and there is rarely an explanation of how, exactly, dignity is infringed in the context reproductive cloning. It is the author's position that the lack of thoughtful analysis of the role of human dignity hurts the broader public debate about reproductive cloning, trivializes the value of human dignity as a normative principle and makes it nearly impossible to critique the actual justifications behind many of the proposed policies.

The Dao of human cloning: utopian/dystopian hype in the British press and popular films.

Science.gov (United States)

Jensen, Eric

2008-04-01

The issue of human cloning has featured in the national science policy agendas in both the United States and the United Kingdom since the announcement in 1997 of Dolly the cloned sheep's birth in Scotland. Such news stories suggesting the imminent cloning of humans have inspired fictional entertainment media over the years, including numerous popular films. Study 1 examines elite British press coverage of human cloning from 1997 to 2004 (n = 857). Study 2 focuses on five human cloning films released between 1978 and 2003. Two sharply divergent discourses emerged from these data. Unqualified hope and habitually hyped claims of future cures permeated the press discourse. In contrast, the films constructed human cloning as an inherently dangerous technology often wielded by hubristic scientists in the tradition of Frankenstein. Both the predominately positive hype in the broadsheet press and the largely negative hype in the films indicate an impoverished and "thin" public debate on the issue of human cloning.

Quantitative Proteomics of Gut-Derived Th1 and Th1/Th17 Clones Reveal the Presence of CD28+ NKG2D- Th1 Cytotoxic CD4+ T cells.

Science.gov (United States)

Riaz, Tahira; Sollid, Ludvig Magne; Olsen, Ingrid; de Souza, Gustavo Antonio

2016-03-01

T-helper cells are differentiated from CD4+ T cells and are traditionally characterized by inflammatory or immunosuppressive responses in contrast to cytotoxic CD8+ T cells. Mass-spectrometry studies on T-helper cells are rare. In this study, we aimed to identify the proteomes of human Th1 and Th1/Th17 clones derived from intestinal biopsies of Crohn's disease patients and to identify differentially expressed proteins between the two phenotypes. Crohn's disease is an inflammatory bowel disease, with predominantly Th1- and Th17-mediated response where cells of the "mixed" phenotype Th1/Th17 have also been commonly found. High-resolution mass spectrometry was used for protein identification and quantitation. In total, we identified 7401 proteins from Th1 and Th1/Th17 clones, where 334 proteins were differentially expressed. Major differences were observed in cytotoxic proteins that were overrepresented in the Th1 clones. The findings were validated by flow cytometry analyses using staining with anti-granzyme B and anti-perforin and by a degranulation assay, confirming higher cytotoxic features of Th1 compared with Th1/Th17 clones. By testing a larger panel of T-helper cell clones from seven different Crohn's disease patients, we concluded that only a subgroup of the Th1 cell clones had cytotoxic features, and these expressed the surface markers T-cell-specific surface glycoprotein CD28 and were negative for expression of natural killer group 2 member D. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Relatively slow stochastic gene-state switching in the presence of positive feedback significantly broadens the region of bimodality through stabilizing the uninduced phenotypic state.

Science.gov (United States)

Ge, Hao; Wu, Pingping; Qian, Hong; Xie, Xiaoliang Sunney

2018-03-01

Within an isogenic population, even in the same extracellular environment, individual cells can exhibit various phenotypic states. The exact role of stochastic gene-state switching regulating the transition among these phenotypic states in a single cell is not fully understood, especially in the presence of positive feedback. Recent high-precision single-cell measurements showed that, at least in bacteria, switching in gene states is slow relative to the typical rates of active transcription and translation. Hence using the lac operon as an archetype, in such a region of operon-state switching, we present a fluctuating-rate model for this classical gene regulation module, incorporating the more realistic operon-state switching mechanism that was recently elucidated. We found that the positive feedback mechanism induces bistability (referred to as deterministic bistability), and that the parameter range for its occurrence is significantly broadened by stochastic operon-state switching. We further show that in the absence of positive feedback, operon-state switching must be extremely slow to trigger bistability by itself. However, in the presence of positive feedback, which stabilizes the induced state, the relatively slow operon-state switching kinetics within the physiological region are sufficient to stabilize the uninduced state, together generating a broadened parameter region of bistability (referred to as stochastic bistability). We illustrate the opposite phenotype-transition rate dependence upon the operon-state switching rates in the two types of bistability, with the aid of a recently proposed rate formula for fluctuating-rate models. The rate formula also predicts a maximal transition rate in the intermediate region of operon-state switching, which is validated by numerical simulations in our model. Overall, our findings suggest a biological function of transcriptional "variations" among genetically identical cells, for the emergence of bistability and

Implications of extreme life span in clonal organisms: millenary clones in meadows of the threatened seagrass Posidonia oceanica.

Directory of Open Access Journals (Sweden)

Sophie Arnaud-Haond

Full Text Available The maximum size and age that clonal organisms can reach remains poorly known, although we do know that the largest natural clones can extend over hundreds or thousands of metres and potentially live for centuries. We made a review of findings to date, which reveal that the maximum clone age and size estimates reported in the literature are typically limited by the scale of sampling, and may grossly underestimate the maximum age and size of clonal organisms. A case study presented here shows the occurrence of clones of slow-growing marine angiosperm Posidonia oceanica at spatial scales ranging from metres to hundreds of kilometres, using microsatellites on 1544 sampling units from a total of 40 locations across the Mediterranean Sea. This analysis revealed the presence, with a prevalence of 3.5 to 8.9%, of very large clones spreading over one to several (up to 15 kilometres at the different locations. Using estimates from field studies and models of the clonal growth of P. oceanica, we estimated these large clones to be hundreds to thousands of years old, suggesting the evolution of general purpose genotypes with large phenotypic plasticity in this species. These results, obtained combining genetics, demography and model-based calculations, question present knowledge and understanding of the spreading capacity and life span of plant clones. These findings call for further research on these life history traits associated with clonality, considering their possible ecological and evolutionary implications.

Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate

Directory of Open Access Journals (Sweden)

Ju-Yeon Yoon

2014-03-01

Full Text Available Chrysanthemum stunt viroid (CSVd, a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1 were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants.

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

Influence of embryo handling and transfer method on pig cloning efficiency.

Science.gov (United States)

Shi, Junsong; Zhou, Rong; Luo, Lvhua; Mai, Ranbiao; Zeng, Haiyu; He, Xiaoyan; Liu, Dewu; Zeng, Fang; Cai, Gengyuan; Ji, Hongmei; Tang, Fei; Wang, Qinglai; Wu, Zhenfang; Li, Zicong

2015-03-01

The somatic cell nuclear transfer (SCNT) technique could be used to produce genetically superior or genetically engineered cloned pigs that have wide application in agriculture and bioscience research. However, the efficiency of porcine SCNT currently is very low. Embryo transfer (ET) is a key step for the success of SCNT. In this study, the effects of several ET-related factors, including cloned embryo culture time, recipient's ovulation status, co-transferred helper embryos and ET position, on the success rate of pig cloning were investigated. The results indicated that transfer of cloned embryos cultured for a longer time (22-24h vs. 4-6h) into pre-ovulatory sows decreased recipient's pregnancy rate and farrowing rate, and use of pre-ovulatory and post-ovulatory sows as recipients for SCNT embryos cultured for 22-24h resulted in a similar porcine SCNT efficiency. Use of insemination-produced in vivo fertilized, parthenogenetically activated and in vitro fertilized embryos as helper embryos to establish and/or maintain pregnancy of SCNT embryos recipients could not improve the success rate of porcine SCNT. Transfer of cloned embryos into double oviducts of surrogates significantly increased pregnancy rate as well as farrowing rate of recipients, and the developmental rate of transferred cloned embryos, as compared to unilateral oviduct transfer. This study provided useful information for optimization of the embryo handling and transfer protocol, which will help to improve the ability to generate cloned pigs. Copyright © 2015 Elsevier B.V. All rights reserved.

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)

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.

Tumor clone dynamics in lethal prostate cancer.

Science.gov (United States)

Carreira, Suzanne; Romanel, Alessandro; Goodall, Jane; Grist, Emily; Ferraldeschi, Roberta; Miranda, Susana; Prandi, Davide; Lorente, David; Frenel, Jean-Sebastien; Pezaro, Carmel; Omlin, Aurelius; Rodrigues, Daniel Nava; Flohr, Penelope; Tunariu, Nina; S de Bono, Johann; Demichelis, Francesca; Attard, Gerhardt

2014-09-17

It is unclear whether a single clone metastasizes and remains dominant over the course of lethal prostate cancer. We describe the clonal architectural heterogeneity at different stages of disease progression by sequencing serial plasma and tumor samples from 16 ERG-positive patients. By characterizing the clonality of commonly occurring deletions at 21q22, 8p21, and 10q23, we identified multiple independent clones in metastatic disease that are differentially represented in tissue and circulation. To exemplify the clinical utility of our studies, we then showed a temporal association between clinical progression and emergence of androgen receptor (AR) mutations activated by glucocorticoids in about 20% of patients progressing on abiraterone and prednisolone or dexamethasone. Resistant clones showed a complex dynamic with temporal and spatial heterogeneity, suggesting distinct mechanisms of resistance at different sites that emerged and regressed depending on treatment selection pressure. This introduces a management paradigm requiring sequential monitoring of advanced prostate cancer patients with plasma and tumor biopsies to ensure early discontinuation of agents when they become potential disease drivers. Copyright © 2014, American Association for the Advancement of Science.

Construction of a full-length infectious bacterial artificial chromosome clone of duck enteritis virus vaccine strain

Science.gov (United States)

2013-01-01

-length genome without any deletions or destruction of the viral coding sequence, and the viruses rescued from the DEV-BAC clone exhibited wild-type phenotypes both in vitro and in vivo. The generated infectious clone will greatly facilitate studies on the individual genes of DEV and applications in gene deletion or live vector vaccines. PMID:24195756

Genomic stability and physiological assessments of live offspring sired by a bull clone, Starbuck II.

Science.gov (United States)

Ortegon, H; Betts, D H; Lin, L; Coppola, G; Perrault, S D; Blondin, P; King, W A

2007-01-01

It appears that overt phenotypic abnormalities observed in some domestic animal clones are not transmitted to their progeny. The current study monitored Holstein heifers sired by a bull clone, Starbuck II, from weaning to puberty. Genomic stability was assessed by telomere length status and chromosomal analysis. Growth parameters, blood profiles, physical exams and reproductive parameters were assessed for 12 months (and compared to age-matched control heifers). Progeny sired by the clone bull did not differ (P>0.05) in weight, length and height compared to controls. However, progeny had lower heart rates (HR) (P=0.009), respiratory rates (RR) (P=0.007) and body temperature (P=0.03). Hematological profiles were within normal ranges and did not differ (P>0.05) between both groups. External and internal genitalia were normal and both groups reached puberty at expected ages. Progeny had two or three ovarian follicular waves per estrous cycle and serum progesterone concentrations were similar (P=0.99) to controls. Telomere lengths of sperm and blood cells from Starbuck II were not different (P>0.05) than those of non-cloned cattle; telomere lengths of progeny were not different (P>0.05) from age-matched controls. In addition, progeny had normal karyotypes in peripheral blood leukocytes compared to controls (89.1% versus 86.3% diploid, respectively). In summary, heifers sired by a bull clone had normal chromosomal stability, growth, physical, hematological and reproductive parameters, compared to normal heifers. Furthermore, they had moderate stress responses to routine handling and restraint.

Cloning of low-temperature induced gene from Morus mongolica CK

African Journals Online (AJOL)

SERVER

2008-03-04

Mar 4, 2008 ... extracted form the stem and was reverse-transcribed into cDNA. Mulberry .... positive clones to obtain engineered Agrobacterium LBA4404/. pIG121/ ..... Comparative experiment for a new cold and drought tolerance mulberry ...

Indexing and Analysis of Fungal Phenotypes Using Morphology and Spectrometry

DEFF Research Database (Denmark)

Hansen, Michael Adsetts Edberg

2005-01-01

and identification of the fungi is considered difficult and laborious. Though visual expressions have been and still is used as phenotype markers in the classification and identification of fungal species, one of the most successful characters used has been the profile of the secondary metabolites. In order...... to evaluate the visual phenotypic characters, a method for visual clone identification of Penicillium commune { the most widespread and most frequently occurring spoilage fungus on cheese { was developed (Papers A, B and C). The method was based on images of fungal colonies acquired after growth on a standard...... extract highly complex and similar ESI-MS mass spectra for identifying fungal extracts in a reference library are being developed and tested (Paper E). Whereas mass spectrometry is one modality used in systematising the fungi, high pressure liquid chromatography combined with an UV diode array detector...

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

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.

Human cloning laws, human dignity and the poverty of the policy making dialogue

Directory of Open Access Journals (Sweden)

Caulfield Timothy

2003-07-01

Full Text Available Abstract Background The regulation of human cloning continues to be a significant national and international policy issue. Despite years of intense academic and public debate, there is little clarity as to the philosophical foundations for many of the emerging policy choices. The notion of "human dignity" is commonly used to justify cloning laws. The basis for this justification is that reproductive human cloning necessarily infringes notions of human dignity. Discussion The author critiques one of the most commonly used ethical justifications for cloning laws – the idea that reproductive cloning necessarily infringes notions of human dignity. He points out that there is, in fact, little consensus on point and that the counter arguments are rarely reflected in formal policy. Rarely do domestic or international instruments provide an operational definition of human dignity and there is rarely an explanation of how, exactly, dignity is infringed in the context reproductive cloning. Summary It is the author's position that the lack of thoughtful analysis of the role of human dignity hurts the broader public debate about reproductive cloning, trivializes the value of human dignity as a normative principle and makes it nearly impossible to critique the actual justifications behind many of the proposed policies.

Human cloning laws, human dignity and the poverty of the policy making dialogue

Science.gov (United States)

Caulfield, Timothy

2003-01-01

Background The regulation of human cloning continues to be a significant national and international policy issue. Despite years of intense academic and public debate, there is little clarity as to the philosophical foundations for many of the emerging policy choices. The notion of "human dignity" is commonly used to justify cloning laws. The basis for this justification is that reproductive human cloning necessarily infringes notions of human dignity. Discussion The author critiques one of the most commonly used ethical justifications for cloning laws – the idea that reproductive cloning necessarily infringes notions of human dignity. He points out that there is, in fact, little consensus on point and that the counter arguments are rarely reflected in formal policy. Rarely do domestic or international instruments provide an operational definition of human dignity and there is rarely an explanation of how, exactly, dignity is infringed in the context reproductive cloning. Summary It is the author's position that the lack of thoughtful analysis of the role of human dignity hurts the broader public debate about reproductive cloning, trivializes the value of human dignity as a normative principle and makes it nearly impossible to critique the actual justifications behind many of the proposed policies. PMID:12887735

Genomic and Phenotypic Characterization of Yeast Biosensor for Deep-space Radiation

Science.gov (United States)

Marina, Diana B.; Santa Maria, Sergio; Bhattacharya, Sharmila

2016-01-01

The BioSentinel mission was selected to launch as a secondary payload onboard NASA Exploration Mission 1 (EM-1) in 2018. In BioSentinel, the budding yeast Saccharomyces cerevisiae will be used as a biosensor to measure the long-term impact of deep-space radiation to living organisms. In the 4U-payload, desiccated yeast cells from different strains will be stored inside microfluidic cards equipped with 3-color LED optical detection system to monitor cell growth and metabolic activity. At different times throughout the 12-month mission, these cards will be filled with liquid yeast growth media to rehydrate and grow the desiccated cells. The growth and metabolic rates of wild-type and radiation-sensitive strains in deep-space radiation environment will be compared to the rates measured in the ground- and microgravity-control units. These rates will also be correlated with measurements obtained from onboard physical dosimeters. In our preliminary long-term desiccation study, we found that air-drying yeast cells in 10% trehalose is the best method of cell preservation in order to survive the entire 18-month mission duration (6-month pre-launch plus 12-month full-mission periods). However, our study also revealed that desiccated yeast cells have decreasing viability over time when stored in payload-like environment. This suggests that the yeast biosensor will have different population of cells at different time points during the long-term mission. In this study, we are characterizing genomic and phenotypic changes in our yeast biosensor due to long-term storage and desiccation. For each yeast strain that will be part of the biosensor, several clones were reisolated after long-term storage by desiccation. These clones were compared to their respective original isolate in terms of genomic composition, desiccation tolerance and radiation sensitivity. Interestingly, clones from a radiation-sensitive mutant have better desiccation tolerance compared to their original isolate

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

Cloning human DNA repair genes

International Nuclear Information System (INIS)

Jeggo, P.A.; Carr, A.M.; Lehmann, A.R.

1994-01-01

Many human genes involved in the repair of UV damage have been cloned using different procedures and they have been of great value in assisting the understanding of the mechanism of nucleotide excision-repair. Genes involved in repair of ionizing radiation damage have proved more difficult to isolate. Positional cloning has localized the XRCC5 gene to a small region of chromosome 2q33-35, and a series of yeast artificial chromosomes covering this region have been isolated. Very recent work has shown that the XRCC5 gene encodes the 80 kDa subunit of the Ku DNA-binding protein. The Ku80 gene also maps to this region. Studies with fission yeast have shown that radiation sensitivity can result not only from defective DNA repair but also from abnormal cell cycle control following DNA damage. Several genes involved in this 'check-point' control in fission yeast have been isolated and characterized in detail. It is likely that a similar checkpoint control mechanism exists in human cells. (author)

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

A potential food-grade cloning vector for Streptococcus thermophilus that uses cadmium resistance as the selectable marker.

Science.gov (United States)

Wong, Wing Yee; Su, Ping; Allison, Gwen E; Liu, Chun-Qiang; Dunn, Noel W

2003-10-01

A potential food-grade cloning vector, pND919, was constructed and transformed into S. thermophilus ST3-1, a plasmid-free strain. The vector contains DNAs from two different food-approved organisms, Streptococcus thermophilus and Lactococcus lactis. The 5.0-kb pND919 is a derivative of the cloning vector pND918 (9.3 kb) and was constructed by deletion of the 4.3-kb region of pND918 which contained DNA from non-food-approved organisms. pND919 carries a heterologous native cadmium resistance selectable marker from L. lactis M71 and expresses the Cd(r) phenotype in S. thermophilus transformants. With the S. thermophilus replicon derived from the shuttle vector pND913, pND919 is able to replicate in the two S. thermophilus industrial strains tested, ST3-1 and ST4-1. Its relatively high retention rate in S. thermophilus further indicates its usefulness as a potential food-grade cloning vector. To our knowledge, this is the first report of a replicative potential food-grade vector for the industrially important organism S. thermophilus.

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

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

Determining Complementary Properties with Quantum Clones

Science.gov (United States)

Thekkadath, G. S.; Saaltink, R. Y.; Giner, L.; Lundeen, J. S.

2017-08-01

In a classical world, simultaneous measurements of complementary properties (e.g., position and momentum) give a system's state. In quantum mechanics, measurement-induced disturbance is largest for complementary properties and, hence, limits the precision with which such properties can be determined simultaneously. It is tempting to try to sidestep this disturbance by copying the system and measuring each complementary property on a separate copy. However, perfect copying is physically impossible in quantum mechanics. Here, we investigate using the closest quantum analog to this copying strategy, optimal cloning. The coherent portion of the generated clones' state corresponds to "twins" of the input system. Like perfect copies, both twins faithfully reproduce the properties of the input system. Unlike perfect copies, the twins are entangled. As such, a measurement on both twins is equivalent to a simultaneous measurement on the input system. For complementary observables, this joint measurement gives the system's state, just as in the classical case. We demonstrate this experimentally using polarized single photons.

How accurate is the phenotype? – An analysis of developmental noise in a cotton aphid clone

Directory of Open Access Journals (Sweden)

Babbitt Gregory A

2008-02-01

Full Text Available Abstract Background The accuracy by which phenotype can be reproduced by genotype potentially is important in determining the stability, environmental sensitivity, and evolvability of morphology and other phenotypic traits. Because two sides of an individual represent independent development of the phenotype under identical genetic and environmental conditions, average body asymmetry (or "fluctuating asymmetry" can estimate the developmental instability of the population. The component of developmental instability not explained by intrapopulational differences in gene or environment (or their interaction can be further defined as internal developmental noise. Surprisingly, developmental noise remains largely unexplored despite its potential influence on our interpretations of developmental stability, canalization, and evolvability. Proponents of fluctuating asymmetry as a bioindicator of environmental or genetic stress, often make the assumption that developmental noise is minimal and, therefore, that phenotype can respond sensitively to the environment. However, biologists still have not measured whether developmental noise actually comprises a significant fraction of the overall environmental response of fluctuating asymmetry observed within a population. Results In a morphometric study designed to partition developmental noise from fluctuating asymmetry in the wing morphology of a monoclonal culture of cotton aphid, Aphis gossipyii, it was discovered that fluctuating asymmetry in the aphid wing was nearly four times higher than in other insect species. Also, developmental noise comprised a surprisingly large fraction (≈ 50% of the overall response of fluctuating asymmetry to a controlled graded temperature environment. Fluctuating asymmetry also correlated negatively with temperature, indicating that environmentally-stimulated changes in developmental instability are mediated mostly by changes in the development time of individuals

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

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

A single amino acid substitution controls DAF-dependent phenotype of echovirus 11 in rhabdomyosarcoma cells.

Science.gov (United States)

Novoselov, Alexey V; Rezaykin, Alexey V; Sergeev, Alexander G; Fadeyev, Fedor A; Grigoryeva, Julia V; Sokolova, Zoya I

2012-06-01

Decay accelerating factor (DAF, CD55) is used by DAF-dependent (Daf+) variants of echovirus 11 (EV11) as a primary cellular receptor. The interaction of EV11 with DAF is completely reversible, therefore DAF-dependent variants require an unidentified coreceptor to initiate uncoating. Daf- variants of EV11, which do not interact with DAF, use an alternative primary cellular receptor. The aim of this study was to test the hypothesis whether the coreceptor, which is necessary for the uncoating of DAF-dependent variants, may act as an alternative primary receptor for the Daf- variants of EV11. By using the model of the two closely related daf+ and daf- clones of EV11 in rhabdomyosarcoma (RD) cell line, it was shown that a single amino acid substitution in the capsid protein VP2 could control the expression of the DAF-dependent phenotype. Anti-DAF monoclonal antibody has blocked the infection of RD cells by the DAF-dependent daf+ clone, but not by the daf- clone of EV11. Since the structural proteins of the two clones differed only in the receptor binding site for DAF, the unidentified non-DAF primary receptor for the daf- clone might have the same conformation as the uncoating coreceptor required for the daf+ clone. Despite the difference in primary receptors, both daf+ and daf- clones were equally inhibited by a monoclonal antibody to beta2-microglobulin. The monoclonal antibody B9.12.1 to class I human leukocyte antigen molecules showed no inhibitory effect in regards to either clone. The hypothesis of convergent intracellular traffic of Daf+ and Daf- variants of EV11 is discussed. Copyright © 2012 Elsevier B.V. All rights reserved.

Phenotypic and genotypic variation of Phragmites australis: Comparison of populations in two human-made lakes of different age and history

Czech Academy of Sciences Publication Activity Database

Čurn, V.; Kubátová, B.; Vávřová, P.; Křiváčková; Suchá, O.; Čížková, Hana

2007-01-01

Roč. 86, - (2007), s. 321-330 ISSN 0304-3770 R&D Projects: GA ČR(CZ) GA526/06/0276 Institutional research plan: CEZ:AV0Z60870520 Keywords : Phragmites * Phenotypic variation * Genotypic variation * Lake * Clone Subject RIV: EF - Botanics Impact factor: 1.497, year: 2007

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?

Gene Cloning of Iranian Leishmania major Mannose-1-Phosphate Guanyltransferase

Directory of Open Access Journals (Sweden)

R Salehi

2009-07-01

Full Text Available "nBackground: Leishmania is an obligatory intracellular protozoan parasite, which infects human be­ings when infected sand fly vector takes a blood meal.  Most efforts are towards designing an effective vaccine to prevent leishmaniasis. In this way, development of candidate antigen for vaccine has spe­cial im­portant. In this study, we cloned mannose-1-phosphate guanyltransferase gene of Iranian L .major in pET32a expression vector. "nMethods: Primers based on L. major mannose-1-phosphate guanyltransferase sequence gene was de­signed and synthesized. DNA of Leishmania promastigotes was extracted and PCR reaction was done. PCR product was cloned into pTZ57R and sub cloned into pET32a expression vector. "nResults: Recombinant plasmid containing 1140 bp as L. major mannose-1-phosphate guanyltrans­ferase gene was extracted and confirmed by restriction analysis. PCR product was sequenced and de­posited to GenBank. There were some differences in amino acid sequences between Iranian L. major mannose-1-phosphate guanyltransferase and others previously accepted in GenBank "nConclusion: We amplified and cloned Iranian L. major mannose-1-phosphate guanyltransferase successfully.

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....

Pattern of Flushing, Cherelle Wilt, and Accuracy of Yield Forecasting of Some Cocoa Clones

Directory of Open Access Journals (Sweden)

Adi Prawoto

2014-08-01

Full Text Available Monthly observation of cocoa flushing, number of cherelle wilt (CW, number of small, medium and large pods of 6 clones was conducted for two years to study its dynamics for one year. A study was conducted in Kaliwining Experimental Station, 45 m asl. and D rainfall type (according to Schmidt & Ferguson, using ICS 13, ICS 60, TSH 858, Sulawesi 1, Sulawesi 2 and KW 165 clones of 8 years old. Each clone was planted intermittently in separate rows, replicated 6 rows. Correlation and regression analysis were done between variables and with rainfall data. The parallel research was conducted in the similar station to assess the accuracy of production estimation method by identify percentage of small pods (length 1—10 cm, medium (11—15 cm and large pods (>15 cm to grow until harvested. The study used 15th years old trees of Sulawesi 1, Sulawesi 2, KW 165, KKM 22, ICS 13 and DR 2 clones. Each clones was replicated 5 times. The result showed that intensive flushing (>50% occured during January, March, September and November meanwhile no flushing during December and February. Correlation between rainfall and flushing was positive (r=0.27. Effect of clones on flushing frequency was similar but for flushing intensity was significant. KW 165 tended to be the lowest but TSH 858 tend to be the highest. CW occured for a year-round but the height level during May and June. Effect of clones was significant, KW 165 showed highest followed by Sulawesi 2. CW level showed positive correlation with number of medium (r=0.71 and big pods (r=0.55, except showed negative correlation with flushing intensity (r=-0.37 and rainfall (r=-0.51. High pod setting happened during May to November and low pod setting during December to March. In this aspect effect of clones were significant, the productive clones were Sulawesi 1, Sulawesi 2 and KW 165, but ICS 60 was the less. CW level during 1st semester was lower than at 2nd semester and clone effect was significant. The

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.

Positional cloning of the PIS mutation in goats and its impact on understanding mammalian sex-differentiation

Directory of Open Access Journals (Sweden)

Pailhoux Eric

2005-12-01

Full Text Available Abstract In goats, the PIS (polled intersex syndrome mutation is responsible for both the absence of horns in males and females and sex-reversal affecting exclusively XX individuals. The mode of inheritance is dominant for the polled trait and recessive for sex-reversal. In XX PIS-/- mutants, the expression of testis-specific genes is observed very precociously during gonad development. Nevertheless, a delay of 4–5 days is observed in comparison with normal testis differentiation in XY males. By positional cloning, we demonstrate that the PIS mutation is an 11.7-kb regulatory-deletion affecting the expression of two genes, PISRT1 and FOXL2 which could act synergistically to promote ovarian differentiation. The transcriptional extinction of these two genes leads, very early, to testis-formation in XX homozygous PIS-/- mutants. According to their expression profiles and bibliographic data, we propose that FOXL2 may be an ovary-differentiating gene, and the non-coding RNA PISRT1, an anti-testis factor repressing SOX9, a key regulator of testis differentiation. Under this hypothesis, SRY, the testis-determining factor would inhibit these two genes in the gonads of XY males, to ensure testis differentiation.

Composite Lymphoma : EBV-positive Classic Hodgkin Lymphoma and Peripheral T-cell Lymphoma A Case Report

NARCIS (Netherlands)

Gualco, Gabriela; Chioato, Lucimara; Van Den Berg, Anke; Weiss, Lawrence M.; Bacchi, Carlos E.

Composite lymphomas are rare and defined as hematopoietic neoplasms with more than I malignant lymphomatous clone showing different phenotypic features. Of all possible combinations between non-Hodgkin lymphomas, B cell or T cell, and Hodgkin lymphoma, the least frequent are the ones combining

Genotypic and Phenotypic Correlations of Multidrug-Resistant Acinetobacter baumannii-A. calcoaceticus Complex Strains Isolated from Patients at the National Naval Medical Center

Science.gov (United States)

Acinetobacter baumannii-calcoaceticus complex (ABC) infections have complicated the care of U.S. combat casualties. In this study, 102 ABC isolates from wounded soldiers treated at National Naval Medical Center (NNMC) were characterized by phenotype and genotype to identify clones in this population...

Emergence of MPLW515 mutation in a patient with CALR deletion: Evidence of secondary acquisition of MPL mutation in the CALR clone.

Science.gov (United States)

Partouche, Nicolas; Conejero, Carole; Barathon, Quentin; Moroch, Julien; Tulliez, Michel; Cordonnier, Catherine; Giraudier, Stephane

2018-02-01

Myeloproliferative neoplasms are characterized by transduction pathway recognized as mutually exclusive molecular abnormalities such as BCR-ABL translocation, JAK2V617F or JAK2 exon 12 mutations, MPL w515, and CALR mutations. However, in some rare cases, associations of such mutations are found in 1 patient. This can be related to 2 pathologies (at least 2 different clones harboring 2 mutations) or associated mutations in 1 clone. We describe here such an association of CALR and MPL mutations in a patient harboring the second mutation in a subclone during the phenotypic evolution of the myeloproliferative neoplasms. Copyright © 2017 John Wiley & Sons, Ltd.

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

A Gateway MultiSite recombination cloning toolkit.

Directory of Open Access Journals (Sweden)

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.

Characterization of the Pseudomonas aeruginosa recA gene: the Les- phenotype

International Nuclear Information System (INIS)

Kokjohn, T.A.; Miller, R.V.

1988-01-01

The Les- phenotype (lysogeny establishment deficient) is a pleiotropic effect of the lesB908 mutation of Pseudomonas aeruginosa PAO. lesB908-containing strains are also (i) deficient in general recombination, (ii) sensitive to UV irradiation, and (iii) deficient in UV-stimulated induction of prophages. The P. aeruginosa recA-containing plasmid pKML3001 complemented each of these pleiotropic characteristics of the lesB908 mutation, supporting the hypothesis that lesB908 is an allele of the P. aeruginosa recA gene. The phenotypic effects of the lesB908 mutation may be best explained by the hypothesis that the lesB908 gene product is altered in such a way that it has lost synaptase activity but possesses intrinsic protease activity in the absence of DNA damage. The Les- phenotype is a result of the rapid destruction of newly synthesized phage repressor, resulting in lytic growth of the infecting virus. This hypothesis is consistent with the observations that increasing the number of copies of the phage repressor gene by increasing the multiplicity of infection (i.e., average number of phage genomes per cell) or by introducing the cloned phage repressor gene into a lesB908 mutant will also suppress the Les- phenotype in a phage-specific fashion

Stability of the phenotypic reversion of x-ray transformed C3H/10T1/2 cells depends on cellular proliferation after subcultivation at low cell density

International Nuclear Information System (INIS)

Brouty-Boye, D.; Gresser, I.; Bandu, M.T.

1982-01-01

Reversion from the transformed to the non-transformed phenotype could be obtained by seeding X-ray transformed C3H/10T1/2 cells at low cell density. Cloned revertant cells of varying degrees of reversion were obtained depending on the time they were isolated after one subculture at low cell density. Most of the revertants isolated 7 and 10 days after seeding at very low cell density eventually returned to the transformed phenotype when passaged serially at high cell density. In contrast, 25-35% of the revertants isolated 17-20 days after seeding at low cell density maintained the non-transformed phenotype despite subsequent serial passages at high cell density. The finding that there was a direct relationship between the time during which transformed cells seeded at low cell density multiplied and the number of stable revertant clones obtained, suggests the possibility that reversion from the transformed to the non-transformed phenotype may be a multistep process. Revertant cells displayed a chromosomal pattern characteristic of the transformed cells rather than that of the parental non-transformed 10T1/2 cells. (author)

Identification, cloning and characterization of sis7 and sis10 sugar-insensitive mutants of Arabidopsis

Directory of Open Access Journals (Sweden)

Biddle Kelly D

2008-10-01

Full Text Available Abstract Background The levels of soluble sugars, such as glucose and sucrose, help regulate many plant metabolic, physiological and developmental processes. Genetic screens are helping identify some of the loci involved in plant sugar response and reveal extensive cross-talk between sugar and phytohormone response pathways. Results A forward genetic screen was performed to identify mutants with increased resistance to the inhibitory effects of high levels of exogenous sugars on early Arabidopsis seedling development. The positional cloning and characterization of two of these sugar insensitive (sis mutants, both of which are also involved in abscisic acid (ABA biosynthesis or response, are reported. Plants carrying mutations in SIS7/NCED3/STO1 or SIS10/ABI3 are resistant to the inhibitory effects of high levels of exogenous Glc and Suc. Quantitative RT-PCR analyses indicate transcriptional upregulation of ABA biosynthesis genes by high concentrations of Glc in wild-type germinating seeds. Gene expression profiling revealed that a significant number of genes that are expressed at lower levels in germinating sis7-1/nced3-4/sto1-4 seeds than in wild-type seeds are implicated in auxin biosynthesis or transport, suggesting cross-talk between ABA and auxin response pathways. The degree of sugar insensitivity of different sis10/abi3 mutant seedlings shows a strong positive correlation with their level of ABA insensitivity during seed germination. Conclusion Mutations in the SIS7/NCED3/STO1 gene, which is primarily required for ABA biosynthesis under drought conditions, confer a sugar-insensitive phenotype, indicating that a constitutive role in ABA biosynthesis is not necessary to confer sugar insensitivity. Findings presented here clearly demonstrate that mutations in ABI3 can confer a sugar-insensitive phenotype and help explain previous, mixed reports on this topic by showing that ABA and sugar insensitivity exhibit a strong positive correlation in

Map-based cloning and expression analysis of BMR-6 in sorghum.

Science.gov (United States)

Li, Jieqin; Wang, Lihua; Zhang, Qiuwen; Liu, Yanlong

2015-09-01

Brown midrib mutants in sorghum are associated with reduced lignin content and increased cell wall digestibility. In this study, we characterized a bmr-6 sorghum mutant, which shows reddish pigment in the midrib and stem after the fifth-leaf stage. Compared to wild type, Kalson lignin content of bmr-6 is decreased significantly. We used histological analysis to determine that the mutant exhibited a modified pattern of lignin staining and found an increased polysaccharide content. We cloned BMR-6 gene, a gene encoded a cinnamyl alcohol dehydrogenase (CAD), using a map-based cloning approach. Genetic complementation confirmed that CAD is responsible for the BMR-6 phenotype. BMR-6 gene was expressed in all tested sorghum tissues, with the highest being in midrib and stem. Transient expression assays in Nicotiana benthamiana leaves demonstrated cytomplasmic localization of BMR-6. We found that the expression level of bmr-6 was significantly decreased in the mutant but expression of SbCAD3 and SbCAD5 were significantly increased. Our results indicate that BMR-6 not only affects the distribution of lignin but also the biosynthesis of lignin in sorghum.

Discovering genes underlying QTL

Energy Technology Data Exchange (ETDEWEB)

Vanavichit, Apichart [Kasetsart University, Kamphaengsaen, Nakorn Pathom (Thailand)

2002-02-01

A map-based approach has allowed scientists to discover few genes at a time. In addition, the reproductive barrier between cultivated rice and wild relatives has prevented us from utilizing the germ plasm by a map-based approach. Most genetic traits important to agriculture or human diseases are manifested as observable, quantitative phenotypes called Quantitative Trait Loci (QTL). In many instances, the complexity of the phenotype/genotype interaction and the general lack of clearly identifiable gene products render the direct molecular cloning approach ineffective, thus additional strategies like genome mapping are required to identify the QTL in question. Genome mapping requires no prior knowledge of the gene function, but utilizes statistical methods to identify the most likely gene location. To completely characterize genes of interest, the initially mapped region of a gene location will have to be narrowed down to a size that is suitable for cloning and sequencing. Strategies for gene identification within the critical region have to be applied after the sequencing of a potentially large clone or set of clones that contains this gene(s). Tremendous success of positional cloning has been shown for cloning many genes responsible for human diseases, including cystic fibrosis and muscular dystrophy as well as plant disease resistance genes. Genome and QTL mapping, positional cloning: the pre-genomics era, comparative approaches to gene identification, and positional cloning: the genomics era are discussed in the report. (M. Suetake)

Spatial constraints govern competition of mutant clones in human epidermis.

Science.gov (United States)

Lynch, M D; Lynch, C N S; Craythorne, E; Liakath-Ali, K; Mallipeddi, R; Barker, J N; Watt, F M

2017-10-24

Deep sequencing can detect somatic DNA mutations in tissues permitting inference of clonal relationships. This has been applied to human epidermis, where sun exposure leads to the accumulation of mutations and an increased risk of skin cancer. However, previous studies have yielded conflicting conclusions about the relative importance of positive selection and neutral drift in clonal evolution. Here, we sequenced larger areas of skin than previously, focusing on cancer-prone skin spanning five decades of life. The mutant clones identified were too large to be accounted for solely by neutral drift. Rather, using mathematical modelling and computational lattice-based simulations, we show that observed clone size distributions can be explained by a combination of neutral drift and stochastic nucleation of mutations at the boundary of expanding mutant clones that have a competitive advantage. These findings demonstrate that spatial context and cell competition cooperate to determine the fate of a mutant stem cell.

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.

Selection of new clones of linalool chemotype from genetic recombination in Lippia alba

Directory of Open Access Journals (Sweden)

Elcio Rodrigo Rufino

2012-01-01

Full Text Available The aromatic and medicinal species Lippia alba is vigorous and rugged native to the South America (Atlantic Rainforest. Because it is an allogamous and self-incompatible species, natural populations have high morphological and chemical variability. This work had as objective to conduct a preliminary screening to identify new promising clones from a novel (recombinant base population of Lippia alba with regard to its agronomic and phytochemical traits, using the linalool oil or chemotype as model. The two superior linalool clones, obtained by collection, were used as controls. Traits evaluated included: dry mass of leaves (DML, oil yield percentage (EOY%, oil production per plant (OP, and linalool percentage (LN%. Forty linalool chemotype clones were evaluated in three experiments, in a random block design with four replicates and four cuttings (clones per plot. Besides means comparisons, multivariate analysis was used in order to aid in the preliminary selection of clones. There were positive correlations from moderate to strong for DML vs. EOY%, OP vs. EOY% and DML vs. OP. Linalool clones superior or similar to both controls were identified for the DML, EOY%, OP, and LN% traits (univariate analyses, aimed at further validating experimentation. Five distinct groups were defined in the cluster analysis (UPGMA, each containing subgroups as well.

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

Protein change in plant evolution: tracing one thread connecting molecular and phenotypic diversity

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Madelaine eBartlett

2013-10-01

Full Text Available Proteins change over the course of evolutionary time. New protein-coding genes and gene families emerge and diversify, ultimately affecting an organism’s phenotype and interactions with its environment. Here we survey the range of structural protein change observed in plants and review the role these changes have had in the evolution of plant form and function. Verified examples tying evolutionary change in protein structure to phenotypic change remain scarce. We will review the existing examples, as well as draw from investigations into domestication, and quantitative trait locus (QTL cloning studies searching for the molecular underpinnings of natural variation. The evolutionary significance of many cloned QTL has not been assessed, but all the examples identified so far have begun to reveal the extent of protein structural diversity tolerated in natural systems. This molecular (and phenotypic diversity could come to represent part of natural selection’s source material in the adaptive evolution of novel traits. Protein structure and function can change in many distinct ways, but the changes we identified in studies of natural diversity and protein evolution were predicted to fall primarily into one of six categories: altered active and binding sites; hypomorphic and hypermorphic alleles; altered protein-protein interactions; altered domain content; altered protein stability; and altered activity as an activator or repressor. Variability was also observed in the evolutionary scale at which particular changes were observed. Some changes were detected at both micro- and macroevolutionary timescales, while others were observed primarily at deep or shallow phylogenetic levels. This variation might be used to determine the trajectory of future investigations in structural molecular evolution.

Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

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Nocarova, Eva; Fischer, Lukas

2009-04-22

Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with

[Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

Science.gov (United States)

Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

2007-01-01

Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

Metal tolerance in emerging clinically relevant multidrug-resistant Salmonella enterica serotype 4,[5],12:i:- clones circulating in Europe.

Science.gov (United States)

Mourão, Joana; Novais, Carla; Machado, Jorge; Peixe, Luísa; Antunes, Patrícia

2015-06-01

The occurrence of acquired metal tolerance genes in emerging MDR Salmonella enterica serotype 4,[5],12:i:- clones was assessed and their associated platforms and tolerance phenotype were characterised. Salmonella 4,[5],12:i:- from different sources belonging to European, Spanish and Southern European clones were studied. Screening for copper (pcoA-pcoD/tcrB), silver/copper (silA-silE), mercury (merA), arsenic (arsB) and tellurite (terF) tolerance genes was performed by PCR/sequencing. CuSO(4)/AgNO(3) MICs were determined in aerobic/anaerobic atmospheres by agar dilution. Conjugation assays, genomic location and plasmid analysis were performed by standard procedures. Most isolates from European (98%) and Spanish (74%) clones carried silA-silE, contrasting with the Southern European clone (26%). merA/62% (European and Spanish clones) and pcoA-pcoD/50% (European clone) were also detected. merA±pco+sil were chromosomally located in the European clone, whereas in Spanish and Southern European clones sil±merA were within plasmids, both with antibiotic resistance genes. The pcoA-pcoD/silA-silE(+) isolates showed higher MICCuSO(4) in anaerobiosis than those without these genes (MIC(50)=24-28 vs. 2 mM). Different MICAgNO(3) of silA-silE(+) (MIC(50)=0.25 mM) and silA-silE(-)(MIC(50)=0.16 mM) isolates were observed in both atmospheres, with an MIC increment after prior exposure to silver (>3 vs. 0.08-0.125 mM) in aerobiosis. A high frequency of copper and silver tolerance, particularly among the two major Salmonella 4,[5],12:i:- MDR clones (European/Spanish) circulating in Europe and causing human infections, might facilitate adaptation/expansion of these strains in metal-contaminated environments, particularly copper in anaerobiosis. Furthermore, metal toxic concentrations in food-animal environments can contribute to persistence of genetic platforms carrying metal/antibiotic resistance genes in this foodborne zoonotic pathogen. Copyright © 2015 Elsevier B.V. and the

Artificial cloning of domestic animals.

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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.

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.

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

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.

c-di-GMP Regulates Various Phenotypes and Insecticidal Activity of Gram-Positive Bacillus thuringiensis

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Yang Fu

2018-02-01

Full Text Available C-di-GMP has been well investigated to play significant roles in the physiology of many Gram-negative bacteria. However, its effect on Gram-positive bacteria is less known. In order to more understand the c-di-GMP functions in Gram-positive bacteria, we have carried out a detailed study on the c-di-GMP-metabolizing enzymes and their physiological functions in Bacillus thuringiensis, a Gram-positive entomopathogenic bacterium that has been applied as an insecticide successfully. We performed a systematic study on the ten putative c-di-GMP-synthesizing enzyme diguanylate cyclases (DGCs and c-di-GMP-degrading enzyme phosphodiesterases (PDEs in B. thuringiensis BMB171, and artificially elevated the intracellular c-di-GMP level in BMB171 by deleting one or more pde genes. We found increasing level of intracellular c-di-GMP exhibits similar activities as those in Gram-negative bacteria, including altered activities in cell motility, biofilm formation, and cell-cell aggregation. Unexpectedly, we additionally found a novel function exhibited by the increasing level of c-di-GMP to promote the insecticidal activity of this bacterium against Helicoverpa armigera. Through whole-genome transcriptome profile analyses, we found that 4.3% of the B. thuringiensis genes were differentially transcribed when c-di-GMP level was increased, and 77.3% of such gene products are involved in some regulatory pathways not reported in other bacteria to date. In summary, our study represents the first comprehensive report on the c-di-GMP-metabolizing enzymes, their effects on phenotypes, and the transcriptome mediated by c-di-GMP in an important Gram-positive bacterium.

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

Phenotypic Plasticity in Reproductive Traits of the Perennial Shrub Ulex europaeus in Response to Shading: A Multi-Year Monitoring of Cultivated Clones.

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Anne Atlan

Full Text Available Phenotypic plasticity may be advantageous for plants to be able to rapidly cope with new and changing environments associated with climate change or during biological invasions. This is especially true for perennial plants, as they may need a longer period to respond genetically to selective pressures than annuals, and also because they are more likely to experience environmental changes during their lifespan. However, few studies have explored the plasticity of the reproductive life history traits of woody perennial species. This study focuses on a woody shrub, Ulex europaeus (common gorse, and on the response of its reproductive traits to one important environmental factor, shading. The study was performed on clones originating from western France (within the native range of this invasive species and grown for seven years. We compared traits of plants grown in a shade treatment (with two successive shade levels vs. full natural light. The traits monitored included flowering onset, pod production and seed predation. All traits studied responded to shading, exhibiting various levels of plasticity. In particular, dense shade induced a radical but reversible decrease in flower and pod production, while moderate shade had little effect on reproductive traits. The magnitude of the response to dense shade depended on the genotype, showing a genetically based polymorphism of plasticity. The level of plasticity also showed substantial variations between years, and the effect of environmental variations was cumulative over time. This suggests that plasticity can influence the lifetime fitness of U. Europaeus and is involved in the capacity of the species to grow under contrasting environmental conditions.

Phenotypic Plasticity in Reproductive Traits of the Perennial Shrub Ulex europaeus in Response to Shading: A Multi-Year Monitoring of Cultivated Clones.

Science.gov (United States)

Atlan, Anne; Hornoy, Benjamin; Delerue, Florian; Gonzalez, Maya; Pierre, Jean-Sébastien; Tarayre, Michèle

2015-01-01

Phenotypic plasticity may be advantageous for plants to be able to rapidly cope with new and changing environments associated with climate change or during biological invasions. This is especially true for perennial plants, as they may need a longer period to respond genetically to selective pressures than annuals, and also because they are more likely to experience environmental changes during their lifespan. However, few studies have explored the plasticity of the reproductive life history traits of woody perennial species. This study focuses on a woody shrub, Ulex europaeus (common gorse), and on the response of its reproductive traits to one important environmental factor, shading. The study was performed on clones originating from western France (within the native range of this invasive species) and grown for seven years. We compared traits of plants grown in a shade treatment (with two successive shade levels) vs. full natural light. The traits monitored included flowering onset, pod production and seed predation. All traits studied responded to shading, exhibiting various levels of plasticity. In particular, dense shade induced a radical but reversible decrease in flower and pod production, while moderate shade had little effect on reproductive traits. The magnitude of the response to dense shade depended on the genotype, showing a genetically based polymorphism of plasticity. The level of plasticity also showed substantial variations between years, and the effect of environmental variations was cumulative over time. This suggests that plasticity can influence the lifetime fitness of U. Europaeus and is involved in the capacity of the species to grow under contrasting environmental conditions.

Diversity of chloroplast genome among local clones of cocoa (Theobroma cacao, L.) from Central Sulawesi

Science.gov (United States)

Suwastika, I. Nengah; Pakawaru, Nurul Aisyah; Rifka, Rahmansyah, Muslimin, Ishizaki, Yoko; Cruz, André Freire; Basri, Zainuddin; Shiina, Takashi

2017-02-01

Chloroplast genomes typically range in size from 120 to 170 kilo base pairs (kb), which relatively conserved among plant species. Recent evaluation on several species, certain unique regions showed high variability which can be utilized in the phylogenetic analysis. Many fragments of coding regions, introns, and intergenic spacers, such as atpB-rbcL, ndhF, rbcL, rpl16, trnH-psbA, trnL-F, trnS-G, etc., have been used for phylogenetic reconstructions at various taxonomic levels. Based on that status, we would like to analysis the diversity of chloroplast genome within species of local cacao (Theobroma cacao L.) from Central Sulawesi. Our recent data showed, there were more than 20 clones from local farming in Central Sulawesi, and it can be detected based on phenotypic and nuclear-genome-based characterization (RAPD- Random Amplified Polymorphic DNA and SSR- Simple Sequences Repeat) markers. In developing DNA marker for this local cacao, here we also included analysis based on the variation of chloroplast genome. At least several regions such as rpl32-TurnL, it can be considered as chloroplast markers on our local clone of cocoa. Furthermore, we could develop phylogenetic analysis in between clones of cocoa.

A resource-based version of the argument that cloning is an affront to human dignity.

Science.gov (United States)

McDougall, R

2008-04-01

The claim that human reproductive cloning constitutes an affront to human dignity became a familiar one in 1997 as policymakers and bioethicists responded to the announcement of the birth of Dolly the sheep. Various versions of the argument that reproductive cloning is an affront to human dignity have been made, most focusing on the dignity of the child produced by cloning. However, these arguments tend to be unpersuasive and strongly criticised in the bioethical literature. In this paper I put forward a different argument that reproductive cloning is an affront to human dignity, one that looks beyond the dignity of the child produced. I suggest that allocating funds to such a pursuit can affront human dignity by diverting resources away from those existing people who lack sufficient health to enable them to exercise basic rights and liberties. This version of the argument posits cloning as an affront to human dignity in particular circumstances, rather than claiming the technology as intrinsically inconsistent with human dignity.

Epigenetic reprogramming in mammalian species after SCNT-based cloning.

Science.gov (United States)

Niemann, Heiner

2016-07-01

The birth of "Dolly," the first mammal cloned from an adult mammary epithelial cell, abolished the decades-old scientific dogma implying that a terminally differentiated cell cannot be reprogrammed into a pluripotent embryonic state. The most dramatic epigenetic reprogramming occurs in SCNT when the expression profile of a differentiated cell is abolished and a new embryo-specific expression profile, involving 10,000 to 12,000 genes, and thus, most genes of the entire genome is established, which drives embryonic and fetal development. The initial release from somatic cell epigenetic constraints is followed by establishment of post-zygotic expression patterns, X-chromosome inactivation, and adjustment of telomere length. Somatic cell nuclear transfer may be associated with a variety of pathologic changes of the fetal and placental phenotype in a proportion of cloned offspring, specifically in ruminants, that are thought to be caused by aberrant epigenetic reprogramming. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realizing the great potential of SCNT for basic research and for important agricultural and biomedical applications. Here, current knowledge on epigenetic reprogramming after use of SCNT in livestock is reviewed, with emphasis on gene-specific and global DNA methylation, imprinting, X-chromosome inactivation, and telomere length restoration in early development. Copyright © 2016 Elsevier Inc. All rights reserved.

Profiling the repertoire of phenotypes influenced by environmental cues that occur during asexual reproduction.

Science.gov (United States)

Dombrovsky, Aviv; Arthaud, Laury; Ledger, Terence N; Tares, Sophie; Robichon, Alain

2009-11-01

The aphid Acyrthosiphon pisum population is composed of different morphs, such as winged and wingless parthenogens, males, and sexual females. The combined effect of reduced photoperiodicity and cold in fall triggers the apparition of sexual morphs. In contrast they reproduce asexually in spring and summer. In our current study, we provide evidence that clonal individuals display phenotypic variability within asexual morph categories. We describe that clones sharing the same morphological features, which arose from the same founder mother, constitute a repertoire of variants with distinct behavioral and physiological traits. Our results suggest that the prevailing environmental conditions influence the recruitment of adaptive phenotypes from a cohort of clonal individuals exhibiting considerable molecular diversity. However, we observed that the variability might be reduced or enhanced by external factors, but is never abolished in accordance with a model of stochastically produced phenotypes. This overall mechanism allows the renewal of colonies from a few adapted individuals that survive drastic episodic changes in a fluctuating environment.

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.

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.

Positive attitude towards life and emotional expression as personality phenotypes for centenarians.

Science.gov (United States)

Kato, Kaori; Zweig, Richard; Barzilai, Nir; Atzmon, Gil

2012-05-01

Centenarians have been reported to share particular personality traits including low neuroticism and high extraversion and conscientiousness. Since these traits have moderate to high heritability and are associated with various health outcomes, personality appears linked to bio-genetic mechanisms which may contribute to exceptional longevity. Therefore, the present study sought to detect genetically-based personality phenotypes in a genetically homogeneous sample of centenarians through developing and examining psychometric properties of a brief measure of the personality of centenarians, the Personality Outlook Profile Scale (POPS). The results generated two personality characteristics/domains, Positive Attitude Towards Life (PATL: optimism, easygoing, laughter, and introversion/outgoing) and Emotional Expression (EE: expressing emotions openly and not bottling up emotions). These domains demonstrated acceptable concurrent validity with two established personality measures, the NEO-Five Factor Inventory and Life Orientation Test-Revised. Additionally, centenarians in both groups had lower neuroticism and higher conscientiousness than the US adult population. Findings suggest that the POPS is a psychometrically sound measure of personality in centenarians and capture personality aspects of extraversion, neuroticism, and conscientiousness, as well as dispositional optimism which may contribute to successful aging.

Use of high throughput qPCR screening to rapidly clone low frequency tumour specific T-cells from peripheral blood for adoptive immunotherapy

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Serrano Oscar K

2008-10-01

Full Text Available Abstract Background The adoptive transfer of autologous tumor reactive lymphocytes can mediate significant tumor regression in some patients with refractory metastatic cancer. However, a significant obstacle for this promising therapy has been the availability of highly efficient methods to rapidly isolate and expand a variety of potentially rare tumor reactive lymphocytes from the natural repertoire of cancer patients. Methods We developed a novel in vitro T cell cloning methodology using high throughput quantitative RT-PCR (qPCR assay as a rapid functional screen to detect and facilitate the limiting dilution cloning of a variety of low frequency T cells from bulk PBMC. In preclinical studies, this strategy was applied to the isolation and expansion of gp100 specific CD8+ T cell clones from the peripheral blood of melanoma patients. Results In optimization studies, the qPCR assay could detect the reactivity of 1 antigen specific T cell in 100,000 background cells. When applied to short term sensitized PBMC microcultures, this assay could detect T cell reactivity against a variety of known melanoma tumor epitopes. This screening was combined with early limiting dilution cloning to rapidly isolate gp100154–162 reactive CD8+ T cell clones. These clones were highly avid against peptide pulsed targets and melanoma tumor lines. They had an effector memory phenotype and showed significant proliferative capacity to reach cell numbers appropriate for adoptive transfer trials (~1010 cells. Conclusion This report describes a novel high efficiency strategy to clone tumor reactive T cells from peripheral blood for use in adoptive immunotherapy.

Complementation of radiation-sensitive Ataxia telangiectasia cells after transfection of cDNA expression libraries and cosmid clones from wildtype cells

International Nuclear Information System (INIS)

Fritz, E.

1994-06-01

In this Ph.D.-thesis, phenotypic complementation of AT-cells (AT5BIVA) by transfection of cDNA-expression-libraries was adressed: After stable transfection of cDNA-expression-libraries G418 resistant clones were selected for enhanced radioresistance by a fractionated X-ray selection. One surviving transfectant clone (clone 514) exhibited enhanced radiation resistance in dose-response experiments and further X-ray selections. Cell cycle analysis revealed complementation of untreated and irradiated 514-cells in cell cycle progression. The rate of DNA synthesis, however, is not diminished after irradiation but shows the reverse effect. A transfected cDNA-fragment (AT500-cDNA) was isolated from the genomic DNA of 514-cells and proved to be an unknown DNA sequence. A homologous sequence could be detected in genomic DNA from human cell lines, but not in DNA from other species. The cDNA-sequence could be localized to human chromosome 11. In human cells the cDNA sequence is part of two large mRNAs. 4 different cosmid clones containing high molecular genomic DNA from normal human cells could be isolated from a library, each hybridizing to the AT500-cDNA. After stable transfection into AT-cells, one cosmid-clone was able to confer enhanced radiation resistance both in X-ray selections and dose-response experiments. The results indicate that the cloned cDNA-fragment is based on an unknown gene from human chromosome 11 which partially complements the radiosensitivity and the defective cell cycle progression in AT5BIVA cells. (orig.) [de

Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction

DEFF Research Database (Denmark)

Fahnøe, Ulrik; Pedersen, Anders Gorm; Dräger, Carolin

2015-01-01

necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from...... the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral...... evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants....

Cloning the enterotoxin gene from Clostridium perfringens type A

OpenAIRE

Iwanejko, Lesley Ann.

1991-01-01

A C. perfringens type A genomic library was constructed in E. coli by banking overlapping 6-10 kbp Hind III fragments of chromosomal DNA from the enterotoxin (CPE) positive strain NCTC 8239 into the pUC derived vector pHG165. The library was screened by colony hybridization with a degenerate 26 bp oligonucleotide probe, derived from the amino acid sequence CPE9_17A. complex mixture of plasmid DNA was isolated from the only hybridization positive clone. A second round of screening picked out a...

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)

Rapid approach for cloning bacterial single-genes directly from soils ...

African Journals Online (AJOL)

Obtaining functional genes of bacteria from environmental samples usually depends on library-based approach which is not favored as its large amount of work with small possibility of positive clones. A kind of bacterial single-gene encoding glutamine synthetase (GS) was selected as example to detect the efficiency of ...

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)

[The variation and clinical significance of paroxysmal nocturnal hemoglobinuria clone in patients with aplastic anemia before and after immunosuppressive therapy].

Science.gov (United States)

Sun, Ying-xin; Zhu, Ming-qing; He, Guang-sheng; Wang, Xiu-li; Fang, Bao-zhi; Lu, Cong; Liu, Zhen-zhen; Wu, Qian; Yang, Yong; Wu, De-pei; Sun, Ai-ning

2013-07-01

To evaluate the evolution of paroxysmal nocturnal hemoglobinuria (PNH) clone and its clinical significance before and after immunosuppressive therapy (IST) in patients with aplastic anemia (AA). A total of 186 patients diagnosed as AA were enrolled in this study. Among them, 55 patients were diagnosed as severe AA (SAA) and treated with cyclosporine (CsA) plus anti-thymocyte globulin (ATG), 131 were diagnosed as non SAA (NSAA) and treated with CsA alone. All patients were screened for PNH clone by flow cytometry before treatment and followed up for 18-76 months, with a median time of 22 months. Positive PNH clones were detected in 10 SAA (18.9%) patients, significantly more than that of NSAA group [9 patients (7.4%), t = 5.041, P = 0.025]. The proportions of PNH clones in SAA group at 6, 12, 24 and > 24 months were 13.38%, 14.88%, 20.00% and 18.85%, respectively, also significantly higher than those of NSAA patients (5.67%, 5.31%, 5.47% and 9.08%, all P values clone was positive or negative. PNH clone are detectable in AA patients either treated with ATG plus CsA or CsA alone, and more significant by ATG plus CsA. Whether PNH clone occurred before or after IST does not affect the therapeutic efficacy.

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.

The ethics of human reproductive cloning: when world views collide.

Science.gov (United States)

Cohen, Cynthia B

2004-01-01

Two camps in bioethics with seemingly opposing world views have staked out conflicting positions regarding the ethics of human reproductive cloning. These camps do not appear to share common concepts or ways of reasoning through which to exchange views and come to a meeting of minds about uses of this technology. Yet analysis of their respective approaches to several issues surrounding reproductive cloning, such as where the ethical limits of individual reproductive choice lie, whether the use of this technology would violate human dignity, whether it would create risks to the resulting fetuses and children that would make its use intolerable, and whether it would challenge certain core social values, reveals that they are not wholly opposed to one another. Indeed, it displays that they hold certain beliefs, values, and concerns in common. Moreover, it indicates that the different world views that they each presuppose, while flawed in certain respects, do not collide in every respect, but can be reconciled in significant ways that provide fertile ground for agreement about several issues related to human reproductive cloning.

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

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.

T-cell clones from Th1, Th17 or Th1/17 lineages and their signature cytokines have different capacity to activate endothelial cells or synoviocytes.

Science.gov (United States)

Lavocat, Fabien; Maggi, Laura; Annunziato, Francesco; Miossec, Pierre

2016-12-01

To compare the direct effect of cytokines on synoviocytes and endothelial cells to the effects of supernatants from Th1, Th17 and Th1/17 clones and the direct cell-cell interactions with the same clones. Th17 and Th1/17 clones were obtained from the CD161+CCR6+ fraction and Th1 clones from the CD161-CCR6- fraction of human CD4+ T-cells. Endothelial cells or synoviocytes were cultured in the presence of either isolated pro-inflammatory cytokines (IL-17 and/or TNF-α) or supernatants from the T-cell clones or co-cultured with T-cell clones themselves. IL-6 and IL-8 expression and production were analyzed. IL-17 and TNF-α induced IL-6 and IL-8 expression, although IL-17 alone had a limited effect on endothelial cells compared to synoviocytes. Supernatants from activated T-helper clones also induced IL-6 and IL-8 expression but with discrepancies between endothelial cells and synoviocytes. Endothelial cells were mostly activated by Th1 clone supernatants whereas synoviocytes were activated by all T-cell subtypes. Finally, cell-cell contact experiments showed a great heterogeneity among cell clones, even from the same lineage. IL-6 expression was mostly induced by contact with Th1 clones both in endothelial and mesenchymal cells whereas IL-8 expression was induced by all T-cell clones whatever their phenotype. We showed that endothelial cells were much more sensitive to Th1 activation whereas synoviocytes were activated by all T-helper lineages. This work highlights the heterogeneity of interactions between T-cells and stromal cells through soluble factors or direct cell contact. Copyright © 2016 Elsevier Ltd. All rights reserved.

Human Chromosome 21: Mapping of the chromosomes and cloning of cDNAs

Energy Technology Data Exchange (ETDEWEB)

Antonarakis, S.E.

1991-09-01

The objective of the research funded by DOE grant DE-FG02-89ER60857 from 6/15/89 to 8/31/91 was to contribute to the physical mapping of human chromosome 21 (HC21) by cloning large fragments of DNA into Yeast Artificial Chromosomes (YACs) and identify YACs that map on HC21. A total of 54 sequence tagged sites (STS) have been developed and mapped in our laboratory to HC21 and can be used as initial reference points for YAC identification and construction of overlapping clones. A small YAC library was constructed which is HC21 specific. DNA from somatic cell hybrid WAV17 or from flow-sorted HC21 was partially digested with EcoRI, ligated into vectors PJS97, PJS98, and YACs have been obtained with average size insert of more than 300 kb. This library has been deposited in D. Patterson's lab for the Joint YAC screening effort. Additional YAC libraries from ICI Pharmaceuticals or from Los Alamos National Laboratories have been screened with several STS and positive YACs have been identified. Work in progress includes screening of YAC libraries in order to construct overlapping clones, characterization of the cloning ends of YACs, characterization of additional STS and cloning of HC21 specific cDNAs. 15 refs., 2 figs., 5 tabs.

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.

Methylotroph cloning vehicle

Science.gov (United States)

Hanson, R.S.; Allen, L.N.

1989-04-25

A cloning vehicle comprising: a replication determinant effective for replicating the vehicle in a non-C[sub 1]-utilizing host and in a C[sub 1]-utilizing host; DNA effective to allow the vehicle to be mobilized from the non-C[sub 1]-utilizing host to the C[sub 1]-utilizing host; DNA providing resistance to two antibiotics to which the wild-type C[sub 1]-utilizing host is susceptible, each of the antibiotic resistance markers having a recognition site for a restriction endonuclease; a cos site; and a means for preventing replication in the C[sub 1]-utilizing host. The vehicle is used for complementation mapping as follows. DNA comprising a gene from the C[sub 1]-utilizing organism is inserted at the restriction nuclease recognition site, inactivating the antibiotic resistance marker at that site. The vehicle can then be used to form a cosmid structure to infect the non-C[sub 1]-utilizing (e.g., E. coli) host, and then conjugated with a selected C[sub 1]-utilizing mutant. Resistance to the other antibiotic by the mutant is a marker of the conjugation. Other phenotypical changes in the mutant, e.g., loss of an auxotrophic trait, is attributed to the C[sub 1] gene. The vector is also used to inactivate genes whose protein products catalyze side reactions that divert compounds from a biosynthetic pathway to a desired product, thereby producing an organism that makes the desired product in higher yields. 3 figs.

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.

Stability and adaptability of early maturing sugarcane clones by AMMI analysis

Directory of Open Access Journals (Sweden)

Edson Perez Guerra

2009-01-01

Full Text Available Stability and adaptability of 14 early maturing sugarcane clones were evaluated at 11 locations in the State ofParaná, in the plant cane and ratoon cycles, by the AMMI method. By AMMI2, 59.44% cumulative variance was explained inplant cane and 54.22% in ratoon cane by the first two principal components of tons of pol per hectare (TPH. For genotypeRB966928 the TPH was medium to high, phenotypic stability high and adaptability general, recommending this early maturingclone with wide adaptability for northern Paraná. The genotype-environment interaction was lowest in Paranavaí andMandaguaçú (most stable locations, where the ranking of genotypes was more reliable than the means of the environmentstested.

Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning.

Science.gov (United States)

Meghdadi, Hossein; Khosravi, Azar D; Ghadiri, Ata A; Sina, Amir H; Alami, Ameneh

2015-01-01

Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39-31.27% for rpoB-PCR, 36.44-60.83% for IS6110- PCR, 75.29-92.93% for nested-rpoB PCR, and 87.98-99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100

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.

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.

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

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.

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.

Geometrical conditions for completely positive trace-preserving maps and their application to a quantum repeater and a state-dependent quantum cloning machine

International Nuclear Information System (INIS)

Carlini, A.; Sasaki, M.

2003-01-01

We address the problem of finding optimal CPTP (completely positive trace-preserving) maps between a set of binary pure states and another set of binary generic mixed state in a two-dimensional space. The necessary and sufficient conditions for the existence of such CPTP maps can be discussed within a simple geometrical picture. We exploit this analysis to show the existence of an optimal quantum repeater which is superior to the known repeating strategies for a set of coherent states sent through a lossy quantum channel. We also show that the geometrical formulation of the CPTP mapping conditions can be a simpler method to derive a state-dependent quantum (anti) cloning machine than the study so far based on the explicit solution of several constraints imposed by unitarity in an extended Hilbert space

[Cloning, mutagenesis and symbiotic phenotype of three lipid transfer protein encoding genes from Mesorhizobium huakuii 7653R].

Science.gov (United States)

Li, Yanan; Zeng, Xiaobo; Zhou, Xuejuan; Li, Youguo

2016-12-04

Lipid transfer protein superfamily is involved in lipid transport and metabolism. This study aimed to construct mutants of three lipid transfer protein encoding genes in Mesorhizobium huakuii 7653R, and to study the phenotypes and function of mutations during symbiosis with Astragalus sinicus. We used bioinformatics to predict structure characteristics and biological functions of lipid transfer proteins, and conducted semi-quantitative and fluorescent quantitative real-time PCR to analyze the expression levels of target genes in free-living and symbiotic conditions. Using pK19mob insertion mutagenesis to construct mutants, we carried out pot plant experiments to observe symbiotic phenotypes. MCHK-5577, MCHK-2172 and MCHK-2779 genes encoding proteins belonged to START/RHO alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) superfamily, involved in lipid transport or metabolism, and were identical to M. loti at 95% level. Gene relative transcription level of the three genes all increased compared to free-living condition. We obtained three mutants. Compared with wild-type 7653R, above-ground biomass of plants and nodulenitrogenase activity induced by the three mutants significantly decreased. Results indicated that lipid transfer protein encoding genes of Mesorhizobium huakuii 7653R may play important roles in symbiotic nitrogen fixation, and the mutations significantly affected the symbiotic phenotypes. The present work provided a basis to study further symbiotic function mechanism associated with lipid transfer proteins from rhizobia.

Genetic variation and correlation of agronomic traits in meadow bromegrass (Bromus riparius Rehm clones

Directory of Open Access Journals (Sweden)

Araújo Marcelo Renato Alves de

2004-01-01

Full Text Available Meadow bromegrass (Bromus riparius Rehm. is a recently introduced pasture grass in western Canada. Its leafy production and rapid regrowth have made it a major grass species for pasturing beef animals in this region. As relatively little breeding work has been done on this species, there is little information on its breeding behaviour. The main objective of this study was to estimate total genetic variability, broad-sense heritability, phenotypic and genetic correlations. Forty-four meadow bromegrass clones were evaluated for agronomic characters. Genetic variation for dry matter yield, seed yield, fertility index, harvest index, plant height, plant spread, crude protein, neutral detergent fiber and acid detergent fiber, was significant. Broad-sense heritability estimates exceeded 50% for all characters. Heritability estimates were at least 3.5 times greater than their standard errors. Phenotypic and genetic correlation between all possible characters were measured. There was general agreement in both sign and magnitude between genetic and phenotypic correlations. Correlations between the different characters demonstrated that it is possible to simultaneously improve seed and forage yield. Based on the results, it appears that the development of higher yielding cultivars with higher crude protein, and lower acid and neutral detergent fibers concentration should be possible.

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

Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation.

Directory of Open Access Journals (Sweden)

Martin Meyer

2016-08-01

Full Text Available We here compared pathogenic (p and non-pathogenic (np isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1-A12 derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1-B12 derived from a pathogenic isolate HM-1:IMSS-B. "Non-pathogenicity" included the induction of small and quickly resolved lesions while "pathogenicity" comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.

Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation.

Science.gov (United States)

Meyer, Martin; Fehling, Helena; Matthiesen, Jenny; Lorenzen, Stephan; Schuldt, Kathrin; Bernin, Hannah; Zaruba, Mareen; Lender, Corinna; Ernst, Thomas; Ittrich, Harald; Roeder, Thomas; Tannich, Egbert; Lotter, Hannelore; Bruchhaus, Iris

2016-08-01

We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1-A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1-B12) derived from a pathogenic isolate HM-1:IMSS-B. "Non-pathogenicity" included the induction of small and quickly resolved lesions while "pathogenicity" comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.

Osteochondritis Dissecans Lesions in Family Members: Does a Positive Family History Impact Phenotypic Potency?

Science.gov (United States)

Gornitzky, Alex L; Mistovich, R Justin; Atuahuene, Brittany; Storey, Eileen P; Ganley, Theodore J

2017-06-01

Although repetitive microtrauma and athletic overuse patterns are most commonly associated with osteochondritis dissecans (OCD), recent studies have identified a potential genetic predisposition for OCD. Several case series have documented family pedigrees that support autosomal-dominant inheritance, but the families in these studies were all selected as a result of unique histories that may not accurately represent OCD inheritance patterns at large. Because there has been little investigation beyond these case reports, we aimed to describe a broader, more representative pattern of OCD inheritance applicable to all affected patients. (1) What proportion of patients treated for OCD of the knee have one or more immediate and/or extended family members with a history of OCD lesions? (2) Do patients with more phenotypically potent lesions, which we defined as patients with bilateral OCD lesions or patients who have undergone multiple procedures for OCD, have a higher frequency of affected relatives than those with less potent lesions? This retrospective study queried patient databases, diagnosis codes (International Classification of Diseases, 9th Revision), and surgical logs at a regional, tertiary care children's hospital to identify all patients treated over a 10-year period (March 2004-March 2014) by the senior author for OCD of the knee. All patients aged 0-18 years at the time of diagnosis were included. At our institution, patients with intact lesions are treated with a trial of conservative therapy; conversely, patients with a break in the articular cartilage and/or loose fragments of bone/cartilage are treated surgically. There were no OCD-specific contraindications to surgery. This search identified 543 patients. After patient identification, a questionnaire was designed that asked for the number, age, and gender of all immediate family members and the history of OCD lesions in any family member (immediate or extended). For all positive family members

Expression of Two N1 Clones with Single Amino Acid Dissimilarity of Avian Influenza H5N1 Virus

Directory of Open Access Journals (Sweden)

RISZA HARTAWAN

2012-12-01

Full Text Available Two clones of N1 gene derived from isolate A/Dk/Tangerang/Bbalitvet-ACIAR-TE11/2007 (H5N1 exhibit single mismatch of amino acid sequence at position 242 that is threonine and methionine for the clone #3 and #5, respectively. In order to evaluate the effect of the amino acid substitution, these clones were inserted into two different expression vectors that are pEGFP-C1 and pcDNA-3.3 TOPO® TA cloning. Subsequently, the respective recombinant clones were transfected into eukaryotic cells, including CEF, RK13 and VERO using Lipofectamine ‘plus’ reagent. As a result, the clone #3 retaining atypical sequence showed lower expression level rather than the clone #15 in both vectors and all type of cells. The 3D conformational modelling revealed that the mutation occurs in the inner part of glycoprotein embedded within envelope or matrix. Therefore, the missense mutation seems has no effect on the antigenic properties of neuraminidase but this substitution by any means causes lethal mutagenesis in the individual gene expression by reducing level of protein transcript.

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...

"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

Golden Haidegg, a new apple mutant clone with improved marketing value

International Nuclear Information System (INIS)

Strempl, F.; Keppl, H.; Brunner, H.

1988-01-01

Full text: Golden Delicious and its derivatives are the leading apple cultivars in Austria. Traits limiting the economic yield are susceptibility to russeting, a heterogenous fruit assortment score and consumer preferences. Mutation breeding was started in 1972. Dormant five bud scions of Golden Delicious were irradiated with 40, 50 or 60 Gy gamma rays at a dose rate of 20 Gy min -1 and grafted on rootstocks M9. M 1 V 1 survival rates were 78% (40 Gy), 36% (50 Gy) and 6% (60 Gy). Surviving scions produced, on the average, two primary shoots from which three to five buds were used for summer budding. Primary shoots were pruned back to force M 1 V 2 shoots from the lower secondary buds. An incidental occurrence of viruses and mycoplasms was overcome by thermotherapy, but delayed completing procedures of selection, re-selection and confirmation of the selected traits till the M 1 V 6 generation. Desirable mutations in shoot vigor, growth type, fruit size and fruit quality characters were obtained from the 40 and 50 Gy treatments only, while 60 Gy produced generally grossly aberrant phenotypes. A mutant with smooth sheen fruits associated with a more flat shape and non-russeting was selected from the 50 Gy treatment. Smooth sheen and non-russeting are evidently independent traits. Among 18 different mutant clones tested in microtrials, only the russet-free, smooth sheen clone was superior to the parent cultivar in market value. This clone, named Golden Haidegg, was tested during four years in different environments, compared with other clones derived from Golden Delicious, i.e. Lysgolden, Belgolden, Supergolden, Cloden, Golden 1972, Golden s.r E9, Golden clone A and B, Golden Shay, Golden Missouri, Charden, Mutsu and Smoothe. All trees were virus free, grafted on rootstock M9 and trained as slender spindle; applied field management conditions were identical. The evaluation concerned yield, russeting, fruit-shape, colour, weight, assortment and cold-storability. Clone

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

Directory of Open Access Journals (Sweden)

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.

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

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

An innovative strategy to clone positive modifier genes of defects caused by mtDNA mutations: MRPS18C as suppressor gene of m.3946G>A mutation in MT-ND1 gene.

Science.gov (United States)

Rodríguez-García, María Elena; Cotrina-Vinagre, Francisco Javier; Carnicero-Rodríguez, Patricia; Martínez-Azorín, Francisco

2017-07-01

We have developed a new functional complementation approach to clone modifier genes which overexpression is able to suppress the biochemical defects caused by mtDNA mutations (suppressor genes). This strategy consists in transferring human genes into respiratory chain-deficient fibroblasts, followed by a metabolic selection in a highly selective medium. We used a normalized expression cDNA library in an episomal vector (pREP4) to transfect the fibroblasts, and a medium with glutamine and devoid of any carbohydrate source to select metabolically. Growing the patient's fibroblasts in this selective medium, the deficient cells rapidly disappear unless they are rescued by the cDNA of a suppressor gene. The use of an episomal vector allows us to carry out several rounds of transfection/selection (cyclical phenotypic rescue) to enrich the rescue with true clones of suppressor genes. Using fibroblasts from a patient with epileptic encephalopathy with the m.3946G>A (p.E214K) mutation in the MT-ND1 gene, several candidate genes were identified and one of them was characterized functionally. Thus, overexpression of MRPS18C gene (that encode for bS18m protein) suppressed the molecular defects produced by this mtDNA mutation, recovering the complex I activity and reducing the ROS produced by this complex to normal levels. We suggest that modulation of bS18m expression may be an effective therapeutic strategy for the patients with this mutation.

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

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.

In vivo localization of cloned IL-2-dependent T cells

International Nuclear Information System (INIS)

Carroll, A.M.; Palladino, M.A.; Oettgen, H.; De Sousa, M.

1983-01-01

The quantitative organ distribution and tissue microenvironment positioning of radioisotopically labeled cloned T cells were characterized. Intravenous (iv) injection of 51chromium ( 51 Cr)-labeled, long-term cultured cloned T-helper cells and cells from several cloned cytolytic T-lymphocyte lines (CTLL) resulted in poor localization of these cells in recipient lymphoid tissues, similar to results reported for activated lymphoblastoid cells. Simultaneous administration of interleukin 2 (IL-2) with labeled cells resulted in enhanced recovery from recipient spleen. By the intraperitoneal (ip) injection route, overall percentage recovery of injected radioactivity was lower than by the iv route, but significant localization to lymph nodes occurred. Examination of autoradiographs of tissue sections from recipients of [ 3 H]adenosine-labeled cells showed most label associated with intact, isolated cells in the liver, lungs, spleen, and small intestine. By 24 hr after iv injection, labeled cells in spleen sections were distributed to both nonlymphoid and T- and B-lymphoid areas. These findings suggest that poor localization of these cells to recipient lymphoid tissue is due both to intrinsic characteristics of cultured lymphocytes and to the possible reduced viability of IL-2-dependent cells in vivo

Natural Reassortants of Potentially Zoonotic Avian Influenza Viruses H5N1 and H9N2 from Egypt Display Distinct Pathogenic Phenotypes in Experimentally Infected Chickens and Ferrets.

Science.gov (United States)

Naguib, Mahmoud M; Ulrich, Reiner; Kasbohm, Elisa; Eng, Christine L P; Hoffmann, Donata; Grund, Christian; Beer, Martin; Harder, Timm C

2017-12-01

The cocirculation of zoonotic highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 and avian influenza virus (AIV) of subtype H9N2 among poultry in Egypt for at least 6 years should render that country a hypothetical hot spot for the emergence of reassortant, phenotypically altered viruses, yet no reassortants have been detected in Egypt. The present investigations proved that reassortants of the Egyptian H5N1 clade 2.2.1.2 virus and H9N2 virus of the G1-B lineage can be generated by coamplification in embryonated chicken eggs. Reassortants were restricted to the H5N1 subtype and acquired between two and all six of the internal segments of the H9N2 virus. Five selected plaque-purified reassortant clones expressed a broad phenotypic spectrum both in vitro and in vivo Two groups of reassortants were characterized to have retarded growth characteristics in vitro compared to the H5N1 parent virus. One clone provoked reduced mortality in inoculated chickens, although the characteristics of a highly pathogenic phenotype were retained. Enhanced zoonotic properties were not predicted for any of these clones, and this prediction was confirmed by ferret inoculation experiments: neither the H5N1 parent virus nor two selected clones induced severe clinical symptoms or were transmitted to sentinel ferrets by contact. While the emergence of reassortants of Egyptian HPAIV of subtype H5N1 with internal gene segments of cocirculating H9N2 viruses is possible in principle, the spread of such viruses is expected to be governed by their fitness to outcompete the parental viruses in the field. The eventual spread of attenuated phenotypes, however, would negatively impact syndrome surveillance on poultry farms and might foster enzootic virus circulation. IMPORTANCE Despite almost 6 years of the continuous cocirculation of highly pathogenic avian influenza virus H5N1 and avian influenza virus H9N2 in poultry in Egypt, no reassortants of the two subtypes have been reported

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.

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)

AMMI analysis to evaluate the adaptability and phenotypic stability of sugarcane genotypes

Directory of Open Access Journals (Sweden)

Luís Cláudio Inácio da Silveira

2013-02-01

Full Text Available Sugarcane (Saccharum sp. is one of the most important crops in Brazil. The high demand for sugarcane-derived products has stimulated the expansion of sugarcane cultivation in recent years, exploring different environments. The adaptability and the phenotypic stability of sugarcane genotypes in the Minas Gerais state, Brazil, were evaluated based on the additive main effects and multiplicative interaction (AMMI method. We evaluated 15 genotypes (13 clones and two checks: RB867515 and RB72454 in nine environments. The average of two cuttings for the variable tons of pol per hectare (TPH measure was used to discriminate genotypes. Besides the check RB867515 (20.44 t ha-1, the genotype RB987935 showed a high average TPH (20.71 t ha-1, general adaptability and phenotypic stability, and should be suitable for cultivation in the target region. The AMMI method allowed for easy visual identification of superior genotypes for each set of environments.

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 ...

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.

Alterations in Mesenteric Lymph Node T Cell Phenotype and Cytokine Secretion are Associated with Changes in Thymocyte Phenotype after LP-BM5 Retrovirus Infection

Directory of Open Access Journals (Sweden)

Maria C. Lopez

2005-01-01

Full Text Available In this study, mouse MLN cells and thymocytes from advanced stages of LP-BM5 retrovirus infection were studied. A decrease in the percentage of IL-7+ cells and an increase in the percentage of IL-16+ cells in the MLN indicated that secretion of these cytokines was also altered after LP-BM5 infection. The percentage of MLN T cells expressing IL-7 receptors was significantly reduced, while the percentage of MLN T cells expressing TNFR-p75 and of B cells expressing TNFR-p55 increased. Simultaneous analysis of surface markers and cytokine secretion was done in an attempt to understand whether the deregulation of IFN-Υ secretion could be ascribed to a defined cell phenotype, concluding that all T cell subsets studied increased IFN-Υ secretion after retrovirus infection. Finally, thymocyte phenotype was further analyzed trying to correlate changes in thymocyte phenotype with MLN cell phenotype. The results indicated that the increase in single positive either CD4+CD8- or CD4- CD8+ cells was due to accumulation of both immature (CD3- and mature (CD3+ single positive thymocytes. Moreover, single positive mature thymocytes presented a phenotype similar to the phenotype previously seen on MLN T cells. In summary, we can conclude that LP-BM5 uses the immune system to reach the thymus where it interferes with the generation of functionally mature T cells, favoring the development of T cells with an abnormal phenotype. These new T cells are activated to secrete several cytokines that in turn will favor retrovirus replication and inhibit any attempt of the immune system to control infection.

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

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.

[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.

Developmental potential of bovine hand-made clone embryos reconstructed by aggregation or fusion with distinct cytoplasmic volumes.

Science.gov (United States)

Ribeiro, Eduardo de Souza; Gerger, Renato Pereira da Costa; Ohlweiler, Lain Uriel; Ortigari, Ivens; Mezzalira, Joana Cláudia; Forell, Fabiana; Bertolini, Luciana Relly; Rodrigues, José Luiz; Ambrósio, Carlos Eduardo; Miglino, Maria Angélica; Mezzalira, Alceu; Bertolini, Marcelo

2009-09-01

Animal cloning has been associated with developmental abnormalities, with the level of heteroplasmy caused by the procedure being one of its potential limiting factors. The aim of this study was to determine the effect of the fusion of hemicytoplasts or aggregation of hemiembryos, varying the final cytoplasmic volume, on development and cell density of embryos produced by hand-made cloning (HMC), parthenogenesis or by in vitro fertilization (IVF). One or two enucleated hemicytoplasts were paired and fused with one skin somatic cell. Activated clone and zona-free parthenote embryos and hemiembryos were in vitro cultured in the well-of-the-well (WOW) system, being allocated to one of six experimental groups, on a per WOW basis: single clone or parthenote hemiembryos (1 x 50%); aggregation of two (2 x 50%), three (3 x 50%), or four (4 x 50%) clone or parthenote hemiembryos; single clone or parthenote embryos (1 x 100%); or aggregation of two clone or parthenote embryos (2 x 100%). Control zona-intact parthenote or IVF embryos were in vitro cultured in four-well dishes. Results indicated that the increase in the number of aggregated structures within each WOW was followed by a linear increase in cleavage, blastocyst rate, and cell density. The increase in cytoplasmic volume, either by fusion or by aggregation, had a positive effect on embryo development, supporting the establishment of pregnancies and the birth of a viable clone calf after transfer to recipients. However, embryo aggregation did not improve development on a hemicytoplast basis, except for the aggregation of two clone embryos.

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

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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

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 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

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

Science.gov (United States)

Wong, J C; Alon, N; Norga, K; Kruyt, F A; Youssoufian, H; Buchwald, M

2000-08-01

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 model system, we cloned and characterized the mouse homolog of the human FANCA cDNA. The mouse cDNA (Fanca) encodes a 161-kDa protein that shares 65% amino acid sequence identity with human FANCA. Fanca is located at the distal region of mouse chromosome 8 and has a ubiquitous pattern of expression in embryonic and adult tissues. Expression of the mouse cDNA in human FA-A cells restores the cellular drug sensitivity to normal levels. Thus, the expression pattern, protein structure, chromosomal location, and function of FANCA are conserved in the mouse. We also isolated a novel zinc finger protein, Zfp276, which has five C(2)H(2) domains. Interestingly, Zfp276 is situated in the Fanca locus, and the 3'UTR of its cDNA overlaps with the last four exons of Fanca in a tail-to-tail manner. Zfp276 is expressed in the same tissues as Fanca, but does not complement the mitomycin C (MMC)-sensitive phenotype of FA-A cells. The overlapping genomic organization between Zfp276 and Fanca may have relevance to the disease phenotype of FA. Copyright 2000 Academic Press.

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

Cloning and expression of the Legionella micdadei "common antigen" in Escherichia coli

DEFF Research Database (Denmark)

Bangsborg, Jette Marie; Collins, M T; Høiby, N

1989-01-01

To study individual Legionella antigens, a Legionella micdadei genomic library in Escherichia coli SC181 was established. Partially Sau3A digested L. micdadei DNA fragments (15-25 kilobase pairs (kb] were cloned into the tetracycline resistance gene of the cosmid vector pHC79. Four thousand...... ampicillin resistant recombinants were obtained; seven hundred were screened for expression of Legionella antigens in Western blot analysis with a polyspecific E. coli-absorbed anti-L. micdadei rabbit antibody. One of the positive clones expressed a 60 kilodalton (K) antigen, which reacted strongly...... will provide important information with respect to genetic vs. antigenic relatedness among Legionellae and other Gram-negative species, as well as to CA structure and possible function....

[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.

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.

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

Clone-Specific Response in Leaf Nitrate Reductase Activity among Unrelated Hybrid Poplars in relation to Soil Nitrate Availability

Directory of Open Access Journals (Sweden)

Julien Fortier

2012-01-01

Full Text Available In this field study, we used in vivo NRA activity in hybrid poplar leaves as an indicator of NO3- assimilation for five unrelated hybrid poplar clones. We also examined if leaf NRA of these clones is influenced to the same extent by different levels of soil NO3- availability in two riparian agroforestry systems located in pastures. Leaf NRA differences of more than one order of magnitude were observed between the clones, clearly showing their different abilities to reduce NO3- in leaves. Clone DxN-3570, a P. deltoides x P. nigra hybrid (Aigeiros intrasectional hybrid, always had the highest leaf NRA during the field assays. This clone was also the only one to increase its leaf NRA with increasing NO3- soil availability, which resulted in a significant Site x Clone interaction and a positive relationship between soil NO3- concentration and NRA. All of the four other clones studied had one or both parental species from the Tacamahaca section. They had relatively low leaf NRA and they did not increase their leaf NRA when grown on the NO3- rich site. These results provide evidence that NO3- assimilation in leaves varies widely among hybrid poplars of different parentages, suggesting potential preferences for N forms.

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.

RESEARCH ARTICLE Molecular cloning and functional ...

Indian Academy of Sciences (India)

Navya

2016-11-25

Nov 25, 2016 ... Molecular cloning and functional characterization of two novel ... Currently, many variants of HMW-GSs have been cloned from bread wheat .... SDS sedimentation tests were conducted using the methods described by Gao et ...

Identification and Preliminary Analysis of Several Centromere-associated Bacterial Artificial Chromosome Clones from a Diploid Wheat Library

Institute of Scientific and Technical Information of China (English)

无

2006-01-01

Although the centromeres of some plants have been investigated previously, our knowledge of the wheat centromere is still very limited. To understand the structure and function of the wheat centromere, we used two centromeric repeats (RCS1 and CCS1-5ab) to obtain some centromere-associated bacterial artificial chromosome (BAC) clones in 32 RCS1-related BAC clones that had been screened out from a diploid wheat (Triticum boeoticum Boiss.; 2n=2x=14) BAC library. Southern hybridization results indicated that, of the 32 candidates,there were 28 RCS1-positive clones. Based on gel blot patterns, the frequency of RCS1 was approximately one copy every 69.4 kb in these 28 RCS1-positive BAC clones. More bands were detected when the same filter was probed with CCS1-5ab. Furthermore, the CCS1 bands covered all the bands detected by RCS1, which suggests that some CCS1 repeats were distributed together with RCS1. The frequency of CCS1 families was once every 35.8 kb, nearly twice that of RCS1. Fluorescence in situ hybridization (FISH) analysis indicated that the five BAC clones containing RCS1 and CCS1 sequences all detected signals at the centromeric regions in hexaploid wheat, but the signal intensities on the A-genome chromosomes were stronger than those on the B- and/or D-genome chromosomes. The FISH analysis among nine Triticeae cereals indicated that there were A-genomespecific (or rich) sequences dispersing on chromosome arms in the BAC clone TbBAC5. In addition, at the interphase cells, the centromeres of diploid species usually clustered at one pole and formed a ring-like allocation in the period before metaphase.

Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype.

Science.gov (United States)

Barnidge, David R; Lundström, Susanna L; Zhang, Bo; Dasari, Surendra; Murray, David L; Zubarev, Roman A

2015-12-04

In our previous work, we showed that electrospray ionization of intact polyclonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift. To determine the origin of the high molecular mass clones, we performed de novo bottom-up mass spectrometry on a purified IgM monoclonal light chain that had a calculated molecular mass of 24 275.03 Da. The entire sequence of the monoclonal light chain was determined using multienzyme digestion and de novo sequence-alignment software and was found to belong to the germline allele IGKV2-30. The alignment of kappa germline sequences revealed ten IGKV2 and one IGKV4 sequences that contained additional amino acids in their CDR1 region, creating the high-molecular-mass phenotype. We also performed an alignment of lambda germline sequences, which showed additional amino acids in the CDR2 region, and the FR3 region of functional germline sequences that result in a high-molecular-mass phenotype. The work presented here illustrates the ability of mass spectrometry to provide information on the diversity of light-chain molecular mass phenotypes in circulation, which reflects the germline sequences selected by the immunoglobulin-secreting B-cell population.

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.

Cloning higher plants from aseptically cultured tissues and cells

Science.gov (United States)

Krikorian, A. D.

1982-01-01

A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

Versican V1 Overexpression Induces a Myofibroblast-Like Phenotype in Cultured Fibroblasts.

Directory of Open Access Journals (Sweden)

Jon M Carthy

Full Text Available Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts.The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle α-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-β signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2.Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

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.

[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.

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

[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)

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

OpenAIRE

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-01-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding t...

[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.

Science.gov (United States)

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.

Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells.

Science.gov (United States)

Fedr, Radek; Pernicová, Zuzana; Slabáková, Eva; Straková, Nicol; Bouchal, Jan; Grepl, Michal; Kozubík, Alois; Souček, Karel

2013-05-01

The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. Copyright © 2013 International Society for Advancement of Cytometry.

Evaluación de patógenos en clones de lulo (Solanum quitoense Lam. Pathogenity evaluation on Solanum quitoense Lam. Clones

Directory of Open Access Journals (Sweden)

Consuelo Montes Rojas

2010-04-01

Full Text Available En el noroccidente de Popayán, Colombia, se evaluó la presencia de plagas causadas por patógenos en 42 clones de lulo (Solanum quitoense Lam.. Los clones fueron plantados en bolsas plásticas, donde se desarrollaron por 3 semanas antes de ser trasplantados al campo. Se utilizó un diseño de bloques completos al azar con cuatro repeticiones, la parcela útil estuvo conformada por 6 plantas, las cuales se sembraron a ‘tresbolillo’ a 2.5 m entre surcos y 2 m entre plantas. Para determinar el efecto de las plagas en el cultivo, se calculó el porcentaje de incidencia y severidad del ataque. La incidencia se evaluó como porcentaje de plantas afectadas, y la severidad como porcentaje de tejido afectado por el patógeno. Las enfermedades más limitantes para los 42 clones fueron: gota (Phytophthora infestans que provocó una mortalidad de plantas superior a 40%; fusarium (Fusarium oxysporum que se presentó en 12 de los clones evaluados; antracnosis (Colletotrichum sp. que afectó 21 clones, los cuales se clasificaron entre tolerantes y medianamente tolerantes; y mancha clorótica (Cladosporium sp. que afectó 21 clones, clasificados como susceptibles. Los clones PL19, PL24, PL11, PL35 fueron medianamente tolerantes. Se seleccionaron por supervivencia los clones: JY E1 (52.2%, PH E 1 (45.8%, VM E2 (45.8%; por supervivencia y por tolerancia a Fusarium oxysporum los clones PL35, PL11, PL24, PL8, PL19, 120052, 120043, ORE1, AGE1. Los clones SER 7, SER 15, SER 9, SEC 31, SEC 27 presentaron alta mortalidad pero se seleccionaron por ser medianamente tolerantes a gota, tolerantes a antracnosis y medianamente resistentes a nematodos, con buen vigor y producción.Presence of plant disease caused by pathogens on 42 clones of Solanum quitoense Lam. were evaluated in the north-western region of Popayán, Colombia. The seed of the clons were planted in plastic bags during three weeks and afterwards transplanted to the field. The statistical design

Elephant grass clones for silage production

Directory of Open Access Journals (Sweden)

Rerisson José Cipriano dos Santos

2013-02-01

Full Text Available Ensiling warm-season grasses often requires wilting due to their high moisture content, and the presence of low-soluble sugars in these grasses usually demands the use of additives during the ensiling process. This study evaluated the bromatological composition of the fodder and silage from five Pennisetum sp. clones (IPA HV 241, IPA/UFRPE Taiwan A-146 2.114, IPA/UFRPE Taiwan A-146 2.37, Elephant B, and Mott. The contents of 20 Polyvinyl chloride (PVC silos, which were opened after 90 days of storage, were used for the bromatological analysis and the evaluation of the pH, nitrogen, ammonia, buffer capacity, soluble carbohydrates, and fermentation coefficients. The effluent losses, gases and dry matter recovery were also calculated. Although differences were observed among the clones (p < 0.05 for the concentrations of dry matter, insoluble nitrogen in acid detergents, insoluble nitrogen in neutral detergents, soluble carbohydrates, fermentation coefficients, and in vitro digestibility in the forage before ensiling, no differences were observed for most of these variables after ensiling. All of the clones were efficient in the fermentation process. The IPA/UFRPE TAIWAN A-146 2.37 clone, however, presented a higher dry matter concentration and the best fermentation coefficient, resulting in a better silage quality, compared to the other clones.

Specific Schistosoma mansoni rat T cell clones. I. Generation and functional analysis in vitro and in vivo.

Science.gov (United States)

Pestel, J; Dissous, C; Dessaint, J P; Louis, J; Engers, H; Capron, A

1985-06-01

In an attempt to determine the role of schistosome-specific T cells in the immune mechanisms developed during schistosomiasis, Schistosoma mansoni-specific T cells and clones were generated in vitro and some of their functions analyzed in vitro and in vivo in the fischer rat model. The data presented here can be summarized as follows: a) Lymph node cells (LNC) from rats primed with the excretory/secretory antigens-incubation products (IPSm) of adult worms proliferate in vitro only in response to the homologous schistosome antigens and not to unrelated antigens (Ag) such as ovalbumin (OVA) or Dipetalonema viteae and Fasciola hepatica parasite extracts. b) After in vitro restimulation of the primed LNC population with IPSm in the presence of antigen-presenting cells (APC) and maintenance in IL 2-containing medium, the frequency of IPSm-specific T cells is increased and the T cells can be restimulated only in the presence of APC possessing the same major histocompatibility complex (MHC) antigens. c) Following appropriate limiting dilution assays (LDA) (1 cell/well), 10 IPSm-specific T cell clones were obtained, and two of four maintained in culture were tested for their helper activity because they expressed only the W3/13+ W3/25+ surface phenotypes. d) The two highly proliferating IPSm-specific T cell clones (G5 and E23) exhibit an IPSm-dependent helper activity, as shown by the increase in IgG production by IPSm-primed B cells. e) IPSm-T cell clone (G5) as well as IPSm-T cell lines when injected in S. mansoni-infested rats can exert an in vivo helper activity, which is characterized by an accelerated production of IgG antibodies specific for the previously identified 30 to 40 kilodaltons (kd) schistosomula surface antigens (Ag). As recent studies have demonstrated that rat monoclonal antibodies recognize some incubation products of adult S. mansoni as well as one of the 30 to 40 kd schistosomula surface antigens, and taking into account the fact that the T cell

Challenges in regulating farm animal cloning

DEFF Research Database (Denmark)

Gunning, Jennifer; Hartlev, Mette; Gamborg, Christian

Report from the project Cloning in Public: A specific support action within the 6th framework programme, priority 5: Food quality and safety......Report from the project Cloning in Public: A specific support action within the 6th framework programme, priority 5: Food quality and safety...

The science and technology of farm animal cloning

DEFF Research Database (Denmark)

Gjerris, Mickey; Vajta, Gábor

, goats, horses, cats, etc. have been cloned with the somatic cell nuclear transfer technique. Although the technology still has relatively low success rates and there seems to be substantial problems with the welfare of some of the cloned animals, cloning is used both within basic research...... include the production of genetically identical animals for research purposes, and also the creation of genetically modified animals. In the agricultural sector, cloning can be used as a tool within farm animal breeding. We do not intend to give an exhaustive review of the all the literature available...

Rapid containment of nosocomial transmission of a rare community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) clone, responsible for the Staphylococcal Scalded Skin Syndrome (SSSS)

OpenAIRE

Lamanna, Onofrio; Bongiorno, Dafne; Bertoncello, Lisa; Grandesso, Stefano; Mazzucato, Sandra; Pozzan, Giovanni Battista; Cutrone, Mario; Chirico, Michela; Baesso, Flavia; Brugnaro, Pierluigi; Cafiso, Viviana; Stefani, Stefania; Campanile, Floriana

2017-01-01

Background The aims of this study were to identify the source and the transmission pathway for a Staphylococcal Scalded Skin Syndrome (SSSS) outbreak in a maternity setting in Italy over 2?months, during 2014; to implement appropriate control measures in order to prevent the epidemic spread within the maternity ward; and to identify the Methicillin-Resistant Staphylococcus aureus (MRSA) epidemic clone. Methods Epidemiological and microbiological investigations, based on phenotyping and genoty...

Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

DEFF Research Database (Denmark)

Lundwall, Åke B; Wetsel, Rick A; Kristensen, Torsten

1985-01-01

DNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence......, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha......'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human...

Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency?

Science.gov (United States)

Wakayama, Teruhiko

2007-02-01

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.

Clone Detection for Graph-Based Model Transformation Languages

DEFF Research Database (Denmark)

Strüber, Daniel; Plöger, Jennifer; Acretoaie, Vlad

2016-01-01

and analytical quality assurance. From these use cases, we derive a set of key requirements. We describe our customization of existing model clone detection techniques allowing us to address these requirements. Finally, we provide an experimental evaluation, indicating that our customization of ConQAT, one......Cloning is a convenient mechanism to enable reuse across and within software artifacts. On the downside, it is also a practice related to significant long-term maintainability impediments, thus generating a need to identify clones in affected artifacts. A large variety of clone detection techniques...... has been proposed for programming and modeling languages; yet no specific ones have emerged for model transformation languages. In this paper, we explore clone detection for graph-based model transformation languages. We introduce potential use cases for such techniques in the context of constructive...

Photonic quantum simulator for unbiased phase covariant cloning

Science.gov (United States)

Knoll, Laura T.; López Grande, Ignacio H.; Larotonda, Miguel A.

2018-01-01

We present the results of a linear optics photonic implementation of a quantum circuit that simulates a phase covariant cloner, using two different degrees of freedom of a single photon. We experimentally simulate the action of two mirrored 1→ 2 cloners, each of them biasing the cloned states into opposite regions of the Bloch sphere. We show that by applying a random sequence of these two cloners, an eavesdropper can mitigate the amount of noise added to the original input state and therefore, prepare clones with no bias, but with the same individual fidelity, masking its presence in a quantum key distribution protocol. Input polarization qubit states are cloned into path qubit states of the same photon, which is identified as a potential eavesdropper in a quantum key distribution protocol. The device has the flexibility to produce mirrored versions that optimally clone states on either the northern or southern hemispheres of the Bloch sphere, as well as to simulate optimal and non-optimal cloning machines by tuning the asymmetry on each of the cloning machines.

Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors.

OpenAIRE

Laine, M J; Nakhei, H; Dreier, J; Lehtilä, K; Meletzus, D; Eichenlaub, R; Metzler, M C

1996-01-01

In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, a...

Culture-positive Pediatric Tuberculosis in Toronto, Ontario: Sources of Infection and Relationship of Birthplace and Mycobacterial Lineage to Phenotype.

Science.gov (United States)

Rayment, Jonathan H; Guthrie, Jennifer L; Lam, Karen; Whelan, Michael; Lee, Brenda; Jamieson, Frances B; Kitai, Ian

2016-01-01

Few data relate Mycobacterium tuberculosis (Mtb) lineage and disease phenotype in the pediatric population or examine the contribution of travel to the tuberculosis (TB)-endemic country in North America. We examined clinical, demographic and Mtb genotype data from patients with TB who were treated in Toronto between 2002 and 2012. Consecutive Mtb culture-positive, pediatric patients were included. Clinical data were collected from a prospectively populated clinical database. Mtb case isolate genotypes were identified using Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeat (MIRU-VNTR) and spoligotyping and were categorized into phylogeographic lineages for analysis. The 77 patients included 30.4% of all culture-positive pediatric TB cases in Ontario from 2002 to 2012. Seventy-six (99%) patients were first or second generation Canadians. Foreign-born patients were more likely to have extrathoracic disease [odds ratios (OR) = 3.0; 95% confidence interval (CI): 1.04-8.71; P < 0.05] and less likely to have a genotype match in the Public Health Ontario Laboratories database [OR = 0.32 (95% CI: 0.11-0.90); P < 0.05] than Canadian-born patients. For those without a known TB contact, Canadian-born patients were more likely to have travelled to a TB-endemic country [OR = 13.0 (95% CI: 2.5-78.5); P < 0.001]. Extrathoracic disease was less likely in patients infected with the East Asian Mtb lineage [OR = 0.1 (95% CI: 0.01-0.9); P < 0.05] and more likely in those infected with the Indo-Oceanic Mtb lineage [OR = 5.4 (95% CI: 1.5-19.2); P < 0.05]. Travel to TB-endemic countries likely plays an important part in the etiology of pediatric TB infection and disease, especially in Canadian-born children. Mtb lineage seems to contribute to disease phenotype in children as it has been described in adults.

Post-death cloning of endangered Jeju black cattle (Korean native cattle): fertility and serum chemistry in a cloned bull and cow and their offspring.

Science.gov (United States)

Kim, Eun Young; Song, Dong Hwan; Park, Min Jee; Park, Hyo Young; Lee, Seung Eun; Choi, Hyun Yong; Moon, Jeremiah Jiman; Kim, Young Hoon; Mun, Seong Ho; Oh, Chang Eon; Ko, Moon Suck; Lee, Dong Sun; Riu, Key Zung; Park, Se Pill

2013-12-17

To preserve Jeju black cattle (JBC; endangered native Korean cattle), a pair of cattle, namely a post-death cloned JBC bull and cow, were produced by somatic cell nuclear transfer (SCNT) in a previous study. In the present study, we examined the in vitro fertilization and reproductive potentials of these post-death cloned animals. Sperm motility, in vitro fertilization and developmental capacity were examined in a post-death cloned bull (Heuk Oll Dolee) and an extinct nuclear donor bull (BK94-13). We assessed reproductive ability in another post-death cloned cow (Heuk Woo Sunee) using cloned sperm for artificial insemination (AI). There were no differences in sperm motility or developmental potential of in vitro fertilized embryos between the post-death cloned bull and its extinct nuclear donor bull; however, the embryo development ratio was slightly higher in the cloned sperm group than in the nuclear donor sperm group. After one attempt at AI, the post-death cloned JBC cow became pregnant, and gestation proceeded normally until day 287. From this post-death cloned sire and dam, a JBC male calf (Heuk Woo Dolee) was delivered naturally (weight, 25 kg). The genetic paternity/maternity of the cloned JBC bull and cow with regard to their offspring was confirmed using International Society for Animal Genetics standard microsatellite markers. Presently, Heuk Woo Dolee is 5 months of age and growing normally. In addition, there were no significant differences in blood chemistry among the post-death cloned JBC bull, the cow, their offspring and cattle bred by AI. This is the first report showing that a pair of cattle, namely, a post-death cloned JBC bull and cow, had normal fertility. Therefore, SCNT can be used effectively to increase the population of endangered JBC.

Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea.

Science.gov (United States)

Kasai, Yuki; Oshima, Kohei; Ikeda, Fukiko; Abe, Jun; Yoshimitsu, Yuya; Harayama, Shigeaki

2015-01-01

Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea. In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator. A self-cloning

Cloning: Past, Present, and the Exciting Future. Breakthroughs in Bioscience.

Science.gov (United States)

Di Berardino, Marie A.

This document explores the history of cloning by focusing on Dolly the Sheep, one of the first large animal clonings. The disadvantages and advantages of transgenic clones are discussed as well as the future implications of cloning from the perspective of human health. (Contains 10 resources.) (YDS)

Transfer of experimental autoimmune thyroiditis with T cell clones

International Nuclear Information System (INIS)

Romball, C.G.; Weigle, W.O.

1987-01-01

We have investigated three T lymphocyte clones isolated from CBA/CaJ mice primed with mouse thyroid extract (MTE) in adjuvant. All three clones are L3T4+, Ig-, and Lyt2- and proliferate to MTE, mouse thyroglobulin (MTG) and rat thyroid extract. Clones A7 and B7 transfer thyroiditis to irradiated (475 rad) syngeneic mice, but not to normal recipients. The thyroid lesion induced by the B7 clone is characterized by the infiltration of both mononuclear and polymorphonuclear cells. The thyroiditis is transient in that lesions are apparent 7 and 14 days after transfer, but thyroids return to normal by day 21. Clone B7 showed helper activity for trinitrophenyl-keyhole limpet hemocyanin-primed B cells in vitro when stimulated with trinitrophenyl-MTG and also stimulated the production of anti-MTG antibody in recipient mice. Clone A7 induced thyroid lesions characterized by infiltration of the thyroid with mononuclear cells, with virtually no polymorphonuclear cell infiltration. This clone has shown no helper activity following stimulation with trinitrophenyl-MTG. The third clone (D2) proliferates to and shows helper activity to MTG, but fails to transfer thyroiditis to syngeneic, irradiated mice. On continuous culture, clone B7 lost its surface Thy. The loss of Thy appears unrelated to the ability to transfer thyroiditis since subclones of B7 with markedly different percentages of Thy+ cells transferred disease equally well

The evaluation of growth dynamics of Lonicera kamtschatica clones

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Ján Matuškovič

2007-01-01

Full Text Available Artickle deals with the evaluation of growth dynamics of selected set of clones Lonicera kamtschatica in the conditions of Nitra. We measured the growth of the shrubs twice a year (in spring and autumn during 2003–2005. Within all clones 5 shrubs were evaluated. On the basis of the obtained results we can claim the highest increase of height in case of LKL 21 followed by clones LKL 16 and LKL 5. The lowest growth increase was typical for LKL 58 and LKL 66.In term of statistical evaluation the year can be considered as a statistically significant factor forming a growth intensity of clones during 2003–2005. The effect of year on growing processes is strong (ε2 = 0.96 while the participation of year with clone influenced the growth increase in medium size (ε2 = 0.42. LKL 21 and LKL 58 in comparison with other clones are the most disperatable in term of growth increase. Within mentioned clones statistically significant differences were recorded in 7 evaluated pairs. In the same way LKL 42 is very different from another clones as well. On the basis of all provided analysis the tested clones from point of wiev perspectivity of planting can be set up in the following order: LKL 21, LKL 16, LKL 5, LKL 42, LKL 49, LKL 96, LKL 6, LKL 60, LKL 66 and LKL 58.

Cloning the Gravity and Shear Stress Related Genes from MG-63 Cells by Subtracting Hybridization

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Zhang, Shu; Dai, Zhong-quan; Wang, Bing; Cao, Xin-sheng; Li, Ying-hui; Sun, Xi-qing

2008-06-01

Background The purpose of the present study was to clone the gravity and shear stress related genes from osteoblast-like human osteosarcoma MG-63 cells by subtractive hybridization. Method MG-63 cells were divided into two groups (1G group and simulated microgravity group). After cultured for 60 h in two different gravitational environments, two groups of MG-63 cells were treated with 1.5Pa fluid shear stress (FSS) for 60 min, respectively. The total RNA in cells was isolated. The gravity and shear stress related genes were cloned by subtractive hybridization. Result 200 clones were gained. 30 positive clones were selected using PCR method based on the primers of vector and sequenced. The obtained sequences were analyzed by blast. changes of 17 sequences were confirmed by RT-PCR and these genes are related to cell proliferation, cell differentiation, protein synthesis, signal transduction and apoptosis. 5 unknown genes related to gravity and shear stress were found. Conclusion In this part of our study, our result indicates that simulated microgravity may change the activities of MG-63 cells by inducing the functional alterations of specific genes.

Duration of gestation in pregnant dogs carrying cloned fetuses.

Science.gov (United States)

Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Park, Eun Jung; Jo, Young Kwang; Lee, Byeong Chun

2013-01-15

The aim of this study was to investigate gestation duration and the physiologic characteristics of pregnant dogs bearing cloned fetuses, especially in the prepartum period. A retrospective study was performed to compare gestation duration in females pregnant with cloned (somatic cell nuclear transfer) fetuses (cloned group) with those bearing noncloned fetuses (control group), and effects of litter size, birth weight, and breed of somatic cell donors on gestation duration in the cloned group were evaluated. Clinical delivery onset signs associated with serum progesterone concentration and rectal temperature were also compared in both groups. The gestation duration calculated from day of ovulation was significantly longer in the cloned (62.8 ± 0.3 days) versus the control group (60.9 ± 0.5 days; P dogs bearing cloned fetuses might be because of the smaller litter size in this group. Also, the weaker drop in serum progesterone levels in the prepartum period in cloned dog pregnancies indicates that the parturition signaling process might be altered resulting in longer gestation periods. Copyright © 2013 Elsevier Inc. All rights reserved.

Towards Clone Detection in UML Domain Models

DEFF Research Database (Denmark)

Störrle, Harald

2013-01-01

Code clones (i.e., duplicate fragments of code) have been studied for long, and there is strong evidence that they are a major source of software faults. Anecdotal evidence suggests that this phenomenon occurs similarly in models, suggesting that model clones are as detrimental to model quality...... as they are to code quality. However, programming language code and visual models have significant differences that make it difficult to directly transfer notions and algorithms developed in the code clone arena to model clones. In this article, we develop and propose a definition of the notion of “model clone” based...... we believe that our approach advances the state of the art significantly, it is restricted to UML models, its results leave room for improvements, and there is no validation by field studies....

Personality consistency analysis in cloned quarantine dog candidates

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Jin Choi

2017-01-01

Full Text Available In recent research, personality consistency has become an important characteristic. Diverse traits and human-animal interactions, in particular, are studied in the field of personality consistency in dogs. Here, we investigated the consistency of dominant behaviours in cloned and control groups followed by the modified Puppy Aptitude Test, which consists of ten subtests to ascertain the influence of genetic identity. In this test, puppies are exposed to stranger, restraint, prey-like object, noise, startling object, etc. Six cloned and four control puppies participated and the consistency of responses at ages 7–10 and 16 weeks in the two groups was compared. The two groups showed different consistencies in the subtests. While the average scores of the cloned group were consistent (P = 0.7991, those of the control group were not (P = 0.0089. Scores of Pack Drive and Fight or Flight Drive were consistent in the cloned group, however, those of the control group were not. Scores of Prey Drive were not consistent in either the cloned or the control group. Therefore, it is suggested that consistency of dominant behaviour is affected by genetic identity and some behaviours can be influenced more than others. Our results suggest that cloned dogs could show more consistent traits than non-cloned. This study implies that personality consistency could be one of the ways to analyse traits of puppies.

Cloning and joint measurements of incompatible components of spin

International Nuclear Information System (INIS)

Brougham, Thomas; Andersson, Erika; Barnett, Stephen M.

2006-01-01

A joint measurement of two observables is a simultaneous measurement of both quantities upon the same quantum system. When two quantum-mechanical observables do not commute, then a joint measurement of these observables cannot be accomplished directly by projective measurements alone. In this paper we shall discuss the use of quantum cloning to perform a joint measurement of two components of spin associated with a qubit system. We introduce cloning schemes which are optimal with respect to this task. The cloning schemes may be thought to work by cloning two components of spin onto their outputs. We compare the proposed cloning machines to existing cloners

Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases.

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Tanaka, T; Kawata, M

1988-08-01

We have isolated a DNA fragment from Bacillus subtilis 168 which, when present in a high-copy plasmid, inhibited production of extracellular alkaline and neutral proteases. The gene responsible for this activity was referred to as iep. The open reading frame of iep was found to be incomplete in the cloned DNA fragment. When the intact iep gene was reconstructed after the missing part of the iep gene had been cloned, it showed an enhancing effect on the production of the extracellular proteases. The open reading frame encodes a polypeptide of 229 amino acids with a molecular weight of ca. 25,866. Deletion of two amino acids from the N-terminal half of the putative iep protein resulted in dual effects, i.e., a decrease in the inhibitory activity shown by the incomplete iep gene and a slight increase in the enhancing activity shown by the complete iep gene. These results show that the iep gene product is a bifunctional protein, containing inhibitory and enhancing activities for the exoprotease production in the N-terminal and C-terminal regions, respectively. It was found by genetic and functional analyses that iep lies very close to sacU.

Genetics of phenotypic plasticity and biomass traits in hybrid willows across contrasting environments and years.

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Berlin, Sofia; Hallingbäck, Henrik R; Beyer, Friderike; Nordh, Nils-Erik; Weih, Martin; Rönnberg-Wästljung, Ann-Christin

2017-07-01

Phenotypic plasticity can affect the geographical distribution of taxa and greatly impact the productivity of crops across contrasting and variable environments. The main objectives of this study were to identify genotype-phenotype associations in key biomass and phenology traits and the strength of phenotypic plasticity of these traits in a short-rotation coppice willow population across multiple years and contrasting environments to facilitate marker-assisted selection for these traits. A hybrid Salix viminalis  × ( S. viminalis × Salix schwerinii ) population with 463 individuals was clonally propagated and planted in three common garden experiments comprising one climatic contrast between Sweden and Italy and one water availability contrast in Italy. Several key phenotypic traits were measured and phenotypic plasticity was estimated as the trait value difference between experiments. Quantitative trait locus (QTL) mapping analyses were conducted using a dense linkage map and phenotypic effects of S. schwerinii haplotypes derived from detected QTL were assessed. Across the climatic contrast, clone predictor correlations for biomass traits were low and few common biomass QTL were detected. This indicates that the genetic regulation of biomass traits was sensitive to environmental variation. Biomass QTL were, however, frequently shared across years and across the water availability contrast. Phenology QTL were generally shared between all experiments. Substantial phenotypic plasticity was found among the hybrid offspring, that to a large extent had a genetic origin. Individuals carrying influential S. schwerinii haplotypes generally performed well in Sweden but less well in Italy in terms of biomass production. The results indicate that specific genetic elements of S. schwerinii are more suited to Swedish conditions than to those of Italy. Therefore, selection should preferably be conducted separately for such environments in order to maximize biomass

Características físicas e sensoriais de clones de batata-doce Physical and sensorial characteristics of sweetpotato clones

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Adriana Dias Cardoso

2007-12-01

Full Text Available Com o objetivo de avaliar propriedades físicas e sensoriais de clones de batata-doce em Vitória da Conquista - BA foi realizado este experimento, composto por 16 clones oriundos de Janaúba- G, Viçosa - MG, Bom Jardim de Minas - MG, Gurupi - TO, Santo Antônio da Platina - PR, Holambra II - SP, Vitória da Conquista - BA e Condeúba - BA. Utilizou-se o delineamento em blocos casualizados, com 16 tratamentos e 3 repetições. Avaliaram-se as características sensoriais: aparência, umidade, doçura, coloração da polpa, dificuldade de deglutição das raízes tuberosas e as características físicas: tempo de cozimento e peso específico. Os dados foram submetidos à análise de variância e teste de Scott-Knott a 5% de probabilidade, entretanto, as características sensoriais foram obtidas apenas em valores de porcentagem. O clone 25 apresentou as melhores características sensoriais e o clone 7 apresentou melhor tempo de cozimento.The aim of this experiment was to evaluate the physical and sensorial characteristics of sweetpotato clones in Vitória da Conquista, Bahia State, Brazil. Sixteen clones were analyzed, originating from Janaúba, MG, Viçosa, MG; Bom Jardim de Minas, MG; Gurupi, TO; Santo Antônio da Platina, PR; Holambra II, SP; Vitória da Conquista, BA; and Condeúba, BA. One utilized randomized blocks with 16 treatments and three repetitions. The following characteristics were analyzed: aspect, humidity, sweetness, color, deglutition difficulty, cooking and specific gravity of the storage roots. The data were submitted to variance analysis using a ScottKnott test with 5% probability. Clone 25 presented the best sensorial characteristics, and clone 7 presented the best cooking time.

Systematic cloning of human minisatellites from ordered array charomid libraries.

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Armour, J A; Povey, S; Jeremiah, S; Jeffreys, A J

1990-11-01

We present a rapid and efficient method for the isolation of minisatellite loci from human DNA. The method combines cloning a size-selected fraction of human MboI DNA fragments in a charomid vector with hybridization screening of the library in ordered array. Size-selection of large MboI fragments enriches for the longer, more variable minisatellites and reduces the size of the library required. The library was screened with a series of multi-locus probes known to detect a large number of hypervariable loci in human DNA. The gridded library allowed both the rapid processing of positive clones and the comparative evaluation of the different multi-locus probes used, in terms of both the relative success in detecting hypervariable loci and the degree of overlap between the sets of loci detected. We report 23 new human minisatellite loci isolated by this method, which map to 14 autosomes and the sex chromosomes.

Growth Indicators of a 48-Clone Sugar Cane Population (Saccharum spp. with Forage Potential

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Yoslen Fernández Gálvez

2016-09-01

Full Text Available The aim of this paper was to determine growth indicators in a 48-clone sugar cane population, with promising phenotypical features for forage production. The following indicators were assessed: leaf area (A, leaf area index (LA1; leaf area ratio (LAR; specific leaf area (SLA; leaf weight ratio (LWR; crop growth rate (CGR; net assimilation rate (NAR; relative growth rate in weight (RGR; biomass production speed (G; leaf area duration (LAD; and biomass duration (Z, monthly (187 - 370 days. The minimum, the mean, the maximum values, and the population variance were determined for all cutting ages and the variables assessed. The results achieved have provided quantitative values that can be used as reference for selection and assessment of forage genotypes for ruminant nutrition.

Chronic exposure to sublethal doses of radiation mimetic ZeocinTM selects for clones deficient in homologous recombination

International Nuclear Information System (INIS)

Delacote, Fabien; Deriano, Ludovic; Lambert, Sarah; Bertrand, Pascale; Saintigny, Yannick; Lopez, Bernard S.

2007-01-01

DNA double-strand breaks (DSBs) are highly toxic lesions leading to genome variability/instability. The balance between homologous recombination (HR) and non-homologous end-joining (NHEJ), two alternative DSB repair systems, is essential to ensure genome maintenance in mammalian cells. Here, we transfected CHO hamster cells with the pcDNA TM 3.1/Zeo plasmid, and selected transfectants with Zeocin TM , a bleomycin analog which produces DSBs. Despite the presence of a Zeocin TM resistance gene in pcDNA TM 3.1/Zeo, Zeocin TM induced 8-10 γ-H2AX foci per cell. This shows that the Zeocin TM resistance gene failed to fully detoxify cells treated with Zeocin TM , and that during selection cells were submitted to a chronic sublethal DSB stress. Selected clones show decreases in both spontaneous and induced intrachromosomal HR. In contrast, in an in vitro assay, these clones show an increase in NHEJ products specific to the KU86 pathway. We selected cells, in the absence of pcDNA TM 3.1/Zeo, with low and sublethal doses of Zeocin TM , producing a mean 8-10 γ-H2AX foci per cell. Newly selected clones exhibited similar phenotypes: HR decrease accompanied by an increase in KU86-dependent NHEJ efficiency. Thus chronic exposure to sublethal numbers of DSBs selects cells whose HR versus NHEJ balance is altered. This may well have implications for radio- and chemotherapy, and for management of environmental hazards

Procreative liberty, enhancement and commodification in the human cloning debate.

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Shapshay, Sandra

2012-12-01

The aim of this paper is to scrutinize a contemporary standoff in the American debate over the moral permissibility of human reproductive cloning in its prospective use as a eugenic enhancement technology. I shall argue that there is some significant and under-appreciated common ground between the defenders and opponents of human cloning. Champions of the moral and legal permissibility of cloning support the technology based on the right to procreative liberty provided it were to become as safe as in vitro fertilization and that it be used only by adults who seek to rear their clone children. However, even champions of procreative liberty oppose the commodification of cloned embryos, and, by extension, the resulting commodification of the cloned children who would be produced via such embryos. I suggest that a Kantian moral argument against the use of cloning as an enhancement technology can be shown to be already implicitly accepted to some extent by champions of procreative liberty on the matter of commodification of cloned embryos. It is in this argument against commodification that the most vocal critics of cloning such as Leon Kass and defenders of cloning such as John Robertson can find greater common ground. Thus, I endeavor to advance the debate by revealing a greater degree of moral agreement on some fundamental premises than hitherto recognized.

Long term presence of a single predominant tyrosinase-specific T-cell clone associated with disease control in a patient with metastatic melanoma.

Science.gov (United States)

Ochsenreither, Sebastian; Fusi, Alberto; Busse, Antonia; Letsch, Anne; Haase, Doreen; Thiel, Eckhard; Scheibenbogen, Carmen; Keilholz, Ulrich

2010-05-15

In an earlier study, we described a patient who developed an anti-tyrosinase T-cell response leading to long-term tumor control. Here we analyzed this response with regard to T-cell receptor (TCR) Vbeta family usage and clonality in order to further elucidate the nature of the T cell response in this patient. For identification of expanded specific cytotoxic T-cell (CTL) clones, tetramer enrichment of tyrosinase reactive T-cells was followed by comparative quantitative reverse transcribed PCR (qRT PCR) quantification of all TCR Vbeta-families and sequencing of family Vbeta4 elevated in the enriched fraction. The predominant specific clone was quantified by clonotypic qRT PCR in multiple samples from blood, bone marrow, and tumor tissue. FACS analyses with staining of TYR.A2 and TCR Vbeta4 were performed. Epitope specific enrichment revealed an isolated increase of Vbeta-family 4. FACS analysis showed a shift of specific CTLs to Vbeta-family 4 during tumor regression with a maximum of 80% of all TYR.A2 specific cells belonging to this family. Sequencing revealed a single predominant clone against polyclonal background coding for identical CDR3 loops. The predominant clone was highly expressed in bone marrow and tumor tissue, and was detectable in blood over a period of ten years. Considering the results of previous studies showing a specific effector phenotype in blood and a specific memory compartment in bone marrow of this patient, this data implicate the predominant clone featured all attributes of a sufficient CTL response including homing capacity and memory formation resulting in long term clonal persistence and tumor control.

Should we clone human beings? Cloning as a source of tissue for transplantation.

Science.gov (United States)

Savulescu, J

1999-01-01

The most publicly justifiable application of human cloning, if there is one at all, is to provide self-compatible cells or tissues for medical use, especially transplantation. Some have argued that this raises no new ethical issues above those raised by any form of embryo experimentation. I argue that this research is less morally problematic than other embryo research. Indeed, it is not merely morally permissible but morally required that we employ cloning to produce embryos or fetuses for the sake of providing cells, tissues or even organs for therapy, followed by abortion of the embryo or fetus. PMID:10226910

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

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Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Lagoumintzis, George; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

454 sequencing of pooled BAC clones on chromosome 3H of barley

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Yamaji Nami

2011-05-01

Full Text Available Abstract Background Genome sequencing of barley has been delayed due to its large genome size (ca. 5,000Mbp. Among the fast sequencing systems, 454 liquid phase pyrosequencing provides the longest reads and is the most promising method for BAC clones. Here we report the results of pooled sequencing of BAC clones selected with ESTs genetically mapped to chromosome 3H. Results We sequenced pooled barley BAC clones using a 454 parallel genome sequencer. A PCR screening system based on primer sets derived from genetically mapped ESTs on chromosome 3H was used for clone selection in a BAC library developed from cultivar "Haruna Nijo". The DNA samples of 10 or 20 BAC clones were pooled and used for shotgun library development. The homology between contig sequences generated in each pooled library and mapped EST sequences was studied. The number of contigs assigned on chromosome 3H was 372. Their lengths ranged from 1,230 bp to 58,322 bp with an average 14,891 bp. Of these contigs, 240 showed homology and colinearity with the genome sequence of rice chromosome 1. A contig annotation browser supplemented with query search by unique sequence or genetic map position was developed. The identified contigs can be annotated with barley cDNAs and reference sequences on the browser. Homology analysis of these contigs with rice genes indicated that 1,239 rice genes can be assigned to barley contigs by the simple comparison of sequence lengths in both species. Of these genes, 492 are assigned to rice chromosome 1. Conclusions We demonstrate the efficiency of sequencing gene rich regions from barley chromosome 3H, with special reference to syntenic relationships with rice chromosome 1.

Genetic analysis of an Escherichia coli urease locus: evidence of DNA rearrangement.

OpenAIRE

Collins, C M; Falkow, S

1988-01-01

Ureolytic Escherichia coli strains are uncommon clinical isolates. The urease phenotype in a large percentage of these isolates is unstable and lost upon storage. We examined two urease-positive uropathogenic E. coli isolates that give off urease-negative segregants and determined that the urease phenotype was chromosomally encoded. The urease phenotype was cloned from E. coli 1021 and found to be encoded on a 9.4-kilobase HindIII restriction fragment. Transposon mutagenesis indicated that at...

Counterfactual quantum cloning without transmitting any physical particles

Science.gov (United States)

Guo, Qi; Zhai, Shuqin; Cheng, Liu-Yong; Wang, Hong-Fu; Zhang, Shou

2017-11-01

We propose a counterfactual 1 →2 economical phase-covariant cloning scheme. Compared with the existing protocols using flying qubits, the main difference of the presented scheme is that the cloning can be achieved without transmitting the photon between the two parties. In addition, this counterfactual scheme does not need to construct controlled quantum gates to perform joint logical operations between the cloned qubit and the blank copy. We also numerically evaluate the performance of the present scheme in the practical experiment, which shows this cloning scheme can be implemented with a high success of probability and the fidelity is close to the optimal value in the ideal asymptotic limit.

Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk.

Science.gov (United States)

Vollrath, D; Feng, W; Duncan, J L; Yasumura, D; D'Cruz, P M; Chappelow, A; Matthes, M T; Kay, M A; LaVail, M M

2001-10-23

The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy (rdy) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.

Decellularized matrix from tumorigenic human mesenchymal stem cells promotes neovascularization with galectin-1 dependent endothelial interaction

DEFF Research Database (Denmark)

Burns, Jorge S; Kristiansen, Malthe; Kristensen, Lars P

2011-01-01

. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells...... of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization....

Cocoa Clone Resistant to Phytophthora Palmivora Pod Borer (CPB) in South Sulawesi

OpenAIRE

Sartika Dewi, Vien

2017-01-01

Helopeltis sp. is one of the main pest in cacao plants. Helopeltis sp. Able to decreasing the production of cacao about 50-60%. This research aims to understand the development of Helopeltis sp. investation in five types of clone cocoa. Collected data have done every week for six weeks in five types of clone cocoa which are clone GBT, clone M01, clone 45, clone s2 and clone BB. Every clone chosen 15 pod sampeles fruit with different size of pod following 5-10cm, 11-13cm and ripe pod which use...

Stability of the JP2 clone of Aggregatibacter actinomycetemcomitans.

Science.gov (United States)

Haubek, D; Ennibi, O-K; Vaeth, M; Poulsen, S; Poulsen, K

2009-09-01

The JP2 clone of Aggregatibacter actinomycetemcomitans is strongly associated with aggressive periodontitis. To obtain information about colonization dynamics of the JP2 clone, we used PCR to examine its presence in 365 Moroccan juveniles from whom periodontal plaque samples were collected at baseline and after one and two years. Periodontal attachment loss was measured at baseline and at the two-year follow-up. At baseline, 43 (12%) carriers of the JP2 clone were found. Nearly half (44 %) of these were persistently colonized with the clone. The relative risk for the development of aggressive periodontitis, adjusted for the concomitant presence of other genotypes of A. actinomycetemcomitans, was highest for individuals continuously infected by the JP2 clone (RR = 13.9; 95% CI, 9.0 to 21.4), indicating a relationship between infectious dose and disease, which further substantiates the evidence for the JP2 clone as a causal factor in aggressive periodontitis.

Differential responses to radiation and hyperthermia of cloned cell lines derived from a single human melanoma xenograft

International Nuclear Information System (INIS)

Rofstad, E.K.; Brustad, T.

1984-01-01

One uncloned and five cloned cell lines were derived from a single human melanoma xenograft. Cells from passages 7-12 were exposed to either radiation or hyperthermia (42.5 0 C, pH = 7.4) under aerobic conditions and the colony forming ability of the cells was assayed in soft agar. The five cloned lines showed individual and characteristic responses to radiation as well as to hyperthermia. The variation in the response to radiation was mainly reflected in the size of the shoulders of the survival curves rather than in the D 0 -values. The variation in the response to hyperthermia was mainly reflected in the terminal slopes of the survival curves. The survival curve of cells from the uncloned line, both when exposed to radiation and hyperthermia, was positioned in the midst of those of the cloned lines. The response of the cloned lines to radiation did not correlate with the response to hyperthermia, indicating that tumor cell subpopulations which are resistant to radiation may respond well to hyperthermia

Regulating (for the benefit of) future persons: a different perspective on the FDA's jurisdiction to regulate human reproductive cloning.

Science.gov (United States)

Javitt, Gail H; Hudson, Kathy

2003-01-01

The Food and Drug Administration (FDA) has taken the position that human reproductive cloning falls within its regulatory jurisdiction. This position has been subject to criticism on both procedural and substantive grounds. Some have contended that the FDA has failed to follow administrative law principles in asserting its jurisdiction, while others claim the FDA is ill suited to the task of addressing the ethical and social implications of human cloning. This Article argues, that, notwithstanding these criticisms, the FDA could plausibly assert jurisdiction over human cloning as a form of human gene therapy, an area in which the FDA is already regarded as having primary regulatory authority. Such an assertion would require that the FDA's jurisdiction extend to products affecting future persons, i.e., those not yet born. This Article demonstrates, for the first time, that such jurisdiction was implicit in the enactment of the 1962 Kefauver-Harris Amendments to the Federal Food, Drug, and Cosmetic Act and that the FDA has historically relied on such authority in promulgating regulations for drugs and devices.

A strategy for clone selection under different production conditions.

Science.gov (United States)

Legmann, Rachel; Benoit, Brian; Fedechko, Ronald W; Deppeler, Cynthia L; Srinivasan, Sriram; Robins, Russell H; McCormick, Ellen L; Ferrick, David A; Rodgers, Seth T; Russo, A Peter

2011-01-01

Top performing clones have failed at the manufacturing scale while the true best performer may have been rejected early in the screening process. Therefore, the ability to screen multiple clones in complex fed-batch processes using multiple process variations can be used to assess robustness and to identify critical factors. This dynamic ranking of clones' strategy requires the execution of many parallel experiments than traditional approaches. Therefore, this approach is best suited for micro-bioreactor models which can perform hundreds of experiments quickly and efficiently. In this study, a fully monitored and controlled small scale platform was used to screen eight CHO clones producing a recombinant monoclonal antibody across several process variations, including different feeding strategies, temperature shifts and pH control profiles. The first screen utilized 240 micro-bioreactors were run for two weeks for this assessment of the scale-down model as a high-throughput tool for clone evaluation. The richness of the outcome data enable to clearly identify the best and worst clone as well as process in term of maximum monoclonal antibody titer. The follow-up comparison study utilized 180 micro-bioreactors in a full factorial design and a subset of 12 clone/process combinations was selected to be run parallel in duplicate shake flasks. Good correlation between the micro-bioreactor predictions and those made in shake flasks with a Pearson correlation value of 0.94. The results also demonstrate that this micro-scale system can perform clone screening and process optimization for gaining significant titer improvements simultaneously. This dynamic ranking strategy can support better choices of production clones. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

Directory of Open Access Journals (Sweden)

Athanasios Niarchos

Full Text Available During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

Trajectories of the healthy ageing phenotype among middle-aged and older Britons, 2004-2013.

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Tampubolon, Gindo

2016-06-01

Since the ageing population demands a response to ensure older people remain healthy and active, we studied the dynamics of a recently proposed healthy ageing phenotype. We drew the phenotype's trajectories and tested whether their levels and rates of change are influenced by health behaviours, comorbidities and socioeconomic positions earlier in the life course. The English Longitudinal Ageing Study, a prospective, nationally representative sample of people aged ≥50 years, measured a set of eight biomarkers which make up the outcome of the healthy ageing phenotype three times over nearly a decade (N2004=5009, N2008=5301, N2013=4455). A cluster of health behaviours, comorbidities and socioeconomic positions were also measured repeatedly. We assessed the phenotype's distribution non-parametrically, then fitted linear mixed models to phenotypic change and further examined time interactions with gender and socioeconomic position. We ran additional analyses to test robustness. Women had a wider distribution of the healthy ageing phenotype than men had. The phenotype declined annually by -0.242 (95% confidence interval [CI]: -0.352, -0.131). However, there was considerable heterogeneity in the levels and rates of phenotypic change. Women started at higher levels, then declined more steeply by -0.293 (CI: -0.403, -0.183) annually, leading to crossover in the trajectories. Smoking and physical activity assessed on the Allied Dunbar scale were strongly associated with the trajectories. Though marked by secular decline, the trajectories of the healthy ageing phenotype showed distinct socioeconomic gradients. The trajectories were also susceptible to variations in health behaviours, strengthening the case for serial interventions to attain healthy and active ageing. Copyright © 2016 The Author. Published by Elsevier Ireland Ltd.. All rights reserved.

T-cell receptor (TCR) phenotype of nodal Epstein-Barr virus (EBV)-positive cytotoxic T-cell lymphoma (CTL): a clinicopathologic study of 39 cases.

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Kato, Seiichi; Asano, Naoko; Miyata-Takata, Tomoko; Takata, Katsuyoshi; Elsayed, Ahmed Ali; Satou, Akira; Takahashi, Emiko; Kinoshita, Tomohiro; Nakamura, Shigeo

2015-04-01

Among Epstein-Barr virus (EBV)-positive cytotoxic T/NK-cell lymphoma, there are only a few reports on the clinicopathologic features of patients with primary nodal presentation (nodal EBV cytotoxic T-cell lymphoma [CTL]). Here, we compared the clinicopathologic profiles of 39 patients with nodal EBV CTL with those of 27 cases of "extranasal" NK/T-cell lymphoma of nasal type (ENKTL), especially addressing their T-cell receptor (TCR) phenotype. Histologically, 22 of 39 nodal EBV CTL cases (56%) were unique in having centroblastoid appearance, which was contrasted with the lower incidence of this feature in ENKTL (15%, P=0.001). In contrast, pleomorphic appearance was more frequently seen in ENKTL than in nodal EBV CTL (67% vs. 23%, P=0.001). Thirty-three of 39 nodal EBV CTL cases (85%) were of T-cell lineage on the basis of TCR expression and/or TCRγ gene rearrangement; in detail, 18 cases (46%) were TCRβ positive (αβ T), 5 (13%) were TCRγ and/or δ positive (γδ T), and 10 (26%) were TCR-silent type with clonal TCRγ gene rearrangement but no expression of TCRβ, γ, or δ. These results were clearly contrasted by a lower incidence of T-cell lineage in ENKTL (7 cases, 26%, P<0.001). Notably, the survival time of the 5 nodal lymphoma patients with γδ T-cell phenotype was within 3 months, which was inferior to those of αβ T and TCR-silent types (P=0.003), and 3 of those with available clinical information were all found to be associated with autoimmune diseases. These data suggest that nodal EBV CTL is distinct from ENKTL.

Early selection of Eucalyptus clones in retrospective nursery test ...

African Journals Online (AJOL)

Within the framework of the eucalyptus breeding programme in the Congo, two retrospective tests were conducted using mature clones in the field and young cuttings under nursery conditions with two hybrids: 13 clones of Eucalyptus tereticornis* Eucalyptus grandis for the test TC 82-1B and 17 clones of Eucalyptus ...

[Human cloning and the protection of women's interests].

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Canabes, Marcela Ahumada

2008-01-01

The Human Cloning, both therapeutic and full birth cloning, involves and affects women in a special way. The United Nation's Declaration on the Cloning of Human Beings includes a special clause referred to them. Also the Spanish law does it. This works pretend to analyse the meaning of the inclusion of women's interests in this document. At the same time, I will consider the foundations and the importance of the reference to the women.

Cloning of cellulase genes using pUC18 and lambda 2001 vectors

International Nuclear Information System (INIS)

Bashir, A.; Ashfaq, S.R.; Rajoka, M.I.; Malik, K.A.; Batt, C.A.

1991-01-01

Chromosomal DNA from cellulomonas biazotea NIAB 442 was used for isolation and cloning of cellulase genes. For this purpose plasmid pUC18 was used for cloning fragments in the range of 109 Kb and phase vector lambda 2001 for cloning fragments in the range of 15-20 Kb respectively. Three restriction enzymes BamHI, Sau3AI and SaII were used for partial restriction of chromosomal DNA to obtain fragment size in the range of 0.5 - 20 Kb. BamHI and SaII were used to linearize pUC18 to obtain compatible ends against the three enzymes used in chromosomal DNA restriction. Linearized pUC18 was then ligated to respective compatible chromosomal DNA fragments and transformed to JM109 competent cells. A total of 6781 recombinants were tested for the production of B-glucosidase and carboxy methyl cellulase (CMC-ase) production. Only one of the recombinants was found to be positive for B-glucosidase production in solid culture. One of the recombinants was found positive for CMC-ase production in solid culture and is being verified and characterized. Larger DNA fragments in the range of 15-20 Kilobase were obtained by partial restriction of chromosomal DNA with BamHI, SaII and Xhol. Lambda 2001 was double digested with BamHI/EcoRI and Xhol/EcoRI for removal of stuffer fragment. Ligation of respective compatible ends was performed between Lambda DNA and chromosomal DNA. Ligation mixture was used for packaging and infection of P2 lysogen. No plaques could be obtained on P2 lysogen due to inefficient packaging. (author)

Experimental demonstration of continuous variable cloning with phase-conjugate inputs

DEFF Research Database (Denmark)

Sabuncu, Metin; Andersen, Ulrik Lund; Leuchs, G.

2007-01-01

We report the first experimental demonstration of continuous variable cloning of phase-conjugate coherent states as proposed by Cerf and Iblisdir [Phys. Rev. Lett. 87, 247903 (2001)]. In contrast to this proposal, the cloning transformation is accomplished using only linear optical components......, homodyne detection, and feedforward. As a result of combining phase conjugation with a joint measurement strategy, superior cloning is demonstrated with cloning fidelities reaching 89%....

Algorithmic Mapping and Characterization of the Drug-Induced Phenotypic-Response Space of Parasites Causing Schistosomiasis.

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Singh, Rahul; Beasley, Rachel; Long, Thavy; Caffrey, Conor R

2018-01-01

Neglected tropical diseases, especially those caused by helminths, constitute some of the most common infections of the world's poorest people. Amongst these, schistosomiasis (bilharzia or 'snail fever'), caused by blood flukes of the genus Schistosoma, ranks second only to malaria in terms of human impact: two hundred million people are infected and close to 800 million are at risk of infection. Drug screening against helminths poses unique challenges: the parasite cannot be cloned and is difficult to target using gene knockouts or RNAi. Consequently, both lead identification and validation involve phenotypic screening, where parasites are exposed to compounds whose effects are determined through the analysis of the ensuing phenotypic responses. The efficacy of leads thus identified derives from one or more or even unknown molecular mechanisms of action. The two most immediate and significant challenges that confront the state-of-the-art in this area are: the development of automated and quantitative phenotypic screening techniques and the mapping and quantitative characterization of the totality of phenotypic responses of the parasite. In this paper, we investigate and propose solutions for the latter problem in terms of the following: (1) mathematical formulation and algorithms that allow rigorous representation of the phenotypic response space of the parasite, (2) application of graph-theoretic and network analysis techniques for quantitative modeling and characterization of the phenotypic space, and (3) application of the aforementioned methodology to analyze the phenotypic space of S. mansoni - one of the etiological agents of schistosomiasis, induced by compounds that target its polo-like kinase 1 (PLK 1) gene - a recently validated drug target. In our approach, first, bio-image analysis algorithms are used to quantify the phenotypic responses of different drugs. Next, these responses are linearly mapped into a low- dimensional space using Principle

cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD)

International Nuclear Information System (INIS)

Matsubara, Y.; Kraus, J.P.; Rosenberg, L.E.; Tanaka, K.

1986-01-01

MCAD is one of three mitochondrial flavoenzymes which catalyze the first step in the β-oxidation of straight chain fatty acids. It is a tetramer with a subunit Mr of 45 kDa. MCAD is synthesized in the cytosol as a 49 kDa precursor polypeptide (pMCAD), imported into mitochondria, and cleaved to the mature form. Genetic deficiency of MCAD causes recurrent episodes of hypoglycemic coma accompanied by medium chain dicarboxylic aciduria. Employing a novel approach, the authors now report isolation of partial rat and human cDNA clones encoding pMCAD. mRNA encoding pMCAD was purified to near homogeneity by polysome immunoadsorption using polyclonal monospecific antibody. Single-stranded [ 32 P]labeled cDNA probe was synthesized using the enriched mRNA as template, and was used to screen directly 16,000 colonies from a total rat liver cDNA library constructed in pBR322. One clone (600 bp) was detected by in situ hybridization. Hybrid-selected translation with this cDNA yielded a 49 kDa polypeptide indistinguishable in size from rat pMCAD and immunoprecipitable with anti-MCAD antibody. Using the rat cDNA as probe, 43,000 colonies from a human liver cDNA library were screened. Four identical positive clones (400 bp) were isolated and positively identified by hybrid-selected translation and immunoprecipitation. The sizes of rat and human mRNAs encoding pMCAD were 2.2 kb and 2.4 kb, respectively, as determined by Northern blotting

Cloning of the rat Waf1/Cip1 gene

International Nuclear Information System (INIS)

Belinsky, S.A.; Middleton, S.K.

1994-01-01

The progression of eukaryotic cells through the cell cycle involves the sequential expression of specific genes. This process is regulated by both external and internal stimuli that prevent the cell from prematurely entering the next phase before all macromolecular events have been completed. The activation and subsequent inactivation of cyclin dependent kinases (Cdks) represent one internal stimuli required to regulate the transit of cells from one stage of the cell cycle to the next. Another member of this regulatory cascade is the p53 tumor suppressor gene, which controls a G 1 checkpoint at which the cell cycle can be arrested prior to the initiation of DNA synthesis. Following DNA damage, p53 protein levels rise, and entry into S phase is delayed, presumably to allow time for repair of the lesions. When p53 function is lost, cells containing damaged DNA template enter S phase leading to fixation and propagation of genetic alterations. Recently, evidence linking the growth-suppressing activity of p53 and inactivation of Cdks has been provided by the cloning of the Waf1/Cip1 gene. Waf1/Cip1 encodes a protein of M r 21,000 (p21), which inhibits Cdks in vitro. The overexpression of Waf1/Cip1 in cells inhibits cell growth, suggesting that p21 is a downstream mediator of p53 function. Loss of Waf1/Cip1 gene function could lead to deregulation of the cell cycle and contribute to the development of the neoplastic phenotype in tumors that do not contain mutations in the p53 gene. The purpose of the present investigation was to clone the rat Waf1/Cip1 gene,then determine the frequency for alteration of this gene in lung tumors induced by X-rays

Cloning animals by somatic cell nuclear transfer – biological factors

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Tian, X Cindy; Kubota, Chikara; Enright, Brian; Yang, Xiangzhong

2003-01-01

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other specie, this review will be focused on somatic cell cloning of cattle. PMID:14614770

Time-Efficient Cloning Attacks Identification in Large-Scale RFID Systems

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Ju-min Zhao

2017-01-01

Full Text Available Radio Frequency Identification (RFID is an emerging technology for electronic labeling of objects for the purpose of automatically identifying, categorizing, locating, and tracking the objects. But in their current form RFID systems are susceptible to cloning attacks that seriously threaten RFID applications but are hard to prevent. Existing protocols aimed at detecting whether there are cloning attacks in single-reader RFID systems. In this paper, we investigate the cloning attacks identification in the multireader scenario and first propose a time-efficient protocol, called the time-efficient Cloning Attacks Identification Protocol (CAIP to identify all cloned tags in multireaders RFID systems. We evaluate the performance of CAIP through extensive simulations. The results show that CAIP can identify all the cloned tags in large-scale RFID systems fairly fast with required accuracy.

[Growth analysis on modules of Cynodon dactylon clones in Yili River Valley Plain of Xinjiang].

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Zhao, Yu; Janar; Li, Hai-Yan; Liu, Ying; Yang, Yun-Fei

2009-04-01

By the method of randomly digging up whole ramet tuft while maintaining natural integrity, large samples of Cynodon dactylon clones were collected from a grape orchard abandoned for 2 years without any management in the Yili River Valley Plain of Xinjiang, aimed to quantitatively analyze the growth patterns of their modules. The results showed that the average ramet number of test 30 clones reached 272.6 +/- 186. 6, among which, vegetative ramets occupied 82.3%, being 4.3 times higher than reproductive ones. The total biomass of the clones was 45.4 +/- 40.0 g, in which, rhizomes accounted for 54.4%, while the vegetative ramets, stolons, and reproductive ramets occupied 21.0%, 14.8%, and 9.4% of the total, respectively. The accumulative length of rhizomes and stolons reached 5.1 + 4.7 m and 3.3 +/- 3.4 m, while the bud number on stolons and rhizomes was 291.5 +/- 246.8 and 78.8 +/- 87.4, respectively. The bud number on stolons and rhizomes was positively correlated to the quantitative characters of vegetative ramets, reproductive ramets, stolons, and rhizomes (P < 0.01), indicating that in Yili River Valley Plain, C. dactylon clone could achieve and maintain its continuous renovation via rhizome buds.

Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors.

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Laine, M J; Nakhei, H; Dreier, J; Lehtilä, K; Meletzus, D; Eichenlaub, R; Metzler, M C

1996-05-01

In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, and pHN216 were stably maintained without antibiotic selection in various strains of C. michiganensis subsp. sepedonicus. We observed that for a single plasmid, different strains of C. michiganensis subsp. sepedonicus showed significantly different transformation efficiencies. We also found unexplained strain-to-strain differences in stability with various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication. We examined the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C. michiganensis subsp. sepedonicus cells were grown on DM agar plates, harvested during the early exponential growth phase, and used fresh (without freezing) for electroporation. The maximal transformation efficiency obtained was 4.6 x 10(4) CFU/microgram of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a beta-1,4-endoglucanase-encoding gene from C. michiganensis subsp. sepedonicus into pHN216. When this construction, pHN216:C8, was electroporated into competent cells of a cellulase-deficient mutant, it restored cellulase production to almost wild-type levels.

A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning.

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Carbonetti, Sara; Oliver, Brian G; Vigdorovich, Vladimir; Dambrauskas, Nicholas; Sack, Brandon; Bergl, Emilee; Kappe, Stefan H I; Sather, D Noah

2017-09-01

Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

Cloning and adoption: a reply to Levy and Lotz.

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Strong, Carson

2008-02-01

In previous articles I discussed the ethics of human reproductive cloning, focusing on a possible future scenario in which reproductive cloning can be accomplished without an elevated risk of anomalies to the children who are created. I argued that in such a scenario it would be ethically permissible for infertile couples to use cloning as a way to have genetically related children and that such use should not be prohibited. In 'Reproductive Cloning and a (Kind of) Genetic Fallacy', Neil Levy and Mianna Lotz raise objections to my conclusions. They disagree with the view, for which I argued, that some couples can have defensible reasons for desiring genetically related children. They also offer several new arguments against reproductive cloning, including an argument that it would diminish the number of adoptions, thereby adversely affecting the welfare of children who need to be adopted. In this paper I point out that Levy and Lotz's criticisms misconstrue my arguments and that there are serious problems with their arguments for prohibiting infertile couples from using cloning, including their argument from adoption.

Information-theoretic limitations on approximate quantum cloning and broadcasting

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Lemm, Marius; Wilde, Mark M.

2017-07-01

We prove quantitative limitations on any approximate simultaneous cloning or broadcasting of mixed states. The results are based on information-theoretic (entropic) considerations and generalize the well-known no-cloning and no-broadcasting theorems. We also observe and exploit the fact that the universal cloning machine on the symmetric subspace of n qudits and symmetrized partial trace channels are dual to each other. This duality manifests itself both in the algebraic sense of adjointness of quantum channels and in the operational sense that a universal cloning machine can be used as an approximate recovery channel for a symmetrized partial trace channel and vice versa. The duality extends to give control of the performance of generalized universal quantum cloning machines (UQCMs) on subspaces more general than the symmetric subspace. This gives a way to quantify the usefulness of a priori information in the context of cloning. For example, we can control the performance of an antisymmetric analog of the UQCM in recovering from the loss of n -k fermionic particles.

Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

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Monica K Akre

Full Text Available Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

Biotechnology. Perseverance leads to cloned pig in Japan.

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Pennisi, E; Normile, D

2000-08-18

Low success rates and unpredictable results have plagued cloning researchers, particularly those trying to clone pigs. Now, on page 1188, Japanese researchers offer the first scientific report of a cloned pig, named Xena, raising hopes that pigs could one day provide an unlimited supply of organs for transplantation thanks to their close physiological relationship to humans. But this week those hopes were dealt a blow by more evidence suggesting that pig retroviruses can infect human cells.

[Media, cloning, and bioethics].

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Costa, S I; Diniz, D

2000-01-01

This article was based on an analysis of three hundred articles from mainstream Brazilian periodicals over a period of eighteen months, beginning with the announcement of the Dolly case in February 1997. There were two main objectives: to outline the moral constants in the press associated with the possibility of cloning human beings and to identify some of the moral assumptions concerning scientific research with non-human animals that were published carelessly by the media. The authors conclude that there was a haphazard spread of fear concerning the cloning of human beings rather than an ethical debate on the issue, and that there is a serious gap between bioethical reflections and the Brazilian media.

Cloning, purification, crystallization and preliminary crystallographic analysis of SecA from Enterococcus faecalis

International Nuclear Information System (INIS)

Meining, Winfried; Scheuring, Johannes; Fischer, Markus; Weinkauf, Sevil

2006-01-01

SecA ATPase from E. faecalis has been cloned, overexpressed, purified and crystallized. Crystals belong to space group C2 and diffract to 2.4 Å resolution. The gene coding for SecA from Enterococcus faecalis was cloned and overexpressed in Escherichia coli. In this protein, the lysine at position 6 was replaced by an asparagine in order to reduce sensitivity towards proteases. The modified protein was purified and crystallized. Crystals diffracting to 2.4 Å resolution were obtained using the vapour-diffusion technique. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 203.4, b = 49.8, c = 100.8 Å, α = γ = 90.0, β = 119.1°. A selenomethionine derivative was prepared and is currently being tested in crystallization trials

Dogs cloned from adult somatic cells.

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Lee, Byeong Chun; Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Yuda, Fibrianto; Kim, Hye Jin; Hossein, M Shamim; Shamim, M Hossein; Kim, Jung Ju; Kang, Sung Keun; Schatten, Gerald; Hwang, Woo Suk

2005-08-04

Several mammals--including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats--have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.

Development of a porcine skeletal muscle cDNA microarray: analysis of differential transcript expression in phenotypically distinct muscles

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Stear Michael

2003-03-01

Full Text Available Abstract Background Microarray profiling has the potential to illuminate the molecular processes that govern the phenotypic characteristics of porcine skeletal muscles, such as hypertrophy or atrophy, and the expression of specific fibre types. This information is not only important for understanding basic muscle biology but also provides underpinning knowledge for enhancing the efficiency of livestock production. Results We report on the de novo development of a composite skeletal muscle cDNA microarray, comprising 5500 clones from two developmentally distinct cDNA libraries (longissimus dorsi of a 50-day porcine foetus and the gastrocnemius of a 3-day-old pig. Clones selected for the microarray assembly were of low to moderate abundance, as indicated by colony hybridisation. We profiled the differential expression of genes between the psoas (red muscle and the longissimus dorsi (white muscle, by co-hybridisation of Cy3 and Cy5 labelled cDNA derived from these two muscles. Results from seven microarray slides (replicates correctly identified genes that were expected to be differentially expressed, as well as a number of novel candidate regulatory genes. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray. Conclusion We have developed a porcine skeletal muscle cDNA microarray and have identified a number of candidate genes that could be involved in muscle phenotype determination, including several members of the casein kinase 2 signalling pathway.

Statement on Human Cloning

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... as our understanding of this technology advances. Support Stem Cell Research (including Research Cloning) AAAS supports stem cell research, including the use of nuclear transplantation techniques (also ...

High-Throughput Cloning and Expression Library Creation for Functional Proteomics

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Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-01-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

Positive health effects of the natural outdoor environment in typical populations in different regions in Europe (PHENOTYPE): a study programme protocol.

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Nieuwenhuijsen, Mark J; Kruize, Hanneke; Gidlow, Christopher; Andrusaityte, Sandra; Antó, Josep Maria; Basagaña, Xavier; Cirach, Marta; Dadvand, Payam; Danileviciute, Asta; Donaire-Gonzalez, David; Garcia, Judith; Jerrett, Michael; Jones, Marc; Julvez, Jordi; van Kempen, Elise; van Kamp, Irene; Maas, Jolanda; Seto, Edmund; Smith, Graham; Triguero, Margarita; Wendel-Vos, Wanda; Wright, John; Zufferey, Joris; van den Hazel, Peter Jan; Lawrence, Roderick; Grazuleviciene, Regina

2014-04-16

Growing evidence suggests that close contact with nature brings benefits to human health and well-being, but the proposed mechanisms are still not well understood and the associations with health remain uncertain. The Positive Health Effects of the Natural Outdoor environment in Typical Populations in different regions in Europe (PHENOTYPE) project investigates the interconnections between natural outdoor environments and better human health and well-being. The PHENOTYPE project explores the proposed underlying mechanisms at work (stress reduction/restorative function, physical activity, social interaction, exposure to environmental hazards) and examines the associations with health outcomes for different population groups. It implements conventional and new innovative high-tech methods to characterise the natural environment in terms of quality and quantity. Preventive as well as therapeutic effects of contact with the natural environment are being covered. PHENOTYPE further addresses implications for land-use planning and green space management. The main innovative part of the study is the evaluation of possible short-term and long-term associations of green space and health and the possible underlying mechanisms in four different countries (each with quite a different type of green space and a different use), using the same methodology, in one research programme. This type of holistic approach has not been undertaken before. Furthermore there are technological innovations such as the use of remote sensing and smartphones in the assessment of green space. The project will produce a more robust evidence base on links between exposure to natural outdoor environment and human health and well-being, in addition to a better integration of human health needs into land-use planning and green space management in rural as well as urban areas.

Metabolic analysis of radioresistant medulloblastoma stem-like clones and potential therapeutic targets.

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Lue Sun

Full Text Available Medulloblastoma is a fatal brain tumor in children, primarily due to the presence of treatment-resistant medulloblastoma stem cells. The energy metabolic pathway is a potential target of cancer therapy because it is often different between cancer cells and normal cells. However, the metabolic properties of medulloblastoma stem cells, and whether specific metabolic pathways are essential for sustaining their stem cell-like phenotype and radioresistance, remain unclear. We have established radioresistant medulloblastoma stem-like clones (rMSLCs by irradiation of the human medulloblastoma cell line ONS-76. Here, we assessed reactive oxygen species (ROS production, mitochondria function, oxygen consumption rate (OCR, energy state, and metabolites of glycolysis and tricarboxylic acid cycle in rMSLCs and parental cells. rMSLCs showed higher lactate production and lower oxygen consumption rate than parental cells. Additionally, rMSLCs had low mitochondria mass, low endogenous ROS production, and existed in a low-energy state. Treatment with the metabolic modifier dichloroacetate (DCA resulted in mitochondria dysfunction, glycolysis inhibition, elongated mitochondria morphology, and increased ROS production. DCA also increased radiosensitivity by suppression of the DNA repair capacity through nuclear oxidization and accelerated the generation of acetyl CoA to compensate for the lack of ATP. Moreover, treatment with DCA decreased cancer stem cell-like characters (e.g., CD133 positivity and sphere-forming ability in rMSLCs. Together, our findings provide insights into the specific metabolism of rMSLCs and illuminate potential metabolic targets that might be exploited for therapeutic benefit in medulloblastoma.

17β-estradiol-induced ACSL4 protein expression promotes an invasive phenotype in estrogen receptor positive mammary carcinoma cells.

Science.gov (United States)

Belkaid, Anissa; Ouellette, Rodney J; Surette, Marc E

2017-04-01

Long chain acyl-CoA synthase-4 (ACSL4) expression has been associated with an aggressive phenotype in breast carcinoma cells, whereas its role in ERα-positive breast cancer has not been studied. ACSL4 prefers 20-carbon polyunsaturated fatty acid (PUFA) substrates, and along with other ACSLs has been associated with cellular uptake of exogenous fatty acids. 17β-estradiol induces proliferation and invasive capacities in ERα+ve breast carcinoma that is associated with modifications of cellular lipid metabolism. In this study, treatment of steroid-starved ERα-positive MCF-7 and T47D mammary carcinoma cells with 17β-estradiol resulted in increased cellular uptake of the PUFA arachidonic acid (AA) and eicosapentaenoic acid (EPA), important building blocks for cellular membranes, and increased ACSL4 protein levels. There was no change in the expression of the ACSL1, ACSL3 and ACSL6 protein isotypes. Increased ACSL4 protein expression was not accompanied by changes in ACSL4 mRNA expression, but was associated with a significant increase in the protein half-life compared to untreated cells. ERα silencing reversed the impact of 17β-estradiol on ACSL4 protein levels and half-life. Silencing of ACSL4 eliminated the 17β-estradiol-induced increase in AA and EPA uptake, as well as the 17β-estradiol-induced cell migration, proliferation and invasion capacities. ASCL4 silencing also prevented the 17β-estradiol induced increases in p-Akt and p-GSK3β, and decrease in E-cadherin expression, important events in epithelial to mesenchymal transition. Taken together, these results demonstrate that ACSL4 is a target of 17β-estradiol-stimulated ERα and is required for the cellular uptake of exogenous PUFA and the manifestation of a more malignant phenotype in ERα+ve breast carcinoma cells. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Clip, connect, clone

DEFF Research Database (Denmark)

Fujima, Jun; Lunzer, Aran; Hornbæk, Kasper

2010-01-01

using three mechanisms: clipping of input and result elements from existing applications to form cells on a spreadsheet; connecting these cells using formulas, thus enabling result transfer between applications; and cloning cells so that multiple requests can be handled side by side. We demonstrate...

Main: Clone Detail [KOME

Lifescience Database Archive (English)

Full Text Available Clone Detail Mapping Pseudomolecule data detail Detail information Mapping to the T...IGR japonica Pseudomolecules kome_mapping_pseudomolecule_data_detail.zip kome_mapping_pseudomolecule_data_detail ...

Piglets born from handmade cloning, an innovative cloning method without micromanipulation

DEFF Research Database (Denmark)

Du, Y.; Kragh, P.M.; Zhang, Y.

2007-01-01

Porcine handmade cloning (HMC), a simplified alternative of micromanipulation based traditional cloning (TC) has been developed in multiple phases during the past years, but the final evidence of its biological value, births of piglets was missing. Here we report the first births of healthy piglets......) of HMC reconstructed embryos developed to blastocysts with an average cell number of 77 ± 3 (n = 26) after 7 days in vitro culture (IVC). According to our knowledge, this is the highest in vitro developmental rate after porcine somatic cell nuclear transfer (SCNT). A total of 416 blastocysts from HMC......, mixed with 150 blastocysts from TC using a cell line from a different breed were transferred surgically to nine synchronized recipients. Out of the four pregnancies (44.4%) two were lost, while two pregnancies went to term and litters of 3 and 10 piglets were delivered by Caesarean section, with live...