Identical substitutions in magnesium chelatase paralogs result in chlorophyll deficient soybean mutants

USDA-ARS?s Scientific Manuscript database

The soybean (Glycine max (L.) Merr.) chlorophyll deficient line MinnGold is a spontaneous mutant characterized by yellow foliage. Map-based cloning and transgenic complementation revealed that the mutant phenotype is caused by a non-synonymous nucleotide substitution in the third exon of a Mg-chelat...

Direct Cloning of Yeast Genes from an Ordered Set of Lambda Clones in Saccharomyces Cerevisiae by Recombination in Vivo

PubMed Central

Erickson, J. R.; Johnston, M.

1993-01-01

We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura(+) transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning. PMID:8514124

Molecular cloning of a ripening-specific lipoxygenase and its expression during wild-type and mutant tomato fruit development.

PubMed Central

Kausch, K D; Handa, A K

1997-01-01

A 94-kD protein that accumulates predominately in tomato (Ly-copersicon esculentum) fruit during ripening was purified, and antibodies specific for the purified protein were used to isolate cDNA clones from a red-ripe fruit cDNA library. A sequence analysis of these cDNAs and cross-reactivity of the 94-kD-specific antibodies to the soybean lipoxygenase (LOX) L-1, L-2, and L-3 proteins and soybean LOX L-1-specific antibodies to the 94-kD protein identified it as a member of the LOX gene family. Maximum levels of the 94-kD LOX mRNA and protein are present in breaker to ripe and red-ripe stages, respectively. Expression of 94-kD LOX in different tissues from mature green and red-ripe tomato fruits was found to be greatest in the radial walls of ripe fruit, but immunocytolocalization using tissue printing suggests that the highest accumulation of its protein occurs in locular jelly. None of 94-kD LOX is expressed in nonripening mutant fruits of any age. Never-ripe mutant fruit accumulate the 94-kD LOX mRNA to levels similar to those obtained in wild-type fruit, but fail to accumulate the 94-kD LOX protein. Collectively, the results show that expression of 94-kD LOX is regulated by the ripening process, and ethylene may play a role in its protein accumulation. PMID:9112767

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

PubMed

Vassbotn, F S; Andersson, M; Westermark, B; Heldin, C H; Ostman, A

1993-07-01

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions.

cDNA cloning of Brassica napus malonyl-CoA:ACP transacylase (MCAT) (fab D) and complementation of an E. coli MCAT mutant.

PubMed

Simon, J W; Slabas, A R

1998-09-18

The GenBank database was searched using the E. coli malonyl CoA:ACP transacylase (MCAT) sequence, for plant protein/cDNA sequences corresponding to MCAT, a component of plant fatty acid synthetase (FAS), for which the plant cDNA has not been isolated. A 272-bp Zea mays EST sequence (GenBank accession number: AA030706) was identified which has strong homology to the E. coli MCAT. A PCR derived cDNA probe from Zea mays was used to screen a Brassica napus (rape) cDNA library. This resulted in the isolation of a 1200-bp cDNA clone which encodes an open reading frame corresponding to a protein of 351 amino acids. The protein shows 47% homology to the E. coli MCAT amino acid sequence in the coding region for the mature protein. Expression of a plasmid (pMCATrap2) containing the plant cDNA sequence in Fab D89, an E. coli mutant, in MCAT activity restores growth demonstrating functional complementation and direct function of the cloned cDNA. This is the first functional evidence supporting the identification of a plant cDNA for MCAT.

A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system

PubMed Central

Suzuki, Yasuhiro; Kagawa, Naoko; Fujino, Toru; Sumiya, Tsuyoshi; Andoh, Taichi; Ishikawa, Kumiko; Kimura, Rie; Kemmochi, Kiyokazu; Ohta, Tsutomu; Tanaka, Shigeo

2005-01-01

There is an increasing demand for easy, high-throughput (HTP) methods for protein engineering to support advances in the development of structural biology, bioinformatics and drug design. Here, we describe an N- and C-terminal cloning method utilizing Gateway cloning technology that we have adopted for chimeric and mutant genes production as well as domain shuffling. This method involves only three steps: PCR, in vitro recombination and transformation. All three processes consist of simple handling, mixing and incubation steps. We have characterized this novel HTP method on 96 targets with >90% success. Here, we also discuss an N- and C-terminal cloning method for domain shuffling and a combination of mutation and chimeragenesis with two types of plasmid vectors. PMID:16009811

Chromosome instability of HPRT-mutant subclones induced by ionising radiation of various LET.

PubMed

Govorun, R D; Koshlan, I V; Koshlan, N A; Krasavin, E A; Shmakova, N L

2002-01-01

The induction of HPRT-mutations and survival of Chinese hamster cells (line B11ii-FAF28, clone 431) were studied after irradiation by 4He and 12C-ions of various LET (20-360 keV/micrometers), produced by the U-200 heavy ion accelerator. The RBE increases with LET up to the maximum at 100-200 keV/micrometers and then decreases. Cytogenetic analysis was performed on the HPRT-mutant subclones selected from unirradiated Chinese hamster V-79 cells and from HPRT-mutant subclones that arose after exposure to gamma-rays, 1 GeV protons and 14N-ions (LET-77 keV/micrometers), produced by the synchrophasotron and the U-400M heavy ion accelerator. Slow growing mutant subclones were observed. The cytogenetic properties of individual clones were highly heterogeneous and chromosome instability was observed in both spontaneous and radiation-induced mutants. Chromosome instability was highest among spontaneous mutants and decreased with increasing LET. c2002 COSPAR. Published by Elsevier Science Ltd. All rights reserved.

Bioactive compounds of fourth generation gamma-irradiated Typhoniumflagelliforme Lodd. mutants based on gas chromatography-mass spectrometry

NASA Astrophysics Data System (ADS)

Sianipar, N. F.; Purnamaningsih, R.; Rosaria

2016-08-01

Rodent tuber (Typhonium flagelliforme Lodd.) is an Indonesian anticancer medicinal plant. The natural genetic diversity of rodent tuber is low due to vegetative propagation. Plant's genetic diversity has to be increased for obtaining clones which contain a high amount of anticancer compounds. In vitro calli were irradiated with 6 Gy of gamma ray to produce in vitro mutant plantlets. Mutant plantlets were acclimated and propagated in a greenhouse. This research was aimed to identify the chemical compounds in the leaves and tubers ofthe fourth generation of rodent tuber's vegetative mutant clones (MV4) and control plantsby using GC- MS method. Leaves and tubers of MV4 each contained 2 and 5 anticancer compounds which quantities were higher compared to control plants. MV4 leaves contained 5 new anticancer compounds while its tubers contained 3 new anticancer compounds which were not found in control. The new anticancer compounds in leaves were hexadecanoic acid, stigmast-5-en-3-ol, ergost-5-en-3-ol, farnesol isomer a, and oleic acid while the new anticancer compounds in tubers were alpha tocopherol, ergost-5-en-3-ol, and beta-elemene. Rodent tuber mutant clones are very potential to be developed into anticancer drugs.

Characteristics of genomic instability in clones of TK6 human lymphoblasts surviving exposure to 56Fe ions

NASA Technical Reports Server (NTRS)

Evans, Helen H.; Horng, Min-Fen; Ricanati, Marlene; Diaz-Insua, Mireya; Jordan, Robert; Schwartz, Jeffrey L.

2002-01-01

Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated 56Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the 56Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to 56Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

PubMed Central

Vassbotn, F S; Andersson, M; Westermark, B; Heldin, C H; Ostman, A

1993-01-01

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions. Images PMID:8321214

Map-based cloning of a gene controlling Omega-3 fatty acid desaturation in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arondel, V.; Lemieux, B.; Hwang, I.

1992-11-20

A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase. 24 refs., 2 figs., 1more » tabs.« less

Study the Expression of marA Gene in Ciprofloxacin and Tetracycline Resistant Mutants of Esherichia coli

PubMed Central

Pourahmad Jaktaji, Razieh; Ebadi, Rayhaneh

2013-01-01

MarA activates two membrane dependent mechanisms of resistance to different antibiotics, such as ciprofloxacin and tetracycline, including promotion of outflux and inhibition of influx of antibiotics. Thus, MarA causes multiple antibiotic resistance phenotype. The activation of these mechanisms needs overexpression of marA. This could happen through mutation in marR. Thus, the aim of this study was to measure marA expression in ciprofloxacin resistant E. coli gyrA mutants and clones with or without marR mutation. For this purpose, real time PCR was used to measure relative expression of marA in above mutants and clones. Results showed that two clones, C14 and C17 overexpressed marA. It is concluded that the level of marA expression is important for activation of above mechanisms. PMID:24523773

Study the Expression of marA Gene in Ciprofloxacin and Tetracycline Resistant Mutants of Esherichia coli.

PubMed

Pourahmad Jaktaji, Razieh; Ebadi, Rayhaneh

2013-01-01

MarA activates two membrane dependent mechanisms of resistance to different antibiotics, such as ciprofloxacin and tetracycline, including promotion of outflux and inhibition of influx of antibiotics. Thus, MarA causes multiple antibiotic resistance phenotype. The activation of these mechanisms needs overexpression of marA. This could happen through mutation in marR. Thus, the aim of this study was to measure marA expression in ciprofloxacin resistant E. coli gyrA mutants and clones with or without marR mutation. For this purpose, real time PCR was used to measure relative expression of marA in above mutants and clones. Results showed that two clones, C14 and C17 overexpressed marA. It is concluded that the level of marA expression is important for activation of above mechanisms.

Dictyostelium discoideum mutants with conditional defects in phagocytosis

PubMed Central

1994-01-01

We have isolated and characterized Dictyostelium discoideum mutants with conditional defects in phagocytosis. Under suspension conditions, the mutants exhibited dramatic reductions in the uptake of bacteria and polystyrene latex beads. The initial binding of these ligands was unaffected, however, indicating that the defect was not in a plasma membrane receptor: Because of the phagocytosis defect, the mutants were unable to grow when cultured in suspensions of heat-killed bacteria. The mutants exhibited normal capacities for fluid phase endocytosis and grew as rapidly as parental (AX4) cells in axenic medium. Both the defects in phagocytosis and growth on bacteria were corrected when the mutant Dictyostelium cells were cultured on solid substrates. Reversion and genetic complementation analysis suggested that the mutant phenotypes were caused by single gene defects. While the precise site of action of the mutations was not established, the mutations are likely to affect an early signaling event because the binding of bacteria to mutant cells in suspension was unable to trigger the localized polymerization of actin filaments required for ingestion; other aspects of actin function appeared normal. This class of conditional phagocytosis mutant should prove to be useful for the expression cloning of the affected gene(s). PMID:7519624

Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

PubMed

Metts, J; West, J; Doares, S H; Matthysse, A G

1991-02-01

Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange. The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome. The effects of the mutations on various steps in tumor formation were examined. All three mutants showed no alteration in binding to carrot cells. However, none of the mutants showed any induction of vir genes by acetosyringone under conditions in which the parent strain showed vir gene induction. When the mutant bacteria were examined for changes in surface components, it was found that all three of the mutants showed a similar alteration in lipopolysaccharide (LPS). LPS from the mutants was larger in size and more heavily saccharide substituted than LPS from the parent strain. Two of the mutants showed no detectable alteration in outer membrane and periplasmic space proteins. The third mutant, Ivr-225, was missing a 79-kDa surface peptide. The reason(s) for the failure of vir gene induction in these mutants and its relationship, if any, to the observed alteration in LPS are unknown.

Using Microarrays to Facilitate Positional Cloning: Identification of Tomosyn as an Inhibitor of Neurosecretion

PubMed Central

Dybbs, Michael; Ngai, John; Kaplan, Joshua M

2005-01-01

Forward genetic screens have been used as a powerful strategy to dissect complex biological pathways in many model systems. A significant limitation of this approach has been the time-consuming and costly process of positional cloning and molecular characterization of the mutations isolated in these screens. Here, the authors describe a strategy using microarray hybridizations to facilitate positional cloning. This method relies on the fact that premature stop codons (i.e., nonsense mutations) constitute a frequent class of mutations isolated in screens and that nonsense mutant messenger RNAs are efficiently degraded by the conserved nonsense-mediated decay pathway. They validate this strategy by identifying two previously uncharacterized mutations: (1) tom-1, a mutation found in a forward genetic screen for enhanced acetylcholine secretion in Caenorhabditis elegans, and (2) an apparently spontaneous mutation in the hif-1 transcription factor gene. They further demonstrate the broad applicability of this strategy using other known mutants in C. elegans, Arabidopsis, and mouse. Characterization of tom-1 mutants suggests that TOM-1, the C. elegans ortholog of mammalian tomosyn, functions as an endogenous inhibitor of neurotransmitter secretion. These results also suggest that microarray hybridizations have the potential to significantly reduce the time and effort required for positional cloning. PMID:16103915

ECB deacylase mutants

DOEpatents

Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

2002-01-01

A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

Molecular mechanisms associated with leukemic transformation of MPL-mutant myeloproliferative neoplasms

PubMed Central

Beer, Philip A.; Ortmann, Christina A.; Stegelmann, Frank; Guglielmelli, Paola; Reilly, John T.; Larsen, Thomas S.; Hasselbalch, Hans C.; Vannucchi, Alessandro M.; Möller, Peter; Döhner, Konstanze; Green, Anthony R.

2010-01-01

Somatic activating mutations in MPL, the thrombopoietin receptor, occur in the myeloproliferative neoplasms, although virtually nothing is known about their role in evolution to acute myeloid leukemia. In this study, the MPL T487A mutation, identified in de novo acute myeloid leukemia, was not detected in 172 patients with a myeloproliferative neoplasm. In patients with a prior MPL W515L-mutant myeloproliferative neoplasm, leukemic transformation was accompanied by MPL-mutant leukemic blasts, was seen in the absence of prior cytoreductive therapy and often involved loss of wild-type MPL by mitotic recombination. Moreover, clonal analysis of progenitor colonies at the time of leukemic transformation revealed the presence of multiple genetically distinct but phylogenetically-related clones bearing different TP53 mutations, implying a mutator-phenotype and indicating that leukemic transformation may be preceded by the parallel expansion of diverse hematopoietic clones. PMID:20823136

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

PubMed

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

Diaminopurine-Resistant Mutants of Cultured, Diploid Human Fibroblasts

PubMed Central

Rappaport, Harriet; DeMars, Robert

1973-01-01

Clones of cells resistant to 2,6-diaminopurine were detected in skin fibroblast cultures derived from 13 of 21 normal humans of both sexes from 17 unrelated families. Almost all of the cultures that yielded mutants were chosen for further study from among a total of 83 surveyed because they displayed a slight resistance to low concentrations of diaminopurine. The incidences of mutant colonies ranged between about 10-5 and 10-4 per cell surviving prior mutagenic treatment with MNNG. The incidences of spontaneous mutants were about 10-7 to 10-5 in three unrelated cultures. Most independent mutants had distinctly reduced activity of adenine phosphoribosyltransferase but some had apparently normal amounts of activity. Two mutants from unrelated boys had little or no detectable enzyme activity and were unable to effectively use exogenous adenine for growth when purine biosynthesis was blocked with azaserine. Most mutants could utilize exogenous adenine, just as most azaguanine-resistant fibroblast mutants can utilize exogenous hypoxanthine, even when their hypoxanthine-guanine phosphoribosyltransferase activity is reduced. Diverse genetic changes conferred diaminopurine resistance but their specific natures are still undefined. Gross numerical or structural chromosome abnormalities were not observed in the mutants examined so far. Since at least one gene responsible for adenine phosphoribosyltransferase activity is on autosome No. 16 our results suggest that at least some of the cultures yielding mutants were heterozygous and that alleles conferring diaminopurine resistance may be frequent enough to comprise a polymorphism. PMID:4358687

Isolation of New Gravitropic Mutants under Hypergravity Conditions.

PubMed

Mori, Akiko; Toyota, Masatsugu; Shimada, Masayoshi; Mekata, Mika; Kurata, Tetsuya; Tasaka, Masao; Morita, Miyo T

2016-01-01

Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 ( eal1 ) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene ( enhancer of eal1 ) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis .

In vitro characterization of felid herpesvirus 1 (FHV-1) mutants generated by recombineering in a recombinant BAC vector

USDA-ARS?s Scientific Manuscript database

Felid herpesvirus 1 (FHV-1) mutants were constructed using two-step Red-mediated recombination techniques based on a virulent full-length FHV-1 BAC clone. The individual mutant viruses generated were deficient in glycoprotein C (gC), glycoprotein E (gE),US3 serine/threonine protein kinase (PK), or b...

Methylotroph cloning vehicle

DOEpatents

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.

Methylotroph cloning vehicle

DOEpatents

Hanson, Richard S.; Allen, Larry 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.

Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coli.

PubMed

Daumy, G O; Williams, J A; McColl, A S; Zuzel, T J; Danley, D

1986-10-01

The penicillin G acylase genes from the Proteus rettgeri wild type and from a hyperproducing mutant which is resistant to succinate repression were cloned in Escherichia coli K-12. Expression of both wild-type and mutant P. rettgeri acylase genes in E. coli K-12 was independent of orientation in the cloning vehicle and apparently resulted from recognition in E. coli of the P. rettgeri promoter sequences. The P. rettgeri acylase was secreted into the E. coli periplasmic space and was composed of subunits electrophoretically identical to those made in P. rettgeri. Expression of these genes in E. coli K-12 was not repressed by succinate as it is in P. rettgeri. Instead, expression of the enzymes was regulated by glucose catabolite repression.

Emergence of a bacterial clone with enhanced virulence by acquisition of a phage encoding a secreted phospholipase A2.

PubMed

Sitkiewicz, Izabela; Nagiec, Michal J; Sumby, Paul; Butler, Stephanie D; Cywes-Bentley, Colette; Musser, James M

2006-10-24

The molecular basis of pathogen clone emergence is relatively poorly understood. Acquisition of a bacteriophage encoding a previously unknown secreted phospholipase A(2) (designated SlaA) has been implicated in the rapid emergence in the mid-1980s of a new hypervirulent clone of serotype M3 group A Streptococcus. Although several lines of circumstantial evidence suggest that SlaA is a virulence factor, this issue has not been addressed experimentally. We found that an isogenic DeltaslaA mutant strain was significantly impaired in ability to adhere to and kill human epithelial cells compared with the wild-type parental strain. The mutant strain was less virulent for mice than the wild-type strain, and immunization with purified SlaA significantly protected mice from invasive disease. Importantly, the mutant strain was significantly attenuated for colonization in a monkey model of pharyngitis. We conclude that transductional acquisition of the ability of a GAS strain to produce SlaA enhanced the spread and virulence of the serotype M3 precursor strain. Hence, these studies identified a crucial molecular event underlying the evolution, rapid emergence, and widespread dissemination of unusually severe human infections caused by a distinct bacterial clone.

Tissue-specific and time-dependent clonal expansion of ENU-induced mutant cells in gpt delta mice.

PubMed

Nakayama, Takafumi; Sawai, Tomoko; Masuda, Ikuko; Kaneko, Shinya; Yamauchi, Kazumi; Blyth, Benjamin J; Shimada, Yoshiya; Tachibana, Akira; Kakinuma, Shizuko

2017-10-01

DNA mutations play a crucial role in the origins of cancer, and the clonal expansion of mutant cells is one of the fundamental steps in multistage carcinogenesis. In this study, we correlated tumor incidence in B6C3F1 mice during the period after exposure to N-ethyl-N-nitrosourea (ENU) with the persistence of ENU-induced mutant clones in transgenic gpt delta B6C3F1 mice. The induced gpt mutations afforded no selective advantage in the mouse cells and could be distinguished by a mutational spectrum that is characteristic of ENU treatment. The gpt mutations were passengers of the mutant cell of origin and its daughter cells and thus could be used as neutral markers of clones that arose and persisted in the tissues. Female B6C3F1 mice exposed for 1 month to 200 ppm ENU in the drinking water developed early thymic lymphomas and late liver and lung tumors. To assay gpt mutations, we sampled the thymus, liver, lung, and small intestine of female gpt delta mice at 3 days, 4 weeks, and 8 weeks after the end of ENU exposure. Our results reveal that, in all four tissues, the ENU-induced gpt mutations persisted for weeks after the end of mutagen exposure. Clonal expansion of mutant cells was observed in the thymus and small intestine, with the thymus showing larger clone sizes. These results indicate that the clearance of mutant cells and the potential for clonal expansion during normal tissue growth depends on tissue type and that these factors may affect the sensitivity of different tissues to carcinogenesis. Environ. Mol. Mutagen. 58:592-606, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Isolation of New Gravitropic Mutants under Hypergravity Conditions

PubMed Central

Mori, Akiko; Toyota, Masatsugu; Shimada, Masayoshi; Mekata, Mika; Kurata, Tetsuya; Tasaka, Masao; Morita, Miyo T.

2016-01-01

Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 (eal1) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene (enhancer of eal1) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis. PMID:27746791

Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans▿

PubMed Central

Xie, Zhoujie; Okinaga, Toshinori; Qi, Fengxia; Zhang, Zhijun; Merritt, Justin

2011-01-01

Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive- and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches. PMID:21948849

[The isolation and characteristics of mutants of the Saccharopolyspora erythraea strain resistant to thiostrepton].

PubMed

Nastasiak, I N; Fedorenko, V A; Danilenko, V N

1997-01-01

The formation of thiostreptone resistant spontaneous and nitrosoguanidine-induced mutants in the erythromycin-producing organism Saccharopolyspora erythraea was investigated. The investigated collection of the mutants was heterogeneous by the level of the thiostreptone resistance (2.5 to 20 micrograms/ml). The thiostreptone resistance mutations had a pleiotropic effect: 17 per cent of the mutants was characterized by the growth thermosensitivity and 26 and 5.8 per cent of the mutants were characterized by loss of the ability to form melanine and aerial mycelium respectively. Such phenotypes were most frequent in the mutants resistant to low concentrations of thiostreptone (2 to 5 micrograms/ml). The absolute majority of the isolated thiostreptone resistant mutants was unstable and formed both the antibiotic resistant and the antibiotic sensitive clones. The greatest portion of the strains with high antibiotic activity (20 per cent) was detected among the S. erythraea spontaneous mutants on the medium with 2.5 micrograms/ ml of thiostreptone. It was shown that the instability of the high antibiotic activity in the mutants was associated with loss of the thiostreptone resistance property.

Identification and cloning of a regulatory gene for nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942.

PubMed Central

Vega-Palas, M A; Madueño, F; Herrero, A; Flores, E

1990-01-01

Twenty-seven mutants that were unable to assimilate nitrate were isolated from Synechococcus sp. strain PCC 7942. In addition to mutants that lacked nitrate reductase or nitrite reductase, seven pleiotropic mutants impaired in both reductases, glutamine synthetase, and methylammonium transport were also isolated. One of the pleiotropic mutants was complemented by transformation with a cosmid gene bank from wild-type strain PCC 7942. Three complementing cosmids were isolated, and a 3.1-kilobase-pair DNA fragment that was still able to complement the mutant was identified. The regulatory gene that was cloned (ntcA) appeared to be required for full expression of proteins subject to ammonium repression in Synechococcus sp. PMID:1967601

Cloning and Sequencing of a Candida albicans Catalase Gene and Effects of Disruption of This Gene†

PubMed Central

Wysong, Deborah R.; Christin, Laurent; Sugar, Alan M.; Robbins, Phillips W.; Diamond, Richard D.

1998-01-01

Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in Candida albicans, a single C. albicans-derived catalase gene, designated CAT1, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of C. albicans ATCC 10261. By using this product as a probe, catalase clones were isolated from genomic libraries of C. albicans. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 487 amino acid residues. Construction of a CAT1-deficient mutant was achieved by using the Ura-blaster technique for sequential disruption of multiple alleles by integrative transformation using URA3 as a selectable marker. Resulting mutants exhibited normal morphology and comparable growth rates of both yeast and mycelial forms. Enzymatic analysis revealed an abundance of catalase in the wild-type strain but decreasing catalase activity in heterozygous mutants and no detectable catalase in a homozygous null mutant. In vitro assays showed the mutant strains to be more sensitive to damage by both neutrophils and concentrations of exogenous peroxide that were sublethal for the parental strain. Compared to the parental strain, the homozygous null mutant strain was far less virulent for mice in an intravenous infection model of disseminated candidiasis. Definitive linkage of CAT1 with virulence would require restoration of activity by reintroduction of the gene into mutants. However, initial results in mice, taken together with the enhanced susceptibility of catalase-deficient hyphae to damage by human neutrophils, suggest that catalase may enhance the pathogenicity of C. albicans. PMID:9573075

Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis.

PubMed

Phillips, A L; Ward, D A; Uknes, S; Appleford, N E; Lange, T; Huttly, A K; Gaskin, P; Graebe, J E; Hedden, P

1995-07-01

Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (GA) 20-oxidase sequence, six different fragments of dioxygenase genes were amplified by polymerase chain reaction from arabidopsis thaliana genomic DNA. One of these was used to isolate two different full-length cDNA clones, At2301 and At2353, from shoots of the GA-deficient Arabidopsis mutant ga1-2. A third, related clone, YAP169, was identified in the Database of Expressed Sequence Tags. The cDNA clones were expressed in Escherichia coli as fusion proteins, each of which oxidized GA12 at C-20 to GA15, GA24, and the C19 compound GA9, a precursor of bioactive GAs; the C20 tricarboxylic acid compound GA25 was formed as a minor product. The expression products also oxidized the 13-hydroxylated substrate GA53, but less effectively than GA12. The three cDNAs hybridized to mRNA species with tissue-specific patterns of accumulation, with At2301 being expressed in stems and inflorescences, At2353 in inflorescences and developing siliques, and YAP169 in siliques only. In the floral shoots of the ga1-2 mutant, transcript levels corresponding to each cDNA decreased dramatically after GA3 application, suggesting that GA biosynthesis may be controlled, at least in part, through down-regulation of the expression of the 20-oxidase genes.

Rapid cloning of genes in hexaploid wheat using cultivar-specific long-range chromosome assembly.

PubMed

Thind, Anupriya Kaur; Wicker, Thomas; Šimková, Hana; Fossati, Dario; Moullet, Odile; Brabant, Cécile; Vrána, Jan; Doležel, Jaroslav; Krattinger, Simon G

2017-08-01

Cereal crops such as wheat and maize have large repeat-rich genomes that make cloning of individual genes challenging. Moreover, gene order and gene sequences often differ substantially between cultivars of the same crop species. A major bottleneck for gene cloning in cereals is the generation of high-quality sequence information from a cultivar of interest. In order to accelerate gene cloning from any cropping line, we report 'targeted chromosome-based cloning via long-range assembly' (TACCA). TACCA combines lossless genome-complexity reduction via chromosome flow sorting with Chicago long-range linkage to assemble complex genomes. We applied TACCA to produce a high-quality (N50 of 9.76 Mb) de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months. Using this assembly we cloned the broad-spectrum Lr22a leaf-rust resistance gene, using molecular marker information and ethyl methanesulfonate (EMS) mutants, and found that Lr22a encodes an intracellular immune receptor homologous to the Arabidopsis thaliana RPM1 protein.

[The Spectrum of Mutations in Genes Associated with Resistance to Rifampicin, Isoniazid, and Fluoroquinolones in the Clinical Strains of M. tuberculosis Reflects the Transmissibility of Mutant Clones].

PubMed

Ergeshov, A; Andreevskaya, S N; Larionova, E E; Smirnova, T G; Chernousova, L N

2017-01-01

To study the transmissibility of drug resistant mutant clones, M. tuberculosis samples were isolated from the patients of the clinical department and the polyclinic of the Central TB Research Institute (n = 1455) for 2011-2014. A number of clones were phenotypically resistant to rifampicin (n = 829), isoniazid (n = 968), and fluoroquinolones (n = 220). We have detected 21 resistance-associated variants in eight codons of rpoB, six variants in three codons of katG, three variants in two positions of inhA, four variants in four positions of ahpC, and nine variants in five codons of gyrA, which were represented in the analyzed samples with varied frequencies. Most common mutations were rpoB 531 Ser→Leu (77.93%), katG 315 (Ser→Thr) (94.11%), and gyrA 94 (Asp→Gly) (45.45%). We found that the mutations at position 15 of inhA (C→T) (frequency of 25.72%) are commonly associated with katG 315 (Ser→Thr). This association of two DNA variants may arise due to the double selection by coexposure of M. tuberculosis to isoniazid and ethionamide. The high transmissibility of mutated strains was observed, which may be explained by the minimal influence of the resistance determinants on strain viability. The high transmissibility of resistant variants may also explain the large populational prevalence of drug-resistant TB strains.

Determination of multiple-clone infection at allelic dimorphism site of Plasmodium vivax merozoite surface protein-1 in the Republic of Korea by pyrosequencing assay.

PubMed

Dinzouna-Boutamba, Sylvatrie-Danne; Lee, Sanghyun; Son, Ui-Han; Yun, Hae Soo; Joo, So-Young; Jeong, Sookwan; Rhee, Man Hee; Kwak, Dongmi; Xuan, Xuenan; Hong, Yeonchul; Chung, Dong-Il; Goo, Youn-Kyoung

2017-12-01

Allelic diversity leading to multiple gene polymorphisms of vivax malaria parasites has been shown to greatly contribute to antigenic variation and drug resistance, increasing the potential for multiple-clone infections within the host. Therefore, to identify multiple-clone infections and the predominant haplotype of Plasmodium vivax in a South Korean population, P. vivax merozoite surface protein-1 (PvMSP-1) was analyzed by pyrosequencing. Pyrosequencing of 156 vivax malaria-infected samples yielded 97 (62.18%) output pyrograms showing two main types of peak patterns of the dimorphic allele for threonine and alanine (T1476A). Most of the samples evaluated (88.66%) carried multiple-clone infections (wild- and mutant-types), whereas 11.34% of the same population carried only the mutant-type (1476A). In addition, each allele showed a high frequency of guanine (G) base substitution at both the first and third positions (86.07% and 81.13%, respectively) of the nucleotide combinations. Pyrosequencing of the PvMSP-1 42-kDa fragment revealed a heterogeneous parasite population, with the mutant-type dominant compared to the wild-type. Understanding the genetic diversity and multiple-clone infection rates may lead to improvements in vivax malaria prevention and strategic control plans. Further studies are needed to improve the efficacy of the pyrosequencing assay with large sample sizes and additional nucleotide positions. Copyright © 2017 Elsevier B.V. All rights reserved.

Recombination Between Guanidine-resistant and Dextran Sulfate-resistant Mutants of Type 1 Poliovirus

PubMed Central

Sergiescu, Dina; Aubert-Combiescu, Andrei; Crainic, Radu

1969-01-01

Mixed infection of monkey kidney cells with two mutants of the LSc2ab strain of poliovirus, one resistant to guanidine and the other resistant to both dextran sulfate and 2-(α-hydroxybenzyl)-benzimidazole (HBB), yielded progeny in which the number of guardexr particles exceeded by a factor of 7 to 10 the expected number of similar particles occurring through spontaneous mutation; recombination would explain the fairly high excess of doubly mutant particles that was obtained. Scoring of HBB resistance in 50 guardexr clones suggested that, during recombination, resistance to dextran sulfate is not associated with HBB resistance. Images PMID:4305674

Mutants of the lactose carrier of Escherichia coli which show altered sugar recognition plus a severe defect in sugar accumulation.

PubMed

Varela, M F; Wilson, T H; Rodon-Rivera, V; Shepherd, S; Dehne, T A; Rector, A C

2000-04-01

Lactose and melibiose are actively accumulated by the wild-type Escherichia coli lactose carrier, which is an integral membrane protein energized by the proton motive force. Mutants of the E. coli lactose carrier were isolated by their ability to grow on minimal plates with succinate plus IPTG in the presence of the toxic lactose analog beta-thio-o-nitrophenylgalactoside (TONPG). TONPG-resistant mutants were streaked on melibiose MacConkey indicator plates, and red clones were picked. These melibiose positive mutants were then streaked on lactose MacConkey plates, and white clones were picked. Transport assays indicated that the mutants had altered sugar recognition and a defect in sugar accumulation. The mutants had a poor apparent K(m) for both lactose and melibiose in transport. One mutant had almost no ability to take up lactose, but melibiose downhill transport was 58% (V(max)) of normal. All of the mutants accumulated methyl-alpha-d-galactopyranoside (TMG) to only 8% or less of normal, and two failed to accumulate. Immunoblot analysis of the mutant lactose carrier proteins indicated that loss of sugar transport activity was not due to loss of expression in the membrane. Nucleotide sequencing of the lacY gene from the mutants revealed changes in the following amino acids of the lactose carrier: M23I, W151L, G257D, A295D and G377V. Two of the mutants (G257D and G377V) are novel in that they represent the first amino acids in periplasmic loops to be implicated with changes in sugar recognition. We conclude that the amino acids M23, W151, G257, A295 and G377 of the E. coli lactose carrier play either a direct or an indirect role in sugar recognition and accumulation.

Production of cloned mice from somatic cells, ES cells, and frozen bodies.

PubMed

Wakayama, Sayaka; Mizutani, Eiji; Wakayama, Teruhiko

2010-01-01

Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, therefore, the nuclear transfer (NT) method has been thought of as a "black box approach" and inadequate to determine the detail of how genomic reprogramming occurs. However, only the NT approach can reveal dynamic and global modifications in the epigenome without using genetic modification, as well as can create live animals. At present, this is the only technique available for the preservation and propagation of valuable genetic resources from mutant mice that are infertile or too old, or recovered from carcasses, without the use of germ cells. This chapter describes a basic protocol for mouse cloning and embryonic stem (ES) cell establishment from cloned embryo using a piezo-actuated micromanipulator. This technique will greatly help not only in mouse cloning but also in other forms of micromanipulation such as intracytoplasmic sperm injection (ICSI) into oocytes or ES cell injection into blastocysts. In addition, we describe a new, more efficient mouse cloning protocol using histone deacetylase inhibitor (HDACi), which increases the success rates of cloned mice or establish rate of ES cells to fivefold. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Cloning

MedlinePlus

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 ...

Generation of a Gaussia luciferase-expressing endotheliotropic cytomegalovirus for screening approaches and mutant analyses.

PubMed

Falk, Jessica J; Laib Sampaio, Kerstin; Stegmann, Cora; Lieber, Diana; Kropff, Barbara; Mach, Michael; Sinzger, Christian

2016-09-01

For many questions in human cytomegalovirus (HCMV) research, assays are desired that allow robust and fast quantification of infection efficiencies under high-throughput conditions. The secreted Gaussia luciferase has been demonstrated as a suitable reporter in the context of a fibroblast-adapted HCMV strain, which however is greatly restricted in the number of cell types to which it can be applied. We inserted the Gaussia luciferase expression cassette into the BAC-cloned virus strain TB40-BAC4, which displays the natural broad cell tropism of HCMV and hence allows application to screening approaches in a variety of cell types including fibroblasts, epithelial, and endothelial cells. Here, we applied the reporter virus TB40-BAC4-IE-GLuc to identify mouse hybridoma clones that preferentially neutralize infection of endothelial cells. In addition, as the Gaussia luciferase is secreted into culture supernatants from infected cells it allows kinetic analyses in living cultures. This can speed up and facilitate phenotypic characterization of BAC-cloned mutants. For example, we analyzed a UL74 stop-mutant of TB40-BAC4-IE-GLuc immediately after reconstitution in transfected cultures and found the increase of luciferase delayed and reduced as compared to wild type. Phenotypic monitoring directly in transfected cultures can minimize the risk of compensating mutations that might occur with extended passaging. Copyright © 2016 Elsevier B.V. All rights reserved.

What is Cloning?

MedlinePlus

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 ...

The First Mutant of the Aequorea victoria Green Fluorescent Protein That Forms a Red Chromophore†

PubMed Central

Mishin, Alexander S.; Subach, Fedor V.; Yampolsky, Ilia V.; King, William; Lukyanov, Konstantin A.; Verkhusha, Vladislav V.

2010-01-01

Green fluorescent protein (GFP) from a jellyfish, Aequorea victoria, and its mutants are widely used in biomedical studies as fluorescent markers. In spite of the enormous efforts of academia and industry toward generating its red fluorescent mutants, no GFP variants with emission maximum at more than 529 nm have been developed during the 15 years since its cloning. Here, we used a new strategy of molecular evolution aimed at generating a red-emitting mutant of GFP. As a result, we have succeeded in producing the first GFP mutant that substantially matures to the red-emitting state with excitation and emission maxima at 555 and 585 nm, respectively. A novel, nonoxidative mechanism for formation of the red chromophore in this mutant that includes a dehydration of the Ser65 side chain has been proposed. Model experiments showed that the novel dual-color GFP mutant with green and red emission is suitable for multicolor flow cytometry as an additional color since it is clearly separable from both green and red fluorescent tags. PMID:18366185

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

PubMed Central

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

2013-01-01

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

Mutant onco-proteins as drug targets: successes, failures, and future prospects.

PubMed

McCormick, Frank

2011-02-01

Mutant onco-proteins play a direct, causal role in cancer and are therefore considered attractive drug targets. Clinical experience has supported this view, with some exceptions. However, clinical benefit has often been restricted by rapid emergence of drug-resistant clones through several distinct mechanisms. This problem can, in principle, be addressed through cocktails containing several drugs. However, the number of tumors whose survival is dependent on a single, druggable mutant onco-protein is currently unknown. The majority of tumors may be driven either by single drivers that are un-druggable, or by combinations of drivers. In both cases, new approaches will be necessary. Development of systemic RNA interference may be a solution to these problems. Copyright © 2010 Elsevier Ltd. All rights reserved.

Screening a wide host-range, waste-water metagenomic library in tryptophan auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different classes of cloned trp genes.

PubMed

Li, Youguo; Wexler, Margaret; Richardson, David J; Bond, Philip L; Johnston, Andrew W B

2005-12-01

A metagenomic cosmid library was constructed, in which the insert DNA was derived from bacteria in a waste-water treatment plant and the vector was the wide host-range cosmid pLAFR3. The library was screened for clones that could correct defined tryptophan auxotrophs of the alpha-proteobacterium Rhizobium leguminosarum and of Escherichia coli. A total of 26 different cosmids that corrected at least one trp mutant in one or both of these species were obtained. Several cosmids corrected the auxotrophy of one or more R. leguminosarum trp mutants, but not the corresponding mutants in E. coli. Conversely, one cosmid corrected trpA, B, C, D and E mutants of E. coli but none of the trp mutants of R. leguminosarum. Two of the Trp+ cosmids were examined in more detail. One contained a trp operon that resembled that of the pathogen Chlamydophila caviae, containing the unusual kynU gene, which specifies kynureninase. The other, whose trp genes functioned in R. leguminosarum but not in E. coli, contained trpDCFBA in an operon that is likely co-transcribed with five other genes, most of which had no known link with tryptophan synthesis. The sequences of these TRP proteins, and the products of nine other genes encoded by this cosmid, failed to affiliate them with any known bacterial lineage. For one metagenomic cosmid, lac reporter fusions confirmed that its cloned trp genes were transcribed in R. leguminosarum, but not in E. coli. Thus, rhizobia, with their many sigma-factors, may be well-suited hosts for metagenomic libraries, cloned in wide host-range vectors.

Isolation of baker's yeast mutants with proline accumulation that showed enhanced tolerance to baking-associated stresses.

PubMed

Tsolmonbaatar, Ariunzaya; Hashida, Keisuke; Sugimoto, Yukiko; Watanabe, Daisuke; Furukawa, Shuhei; Takagi, Hiroshi

2016-12-05

During bread-making processes, yeast cells are exposed to baking-associated stresses such as freeze-thaw, air-drying, and high-sucrose concentrations. Previously, we reported that self-cloning diploid baker's yeast strains that accumulate proline retained higher-level fermentation abilities in both frozen and sweet doughs than the wild-type strain. Although self-cloning yeasts do not have to be treated as genetically modified yeasts, the conventional methods for breeding baker's yeasts are more acceptable to consumers than the use of self-cloning yeasts. In this study, we isolated mutants resistant to the proline analogue azetidine-2-carboxylate (AZC) derived from diploid baker's yeast of Saccharomyces cerevisiae. Some of the mutants accumulated a greater amount of intracellular proline, and among them, 5 mutants showed higher cell viability than that observed in the parent wild-type strain under freezing or high-sucrose stress conditions. Two of them carried novel mutations in the PRO1 gene encoding the Pro247Ser or Glu415Lys variant of γ-glutamyl kinase (GK), which is a key enzyme in proline biosynthesis in S. cerevisiae. Interestingly, we found that these mutations resulted in AZC resistance of yeast cells and desensitization to proline feedback inhibition of GK, leading to intracellular proline accumulation. Moreover, baker's yeast cells expressing the PRO1 P247S and PRO1 E415K gene were more tolerant to freezing stress than cells expressing the wild-type PRO1 gene. The approach described here could be a practical method for the breeding of proline-accumulating baker's yeasts with higher tolerance to baking-associated stresses. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization and gene cloning of the rice (Oryza sativa L.) dwarf and narrow-leaf mutant dnl3.

PubMed

Shi, L; Wei, X J; Adedze, Y M N; Sheng, Z H; Tang, S Q; Hu, P S; Wang, J L

2016-09-16

The dwarf and narrow-leaf rice (Oryza sativa L.) mutant dnl3 was isolated from the Japonica cultivar Zhonghua 11 (wild-type). dnl3 exhibited pleiotropic developmental defects. The narrow-leaf phenotype resulted from a marked reduction in the number of vascular bundles, while the dwarf stature was caused by the formation of foreshortened internodes and a reduced number of parenchyma cells. The suggestion that cell division is impaired in the mutant was consistent with the transcriptional behavior of various genes associated with cell division. The mutant was less responsive to exogenously supplied gibberellic acid than the wild-type, and profiling the transcription of genes involved in gibberellin synthesis and response revealed that a lesion in the mutant affected gibberellin signal transduction. The dnl3 phenotype was inherited as a single-dominant gene, mapping within a 19.1-kb region of chromosome 12, which was found to harbor three open reading frames. Resequencing the open reading frames revealed that the mutant carried an allele at one of the three genes that differed from the wild-type sequence by 2-bp deletions; this gene encoded a cellulose synthase-like D4 (CSLD4) protein. Therefore, OsCSLD4 is a candidate gene for DNL3. DNL3 was expressed in all of the rice organs tested at the heading stage, particularly in the leaves, roots, and culms. These results suggest that DNL3 plays important roles in rice leaf morphogenesis and vegetative development.

Functional Loss of Bmsei Causes Thermosensitive Epilepsy in Contractile Mutant Silkworm, Bombyx mori

PubMed Central

Nie, Hongyi; Cheng, Tingcai; Huang, Xiaofeng; Zhou, Mengting; Zhang, Yinxia; Dai, Fangyin; Mita, Kazuei; Xia, Qingyou; Liu, Chun

2015-01-01

The thermoprotective mechanisms of insects remain largely unknown. We reported the Bombyx mori contractile (cot) behavioral mutant with thermo-sensitive seizures phenotype. At elevated temperatures, the cot mutant exhibit seizures associated with strong contractions, rolling, vomiting, and a temporary lack of movement. We narrowed a region containing cot to ~268 kb by positional cloning and identified the mutant gene as Bmsei which encoded a potassium channel protein. Bmsei was present in both the cell membrane and cytoplasm in wild-type ganglia but faint in cot. Furthermore, Bmsei was markedly decreased upon high temperature treatment in cot mutant. With the RNAi method and injecting potassium channel blockers, the wild type silkworm was induced the cot phenotype. These results demonstrated that Bmsei was responsible for the cot mutant phenotype and played an important role in thermoprotection in silkworm. Meanwhile, comparative proteomic approach was used to investigate the proteomic differences. The results showed that the protein of Hsp-1 and Tn1 were significantly decreased and increased on protein level in cot mutant after thermo-stimulus, respectively. Our data provide insights into the mechanism of thermoprotection in insect. As cot phenotype closely resembles human epilepsy, cot might be a potential model for the mechanism of epilepsy in future. PMID:26198671

Functional Loss of Bmsei Causes Thermosensitive Epilepsy in Contractile Mutant Silkworm, Bombyx mori

NASA Astrophysics Data System (ADS)

Nie, Hongyi; Cheng, Tingcai; Huang, Xiaofeng; Zhou, Mengting; Zhang, Yinxia; Dai, Fangyin; Mita, Kazuei; Xia, Qingyou; Liu, Chun

2015-07-01

The thermoprotective mechanisms of insects remain largely unknown. We reported the Bombyx mori contractile (cot) behavioral mutant with thermo-sensitive seizures phenotype. At elevated temperatures, the cot mutant exhibit seizures associated with strong contractions, rolling, vomiting, and a temporary lack of movement. We narrowed a region containing cot to ~268 kb by positional cloning and identified the mutant gene as Bmsei which encoded a potassium channel protein. Bmsei was present in both the cell membrane and cytoplasm in wild-type ganglia but faint in cot. Furthermore, Bmsei was markedly decreased upon high temperature treatment in cot mutant. With the RNAi method and injecting potassium channel blockers, the wild type silkworm was induced the cot phenotype. These results demonstrated that Bmsei was responsible for the cot mutant phenotype and played an important role in thermoprotection in silkworm. Meanwhile, comparative proteomic approach was used to investigate the proteomic differences. The results showed that the protein of Hsp-1 and Tn1 were significantly decreased and increased on protein level in cot mutant after thermo-stimulus, respectively. Our data provide insights into the mechanism of thermoprotection in insect. As cot phenotype closely resembles human epilepsy, cot might be a potential model for the mechanism of epilepsy in future.

Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

PubMed Central

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

Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology.

PubMed Central

Siehnel, R J; Worobec, E A; Hancock, R E

1988-01-01

The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes. Images PMID:2834340

A Genetic Screen for Mutants with Supersized Lipid Droplets in Caenorhabditis elegans

PubMed Central

Li, Shiwei; Xu, Shibin; Ma, Yanli; Wu, Shuang; Feng, Yu; Cui, Qingpo; Chen, Lifeng; Zhou, Shuang; Kong, Yuanyuan; Zhang, Xiaoyu; Yu, Jialei; Wu, Mengdi; Zhang, Shaobing O.

2016-01-01

To identify genes that regulate the dynamics of lipid droplet (LD) size, we have used the genetically tractable model organism Caenorhabditis elegans, whose wild-type LD population displays a steady state of size with an upper limit of 3 μm in diameter. From a saturated forward genetic screen of 6.7 × 105 mutagenized haploid genomes, we isolated 118 mutants with supersized intestinal LDs often reaching 10 μm. These mutants define nine novel complementation groups, in addition to four known genes (maoc-1, dhs-28, daf-22, and prx-10). The nine groups are named drop (lipid droplet abnormal) and categorized into four classes. Class I mutants drop-5 and drop-9, similar to prx-10, are up-regulated in ACS-22-DGAT-2-dependent LD growth, resistant to LD hydrolysis, and defective in peroxisome import. Class II mutants drop-2, drop-3, drop-6, and drop-7 are up-regulated in LD growth, are resistant to LD hydrolysis, but are not defective in peroxisome import. Class III mutants drop-1 and drop-8 are neither up-regulated in LD growth nor resistant to LD hydrolysis, but seemingly up-regulated in LD fusion. Class IV mutant drop-4 is cloned as sams-1 and, different to the other three classes, is ACS-22-independent and hydrolysis-resistant. These four classes of supersized LD mutants should be valuable for mechanistic studies of LD cellular processes including growth, hydrolysis, and fusion. PMID:27261001

Method for rapid isolation of sensitive mutants

DOEpatents

Freyer, J.P.

1997-07-29

Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned. 15 figs.

Method for rapid isolation of sensitive mutants

DOEpatents

Freyer, James P.

1997-01-01

Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned.

Dwarfism and increased adiposity in the gh1 mutant zebrafish vizzini.

PubMed

McMenamin, Sarah K; Minchin, James E N; Gordon, Tiffany N; Rawls, John F; Parichy, David M

2013-04-01

Somatic growth and adipogenesis are closely associated with the development of obesity in humans. In this study, we identify a zebrafish mutant, vizzini, that exhibits both a severe defect in somatic growth and increased accumulation of adipose tissue. Positional cloning of vizzini revealed a premature stop codon in gh1. Although the effects of GH are largely through igfs in mammals, we found no decrease in the expression of igf transcripts in gh1 mutants during larval development. As development progressed, however, we found overall growth to be progressively retarded and the attainment of specific developmental stages to occur at abnormally small body sizes relative to wild type. Moreover, both subcutaneous (sc) and visceral adipose tissues underwent precocious development in vizzini mutants, and at maturity, the sizes of different fat deposits were greatly expanded relative to wild type. In vivo confocal imaging of sc adipose tissue (SAT) expansion revealed that vizzini mutants exhibit extreme enlargement of adipocyte lipid droplets without a corresponding increase in lipid droplet number. These findings suggest that GH1 signaling restricts SAT hypertrophy in zebrafish. Finally, nutrient deprivation of vizzini mutants revealed that SAT mobilization was greatly diminished during caloric restriction, further implicating GH1 signaling in adipose tissue homeostasis. Overall, the zebrafish gh1 mutant, vizzini, exhibits decreased somatic growth, increased adipose tissue accumulation, and disrupted adipose plasticity after nutrient deprivation and represents a novel model to investigate the in vivo dynamics of vertebrate obesity.

Dwarfism and Increased Adiposity in the gh1 Mutant Zebrafish vizzini

PubMed Central

McMenamin, Sarah K.; Minchin, James E.N.; Gordon, Tiffany N.

2013-01-01

Somatic growth and adipogenesis are closely associated with the development of obesity in humans. In this study, we identify a zebrafish mutant, vizzini, that exhibits both a severe defect in somatic growth and increased accumulation of adipose tissue. Positional cloning of vizzini revealed a premature stop codon in gh1. Although the effects of GH are largely through igfs in mammals, we found no decrease in the expression of igf transcripts in gh1 mutants during larval development. As development progressed, however, we found overall growth to be progressively retarded and the attainment of specific developmental stages to occur at abnormally small body sizes relative to wild type. Moreover, both subcutaneous (sc) and visceral adipose tissues underwent precocious development in vizzini mutants, and at maturity, the sizes of different fat deposits were greatly expanded relative to wild type. In vivo confocal imaging of sc adipose tissue (SAT) expansion revealed that vizzini mutants exhibit extreme enlargement of adipocyte lipid droplets without a corresponding increase in lipid droplet number. These findings suggest that GH1 signaling restricts SAT hypertrophy in zebrafish. Finally, nutrient deprivation of vizzini mutants revealed that SAT mobilization was greatly diminished during caloric restriction, further implicating GH1 signaling in adipose tissue homeostasis. Overall, the zebrafish gh1 mutant, vizzini, exhibits decreased somatic growth, increased adipose tissue accumulation, and disrupted adipose plasticity after nutrient deprivation and represents a novel model to investigate the in vivo dynamics of vertebrate obesity. PMID:23456361

Novel mutations in β-tubulin gene in Trichoderma harzianum mutants resistant to methyl benzimidazol-2-yl carbamate.

PubMed

Li, M; Zhang, H Y; Liang, B

2013-01-01

Twelve-low resistant (LR) mutants of Trichoderma harzianum with the capability of grow fast at 0.8 μg/mL methyl benzimidazol-2-yl carbamate (MBC) were obtained using UV mutagenesis. MR and HR mutants which could grow fast at 10 and 100 μg/mL MBC, respectively, were isolated by step-up selection protocols in which UV-treated mutants were induced and mycelial sector screening was made in plates with growth medium. Subsequently, β-tubulin genes of 14 mutants were cloned to describe-the molecular lesion likely to be responsible-for MBC resistance. Comparison of the β-tubulin sequences of the mutant and sensitive strains of T. harzianum revealed 2 new MBC-binding sites differed from those in other plant pathogens. A single mutation at-amino acid 168, having Phe (TTC) instead of Ser (TCC)', was demonstrated for the HR mutant; a double mutation in amino acid 13 resulting in the substitution of Gly (GGC) by Val (GTG) was observed in β-tubulin gene of MR mutant. On the other hand, no substitutions were identified in the β-tubulin gene and its 5'-flanking regions in 12 LR mutants of T. harzianum.

Positional cloning in mice and its use for molecular dissection of inflammatory arthritis.

PubMed

Abe, Koichiro; Yu, Philipp

2009-02-01

One of the upcoming next quests in the field of genetics might be molecular dissection of the genetic and environmental components of human complex diseases. In humans, however, there are certain experimental limitations for identification of a single component of the complex interactions by genetic analyses. Experimental animals offer simplified models for genetic and environmental interactions in human complex diseases. In particular, mice are the best mammalian models because of a long history and ample experience for genetic analyses. Forward genetics, which includes genetic screen and subsequent positional cloning of the causative genes, is a powerful strategy to dissect a complex phenomenon without preliminarily molecular knowledge of the process. In this review, first, we describe a general scheme of positional cloning in mice. Next, recent accomplishments on the patho-mechanisms of inflammatory arthritis by forward genetics approaches are introduced; Positional cloning effort for skg, Ali5, Ali18, cmo, and lupo mutants are provided as examples for the application to human complex diseases. As seen in the examples, the identification of genetic factors by positional cloning in the mouse have potential in solving molecular complexity of gene-environment interactions in human complex diseases.

Cloning, clones and clonal disease.

PubMed

Luzzatto, L

2000-01-01

In the past, cloning has been familiar to plant breeders because many plants can be easily reproduced in this way, bypassing the lengthy process of cross-fertilisation. Recently, the concept of cloning has become popular in human biology and medicine on two accounts. First, individual genes can be cloned from the enormous complexity of the DNA that makes up the human genetic material. It is expected that, within a few years, all the estimated 100,000 human genes will be isolated by this approach. This should make it possible to identify all the genes that determine the individual characteristics of human beings, including those responsible for causing human diseases or for making people more or less susceptible to pick up diseases from the environment. Cloned genes made into pharmaceutical products are already in use for treating a variety of diseases, from hormonal deficiencies to certain types of anaemia.

Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila.

PubMed

Varshney, Nishant Kumar; Ramasamy, Sureshkumar; Brannigan, James A; Wilkinson, Anthony J; Suresh, C G

2013-08-01

Kluyvera citrophila penicillin G acylase (KcPGA) has recently attracted increased attention relative to the well studied and commonly used Escherichia coli PGA (EcPGA) because KcPGA is more resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin (6-APA) nucleus, which is the starting material for semi-synthetic antibiotic production. Like other penicillin acylases, KcPGA is synthesized as a single-chain inactive pro-PGA, which upon autocatalytic processing becomes an active heterodimer of α and β chains. Here, the cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported. The purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described. The protein crystallized in two different space groups, P1, with unit-cell parameters a = 54.0, b = 124.6, c = 135.1 Å, α = 104.1, β = 101.4, γ = 96.5°, and C2, with unit-cell parameters a = 265.1, b = 54.0, c = 249.2 Å, β = 104.4°, using the sitting-drop vapour-diffusion method. Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method. The initial maps revealed electron density for the spacer peptide.

Map-Based Cloning of Genes Important for Maize Anther Development

NASA Astrophysics Data System (ADS)

Anaya, Y.; Walbot, V.; Nan, G.

2012-12-01

Map-Based cloning for maize mutant MS13 . Scientists still do not understand what decides the fate of a cell in plants. Many maize genes are important for anther development and when they are disrupted, the anthers do not shed pollen, i.e. male sterile. Since the maize genome has been fully sequenced, we conduct map-based cloning using a bulk segregant analysis strategy. Using PCR (polymerase chain reaction), we look for biomarkers that are linked to our gene of interest, Male Sterile 13 (MS13). Recombinations occur more often if the biomarkers are further away from the gene, therefore we can estimate where the gene is and design more PCR primers to get closer to our gene. Genetic and molecular analysis will help distinguish the role of key genes in setting cell fates before meiosis and for being in charge of the switch from mitosis to meiosis.

Human cloning 2001.

PubMed

Healy, David L; Weston, Gareth; Pera, Martin F; Rombauts, Luk; Trounson, Alan O

2002-05-01

This review summaries human cloning from a clinical perspective. Natural human clones, that is, monozygotic twins, are increasing in the general community. Iatrogenic human clones have been produced for decades in infertile couples given fertility treatment such as ovulation induction. A clear distinction must be made between therapeutic cloning using embryonic stem cells and reproductive cloning attempts. Unlike the early clinical years of in vitro fertilization, with cloning there is no animal model that is safe and dependable. Until there is such a model, 'Dolly'-style human cloning is medically unacceptable.

cDNA Clones with Rare and Recurrent Mutations Found in Cancers | Office of Cancer Genomics

Cancer.gov

The CTD2 Center at UT- MD Anderson Cancer Center has developed High-Throughput Mutagenesis and Molecular Barcoding (HiTMMoB)1,2 pipeline to construct mutant alleles open reading frame expression clones that are either recurrent or rare in cancers. These barcoded genes can be used for context-specific functional validation, detection of novel biomarkers (pathway activation) and targets (drug sensitivity).

Nonselective enrichment for yeast adenine mutants by flow cytometry

NASA Technical Reports Server (NTRS)

Bruschi, C. V.; Chuba, P. J.

1988-01-01

The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

Nitrogen fixation in transposon mutants from Bradyrhizobium japonicum USDA 110 impaired in nitrate reductase.

PubMed

Camacho, María; Burgos, Araceli; Chamber-Pérez, Manuel A

2003-04-01

Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants. From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules. Tn5 insertions were checked previously and mutants were arranged into four different groups. Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR. The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors. PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes. Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE). Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N. Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids. Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain. These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA.

Cloning and Characterization of the Lipooligosaccharide Galactosyltransferase II Gene of Haemophilus ducreyi

PubMed Central

Sun, Shuhua; Schilling, Birgit; Tarantino, Laurie; Tullius, Michael V.; Gibson, Bradford W.; Munson, Robert S.

2000-01-01

Haemophilus ducreyi is the etiologic agent of chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is considered to be a major virulence determinant and has been implicated in the adherence of H. ducreyi to keratinocytes. Strain A77, an isolate from the Paris collection, is serum sensitive, poorly adherent to fibroblasts, and deficient in microcolony formation. Structural analysis indicates that the LOS of strain A77 lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS as well as the sialic acid substitution. From an H. ducreyi 35000HP genomic DNA library, a clone complementing the defect in A77 was identified by immunologic screening with monoclonal antibody (MAb) 3F11, a MAb which recognizes the N-acetyllactosamine portion of strain 35000HP LOS. The clone contained a 4-kb insert that was sequenced. One open reading frame which encodes a protein with a molecular weight of 33,400 was identified. This protein has homology to glycosyltransferases of Haemophilus influenzae, Haemophilus somnus, Neisseria species, and Pasteurella haemolytica. The putative H. ducreyi glycosyltransferase gene was insertionally inactivated, and an isogenic mutant of strain 35000HP was constructed. The most complex LOS glycoform produced by the mutant has a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to that of the LOS of strain A77 and lacks the 3F11-binding epitope. Structural studies confirm that the most complex glycoform of the LOS isolated from the mutant lacks the galactose residue found in the N-acetyllactosamine portion of the strain 35000HP LOS. Although previously published data suggested that the serum-sensitive phenotype of A77 was due to the LOS mutation, we observed that the complemented A77 strain retained its serum-sensitive phenotype and that the galactosyltransferase mutant retained its serum-resistant phenotype. Thus, the serum sensitivity of strain A77 cannot be attributed to the

Characterization of the mutant spectra of a fish RNA virus within individual hosts during natural infections

USGS Publications Warehouse

Emmenegger, Eveline J.; Troyer, Ryan M.; Kurath, Gael

2003-01-01

Infectious hematopoietic necrosis virus (IHNV) is an RNA virus that causes significant mortalities of salmonids in the Pacific Northwest of North America. RNA virus populations typically contain genetic variants that form a heterogeneous virus pool, referred to as a quasispecies or mutant spectrum. This study characterized the mutant spectra of IHNV populations within individual fish reared in different environmental settings by RT–PCR of genomic viral RNA and determination of partial glycoprotein gene sequences of molecular clones. The diversity of the mutant spectra from ten in vivo populations was low and the average mutation frequencies of duplicate populations did not significantly exceed the background mutation level expected from the methodology. In contrast, two in vitro populations contained variants with an identical mutational hot spot. These results indicated that the mutant spectra of natural IHNV populations is very homogeneous, and does not explain the different magnitudes of genetic diversity observed between the different IHNV genogroups. Overall the mutant frequency of IHNV within its host is one of the lowest reported for RNA viruses.

In vitro capability of faropenem to select for resistant mutants of Streptococcus pneumoniae and Haemophilus influenzae.

PubMed

Kosowska-Shick, Klaudia; Clark, Catherine; Credito, Kim; Dewasse, Bonifacio; Beachel, Linda; Ednie, Lois; Appelbaum, Peter C

2008-02-01

When tested against nine strains of pneumococci and six of Haemophilus influenzae of various resistotypes, faropenem failed to select for resistant mutants after 50 days of consecutive subculture in subinhibitory concentrations. Faropenem also yielded low rates of spontaneous mutations against all organisms of both species. By comparison, resistant clones were obtained with macrolides, ketolides, and quinolones.

Impairment of Infectivity and Immunoprotective Effect of a LYT1 Null Mutant of Trypanosoma cruzi▿

PubMed Central

Zago, M. Paola; Barrio, Alejandra B.; Cardozo, Rubén M.; Duffy, Tomás; Schijman, Alejandro G.; Basombrío, Miguel A.

2008-01-01

Trypanosoma cruzi infection of host cells is a complex process in which many proteins participate but only a few of these proteins have been identified experimentally. One parasite factor likely to be involved is the protein product of LYT1, a single-copy gene cloned, sequenced, and characterized by Manning-Cela et al. (Infect. Immun. 69:3916-3923, 2001). This gene was potentially associated with infectivity, since the deletion of both LYT1 alleles in the CL Brenner strain (the wild type [WT]) resulted in a null mutant T. cruzi clone (L16) that shows an attenuated phenotype in cell culture models. The aim of this work was to characterize the infective behavior of L16 in the insect vector and murine models. The infection of adult Swiss mice with 103 trypomastigotes of both clones revealed a significant reduction in infective behavior of L16, as shown by direct parasitemia, spleen index, and quantitation of tissue parasite burden, suggesting the loss of virulence in the null mutant clone. Although L16 blood counts were almost undetectable, blood-based PCRs indicated the presence of latent and persistent infection during all of the study period and epimastigotes were reisolated from hemocultures until 12 months postinfection. Nevertheless, virulence was not restored in L16 by serial passages in mice, and reisolated parasites lacking the LYT1 gene and bearing the antibiotic resistance genes revealed the stability of the genetic manipulation. Histopathological studies showed a strong diminution in the muscle inflammatory response triggered by L16 compared to that triggered by the WT group, consistent with a lower tissue parasite load. A strong protection against a virulent challenge in both L16- and WT-infected mice was observed; however, the immunizing infection by the genetically modified parasite was highly attenuated. PMID:17938222

RESISTANCE AND CROSS-RESISTANCE OF ESCHERICHIA COLI S MUTANTS TO THE RADIOMIMETIC AGENT PROFLAVINE

PubMed Central

Woody-Karrer, Pearl; Greenberg, Joseph

1964-01-01

Woody-Karrer, Pearl (Palo Alto Medical Research Foundation, Palo Alto, Calif.), and Joseph Greenberg. Resistance and cross-resistance of Escherichia coli S mutants to the radiomimetic agent proflavine. J. Bacteriol. 87:536–542. 1964.—All 50 of the first-step mutants of Escherichia coli S selected for resistance to proflavine were resistant to ultraviolet light and each of five different radiomimetic chemicals. The mutants were classified into eight types on the basis of their relative resistance to six different radiomimetic drugs and on the basis of the shape of their ultraviolet survival curves. Three of these types are identical to types previously isolated with other radiomimetic drugs; five of the types are new. A high proportion of the clones surviving proflavine treatment were phenotypically but not genetically resistant, and no strains were isolated which were resistant to proflavine but were not resistant to radiation. PMID:14129667

To clone or not to clone--a Jewish perspective.

PubMed Central

Lipschutz, J H

1999-01-01

Many new reproductive methods such as artificial insemination, in vitro fertilisation, freezing of human embryos, and surrogate motherhood were at first widely condemned but are now seen in Western society as not just ethically and morally acceptable, but beneficial in that they allow otherwise infertile couples to have children. The idea of human cloning was also quickly condemned but debate is now emerging. This article examines cloning from a Jewish perspective and finds evidence to support the view that there is nothing inherently wrong with the idea of human cloning. A hypothesis is also advanced suggesting that even if a body was cloned, the brain, which is the essence of humanity, would remain unique. This author suggests that the debate should be changed from "Is cloning wrong?" to "When is cloning wrong?". PMID:10226913

RESISTANCE AND CROSS-RESISTANCE OF ESCHERICHIA COLI S MUTANTS TO THE RADIOMIMETIC AGENT PROFLAVINE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woody-Karrer, P.; Greenberg, J.

1964-03-01

All 50 of the first-step mutants of Escherichia coli S selected for resistance to proflavine were resistant to uv light and each of five different radiomimetic chemicals. The mutants were classified into eight types on the basis of their relative resistance to six different radiomimetic drugs and on the basis of the shape of their uv survival curves. Three of these types are identical to types previously isolated with other radiomimetic drugs; five of the types are new. A high proportion of the clones surviving proflavine treatment were phenotypically but not genetically resistant, and no strains were isolated which weremore » resistant to proflavine but were not resistant to radiation. (auth)« less

Cloning and sequence analysis of the invertase gene INV 1 from the yeast Pichia anomala.

PubMed

Pérez, J A; Rodríguez, J; Rodríguez, L; Ruiz, T

1996-02-01

A genomic library from the yeast Pichia anomala has been constructed and employed to clone the gene encoding the sucrose-hydrolysing enzyme invertase by complementation of a sucrose non-fermenting mutant of Saccharomyces cerevisiae. The cloned gene, INV1, was sequenced and found to encode a polypeptide of 550 amino acids which contained a 22 amino-acid signal sequence and ten potential glycosylation sites. The amino-acid sequence shows significant identity with other yeast invertases and also with Kluyveromyces marxianus inulinase, a yeast beta-fructofuranosidase which has a different substrate specificity. The nucleotide sequences of the 5' and 3' non-coding regions were found to contain several consensus motifs probably involved in the initiation and termination of gene transcription.

Study the Expression of ompf Gene in Esherichia coli Mutants.

PubMed

Jaktaji, R Pourahmad; Heidari, F

2013-09-01

The outer membrane porin proteins are the major factors in controlling the permeability of cell membrane. OmpF is an example of porin proteins in Esherichia coli. In normal growth condition a large amount of this protein is synthesised, but under stress condition, such as the presence of antibiotics in environment its expression is decreased inhibiting the entrance of antibiotics into cell. The expression of ompF is inhibited by antisense RNA transcribed from micF. In normal condition the expression of micF is low, but in the presence of antibiotics its expression is increased and causes multiple resistances to irrelevant antibiotics. The aims of this research were to study first, the intactness of micF and then quantify the expression of ompF in ciprofloxacin and tetracycline resistant mutants of E. coli. For this purpose the 5' end of micF was amplified and then sequenced. None of these mutants except one and its clone has a mutation in this gene. Then the relative expression of ompF in these mutants was quantified by real time PCR. There was no significant difference between ompF transcription of mutants and wild type strain. Based on this study and previous study it is concluded that low to intermediate levels of resistance to ciprofloxacin and tetracycline does not decrease ompF transcription.

Study the Expression of ompf Gene in Esherichia coli Mutants

PubMed Central

Jaktaji, R. Pourahmad; Heidari, F.

2013-01-01

The outer membrane porin proteins are the major factors in controlling the permeability of cell membrane. OmpF is an example of porin proteins in Esherichia coli. In normal growth condition a large amount of this protein is synthesised, but under stress condition, such as the presence of antibiotics in environment its expression is decreased inhibiting the entrance of antibiotics into cell. The expression of ompF is inhibited by antisense RNA transcribed from micF. In normal condition the expression of micF is low, but in the presence of antibiotics its expression is increased and causes multiple resistances to irrelevant antibiotics. The aims of this research were to study first, the intactness of micF and then quantify the expression of ompF in ciprofloxacin and tetracycline resistant mutants of E. coli. For this purpose the 5’ end of micF was amplified and then sequenced. None of these mutants except one and its clone has a mutation in this gene. Then the relative expression of ompF in these mutants was quantified by real time PCR. There was no significant difference between ompF transcription of mutants and wild type strain. Based on this study and previous study it is concluded that low to intermediate levels of resistance to ciprofloxacin and tetracycline does not decrease ompF transcription. PMID:24403654

Construction of Signature-tagged Mutant Library in Mesorhizobium loti as a Powerful Tool for Functional Genomics

PubMed Central

Shimoda, Yoshikazu; Mitsui, Hisayuki; Kamimatsuse, Hiroko; Minamisawa, Kiwamu; Nishiyama, Eri; Ohtsubo, Yoshiyuki; Nagata, Yuji; Tsuda, Masataka; Shinpo, Sayaka; Watanabe, Akiko; Kohara, Mitsuyo; Yamada, Manabu; Nakamura, Yasukazu; Tabata, Satoshi; Sato, Shusei

2008-01-01

Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29 330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G+C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology. PMID:18658183

Molecular cloning of the plasma membrane H(+)-ATPase from Kluyveromyces lactis: a single nucleotide substitution in the gene confers ethidium bromide resistance and deficiency in K+ uptake.

PubMed Central

Miranda, M; Ramírez, J; Peña, A; Coria, R

1995-01-01

A Kluyveromyces lactis strain resistant to ethidium bromide and deficient in potassium uptake was isolated. Studies on the proton-pumping activity of the mutant strain showed that a decreased H(+)-ATPase specific activity was responsible for the observed phenotypes. The putative K. lactis PMA1 gene encoding the plasma membrane H(+)-ATPase was cloned by its ability to relieve the potassium transport defect of this mutant and by reversing its resistance to ethidium bromide. Its deduced amino acid sequence predicts a protein 899 residues long that is structurally colinear in its full length to H(+)-ATPases cloned from different yeasts, except for the presence of a variable N-terminal domain. By PCR-mediated amplification, we identified a transition from G to A that rendered the substitution of the fully conserved methionine at position 699 by isoleucine. We attribute to this amino acid change the low capacity of the mutant H(+)-ATPase to pump out protons. PMID:7730265

A herpes simplex virus 2 glycoprotein D mutant generated by bacterial artificial chromosome mutagenesis is severely impaired for infecting neuronal cells and infects only Vero cells expressing exogenous HVEM.

PubMed

Wang, Kening; Kappel, Justin D; Canders, Caleb; Davila, Wilmer F; Sayre, Dean; Chavez, Mayra; Pesnicak, Lesley; Cohen, Jeffrey I

2012-12-01

We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine.

A Herpes Simplex Virus 2 Glycoprotein D Mutant Generated by Bacterial Artificial Chromosome Mutagenesis Is Severely Impaired for Infecting Neuronal Cells and Infects Only Vero Cells Expressing Exogenous HVEM

PubMed Central

Kappel, Justin D.; Canders, Caleb; Davila, Wilmer F.; Sayre, Dean; Chavez, Mayra; Pesnicak, Lesley; Cohen, Jeffrey I.

2012-01-01

We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine. PMID:22993162

Analysis of Lactobacillus sakei Mutants Selected after Adaptation to the Gastrointestinal Tracts of Axenic Mice▿ †

PubMed Central

Chiaramonte, Fabrizio; Anglade, Patricia; Baraige, Fabienne; Gratadoux, Jean-Jacques; Langella, Philippe; Champomier-Vergès, Marie-Christine; Zagorec, Monique

2010-01-01

We recently showed that Lactobacillus sakei, a natural meat-borne lactic acid bacterium, can colonize the gastrointestinal tracts (GIT) of axenic mice but that this colonization in the intestinal environment selects L. sakei mutants showing modified colony morphology (small and rough) and cell shape, most probably resulting from the accumulation of various mutations that confer a selective advantage for persistence in the GIT. In the present study, we analyzed such clones, issued from three different L. sakei strains, in order to determine which functions were modified in the mutants. In the elongated filamentous cells of the rough clones, transmission electron microscopy (TEM) analysis showed a septation defect and dotted and slanted black bands, suggesting the presence of a helical structure around the cells. Comparison of the cytoplasmic and cell wall/membrane proteomes of the meat isolate L. sakei 23K and of one of its rough derivatives revealed a modified expression for 38 spots. The expression of six oxidoreductases, several stress proteins, and four ABC transporters was strongly reduced in the GIT-adapted strain, while the actin-like MreB protein responsible for cell shaping was upregulated. In addition, the expression of several enzymes involved in carbohydrate metabolism was modified, which may correlate with the observation of modified growth of mutants on various carbon sources. These results suggest that the modifications leading to a better adaptation to the GIT are pleiotropic and are characterized in a rough mutant by a different stress status, a cell wall modification, and modified use of energy sources, leading to an improved fitness for the colonization of the GIT. PMID:20208026

Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum.

PubMed

Woodcock, M Ryan; Vaughn-Wolfe, Jennifer; Elias, Alexandra; Kump, D Kevin; Kendall, Katharina Denise; Timoshevskaya, Nataliya; Timoshevskiy, Vladimir; Perry, Dustin W; Smith, Jeramiah J; Spiewak, Jessica E; Parichy, David M; Voss, S Randal

2017-01-31

The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning-candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr a ) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr a has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr a significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

[Construction of Plasmodium falciparum signal peptide peptidase-GFP mutant and its expression analysis in the malaria parasite].

PubMed

Li, Xue-rong; Wu, Yin-juan; Shang, Mei; Li, Ye; Xu, Jin; Yu, Xin-bing; Athar, Chishti

2014-08-01

To construct recombinant plasmid pSPPcGT which contains signal peptide peptidase gene of Plasmodium falciparum (PJSPP) and GFP, and transfect into P. falciparum (3D7 strain) to obtain mutant parasites which can express PJSPP-GFP. Plasmodium falciparum(3D7 strain) genomic DNA was extracted from cultured malaria parasites. The C-terminal region of PJSPP, an 883 bp gene fragment was amplified by PCR, and then cloned into pPM2GT vector to get recombinant vector pSPPcGT. The recombinant vectors were identified by PCR, double restriction enzyme digestion and DNA sequencing. pSPPcGT vector was transfected into malaria parasites. The positive clones were selected by adding inhibitor of Plasmodium falciparum dihydrofolate reductase WR99210 to the culture medium. The pSPP-GFP-transfected parasites were fixed with methanol, stained with DAPI, and observed under immunofluorescence microscope. The PJSPP-GFP expression in P. falciparum was identified by SDS-PAGE and Western blotting. The C-terminal region of PJSPP was amplified from P.falciparum (3D7 strain) genomic DNA by PCR with the length of 883 bp. The constructed recombinant vectors were identified by PCR screening, double restriction enzyme digestion and DNA sequencing. The pSPPcGT vector was transfected into P. falciparum and the positive clones were selected by WR99210. GFP fluorescence was observed in transfected parasites by immunofluorescence microscopy, and mainly located in the cytoplasm. The PJSPP-GFP expression in malaria parasites was confirmed by Western blotting with a relative molecular mass of Mr 64,000. Recombinant vector PJSPP-GFP is constructed and transfected into P. falciparum to obtain P. falciparum mutant clone which can express PfSPP-GFP.

In Vitro Capability of Faropenem To Select for Resistant Mutants of Streptococcus pneumoniae and Haemophilus influenzae▿ †

PubMed Central

Kosowska-Shick, Klaudia; Clark, Catherine; Credito, Kim; Dewasse, Bonifacio; Beachel, Linda; Ednie, Lois; Appelbaum, Peter C.

2008-01-01

When tested against nine strains of pneumococci and six of Haemophilus influenzae of various resistotypes, faropenem failed to select for resistant mutants after 50 days of consecutive subculture in subinhibitory concentrations. Faropenem also yielded low rates of spontaneous mutations against all organisms of both species. By comparison, resistant clones were obtained with macrolides, ketolides, and quinolones. PMID:18086853

Identification and Cloning of gusA, Encoding a New β-Glucuronidase from Lactobacillus gasseri ADH†

PubMed Central

Russell, W. M.; Klaenhammer, T. R.

2001-01-01

The gusA gene, encoding a new β-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a β-glucuronidase gene cloned from a bacterial source other than Escherichia coli. A plasmid library of L. gasseri chromosomal DNA was screened for complementation of an E. coli gus mutant. Two overlapping clones that restored β-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to β-glucuronidase (GusA) of E. coli K-12 (EC 3.2.1.31). The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a β-glucuronidase-negative L. gasseri strain by expressing the gusA gene, subcloned onto a low-copy-number shuttle vector, from the strong Lactobacillus P6 promoter. GusA was also expressed in E. coli from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified β-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in other Lactobacillus species tested. PMID:11229918

Limited number of immunoglobulin VH regions expressed in the mutant rabbit "Alicia".

PubMed

DiPietro, L A; Short, J A; Zhai, S K; Kelus, A S; Meier, D; Knight, K L

1990-06-01

A unique feature of rabbit Ig is the presence of VH region allotypic specificities. In normal rabbits, more than 80% of circulating immunoglobulin molecules bear the VHa allotypic specificities, al, a2 or a3; the remaining 10% to 20% of immunoglobulin molecules lack VHa allotypic specificities and are designated VHa-. A mutant rabbit designated Alicia, in contrast, has predominantly serum immunoglobulin molecules that lack the VHa allotypic specificities (Kelus and Weiss, Proc. Natl. Acad. Sci. USA 1986. 83: 4883). To study the nature and molecular complexity of VHa- molecules, we cloned and determined the nucleotide sequence of seven cDNA prepared from splenic RNA of an Alicia rabbit. Six of the clones appeared to encode VHa- molecules; the framework regions encoded by these clones were remarkably similar to each other, each having an unusual insertion of four amino acids at position 10. This insertion of four amino acids has been seen in only 2 of 54 sequenced rabbit VH genes. The similarity of the sequences of the six VHa- clones to each other and their dissimilarity to most other VH genes leads us to suggest that the VHa- molecules in Alicia rabbits are derived predominantly from one or a small number of very similar VH genes. Such preferential utilization of a small number of VH genes may explain the allelic inheritance of VH allotypes.

A Clonal Genetic Screen for Mutants Causing Defects in Larval Tracheal Morphogenesis in Drosophila

PubMed Central

Baer, Magdalena M.; Bilstein, Andreas; Leptin, Maria

2007-01-01

The initial establishment of the tracheal network in the Drosophila embryo is beginning to be understood in great detail, both in its genetic control cascades and in its cell biological events. By contrast, the vast expansion of the system during larval growth, with its extensive ramification of preexisting tracheal branches, has been analyzed less well. The mutant phenotypes of many genes involved in this process are probably not easy to reveal, as these genes may be required for other functions at earlier developmental stages. We therefore conducted a screen for defects in individual clonal homozygous mutant cells in the tracheal network of heterozygous larvae using the mosaic analysis with a repressible cell marker (MARCM) system to generate marked, recombinant mitotic clones. We describe the identification of a set of mutants with distinct phenotypic effects. In particular we found a range of defects in terminal cells, including failure in lumen formation and reduced or extensive branching. Other mutations affect cell growth, cell shape, and cell migration. PMID:17603107

Rha1, a new mutant of Arabidopsis disturbed in root slanting, gravitropism and auxin physiology.

PubMed

Fortunati, Alessio; Piconese, Silvia; Tassone, Paola; Ferrari, Simone; Migliaccio, Fernando

2008-11-01

A new Arabidopsis mutant is characterized (rha1) that shows, in the roots, reduced right-handed slanting, reduced gravitropism and resistance to 2,4-D, TIBA, NPA and ethylene. It also shows reduced length in the shoot and root, reduced number of lateral roots and shorter siliques. The gene was cloned through TAIL-PCR and resulted in a HSF. Because none of the known gravitropic and auxinic mutants result from damage in a HSF, rha1 seems to belong to a new class of this group of mutants. Quantitative PCR analysis showed that the expression of the gene is increased by heat and cold shock, and by presence of 2,4-D in the media. Study of the expression through the GUS reporter gene revealed increased expression in clinostated and gravistimulated plants, but only in adult tissues, and not in the apical meristems of shoots and roots.

Rha1, a new mutant of Arabidopsis disturbed in root slanting, gravitropism and auxin physiology

PubMed Central

Fortunati, Alessio; Piconese, Silvia; Tassone, Paola; Ferrari, Simone

2008-01-01

A new Arabidopsis mutant is characterized (rha1) that shows, in the roots, reduced right-handed slanting, reduced gravitropism and resistance to 2,4-D, TIBA, NPA and ethylene. It also shows reduced length in the shoot and root, reduced number of lateral roots and shorter siliques. The gene was cloned through TAIL-PCR and resulted in a HSF. Because none of the known gravitropic and auxinic mutants result from damage in a HSF, rha1 seems to belong to a new class of this group of mutants. Quantitative PCR analysis showed that the expression of the gene is increased by heat and cold shock, and by presence of 2,4-D in the media. Study of the expression through the GUS reporter gene revealed increased expression in clinostated and gravistimulated plants, but only in adult tissues, and not in the apical meristems of shoots and roots. PMID:19704429

The Clone Factory

ERIC Educational Resources Information Center

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…

Binding-, intracellular transport-, and biosynthesis-defective mutants of vasopressin type 2 receptor in patients with X-linked nephrogenic diabetes insipidus.

PubMed Central

Tsukaguchi, H; Matsubara, H; Taketani, S; Mori, Y; Seido, T; Inada, M

1995-01-01

Nephrogenic diabetes insipidus (NDI) is most often an X-linked disorder in which urine is not concentrated due to renal resistance to arginine vasopressin. We recently identified four vasopressin type 2 receptor gene mutations in unrelated X-linked NDI families, including R143P, delta V278, R202C, and 804insG. All these mutations reduced ligand binding activity to < 10% of the normal without affecting mRNA accumulation. To elucidate whether the receptors are expressed on the cell surface, we analyzed biosynthesis and localization of tagged or untagged receptors stably expressed in Chinese hamster ovary (CHO) cells, using two antibodies directed against distinct termini. Whole-cell and surface labeling studies revealed that the R202C clone had both surface-localized (50-55 kD) and intracellular proteins (40 and 75 kD), similar to the wild-type AVPR2 clone, whereas the R143P and delta V278 clones lacked the surface receptors, despite relatively increased intracellular components. The 804insG mutant cell produced no proteins despite an adequate mRNA level. Immunofluorescence staining confirmed that the R202C mutant reaches the cell surface, whereas the R143P and delta V278 mutants are retained within the cytoplasmic compartment. Thus, R202C, R143P/delta V278, and 804insG result in three distinct phenotypes, that is, a simple binding impairment at the cell surface, blocked intracellular transport, and ineffective biosynthesis or/and accelerated degradation of the receptor, respectively, and therefore are responsible for NDI. This phenotypic classification will help understanding of molecular pathophysiology of this disorder. Images PMID:7560098

Dictyostelium discoideum mutants with temperature-sensitive defects in endocytosis

PubMed Central

1994-01-01

We have isolated and characterized temperature-sensitive endocytosis mutants in Dictyostelium discoideum. Dictyostelium is an attractive model for genetic studies of endocytosis because of its high rates of endocytosis, its reliance on endocytosis for nutrient uptake, and tractable molecular genetics. Endocytosis-defective mutants were isolated by a fluorescence-activated cell sorting (FACS) as cells unable to take up a fluorescent marker. One temperature-sensitive mutant (indy1) was characterized in detail and found to exhibit a complete block in fluid phase endocytosis at the restrictive temperature, but normal rates of endocytosis at the permissive temperature. Likewise, a potential cell surface receptor that was rapidly internalized in wild-type cells and indy1 cells at the permissive temperature was poorly internalized in indy1 under restrictive conditions. Growth was also completely arrested at the restrictive temperature. The endocytosis block was rapidly induced upon shift to the restrictive temperature and reversed upon return to normal conditions. Inhibition of endocytosis was also specific, as other membrane-trafficking events such as phagocytosis, secretion of lysosomal enzymes, and contractile vacuole function were unaffected at the restrictive temperature. Because recycling and transport to late endocytic compartments were not affected, the site of the defect's action is probably at an early step in the endocytic pathway. Additionally, indy1 cells were unable to proceed through the normal development program at the restrictive temperature. Given the tight functional and growth phenotypes, the indy1 mutant provides an opportunity to isolate genes responsible for endocytosis in Dictyostelium by complementation cloning. PMID:7929583

A Medicago truncatula tobacco retrotransposon insertion mutant collection with defects in nodule development and symbiotic nitrogen fixation.

PubMed

Pislariu, Catalina I; Murray, Jeremy D; Wen, JiangQi; Cosson, Viviane; Muni, RajaSekhara Reddy Duvvuru; Wang, Mingyi; Benedito, Vagner A; Andriankaja, Andry; Cheng, Xiaofei; Jerez, Ivone Torres; Mondy, Samuel; Zhang, Shulan; Taylor, Mark E; Tadege, Million; Ratet, Pascal; Mysore, Kirankumar S; Chen, Rujin; Udvardi, Michael K

2012-08-01

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod-), 51 mutants with totally ineffective nodules (Nod+ Fix-), 17 mutants with partially ineffective nodules (Nod+ Fix+/-), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/- Fix-), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/- Fix+), and 11 supernodulating mutants (Nod++Fix+/-). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN'T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod- lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging.

Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli.

PubMed

Triman, K; Becker, E; Dammel, C; Katz, J; Mori, H; Douthwaite, S; Yapijakis, C; Yoast, S; Noller, H F

1989-10-20

Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance alleles, originally identified by Morgan and co-workers, enable us to follow expression of cloned rRNA genes in vivo. Recessive mutations causing the loss of expression of the cloned 16 S rRNA gene were identified by the loss of the ability of cells to survive on media containing spectinomycin. The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis. We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature-sensitive spectinomycin resistance and two that produce unconditional loss of resistance. In each case, loss of ribosomal function can be accounted for by disruption of base-pairing in the secondary structure of 16 S rRNA. For the temperature-sensitive mutants, there is a lag period of about two generations between a shift to the restrictive temperature and cessation of growth, implying that the structural defects cause impairment of ribosome assembly.

Elucidating the genomic architecture of Asian EGFR-mutant lung adenocarcinoma through multi-region exome sequencing.

PubMed

Nahar, Rahul; Zhai, Weiwei; Zhang, Tong; Takano, Angela; Khng, Alexis J; Lee, Yin Yeng; Liu, Xingliang; Lim, Chong Hee; Koh, Tina P T; Aung, Zaw Win; Lim, Tony Kiat Hon; Veeravalli, Lavanya; Yuan, Ju; Teo, Audrey S M; Chan, Cheryl X; Poh, Huay Mei; Chua, Ivan M L; Liew, Audrey Ann; Lau, Dawn Ping Xi; Kwang, Xue Lin; Toh, Chee Keong; Lim, Wan-Teck; Lim, Bing; Tam, Wai Leong; Tan, Eng-Huat; Hillmer, Axel M; Tan, Daniel S W

2018-01-15

EGFR-mutant lung adenocarcinomas (LUAD) display diverse clinical trajectories and are characterized by rapid but short-lived responses to EGFR tyrosine kinase inhibitors (TKIs). Through sequencing of 79 spatially distinct regions from 16 early stage tumors, we show that despite low mutation burdens, EGFR-mutant Asian LUADs unexpectedly exhibit a complex genomic landscape with frequent and early whole-genome doubling, aneuploidy, and high clonal diversity. Multiple truncal alterations, including TP53 mutations and loss of CDKN2A and RB1, converge on cell cycle dysregulation, with late sector-specific high-amplitude amplifications and deletions that potentially beget drug resistant clones. We highlight the association between genomic architecture and clinical phenotypes, such as co-occurring truncal drivers and primary TKI resistance. Through comparative analysis with published smoking-related LUAD, we postulate that the high intra-tumor heterogeneity observed in Asian EGFR-mutant LUAD may be contributed by an early dominant driver, genomic instability, and low background mutation rates.

Mutant Cells That Do Not Respond to Interleukin-1 (IL-1) Reveal a Novel Role for IL-1 Receptor-Associated Kinase

PubMed Central

Li, Xiaoxia; Commane, Mairead; Burns, Carmel; Vithalani, Kalpa; Cao, Zhaodan; Stark, George R.

1999-01-01

Mutagenized human 293 cells containing an interleukin-1 (IL-1)-regulated herpes thymidine kinase gene, selected in IL-1 and gancyclovir, have yielded many independent clones that are unresponsive to IL-1. The four clones analyzed here carry recessive mutations and represent three complementation groups. Mutant A in complementation group I1 lacks IL-1 receptor-associated kinase (IRAK), while the mutants in the other two groups are defective in unknown components that function upstream of IRAK. Expression of exogenous IRAK in I1A cells (I1A-IRAK) restores their responsiveness to IL-1. Neither NFκB nor Jun kinase is activated in IL-1-treated I1A cells, but these responses are restored in I1A-IRAK cells, indicating that IRAK is required for both. To address the role of the kinase activity of IRAK in IL-1 signaling, its ATP binding site was mutated (K239A), completely abolishing kinase activity. In transfected I1A cells, IRAK-K239A was still phosphorylated upon IL-1 stimulation and, surprisingly, still complemented all the defects in the mutant cells. Therefore, IRAK must be phosphorylated by a different kinase, and phospho-IRAK must play a role in IL-1-mediated signaling that does not require its kinase activity. PMID:10373513

Transcription analysis of peloric mutants of Phalaenopsis orchids derived from tissue culture.

PubMed

Chen, Ya Huei; Tsai, Yi Jung; Huang, Jian Zhi; Chen, Fure Chyi

2005-08-01

Tissue culture has been widely used for mass propagation of Phalaenopsis. However, somaclonal variation occurred during micropropagation process posed a severe problem by affecting product quality. In this study, wild type and peloric flower buds of Phalaenopsis hybrids derived from flower stalk nodal culture were used for cDNA-RAPD and cDNA suppression subtractive hybridization analyses in order to study their genetic difference in terms of expressed sequence tags. A total of 209 ESTs from normal flower buds and 230 from mutants were sequenced. These ESTs sequences can be grouped into several functional categories involved in different cellular processes including metabolism, signal transduction, transcription, cell growth and division, protein synthesis, and protein localization, and into a subcategory of proteins with unknown function. Cymbidium mosaic virus transcript was surprisingly found expressed frequently in the peloric mutant of P. Little Mary. Real-time RT-PCR analysis on selected ESTs showed that in mutant flower buds, a bZIP transcription factor (TGA1a-like protein) was down-regulated, while up-regulated genes include auxin-regulated protein kinase, cyclophilin, and TCP-like genes. A retroelement clone was also preferentially expressed in the peloric mutant flowers. On the other hand, ESTs involved in DNA methylation, chromatin remodeling and post-transcriptional regulation, such as DNA methyltransferase, histone acetyltransferase, ERECTA, and DEAD/DEAH RNA helicase, were enriched in normal flower buds than the mutants. The enriched transcripts in the wild type indicate the down regulation of these transcripts in the mutants, and vice versa. The potential roles of the analyzed transcripts in the development of Phalaenopsis flowers are discussed.

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

PubMed

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.

Cloning of the Pichia anomala SEC61 gene and its expression in a Saccharomyces cerevisiae sec61 mutant.

PubMed

Ruíz, Teresa; De la Rosa, José M; Domínguez, Angel; Rodríguez, Luis

2003-05-01

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene.

Sharing mutants and experimental information prepublication using FgMutantDb (https://scabusa.org/FgMutantDb).

PubMed

Baldwin, Thomas T; Basenko, Evelina; Harb, Omar; Brown, Neil A; Urban, Martin; Hammond-Kosack, Kim E; Bregitzer, Phil P

2018-06-01

There is no comprehensive storage for generated mutants of Fusarium graminearum or data associated with these mutants. Instead, researchers relied on several independent and non-integrated databases. FgMutantDb was designed as a simple spreadsheet that is accessible globally on the web that will function as a centralized source of information on F. graminearum mutants. FgMutantDb aids in the maintenance and sharing of mutants within a research community. It will serve also as a platform for disseminating prepublication results as well as negative results that often go unreported. Additionally, the highly curated information on mutants in FgMutantDb will be shared with other databases (FungiDB, Ensembl, PhytoPath, and PHI-base) through updating reports. Here we describe the creation and potential usefulness of FgMutantDb to the F. graminearum research community, and provide a tutorial on its use. This type of database could be easily emulated for other fungal species. Published by Elsevier Inc.

Why Clone?

MedlinePlus

... have been cloned already, including two relatives of cattle called the guar and the banteng, mouflon sheep, ... are underway to clone agricultural animals, such as cattle and pigs, that are efficient producers of high- ...

Derivation of Mutants of Erwinia carotovora subsp. betavasculorum Deficient in Export of Pectolytic Enzymes with Potential for Biological Control of Potato Soft Rot

PubMed Central

Costa, José M.; Loper, Joyce E.

1994-01-01

Erwinia carotovora subsp. betavasculorum Ecb168 produces an antibiotic(s) that suppresses growth of the related bacterium Erwinia carotovora subsp. carotovora in culture and in wounds of potato tubers. Strain Ecb168 also produces and secretes pectolytic enzymes and causes a vascular necrosis and root rot of sugar beet. Genes (out) involved in secretion of pectolytic enzymes by Ecb168 were localized to two HindIII fragments (8.5 and 10.5 kb) of Ecb168 genomic DNA by hybridization to the cloned out region of E. carotovora subsp. carotovora and by complementation of Out- mutants of E. carotovora subsp. carotovora. Out- mutants of Ecb168, which did not secrete pectate lyase into the culture medium, were obtained when deletions internal to either HindIII fragment were introduced into the genome of Ecb168 through marker exchange mutagenesis. Out- mutants of Ecb168 were complemented to the Out+ phenotype by introduction of the corresponding cloned HindIII fragment. Out- mutants of Ecb168 were less virulent than the Out+ parental strain on potato tubers. Strain Ecb168 and Out- derivatives inhibited the growth of E. carotovora subsp. carotovora in culture, indicating that the uncharacterized antibiotic(s) responsible for antagonism was exported through an out-independent mechanism. Strain Ecb168 and Out- derivatives reduced the establishment of large populations of E. carotovora subsp. carotovora in wounds of potato tubers and suppressed tuber soft rot caused by E. carotovora subsp. carotovora. PMID:16349316

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

PubMed

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.

Generating an Open Reading Frame (ORF) Entry Clone and Destination Clone.

PubMed

Reece-Hoyes, John S; Walhout, Albertha J M

2018-01-02

This protocol describes using the Gateway recombinatorial cloning system to create an Entry clone carrying an open reading frame (ORF) and then to transfer the ORF into a Destination vector. In this example, BP recombination is used to clone an ORF from a cDNA source into the Donor vector pDONR 221. The ORF from the resulting Entry clone is then transferred into the Destination vector pDEST-15; the product (the Destination clone) will express the ORF as an amino-terminal GST-fusion. The technique can be used as a guide for cloning any other DNA fragment of interest-a promoter sequence or 3' untranslated region (UTR), for example-with substitutions of different genetic material such as genomic DNA, att sites, and vectors as required. The series of constructions and transformations requires 9-15 d, not including time that may be required for sequence confirmation, if desired/necessary. © 2018 Cold Spring Harbor Laboratory Press.

Identification and Characterization of an Arabidopsis thaliana Mutant lbt With High Tolerance to Boron Deficiency

PubMed Central

Huai, Zexun; Peng, Lishun; Wang, Sheliang; Zhao, Hua; Shi, Lei; Xu, Fangsen

2018-01-01

Boron (B) is an essential micronutrient of plants. In the present study, we characterized an Arabidopsis mutant lbt with significant low-boron tolerance that was identified based on our previous mapping of QTL for B efficiency in Arabidopsis. Multiple nutrient-deficiency analyses point out that lbt mutant is insensitive to only B-limitation stress. Compared with wild-type Col-0, the fresh weight, leaf area, root length and root elongation rate of lbt mutant were significantly improved under B deficiency during vegetative growth. lbt mutant also showed the improvements in plant height, branches and inflorescences compared with Col-0 during the reproductive stage under B limitation. Ultrastructure analysis of the leaves showed that starch accumulation in lbt mutant was significantly diminished compared with Col-0. Furthermore, there were no significant differences in the expression of transporter-related genes and B concentrations between Col-0 and lbt mutant under both normal B and low-B conditions. These results suggest that lbt mutant has a lower B demand than Col-0. Genetic analysis suggests that the low-B tolerant phenotype of lbt mutant is under the control of a monogenic recessive gene. Based on the high-density SNP linkage genetic map, only one QTL for low-B tolerance was mapped on chromosome 4 between 10.4 and 14.8 Mb. No any reported B-relative genes exist in the QTL interval, suggesting that a gene with unknown function controls the tolerance of lbt to B limitation. Taken together, lbt is a low-B tolerant mutant that does not depend on the uptake or transport of B and is controlled by a monogenic recessive gene mapped on chromosome 4, and cloning and functional analysis of LBT gene are expected to reveal novel mechanisms for plant resistance to B deficiency.

Isolation of parafluorophenylalanine-resistant mutants from HeLa cell cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yim, L.K.; Stuart, W.D.

This report describes a method to isolate temperature-conditional phenylalanine transport mutants from the transformed human cell line HeLa. Using ultraviolet light as a mutagenic agent and DL-parafluorophenylalanine (PFPA), a poisonous analogue of L-phenylalanine, as a selective agent, mutagenized cells were selected for survival in the presence of PFPA at a temperature of 39 degrees C. Survivors of the mutagenesis and selection procedures were removed from the culture dishes by trypsin and cloned at a temperature of 35 degrees C. Seven of these lines isolated demonstrated continued resistance to PFPA at 39 degrees C. These lines were tested for uptake ofmore » L-phenylalanine at an external concentration of 100 microM and for continued resistance to PFPA at two concentrations. Cells were tested at 35 and at 39 degrees C. The data were compared to those obtained for the parental HeLa cell line under identical conditions. The seven mutant cell lines demonstrated varying resistances to PFPA and varying levels of accumulation of L-phenylalanine when tested at 35 and 39 degrees C. Three mutant lines were additionally tested for L-phenylalanine tRNA charging levels and for transport of L-arginine. The lines had parental cell levels of tRNA charging and L-arginine transport which suggest that the induced genetic defect affects a specific L-phenylalanine transport system.« less

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

USGS Publications Warehouse

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

2001-01-01

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

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

PubMed Central

2012-01-01

Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches. PMID:22554201

Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe.

PubMed

Chen, Bo-Ruei; Hale, Devin C; Ciolek, Peter J; Runge, Kurt W

2012-05-03

Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches.

Zebrafish pit1 mutants lack three pituitary cell types and develop severe dwarfism.

PubMed

Nica, Gabriela; Herzog, Wiebke; Sonntag, Carmen; Hammerschmidt, Matthias

2004-05-01

The Pou domain transcription factor Pit-1 is required for lineage determination and cellular commitment processes during mammalian adenohypophysis development. Here we report the cloning and mutational analysis of a pit1 homolog from zebrafish. Compared with mouse, zebrafish pit1 starts to be expressed at a much earlier stage of adenohypophysis development. However, as in the mouse, expression is restricted to a subset of pituitary cell types, excluding proopiomelanocortin (pomc)-expressing cells (corticotropes, melanotropes) and possibly gonadotropes. We could identify two N-ethyl-N-nitrosourea-induced zebrafish pit1 null mutants. Most mutants die during larval stages, whereas survivors develop severe dwarfism. Mutant larvae lack lactotropes, somatotropes, and thyrotropes, although the adenohypophysis is of normal size, without any sign of increased apoptosis rates. Instead, mutant embryos initiate ectopic expression of pomc in pit1-positive cells, leading to an expansion of the Pomc lineage. Similarly, the number of gonadotropes seems increased, as indicated by the expression of gsualpha, a marker for thyrotropes and gonadotropes. In pit1 mutants, the total number of gsualpha-positive cells is normal despite the loss of gsualpha and tshbeta coexpressing cells. Together, these data suggest a transfating of the Pit1 lineage to the Pomc and possibly the gonadotroph lineages in the mutant, and a pomc- and gonadotropin-repressive role of Pit1 during normal zebrafish development. This is different from mouse, for which a repressive role of Pit-1 has only been reported for the gonadotropin Lhbeta, but not for Pomc. In sum, our data point to both conserved and class-specific aspects of Pit1 function during pituitary development in different vertebrate species.

Cloning and characterization of a Candida albicans gene homologous to fructose-1,6-bisphosphatase genes.

PubMed

De la Rosa, J M; Ruíz, T; Rodríguez, L

2000-12-01

By sequencing of the DNA adjacent to the Candida albicans SEC61 gene, an open reading frame encoding a polypeptide of 331 amino acids was found. The predicted protein showed a strong homology with the fructose-1,6-bisphosphatase [FbPase] from other organisms, and conserved regions included the catalytic motif found in all known FbPases. Although the cloned gene did not complement the growth failure of a Saccharomyces cerevisiae fbp1 mutant in media with gluconeogenic carbon sources, it was transcribed in the transformants in a fashion that indicates a partial repression by glucose. A similar control on the transcription of this gene and on FbPase activity was found in wild-type C. albicans, where the cloned gene (CaFBP1) was shown to be localized in a single chromosomal locus in the genome.

Quick and clean cloning.

PubMed

Thieme, Frank; Marillonnet, Sylvestre

2014-01-01

Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific products, the quick and clean (QC) cloning procedure was developed to clone specific products only. QC cloning is a ligation-independent cloning procedure that relies on the exonuclease activity of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. A specific feature of QC cloning is the use of vectors that contain a sequence called catching sequence that allows cloning specific products only. QC cloning is performed by a one-pot incubation of insert and vector in the presence of T4 DNA polymerase at room temperature for 10 min followed by direct transformation of the incubation mix in chemo-competent Escherichia coli cells.

Ethical issues in animal cloning.

PubMed

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.

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

PubMed

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.

A journey through horse cloning.

PubMed

Gambini, Andrés; Maserati, Marc

2017-01-01

Interest in equine somatic cell nuclear transfer technology has increased significantly since the first equid clones were produced in 2003. This is demonstrated by the multiple commercial equine cloning companies having produced numerous cloned equids to date; worldwide, more than 370 cloned horses have been produced in at least six different countries. Equine cloning can be performed using several different approaches, each with different rates of success. In this review we cover the history and applications of equine cloning and summarise the major scientific advances in the development of this technology in horses. We explain the advantages and disadvantages of different procedures to produce cloned equine embryos and describe the current status of equine clone commercialisation, along with observations of differences in regional breed association registration regulations.

The topsy-turvy cloning law.

PubMed

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.

Academic Cloning.

ERIC Educational Resources Information Center

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…

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.

PubMed

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, P<0.0001) and in non-cloned control pigs (r=0.9, P<0.0001). Shannon Weaver and principal component analysis (PCA) of the terminal restriction fragments (T-RFs) revealed no differences in the bacterial composition or variability of the fecal microbiota 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; P<0.02) and non cloned-control pigs (r=0.45; P<0.006), and negatively with the abundance of Bacteroidetes in cloned pigs (r=-0.33, P<0.04), but not in the non-cloned control pigs. The cloned pigs did not have reduced inter-individual variation as compared to non-cloned pigs in regard to their gut microbiota in neither the obese nor the lean state. Diet-induced obesity was associated with an

Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction

PubMed Central

Mochizuki, Nobuyoshi; Brusslan, Judy A.; Larkin, Robert; Nagatani, Akira; Chory, Joanne

2001-01-01

A plastid-derived signal plays an important role in the coordinated expression of both nuclear- and chloroplast-localized genes that encode photosynthesis-related proteins. Arabidopsis GUN (genomes uncoupled) loci have been identified as components of plastid-to-nucleus signal transduction. Unlike wild-type plants, gun mutants have nuclear Lhcb1 expression in the absence of chloroplast development. We observed a synergistic phenotype in some gun double-mutant combinations, suggesting there are at least two independent pathways in plastid-to-nucleus signal transduction. There is a reduction of chlorophyll accumulation in gun4 and gun5 mutant plants, and a gun4gun5 double mutant shows an albino phenotype. We cloned the GUN5 gene, which encodes the ChlH subunit of Mg-chelatase. We also show that gun2 and gun3 are alleles of the known photomorphogenic mutants, hy1 and hy2, which are required for phytochromobilin synthesis from heme. These findings suggest that certain perturbations of the tetrapyrrole biosynthetic pathway generate a signal from chloroplasts that causes transcriptional repression of nuclear genes encoding plastid-localized proteins. The comparison of mutant phenotypes of gun5 and another Mg-chelatase subunit (ChlI) mutant suggests a specific function for ChlH protein in the plastid-signaling pathway. PMID:11172074

Cloning and characterization of ftsZ and pyrF from the archaeon Thermoplasma acidophilum

NASA Technical Reports Server (NTRS)

Yaoi, T.; Laksanalamai, P.; Jiemjit, A.; Kagawa, H. K.; Alton, T.; Trent, J. D.

2000-01-01

To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced. In T. acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5'-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis. Both ftsZ and pyrF from T. acidophilum were expressed in Escherichia coli and formed functional proteins. FtsZ expression in wild-type E. coli resulted in the filamentous phenotype characteristic of ftsZ mutants. T. acidophilum pyrF expression in an E. coli mutant lacking pyrF complemented the mutation and rescued the strain. Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif. Copyright 2000 Academic Press.

Rapid one-step recombinational cloning

PubMed Central

Fu, Changlin; Wehr, Daniel R.; Edwards, Janice; Hauge, Brian

2008-01-01

As an increasing number of genes and open reading frames of unknown function are discovered, expression of the encoded proteins is critical toward establishing function. Accordingly, there is an increased need for highly efficient, high-fidelity methods for directional cloning. Among the available methods, site-specific recombination-based cloning techniques, which eliminate the use of restriction endonucleases and ligase, have been widely used for high-throughput (HTP) procedures. We have developed a recombination cloning method, which uses truncated recombination sites to clone PCR products directly into destination/expression vectors, thereby bypassing the requirement for first producing an entry clone. Cloning efficiencies in excess of 80% are obtained providing a highly efficient method for directional HTP cloning. PMID:18424799

Positional cloning of the sex-linked giant egg (Ge) locus in the silkworm, Bombyx mori.

PubMed

Fujii, T; Abe, H; Kawamoto, M; Banno, Y; Shimada, T

2015-04-01

The giant egg (Ge) locus is a Z-linked mutation that leads to the production of large eggs. Cytological observations suggest that an unusual translocation of a large fragment of the W chromosome bearing a putative egg size-determining gene, Esd, gave rise to giant egg mutants. However, there is currently no molecular evidence confirming either a W-Z translocation or the presence of Esd on the W chromosome. To elucidate the origin of giant egg mutants, we performed positional cloning. We observed that the Bombyx mori. orthologue of the human Phytanoyl-CoA dioxygenase domain containing 1 gene (PHYHD1) is disrupted in giant egg mutants. PHYHD1 is highly conserved in eukaryotes and is predicted to be a Fe(II) and 2-oxoglutarate-dependent oxygenase. Exon skipping in one of the two available Ge mutants is probably caused by the insertion of a non-long terminal repeat transposon into intron 4 in the vicinity of the 5' splice site. Segmental duplication in Ge(2) , an independent allele, was caused by unequal recombination between short interspersed elements inserted into introns 3 and 5. Our results indicate that (1) Bombyx PHYHD1 is responsible for the Ge mutants and that (2) the Ge locus is unrelated to the W-linked putative Esd. To our knowledge, this is the first report describing the phenotypic defects caused by mutations in PHYHD1 orthologues. © 2014 The Royal Entomological Society.

Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line

PubMed Central

Schromm, Andra B.; Lien, Egil; Henneke, Philipp; Chow, Jesse C.; Yoshimura, Atsutoshi; Heine, Holger; Latz, Eicke; Monks, Brian G.; Schwartz, David A.; Miyake, Kensuke; Golenbock, Douglas T.

2001-01-01

Somatic cell mutagenesis is a powerful tool for characterizing receptor systems. We reported previously two complementation groups of mutant cell lines derived from CD14-transfected Chinese hamster ovary–K1 fibroblasts defective in responses to bacterial endotoxin. Both classes of mutants expressed a normal gene product for Toll-like receptor (TLR)4, and fully responded to stimulation by tumor necrosis factor (TNF)-α or interleukin (IL)-1β. We identified the lesion in one of the complementation groups in the gene for MD-2, a putative TLR4 coreceptor. The nonresponder phenotype of this mutant was reversed by transfection with MD-2. Cloning of MD-2 from the nonresponder cell line revealed a point mutation in a highly conserved region resulting in a C95Y amino acid exchange. Both forms of MD-2 colocalized with TLR4 on the cell surface after transfection, but only the wild-type cDNA reverted the lipopolysaccharide (LPS) nonresponder phenotype. Furthermore, soluble MD-2, but not soluble MD-2C95Y, functioned to enable LPS responses in cells that expressed TLR4. Thus, MD-2 is a required component of the LPS signaling complex and can function as a soluble receptor for cells that do not otherwise express it. We hypothesize that MD-2 conformationally affects the extracellular domain of TLR4, perhaps resulting in a change in affinity for LPS or functioning as a portion of the true ligand for TLR4. PMID:11435474

Saccharomyces Cerevisiae Cho2 Mutants Are Deficient in Phospholipid Methylation and Cross-Pathway Regulation of Inositol Synthesis

PubMed Central

Summers, E. F.; Letts, V. A.; McGraw, P.; Henry, S. A.

1988-01-01

Five allelic Saccharomyces cerevisiae mutants deficient in the methylation of phosphatidylethanolamine (PE) have been isolated, using two different screening techniques. Biochemical analysis suggested that these mutants define a locus, designated CHO2, that may encode a methyltransferase. Membranes of cho2 mutant cells grown in defined medium contain approximately 10% phosphatidylcholine (PC) and 40-50% PE as compared to wild-type levels of 40-45% PC and 15-20% PE. In spite of this greatly altered phospholipid composition, cho2 mutant cells are viable in defined medium and are not auxotrophic for choline or other phospholipid precursors such as monomethylethanolamine (MME). However, analysis of yeast strains carrying more than one mutation affecting phospholipid biosynthesis indicated that some level of methylated phospholipid is essential for viability. The cho2 locus was shown by tetrad analysis to be unlinked to other loci affecting phospholipid synthesis. Interestingly, cho2 mutants and other mutant strains that produce reduced levels of methylated phospholipids are unable to properly repress synthesis of the cytoplasmic enzyme inositol-1-phosphate synthase. This enzyme was previously shown to be regulated at the level of mRNA abundance in response to inositol and choline in the growth medium. We cloned the CHO2 gene on a 3.6-kb genomic DNA fragment and created a null allele of cho2 by disrupting the CHO2 gene in vivo. The cho2 disruptant, like all other cho2 mutants, is viable, exhibits altered regulation of inositol biosynthesis and is not auxotrophic for choline or MME. PMID:3066687

[Nuclear transfer and therapeutic cloning].

PubMed

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.

A Medicago truncatula Tobacco Retrotransposon Insertion Mutant Collection with Defects in Nodule Development and Symbiotic Nitrogen Fixation1[W][OA

PubMed Central

Pislariu, Catalina I.; D. Murray, Jeremy; Wen, JiangQi; Cosson, Viviane; Muni, RajaSekhara Reddy Duvvuru; Wang, Mingyi; A. Benedito, Vagner; Andriankaja, Andry; Cheng, Xiaofei; Jerez, Ivone Torres; Mondy, Samuel; Zhang, Shulan; Taylor, Mark E.; Tadege, Million; Ratet, Pascal; Mysore, Kirankumar S.; Chen, Rujin; Udvardi, Michael K.

2012-01-01

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod−), 51 mutants with totally ineffective nodules (Nod+ Fix−), 17 mutants with partially ineffective nodules (Nod+ Fix+/−), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/− Fix−), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/− Fix+), and 11 supernodulating mutants (Nod++Fix+/−). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN’T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod− lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging. PMID:22679222

Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

PubMed

Reed, Matthew D; Wilder, Julie A; Mega, William M; Hutt, Julie A; Kuehl, Philip J; Valderas, Michelle W; Chew, Lawrence L; Liang, Bertrand C; Squires, Charles H

2015-01-01

Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg.

Geranylgeranyl diphosphate synthases from Scoparia dulcis and Croton sublyratus. cDNA cloning, functional expression, and conversion to a farnesyl diphosphate synthase.

PubMed

Kojima, N; Sitthithaworn, W; Viroonchatapan, E; Suh, D Y; Iwanami, N; Hayashi, T; Sankaw, U

2000-07-01

cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene producing plants, Scoparia dulcis and Croton sublyratus, were isolated using the homology-based polymerase chain reaction method. Both cloned genes showed high amino acid sequence homology (60-70%) to other plant GGPPSs and contained highly conserved aspartate-rich motifs. The obtained clones were functionally expressed in Escherichia coli and showed sufficient GGPPS activity to catalyze the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate to form geranylgeranyl diphosphate. To investigate the factor determining the product chain length of plant GGPPSs, S. dulcis GGPPS mutants in which either the small amino acids at the fourth and fifth positions before the first aspartate-rich motif (FARM) were replaced with aromatic amino acids or in which two additional amino acids in FARM were deleted were constructed. Both mutants behaved like FPPS-like enzymes and almost exclusively produced FPP when dimethylallyl diphosphate was used as a primer substrate, and failed to accept FPP as a primer substrate. These results indicate that both small amino acids at the fourth and fifth positions before FARM and the amino acid insertion in FARM play essential roles in product length determination in plant GGPPSs.

bdhA-patD operon as a virulence determinant, revealed by a novel large-scale approach for identification of Legionella pneumophila mutants defective for amoeba infection.

PubMed

Aurass, P; Pless, B; Rydzewski, K; Holland, G; Bannert, N; Flieger, A

2009-07-01

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular parasite of eukaryotic cells. In the environment, it colonizes amoebae. After being inhaled into the human lung, the bacteria infect and damage alveolar cells in a way that is mechanistically similar to the amoeba infection. Several L. pneumophila traits, among those the Dot/Icm type IVB protein secretion machinery, are essential for exploiting host cells. In our search for novel Legionella virulence factors, we developed an agar plate assay, designated the scatter screen, which allowed screening for mutants deficient in infecting Acanthamoeba castellanii amoebae. Likewise, an L. pneumophila clone bank consisting of 23,000 transposon mutants was investigated here, and 19 different established Legionella virulence genes, for example, dot/icm genes, were identified. Importantly, 70 novel virulence-associated genes were found. One of those is L. pneumophila bdhA, coding for a protein with homology to established 3-hydroxybutyrate dehydrogenases involved in poly-3-hydroxybutyrate metabolism. Our study revealed that bdhA is cotranscribed with patD, encoding a patatin-like protein of L. pneumophila showing phospholipase A and lysophospholipase A activities. In addition to strongly reduced lipolytic activities and increased poly-3-hydroxybutyrate levels, the L. pneumophila bdhA-patD mutant showed a severe replication defect in amoebae and U937 macrophages. Our data suggest that the operon is involved in poly-3-hydroxybutyrate utilization and phospholipolysis and show that the bdhA-patD operon is a virulence determinant of L. pneumophila. In summary, the screen for amoeba-sensitive Legionella clones efficiently isolated mutants that do not grow in amoebae and, in the case of the bdhA-patD mutant, also human cells.

Characterization and fine mapping of a light-dependent leaf lesion mimic mutant 1 in rice.

PubMed

Wang, Jing; Ye, Bangquan; Yin, Junjie; Yuan, Can; Zhou, Xiaogang; Li, Weitao; He, Min; Wang, Jichun; Chen, Weilan; Qin, Peng; Ma, Bintian; Wang, Yuping; Li, Shigui; Chen, Xuewei

2015-12-01

Plants that spontaneously produce lesion mimics or spots, without any signs of obvious adversity, such as pesticide and mechanical damage, or pathogen infection, are so-called lesion mimic mutants (lmms). In rice, many lmms exhibit enhanced resistance to pathogens, which provides a unique opportunity to uncover the molecular mechanism underlying lmms. We isolated a rice light-dependent leaf lesion mimic mutant 1 (llm1). Lesion spots appeared in the leaves of the llm1 mutant at the tillering stage. Furthermore, the mutant llm1 had similar agronomic traits to wild type rice. Trypan blue and diamiobenzidine staining analyses revealed that the lesion spot formation on the llm1 mutant was due to programmed cell death and reactive oxygen species. The chloroplasts were severely damaged in the llm1 mutant, suggesting that chloroplast damage was associated with the formation of lesion spots in llm1. More importantly, llm1 exhibited enhanced resistance to bacterial blight pathogens within increased expression of pathogenesis related genes (PRs). Using a map-based cloning approach, we delimited the LLM1 locus to a 121-kb interval between two simple sequence repeat markers, RM17470 and RM17473, on chromosome 4. We sequenced the candidate genes on the interval and found that a base mutation had substituted adenine phosphate for thymine in the last exon of LOC_Os04g52130, which led to an amino acid change (Asp(388) to Val) in the llm1 mutant. Our investigation showed that the putative coproporphyrinogen III oxidase (CPOX) encoded by LOC_Os04g52130 was produced by LLM1 and that amino acid Asp(388) was essential for CPOX function. Our study provides the basis for further investigations into the mechanism underlying lesion mimic initiation associated with LLM1. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

The tillering phenotype of the rice plastid terminal oxidase (PTOX) loss-of-function mutant is associated with strigolactone deficiency.

PubMed

Tamiru, Muluneh; Abe, Akira; Utsushi, Hiroe; Yoshida, Kakoto; Takagi, Hiroki; Fujisaki, Koki; Undan, Jerwin R; Rakshit, Sujay; Takaichi, Shinichi; Jikumaru, Yusuke; Yokota, Takao; Terry, Matthew J; Terauchi, Ryohei

2014-04-01

The significance of plastid terminal oxidase (PTOX) in phytoene desaturation and chloroplast function has been demonstrated using PTOX-deficient mutants, particularly in Arabidopsis. However, studies on its role in monocots are lacking. Here, we report cloning and characterization of the rice (Oryza sativa) PTOX1 gene. Using Ecotype Targeting Induced Local Lesions IN Genomes (EcoTILLING) and TILLING as forward genetic tools, we identified the causative mutation of an EMS mutant characterized by excessive tillering, semi-dwarfism and leaf variegation that corresponded to the PTOX1 gene. The tillering and semi-dwarf phenotypes of the ptox1 mutant are similar to phenotypes of known strigolactone (SL)-related rice mutants, and both phenotypic traits could be rescued by application of the synthetic SL GR24. The ptox1 mutant accumulated phytoene in white leaf sectors with a corresponding deficiency in β-carotene, consistent with the expected function of PTOX1 in promoting phytoene desaturase activity. There was also no accumulation of the carotenoid-derived SL ent-2'-epi-5-deoxystrigol in root exudates. Elevated concentrations of auxin were detected in the mutant, supporting previous observations that SL interaction with auxin is important in shoot branching control. Our results demonstrate that PTOX1 is required for both carotenoid and SL synthesis resulting in SL-deficient phenotypes in rice. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Ethical issues in livestock cloning.

PubMed

Thompson, P B

1999-01-01

Although cloning may eventually become an important technology for livestock production, four ethical issues must be addressed before the practice becomes widespread. First, researchers must establish that the procedure is not detrimental to the health or well-being of affected animals. Second, animal research institutions should evaluate the net social benefits to livestock producers by weighing the benefits to producers against the opportunity cost of research capacity lost to biomedical projects. Third, scientists should consider the indirect effects of cloning research on the larger ethical issues surrounding human cloning. Finally, the market structure for products of cloned animals should protect individual choice, and should recognize that many individuals find the prospect of cloning (or consuming cloned animals) repugnant. Analysis of these four issues is complicated by spurious arguments alleging that cloning will have a negative impact on environment and genetic diversity.

[Human cloning or cannibalism].

PubMed

Sokolowski, L M

2001-01-01

In this article I develop the idea presented in my previous work that human cloning would be of little practical use since almost any aim that one would like to attain by multiple cloning of a concrete man or a group of people, are unattainable or it might be achieved by easier, cheaper and more efficient traditional methods. For this reason cloning of a man is unlikely to occur on a larger scale and only few people will decide to clone themselves. In this sense no social effects of human cloning will be disastrous for the human population. Yet investigations in human genetics are very important since they may provide medical applications far more important than human cloning. It is argued that the main trend of modern medicine: organ transplantation from an alien donor, will become socially dangerous in near future since the number of donors will be drastically smaller than the number of potential patients waiting for transplantations. This in turn may cause social conflicts and a form of medical cannibalism may arise. These problems and conflicts will be avoided if organ transplantation from an alien donor is replaced by organ cloning, i.e. by transplanting an organ developed from the patient.

Recombination-assisted megaprimer (RAM) cloning

PubMed Central

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.

PubMed Central

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

Map-based cloning and characterization of Zea mays male sterility33 (ZmMs33) gene, encoding a glycerol-3-phosphate acyltransferase.

PubMed

Xie, Ke; Wu, Suowei; Li, Ziwen; Zhou, Yan; Zhang, Danfeng; Dong, Zhenying; An, Xueli; Zhu, Taotao; Zhang, Simiao; Liu, Shuangshuang; Li, Jinping; Wan, Xiangyuan

2018-06-01

Map-based cloning of maize ms33 gene showed that ZmMs33 encodes a sn-2 glycerol-3-phosphate acyltransferase, the ortholog of rice OsGPAT3, and it is essential for male fertility in maize. Genetic male sterility has been widely studied for its biological significance and commercial value in hybrid seed production. Although many male-sterile mutants have been identified in maize (Zea mays L.), it is likely that most genes that cause male sterility are unknown. Here, we report a recessive genetic male-sterile mutant, male sterility33 (ms33), which displays small, pale yellow anthers, and complete male sterility. Using a map-based cloning approach, maize GRMZM2G070304 was identified as the ms33 gene (ZmMs33). ZmMs33 encodes a novel sn-2 glycerol-3-phosphate acyltransferase (GPAT) in maize. A functional complementation experiment showed that GRMZM2G070304 can rescue the male-sterile phenotype of the ms33-6029 mutant. GRMZM2G070304 was further confirmed to be the ms33 gene via targeted knockouts induced by the clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system. ZmMs33 is preferentially expressed in the immature anther from the quartet to early-vacuolate microspore stages and in root tissues at the fifth leaf growth stage. Phylogenetic analysis indicated that ZmMs33 and OsGPAT3 are evolutionarily conserved for anther and pollen development in monocot species. This study reveals that the monocot-specific GPAT3 protein plays an important role in male fertility in maize, and ZmMs33 and mutants in this gene may have value in maize male-sterile line breeding and hybrid seed production.

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

PubMed

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

Optimally cloned binary coherent states

NASA Astrophysics Data System (ADS)

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.

Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant.

PubMed Central

Tsuchimoto, S; van der Krol, A R; Chua, N H

1993-01-01

We cloned a MADS-box gene, pMADS3, from Petunia hybrida, which shows high sequence homology to the Arabidopsis AGAMOUS and Antirrhinum PLENA. pMADS3 is expressed exclusively in stamens and carpels of wild-type petunia plants. In the petunia mutant blind, which shows homeotic conversions of corolla limbs into antheroid structures with pollen grains and small parts of sepals into carpelloid tissue, pMADS3 is expressed in all floral organs as well as in leaves. Ectopic expression of pMADS3 in transgenic petunia leads to phenocopies of the blind mutant, i.e., the formation of antheroid structures on limbs and carpelloid tissue on sepals. Transgenic tobacco plants that overexpress pMADS3 exhibit an even more severe phenotype, with the sepals forming a carpel-like structure encasing the interior floral organs. Our results identify BLIND as a negative regulator of pMADS3, which specifies stamens and carpels during petunia flower development. PMID:8104573

Recombination Is Responsible for the Increased Recovery of Drug-Resistant Mutants with Hypermutated Genomes in Resting Yeast Diploids Expressing APOBEC Deaminases

PubMed Central

Lada, Artem G.; Stepchenkova, Elena I.; Zhuk, Anna S.; Kliver, Sergei F.; Rogozin, Igor B.; Polev, Dmitrii E.; Dhar, Alok; Pavlov, Youri I.

2017-01-01

DNA editing deaminases (APOBECs) are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters (kataegis). We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH) leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive mutations induced by

Recombination Is Responsible for the Increased Recovery of Drug-Resistant Mutants with Hypermutated Genomes in Resting Yeast Diploids Expressing APOBEC Deaminases.

PubMed

Lada, Artem G; Stepchenkova, Elena I; Zhuk, Anna S; Kliver, Sergei F; Rogozin, Igor B; Polev, Dmitrii E; Dhar, Alok; Pavlov, Youri I

2017-01-01

DNA editing deaminases (APOBECs) are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters ( kataegis ). We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH) leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive mutations induced by

Three concepts of cloning in human beings.

PubMed

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.

A new mutant of Arabidopsis disturbed in its roots, right-handed slanting, and gravitropism defines a gene that encodes a heat-shock factor.

PubMed

Fortunati, A; Piconese, S; Tassone, P; Ferrari, S; Migliaccio, F

2008-01-01

A new mutant of Arabidopsis named rha1 is characterized and the gene involved cloned. In roots, the mutant shows minimal right-handed slanting, reduced gravitropic response, notable resistance to 2,4-D, but scarce resistance to IAA and NAA. The roots also show a clear resistance to the auxin transport inhibitors TIBA and NPA, and to ethylene. Other characteristics are a reduced number of lateral roots and reduced size of shoot and root in the seedlings. The gene, cloned through TAIL-PCR, was found to be a heat-shock factor that maps on chromosome 5, close to and above the RFLP marker m61. The rha1 structure, mRNA, and translation product are reported. Since, so far, no other gravitropic mutant has been described as mutated in a heat-shock factor, rha1 belongs to a new group of mutants disturbed in slanting, gravitropism, and auxin physiology. As shown through the RT-PCR analyses of its expression, the gene retains the function connected with heat shock. If the characteristics connected with auxin physiology are considered, however, it is also likely that the gene, as a transcription factor, could be involved in root circumnutation, gravitropic response, and hormonal control of differentiation. Since GUS staining under the gene promoter was localized mainly in the mature tissues, rha1 does not seem to be involved in the first steps of gravitropism, but is rather related to the general response to auxin. The alterations in slanting (mainly due to reduced chiral circumnutation) and gravitropism lead to the supposition that the two processes may have, at least in part, common origins.

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

PubMed

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

2013-02-01

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

Construction of broad-host-range cosmid cloning vectors: identification of genes necessary for growth of Methylobacterium organophilum on methanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

Four new cloning vectors have been constructed from the broad-host-range cloning vector pRK290. These vectors, pLA2901, pLA2905, pLA2910, and pLA2917, confer resistance to kanamycin and tetracycline. The latter two are cosmid derivatives of pLA2901. The new vectors can be mobilized into, and are stably maintained in, a variety of gram-negative bacteria. A Sau3A genomic bank of Methylobacterium organophilum strain xx DNA has been constructed in pLA2917, and complementation analysis, with a variety of mutants unable to grow on methanol, revealed at least five separate regions necessary for growth on methanol. Complementation analysis and Tn5 mutagenesis data suggest that at leastmore » three genes are responsible for expression of active methanol dehydrogenase.« less

Cloning and characterization of an RNase-related protein gene preferentially expressed in rice stems.

PubMed

Wei, Jun-Ya; Li, An-Ming; Li, Yin; Wang, Jing; Liu, Xiao-Bin; Liu, Liang-Shi; Xu, Zeng-Fu

2006-04-01

RNase-related proteins (RRPs) are S- and S-like RNase homologs lacking the active site required for RNase activity. Here we describe the cloning and characterization of the rice (Oryza sativa) RRP gene (OsRRP). A single copy of OsRRP occurs in the rice genome. OsRRP contains three introns and an open reading frame encoding 252 amino acids, with the replacement of two histidines involved in the active site of RNase by lysine and tyrosine respectively. OsRRP is preferentially expressed in stems of wild-type rice and is significantly down-regulated in an increased tillering dwarf mutant ext37.

Isolation and complementation analysis of 10 methanol oxidation mutant classes and identification of the methanol dehydrogenase structural gene of Methylobacterium sp. strain AM1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nunn, D.N.; Lidstrom, M.E.

A method has been developed for the direct selection of methanol oxidation mutants of the facultative methylotroph Methylobacterium sp. strain AM1 (formerly Pseudomonas sp. strain AM1). Using this direct selection technique, we have isolated mutants of Methylobacterium sp. strain AM1 that are no longer capable of growth on methanol but retain the ability to grow on methylamine. These methanol oxidation (Mox) mutants were complemented with a genomic clone bank of this organism constructed in the broad-host-range cosmid pVK100, and subcloning and Tn5 mutagenesis experiments have assigned the Mox mutants to 10 distinct complementation groups. Using an open reading frame beta-galactosidasemore » fusion vector and antibodies specific for Methylobacterium sp. strain AM1 methanol dehydrogenase, we have identified the methanol dehydrogenase structural gene and determined the direction of transcription. The results suggest that the synthesis and utilization of an active methanol dehydrogenase in this organism requires at least 10 different gene functions.« less

Lessons learned from cloning dogs.

PubMed

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.

PubMed

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.

Effects of Donor Fibroblast Cell Type and Transferred Cloned Embryo Number on the Efficiency of Pig Cloning

PubMed Central

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

2013-01-01

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

In vivo analysis of replication and immunogenicity of proviral clones of human T-lymphotropic virus type 1 with selective envelope surface-unit mutations

PubMed Central

Silverman, Lee R.; Phipps, Andrew J.; Montgomery, Andy; Fernandez, Soledad; Tsukahara, Tomonori; Ratner, Lee; Lairmore, Michael D.

2005-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell lymphoma/leukemia (ATL). The HTLV-1 envelope gene exhibits limited variability when examined from infected individuals, but has not been tested using infectious clones of the virus in animal models. In vitro assays indicate that HTLV-1 envelope (Env) Ser75Ile, Asn95Asp, and Asn195Asp surface unit (SU) mutants are able to replicate in and immortalize lymphocytes. Herein, we examined the effects of these Env mutants in rabbits inoculated with HTLV-1 immortalized ACH.75, ACH.95, or ACH.195 cell lines (expressing full-length molecular clones with the SU mutations) or the ACH.1 cell line (expressing wild-type SU). All rabbits became infected, and the fidelity of the mutations was maintained throughout the 8-week study. However, SU point mutations resulted in decreased antibody responses to viral group-associated antigen (Gag) and Env antigens. ACH.195 rabbits had a selective decreased antibody response to SU, and one ACH.195 rabbit had an antibody response to both HTLV-1 and HTLV-2 SUs. Some mutant inoculation groups had altered proviral loads. However, peripheral-blood mononuclear cell (PBMC) proviral loads did not correlate with antibody responses. Our data are the first to demonstrate that mutations in critical determinants of HTLV-1 Env SU altered antibody responses and proviral loads, but do not prevent viral replication in vivo. PMID:16046523

Isolation and characterization of pediocin AcH chimeric protein mutants with altered bactericidal activity.

PubMed

Miller, K W; Schamber, R; Osmanagaoglu, O; Ray, B

1998-06-01

A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an Escherichia coli malE plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14-20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains L. plantarum NCDO955, Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc mesenteroides Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to approximately 60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed approximately 2. 8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.

Identification of aflatoxin biosynthesis genes by genetic complementation in an Aspergillus flavus mutant lacking the aflatoxin gene cluster.

PubMed Central

Prieto, R; Yousibova, G L; Woloshuk, C P

1996-01-01

Aspergillus flavus mutant strain 649, which has a genomic DNA deletion of at least 120 kb covering the aflatoxin biosynthesis cluster, was transformed with a series of overlapping cosmids that contained DNA harboring the cluster of genes. The mutant phenotype of strain 649 was rescued by transformation with a combination of cosmid clones 5E6, 8B9, and 13B9, indicating that the cluster of genes involved in aflatoxin biosynthesis resides in the 90 kb of A. flavus genomic DNA carried by these clones. Transformants 5E6 and 20B11 and transformants 5E6 and 8B9 accumulated intermediate metabolites of the aflatoxin pathway, which were identified as averufanin and/or averufin, respectively.These data suggest that avf1, which is involved in the conversion of averufin to versiconal hemiacetal acetate, was present in the cosmid 13B9. Deletion analysis of 13B9 located the gene on a 7-kb DNA fragment of the cosmid. Transformants containing cosmid 8B9 converted exogenously supplied O-methylsterigmatocystin to aflatoxin, indicating that the oxidoreductase gene (ord1), which mediates the conversion of O-methylsterigmatocystin to aflatoxin, is carried by this cosmid. The analysis of transformants containing deletions of 8B9 led to the localization of ord1 on a 3.3-kb A. flavus genomic DNA fragment of the cosmid. PMID:8967772

Sharing mutants and experimental information prepublication using FgMutantDB

USDA-ARS?s Scientific Manuscript database

There has been no central location for storing generated mutants of Fusarium graminearum or for data associated with these mutants. Instead researchers relied on several independent, non-integrated databases. FgMutantDB was designed as a simple spreadsheet that is accessible globally on the web th...

A novel candidate gene for mouse and human preaxial polydactyly with altered expression in limbs of Hemimelic extra-toes mutant mice.

PubMed

Clark, R M; Marker, P C; Kingsley, D M

2000-07-01

Polydactyly is a common malformation of vertebrate limbs. In humans a major locus for nonsyndromic pre-axial polydactyly (PPD) has been mapped previously to 7q36. The mouse Hemimelic extra-toes (Hx) mutation maps to a homologous chromosome segment and has been proposed to affect a homologous gene. To understand the molecular changes underlying PPD, we used a positional cloning approach to identify the gene or genes disrupted by the Hx mutation and a closely linked limb mutation, Hammertoe (Hm). High resolution genetic mapping identified a small candidate interval for the mouse mutations located 1.2 cM distal to the Shh locus. The nonrecombinant interval was completely cloned in bacterial artificial chromosomes and searched for genes using a combination of exon trapping, sample sequencing, and mapping of known genes. Two novel genes, Lmbr1 and Lmbr2, are entirely within the candidate interval we defined genetically. The open reading frame of both genes is intact in mutant mice, but the expression of the Lmbr1 gene is dramatically altered in developing limbs of Hx mutant mice. The correspondence between the spatial and temporal changes in Lmbr1 expression and the embryonic onset of the Hx mutant phenotype suggests that the mouse Hx mutation may be a regulatory allele of Lmbr1. The human ortholog of Lmbr1 maps within the recently described interval for human PPD, strengthening the possibility that both mouse and human limb abnormalities are due to defects in the same highly conserved gene.

Cloning, killing, and identity.

PubMed Central

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

Twins: A cloning experience.

PubMed

Prainsack, Barbara; Spector, Tim D

2006-11-01

Drawing upon qualitative interviews with monozygotic (identical) twins sharing 100% of their genes, and with dizygotic (fraternal) twins and singletons as control groups, this paper explores what it means to be genetically identical. (The twins interviewed were from the TwinsUK register in London.) In the context of the ongoing debate on human reproductive cloning, it examines questions such as: To what extent do identical twins perceive their emotional and physical bond to be a result of their genetic makeup? What would they think if they had been deliberately created genetically identical? How would they feel about being genetically identical to a person who was born a few years earlier or later? First, our respondents ascribed no great significance to the role of genes in their understanding of what it means to be identical twins. Second, the opinion that human reproductive cloning would "interfere with nature", or "contradict God's will", was expressed by our respondents exclusively on the abstract level. The more our respondents were able to relate a particular invented cloning scenario to their own life-worlds, the lower the prevalence of the argument. Third, for all three groups of respondents, the scenario of having been born in one of the other groups was perceived as strange. Fourth, the aspect that our respondents disliked about cloning scenarios was the potential motives of the cloners. Without equating monozygotic twins directly with "clones", these results from "naturally" genetically identical individuals add a new dimension to what a future cloning situation could entail: The cloned person might possibly (a) perceive a close physical and emotional connection to the progenitor as a blessing; (b) suffer from preconceptions of people who regard physical likeness as a sign of incomplete individuality; and (c) perceive the idea of not having been born a clone of a particular person as unpleasant.

[Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants].

PubMed

Yue, Chen-Li; Shi, Jie-Ran; Shi, Chang-Hong; Zhang, Hai; Zhao, Lei; Zhang, Ting-Fen; Zhao, Yong; Xi, Li

2008-10-01

To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity. The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis. The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity. Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.

Map-based cloning and characterization of the novel yellow-green leaf gene ys83 in rice (Oryza sativa).

PubMed

Ma, Xiaozhi; Sun, Xiaoqiu; Li, Chunmei; Huan, Rui; Sun, Changhui; Wang, Yang; Xiao, Fuliang; Wang, Qian; Chen, Purui; Ma, Furong; Zhang, Kuan; Wang, Pingrong; Deng, Xiaojian

2017-02-01

Leaf-color mutants have been extensively studied in rice, and many corresponding genes have been identified up to now. However, leaf-color mutation mechanisms are diverse and still need further research through identification of novel genes. In the present paper, we isolated a leaf-color mutant, ys83, in rice (Oryza sativa). The mutant displayed a yellow-green leaf phenotype at seedling stage, and then slowly turned into light-green leaf from late tillering stage. In its yellow leaves, photosynthetic pigment contents significantly decreased and the chloroplast development was retarded. The mutant phenotype was controlled by a recessive mutation in a nuclear gene on the short arm of rice chromosome 2. Map-based cloning and sequencing analysis suggested that the candidate gene was YS83 (LOC_Os02g05890) encoding a protein containing 165 amino acid residues. Gene YS83 was expressed in a wide range of tissues, and its encoded protein was targeted to the chloroplast. In the mutant, a T-to-A substitution occurred in coding sequence of gene YS83, which caused a premature translation of its encoded product. By introduction of the wild-type gene, the ys83 mutant recovered to normal green-leaf phenotype. Taken together, we successfully identified a novel yellow-green leaf gene YS83. In addition, number of productive panicles per plant and number of spikelets per panicle only reduced by 6.7% and 7.6%, respectively, meanwhile its seed setting rate and 1000-grain weight (seed size) were not significantly affected in the mutant, so leaf-color mutant gene ys83 could be used as a trait marker gene in commercial hybrid rice production. Copyright © 2016. Published by Elsevier Masson SAS.

Cloning and sequence analysis of the LEU2 homologue gene from Pichia anomala.

PubMed

De la Rosa, J M; Pérez, J A; Gutiérrez, F; González, J M; Ruiz, T; Rodríguez, L

2001-11-01

The Pichia anomala LEU2 gene (PaLEU2) was isolated by complementation of a leu2 Saccharomyces cerevisiae mutant. The cloned gene also allowed growth of a Escherichia coli leuB mutant in leucine-lacking medium, indicating that it encodes a product able to complement the beta-isopropylmalate dehydrogenase deficiency of the mutants. The sequenced DNA fragment contains a complete ORF of 1092 bp, and the deduced polypeptide shares significant homologies with the products of the LEU2 genes from S. cerevisiae (84% identity) and other yeast species. A sequence resembling the GC-rich palindrome motif identified in the 5' region of S. cerevisiae LEU2 gene as the binding site for the transcription activating factor encoded by the LEU3 gene was found at the promoter region. In addition, upstream of the PaLEU2 the 3'-terminal half of a gene of the same orientation, encoding a homologue of the S. cerevisiae NFS1/SPL1 gene that encodes a mitochondrial cysteine desulphurase involved in both tRNA processing and mitochondrial metabolism, was found. The genomic organization of the PaNFS1-PaLEU2 gene pair is similar to that found in several other yeast species, including S. cerevisiae and Candida albicans, except that in some of them the LEU2 gene appears in the reverse orientation. Copyright 2001 John Wiley & Sons, Ltd.

Therapeutic cloning and reproductive liberty.

PubMed

Sparrow, Robert

2009-04-01

Concern for "reproductive liberty" suggests that decisions about embryos should normally be made by the persons who would be the genetic parents of the child that would be brought into existence if the embryo were brought to term. Therapeutic cloning would involve creating and destroying an embryo, which, if brought to term, would be the offspring of the genetic parents of the person undergoing therapy. I argue that central arguments in debates about parenthood and genetics therefore suggest that therapeutic cloning would be prima facie unethical unless it occurred with the consent of the parents of the person being cloned. Alternatively, if therapeutic cloning is thought to be legitimate, this undermines the case for some uses of reproductive cloning by implying that the genetic relation it establishes between clones and DNA donors does not carry the same moral weight as it does in cases of normal reproduction.

Animal cloning: problems and prospects.

PubMed

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

Localization of causal locus in the genome of the brown macroalga Ectocarpus: NGS-based mapping and positional cloning approaches

PubMed Central

Billoud, Bernard; Jouanno, Émilie; Nehr, Zofia; Carton, Baptiste; Rolland, Élodie; Chenivesse, Sabine; Charrier, Bénédicte

2015-01-01

Mutagenesis is the only process by which unpredicted biological gene function can be identified. Despite that several macroalgal developmental mutants have been generated, their causal mutation was never identified, because experimental conditions were not gathered at that time. Today, progresses in macroalgal genomics and judicious choices of suitable genetic models make mutated gene identification possible. This article presents a comparative study of two methods aiming at identifying a genetic locus in the brown alga Ectocarpus siliculosus: positional cloning and Next-Generation Sequencing (NGS)-based mapping. Once necessary preliminary experimental tools were gathered, we tested both analyses on an Ectocarpus morphogenetic mutant. We show how a narrower localization results from the combination of the two methods. Advantages and drawbacks of these two approaches as well as potential transfer to other macroalgae are discussed. PMID:25745426

Arabidopsis Brassinosteroid-Insensitive dwarf12 Mutants Are Semidominant and Defective in a Glycogen Synthase Kinase 3β-Like Kinase1

PubMed Central

Choe, Sunghwa; Schmitz, Robert J.; Fujioka, Shozo; Takatsuto, Suguru; Lee, Mi-Ok; Yoshida, Shigeo; Feldmann, Kenneth A.; Tax, Frans E.

2002-01-01

Mutants defective in the biosynthesis or signaling of brassinosteroids (BRs), plant steroid hormones, display dwarfism. Loss-of-function mutants for the gene encoding the plasma membrane-located BR receptor BRI1 are resistant to exogenous application of BRs, and characterization of this protein has contributed significantly to the understanding of BR signaling. We have isolated two new BR-insensitive mutants (dwarf12-1D and dwf12-2D) after screening Arabidopsis ethyl methanesulfonate mutant populations. dwf12 mutants displayed the characteristic morphology of previously reported BR dwarfs including short stature, short round leaves, infertility, and abnormal de-etiolation. In addition, dwf12 mutants exhibited several unique phenotypes, including severe downward curling of the leaves. Genetic analysis indicates that the two mutations are semidominant in that heterozygous plants show a semidwarf phenotype whose height is intermediate between wild-type and homozygous mutant plants. Unlike BR biosynthetic mutants, dwf12 plants were not rescued by high doses of exogenously applied BRs. Like bri1 mutants, dwf12 plants accumulated castasterone and brassinolide, 43- and 15-fold higher, respectively, providing further evidence that DWF12 is a component of the BR signaling pathway that includes BRI1. Map-based cloning of the DWF12 gene revealed that DWF12 belongs to a member of the glycogen synthase kinase 3β family. Unlike human glycogen synthase kinase 3β, DWF12 lacks the conserved serine-9 residue in the auto-inhibitory N terminus. In addition, dwf12-1D and dwf12-2D encode changes in consecutive glutamate residues in a highly conserved TREE domain. Together with previous reports that both bin2 and ucu1 mutants contain mutations in this TREE domain, this provides evidence that the TREE domain is of critical importance for proper function of DWF12/BIN2/UCU1 in BR signal transduction pathways. PMID:12428015

Quantum cloning by cellular automata

NASA Astrophysics Data System (ADS)

D'Ariano, G. M.; Macchiavello, C.; Rossi, M.

2013-03-01

We introduce a quantum cellular automaton that achieves approximate phase-covariant cloning of qubits. The automaton is optimized for 1→2N economical cloning. The use of the automaton for cloning allows us to exploit different foliations for improving the performance with given resources.

Isolation and characterization of two overlapping cosmid clones from the 4q35 region, near the facioscapulohumeral muscular dystrophy locus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deidda, G.; Grisanti, P.; Vigneti, E.

1994-09-01

The gene for facioscapulohumeral muscular dystrophy (FSHD) has been localized by linkage analysis to the 4q35 region. The most telomeric p13E-11 prove has been shown to detect 4q35 DNA rearrangements in both sporadic and familial cases of the disease. With the aim of constructing a detailed physical map of the 4q35 region and searching for the mutant gene, we used p13E-11 probe to isolate cosmid clones from a human genomic library in a pCos-EMBL 2 vector. Two positive clones were isolated, clones 3 and 5, which partially overlap and carry human genomic inserts of 42 and 45 kb, respectively. Themore » cosmids share a common region containing the p13E-11 region and a stretch of KpnI units consisting of 3.2 kb tandemly repeated sequences (about 10). The restriction maps were constructed using the following enzymes: Bam HI, BgIII, Eco RI, EcoRV, KpnI and Sfi I. Clone 3 extends 4 kb upstream of C5 and stops within the Kpn repeats. Clone 5 extends 4 kb downstream from the Kpn repeats and it presents an additional EcoRI site. Clone 5 contains a stretch of Kpn sequences of nearly 32 kb, corresponding to 10 Kpn repeats; clone 3 contains a stretch of 29 kb corresponding to 9 Kpn repeats, as determined by PFGE analysis of partial digestion of the clones. Clone 5 seems to contain the entire Eco RI region prone to rearrangements in FSHD patients. From clone 5 several subclones were obtained, from the Kpn region and from the region spanning from the last Kpn repeat to the cloning site. No single copy sequences were detected. Subclones from the 3{prime} end region contain beta-satellite or Sau3A-like sequences. In situ hybridization with the whole C5 cosmid shows hybridization signals at the tip of chromosome 4 (4q35) and chromosome 10 (10q26), in the pericentromeric region of chromosome 1 (1q12) and in the p12 region of the acrocentric chromosomes (chr. 21, 22, 13, 14, 15).« less

Gene I mutants of peanut chlorotic streak virus, a caulimovirus, replicate in plants but do not move from cell to cell.

PubMed Central

Ducasse, D A; Mushegian, A R; Shepherd, R J

1995-01-01

Gene I of peanut chlorotic streak virus (PCISV), a caulimovirus, is homologous to gene I of other caulimoviruses and may encode a protein for virus movement. To evaluate the function of gene I, several mutations were created in this gene of an infectious, partially redundant clone of PCISV. Constructs with an in-frame deletion and a single amino acid substitution in gene I were not infectious. To test for replication of these mutants in primarily infected cells, an immunosorbent PCR technique was devised. Virus particles formed by mutants in plants were recovered by binding to antivirus antibodies on a solid matrix and DNase treated to discriminate against residual inoculum, and DNA of trapped virions was subjected to PCR amplification. Gene I mutants were shown to direct formation of encapsidated DNA as revealed by a PCR product. Control gene V mutants (reverse transcriptase essential for replication) did not yield a PCR product. Quantitative PCR allowed estimation of the proportion of cells initially infected by gene I mutants and the amount of extractable virus per cell. It is concluded that PCISV gene I encodes a movement protein and that the immunoselection-PCR technique is useful in studying subliminal virus infection in plants. PMID:7543587

Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1.

PubMed

Agarwal, Shubhra; Baccam, Prasith; Aggarwal, Rakesh; Veerapu, Naga Suresh

2018-02-15

Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 ( ORF1 ) or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10 -3 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases. IMPORTANCE Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud in vitro We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a single-stranded (+)-RNA [ss(+) RNA] virus, with various and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effects of cloud size and

Cloning goes to the movies.

PubMed

Cormick, Craig

2006-10-01

Public attitude research conducted by Biotechnology Australia shows that one of the major sources of information on human reproductive cloning is movies. Traditionally, understanding of new and emerging technologies has come through the mass media but human cloning, being so widely addressed through the popular culture of movies, is more effectively defined by Hollywood than the news media or science media. But how well are the science and social issues of cloning portrayed in box office hits such as The Island, Multiplicity, Star Wars: Attack of the Clones and Jurassic Park? These movies have enormous reach and undoubted influence, and are therefore worth analyzing in some detail. This study looks at 33 movies made between 1971 and 2005 that address human reproductive cloning, and it categorizes the films based on their genre and potential influence. Yet rather than simply rating the quality of the science portrayed, the study compares the key messages in these movies with public attitudes towards cloning, to examine the correlations.

[Therapeutic cloning in debate].

PubMed

de Wert, G

2001-11-03

Human embryos can be conceived by cell nuclear transfer in order to isolate human embryonic stem cells (hES cells) for research into autologous cell therapy (therapeutic cloning). However, this technique broaches the major ethical problem concerning the instrumental use of human preimplantation embryos. From the viewpoint of subsidiarity, it is argued that various potential alternatives for therapeutic cloning should first be investigated further. The question as to whether therapeutic cloning should be allowed only becomes apparent when research with surplus embryos obtained in the course of in-vitro fertilization suggests that usable transplants can be obtained in vitro from hES cells, and when the potential alternatives for therapeutic cloning are either less promising or need more time for development than is currently expected.

Molecular cloning and characterization of a tomato cDNA encoding a systemically wound-inducible bZIP DNA-binding protein

NASA Technical Reports Server (NTRS)

Stankovic, B.; Vian, A.; Henry-Vian, C.; Davies, E.

2000-01-01

Localized wounding of one leaf in intact tomato (Lycopersicon esculentum Mill.) plants triggers rapid systemic transcriptional responses that might be involved in defense. To better understand the mechanism(s) of intercellular signal transmission in wounded tomatoes, and to identify the array of genes systemically up-regulated by wounding, a subtractive cDNA library for wounded tomato leaves was constructed. A novel cDNA clone (designated LebZIP1) encoding a DNA-binding protein was isolated and identified. This clone appears to be encoded by a single gene, and belongs to the family of basic leucine zipper domain (bZIP) transcription factors shown to be up-regulated by cold and dark treatments. Analysis of the mRNA levels suggests that the transcript for LebZIP1 is both organ-specific and up-regulated by wounding. In wounded wild-type tomatoes, the LebZIP1 mRNA levels in distant tissue were maximally up-regulated within only 5 min following localized wounding. Exogenous abscisic acid (ABA) prevented the rapid wound-induced increase in LebZIP1 mRNA levels, while the basal levels of LebZIP1 transcripts were higher in the ABA mutants notabilis (not), sitiens (sit), and flacca (flc), and wound-induced increases were greater in the ABA-deficient mutants. Together, these results suggest that ABA acts to curtail the wound-induced synthesis of LebZIP1 mRNA.

[Cloning: applications in humans 2. Ethical considerations].

PubMed

de Wert, G M; Geraedts, J P

2001-05-01

Reproductive cloning in adults/children evokes unfavourable reactions. Direct objections are that cloning is unnatural, that it affects human dignity and violates the individual's right to genetic uniqueness. Consequential objections concern unjustified health risks for the progeny, unjustified psychosocial risks for the clone child and the risk of cloning for eugenetic purposes. There is consensus that reproductive cloning of existing persons is unjustify as yet because of the health risks for the offspring. Reproductive cloning of embryos is possible by means of nucleus transplantation and of embryo splitting. The ethical analysis of reproductive cloning of embryos depends on the purposes and applications. At least some of the moral objections against cloning of adults/children are not or not completely applicable to reproductive cloning of embryos. Conditions to be put to reproductive cloning of embryos are efficacy, safety and, at least for the time being, avoidance of asynchrony in transferring identical embryos. The ethical aspects of its application in the context of genetical reproductive techniques must be evaluated separately. Therapeutic cloning may be acceptable if alternatives are lacking.

[Cloning: applications in humans. II. Ethical considerations].

PubMed

de Wert, G M; Geraedts, J P

2000-05-13

Reproductive cloning in adults/children evokes unfavourable reactions. Direct objections are that cloning is unnatural, that it affects human dignity and violates the individual's right to genetic uniqueness. Consequential objections concern unjustified health risks for the progeny, unjustified psychosocial risks for the clone child and the risk of cloning for eugenetic purposes. There is consensus that reproductive cloning of existing persons is unjustifiable as yet because of the health risks for the offspring. Reproductive cloning of embryos is possible by means of nucleus transplantation and of embryo splitting. The ethical analysis of reproductive cloning of embryos depends on the purposes and applications. At least some of the moral objections against cloning of adults/children are not or not completely applicable to reproductive cloning of embryos. Conditions to be put to reproductive cloning of embryos are efficacy, safety and, at least for the time being, avoidance of asynchrony in transferring identical embryos. The ethical aspects of its application in the context of genetical reproductive techniques must be evaluated separately. Therapeutic cloning may be acceptable if alternatives are lacking.

CRISPR-Cas9-Edited Site Sequencing (CRES-Seq): An Efficient and High-Throughput Method for the Selection of CRISPR-Cas9-Edited Clones.

PubMed

Veeranagouda, Yaligara; Debono-Lagneaux, Delphine; Fournet, Hamida; Thill, Gilbert; Didier, Michel

2018-01-16

The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells). Here we described a next-generation sequencing method called CRISPR-Cas9 Edited Site Sequencing (CRES-Seq) for the efficient and high-throughput screening of CRISPR-Cas9-edited clones. CRES-Seq facilitates the precise genotyping up to 96 CRISPR-Cas9-edited sites (CRES) in a single MiniSeq (Illumina) run with an approximate sequencing cost of $6/clone. CRES-Seq is particularly useful when multiple genes are simultaneously targeted by CRISPR-Cas9, and also for screening of clones generated from multiallelic genes/polyploid cells. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Human cloning: can it be made safe?

PubMed

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?

Isolation and genetic study of p-fluoro-DL-phenylalanine-resistant mutants overproducing beta-phenethyl-alcohol in Saccharomyces cerevisiae.

PubMed

Fukuda, K; Watanabe, M; Asano, K; Ouchi, K; Takasawa, S

1991-12-01

p-Fluoro-DL-phenylalanine (PFP)-resistant mutants which produce a large amount of beta-phenethyl-alcohol, a rose-like flavor component, were isolated from the isogenic strains X2180-1A and X2180-1B of Saccharomyces cerevisiae. Cells of these mutants accumulated phenylalanine and tryptophan more than 3-fold times that of wild-type cells, while they accumulated less than half the tyrosine. The activity of prephenate dehydrogenase (PDG) (EC 1.3.1.12) was markedly decreased while that of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) was increased. Genetic analysis revealed that the mutation occurred at the TYR1 locus, encoding PDG, and that the mutated TYR1 gene, try1-pfp, caused both PFP resistance and beta-phenethyl-alcohol overproduction. This was supported by molecular genetic studies with cloned tyr1-pfp DNA.

Agro-economic impact of cattle cloning.

PubMed

Faber, D C; Ferre, L B; Metzger, J; Robl, J M; Kasinathan, P

2004-01-01

The purpose of this paper is to review the economic and social implications of cloned cattle, their products, and their offspring as related to production agriculture. Cloning technology in cattle has several applications outside of traditional production agriculture. These applications can include bio-medical applications, such as the production of pharmaceuticals in the blood or milk of transgenic cattle. Cloning may also be useful in the production of research models. These models may or may not include genetic modifications. Uses in agriculture include many applications of the technology. These include making genetic copies of elite seed stock and prize winning show cattle. Other purposes may range from "insurance" to making copies of cattle that have sentimental value, similar to cloning of pets. Increased selection opportunities available with cloning may provide for improvement in genetic gain. The ultimate goal of cloning has often been envisioned as a system for producing quantity and uniformity of the perfect dairy cow. However, only if heritability were 100%, would clone mates have complete uniformity. Changes in the environment may have significant impact on the productivity and longevity of the resulting clones. Changes in consumer preferences and economic input costs may all change the definition of the perfect cow. The cost of producing such animals via cloning must be economically feasible to meet the intended applications. Present inefficiencies limit cloning opportunities to highly valued animals. Improvements are necessary to move the applications toward commercial application. Cloning has additional obstacles to conquer. Social and regulatory acceptance of cloning is paramount to its utilization in production agriculture. Regulatory acceptance will need to address the animal, its products, and its offspring. In summary, cloning is another tool in the animal biotechnology toolbox, which includes artificial insemination, sexing of semen, embryo

Cloning, Site-Directed Mutagenesis, and Functional Analysis of Active Residues in Lymantria dispar Chitinase.

PubMed

Fan, Xiao-Jun; Yang, Chun; Zhang, Chang; Ren, Hui; Zhang, Jian-Dong

2018-01-01

Chitinases are glycosyl hydrolases that catalyze the hydrolysis of β-(1,4)-glycosidic bonds in chitin, the major structural polysaccharide presented in the cuticle and gut peritrophic matrix of insects. Two aspartate residues (D143, D145) and one tryptophan (W146) in the Lymantria dispar chitinase are highly conserved residues observed within the second conserved motif of the family 18 chitinase catalytic region. In this study, a chitinase cDNA, LdCht5, was cloned from L. dispar, and the roles of the three residues were investigated using site-directed mutagenesis and substituting them with three other amino acids. Seven mutant proteins, D143E, D145E, W146G, D143E/D145E, D143E/W146G, D145E/W146G, and D143E/D145E/W146G, as well as the wild-type enzyme, were produced using the baculovirus-insect cell line expression system. The enzymatic and kinetic properties of these mutant enzymes were measured using the oligosaccharide substrate MU-(GlcNAc) 3 . Among the seven mutants, the D145E, D143E/D145E, and D145E/W146G mutations kept some extant catalytic activity toward MU-(GlcNAc) 3 , while the D143E, W146G, D143E/W146G, and D143E/D145E/W146G mutant enzymes were inactivated. Compared with the mutant enzymes, the wild-type enzyme had higher values of k cat and k cat / K m . A study of the multiple point mutations in the second conserved catalytic region would help to elucidate the role of the critical residues and their relationships.

Wildlife conservation and reproductive cloning.

PubMed

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.

Statement on Human Cloning

MedlinePlus

... form Search American Association for the Advancement of Science Statement on Human Cloning Tweet The American Association ... to implant a human cloned embryo for the purpose of reproduction. The scientific evidence documenting the serious ...

Analyses of spontaneous pink mutant events in the stamen hairs of Tradescantia clone BNL 4430 cultivated in a nutrient solution circulating growth chamber.

PubMed

Ichikawa, S; Wushur, S

2000-12-20

In order to obtain more fundamental data on Tradescantia clone BNL 4430, one of the most suitable testers for environmental mutagens, the occurrences of spontaneous somatic pink mutations in the stamen hairs were scored for 52 weeks from 12 December 1998 to 10 December 1999, cultivating the young inflorescence-bearing shoots with roots in a nutrient solution circulating (NSC) growth chamber. The environmental conditions in the chamber were 22.0+/-0.5 degrees C during the 16h day with the light intensity of 7.5klx from white fluorescent tubes, and 20.0+/-0.5 degrees C at night. During the scoring period, 697,443 stamen hairs with an average cell number of 25.36 were observed and 2642 pink mutant events (PMEs) were detected. The overall spontaneous mutation frequency was 1.56+/-0.03 PMEs per 10(4) hair-cell divisions, and the frequency was significantly lower in May, July and August and significantly higher in November and December. By analyzing the sectoring patterns of 1856 PMEs (70.25% of PMEs detected), the most of 172 cases of multiple (two to five) pink sectors observed in the same hairs (scored as 232 PMEs for calculating mutation frequency) were found to be the results of events involving somatic recombinations occurred in single cells or cell lineages, rather than those of two or more independent somatic mutations occurred in different cells. This finding clearly shows the significance of somatic recombinations in producing such multiple sectors (382 sectors in total) which occupied 19.0% of the 2006 pink sectors in total analyzed. Somatic recombinations were considered to be playing a significant role also in producing single PMEs in the stamen hairs.

A GA-insensitive dwarf mutant of Brassica napus L. correlated with mutation in pyrimidine box in the promoter of GID1.

PubMed

Li, Huapeng; Wang, Yun; Li, Xiaocheng; Gao, Yong; Wang, Zhijun; Zhao, Yun; Wang, Maolin

2011-01-01

A dwarf mutant from Brassica napus, namely NDF-1, which was derived from a high doubled haploid (DH) line '3529'(Brassica napus L.) of which seeds were jointly treated with chemical inducers and fast neutron bombardment, was revealed that dwarfism is under the control of a major gene(designated as ndf1) with a mainly additive effect and non-significant dominance effect. The germination and hypocotyls elongation response of dwarf mutants after exogenous GA and uniconazol application showed NDF-1 was a gibberellin insensitive dwarf. We cloned the Brassica napus GID1 gene, named BnGID1, and found it was the ortholog of AtGID1a. The sequence blasting of the BnGID1 genes from NDF-1 and wild type showed there was no mutant in the gene. But the quantitative RT-PCR analysis of GID1 EST pointed out the mutation was caused by the low-level expression of BnGID1 gene. After sequenced the BnGID1 gene's upstream, we found three bases mutated in the pyrimidine box (P-box) of the BnGID1 promoter, which is linkage with the dwarf mutant.

Impaired Photosynthesis in Phosphatidylglycerol-Deficient Mutant of Cyanobacterium Anabaena sp. PCC7120 with a Disrupted Gene Encoding a Putative Phosphatidylglycerophosphatase1

PubMed Central

Wu, Feng; Yang, Zhenle; Kuang, Tingyun

2006-01-01

Phosphatidylglycerol (PG) is a ubiquitous phospholipid in thylakoid membranes of cyanobacteria and chloroplasts and plays an important role in the structure and function of photosynthetic membranes. The last step of the PG biosynthesis is dephosphorylation of phosphatidylglycerophosphate (PGP) catalyzed by PGP phosphatase. However, the gene-encoding PGP phosphatase has not been identified and cloned from cyanobacteria or higher plants. In this study, we constructed a PG-deficient mutant from cyanobacterium Anabaena sp. PCC7120 with a disrupted gene (alr1715, a gene for Alr1715 protein, GenBank accession no. BAB78081) encoding a putative PGP phosphatase. The obtained mutant showed an approximately 30% reduction in the cellular content of PG. Following the reduction in the PG content, the photoautotrophical growth of the mutant was restrained, and the cellular content of chlorophyll was decreased. The decreases in net photosynthetic and photosystem II (PSII) activities on a cell basis also occurred in this mutant. Simultaneously, the photochemical efficiency of PSII was considerably declined, and less excitation energy was transferred toward PSII. These findings demonstrate that the alr1715 gene of Anabaena sp. PCC7120 is involved in the biosynthesis of PG and essential for photosynthesis. PMID:16815953

Islamic perspectives on human cloning.

PubMed

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.

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.

PubMed

Xu, Y L; Li, L; Wu, K; Peeters, A J; Gage, D A; Zeevaart, J A

1995-07-03

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.

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

PubMed

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.

Cloning

MedlinePlus

... Sheets A Brief Guide to Genomics About NHGRI Research About the International HapMap Project Biological Pathways Chromosome Abnormalities Chromosomes Cloning Comparative Genomics DNA Microarray Technology DNA Sequencing Deoxyribonucleic Acid ( ...

Characterization of mutant tobacco mosaic virus coat protein that interferes with virus cell-to-cell movement.

PubMed

Bendahmane, Mohammed; Szecsi, Judit; Chen, Iju; Berg, R Howard; Beachy, Roger N

2002-03-19

Expression of tobacco mosaic virus (TMV) coat protein (CP) in plants confers resistance to infection by TMV and related tobamoviruses. Certain mutants of the CP (CP(T42W)) provide much greater levels of resistance than wild-type (wt) CP. In the present work, infection induced by RNA transcripts of TMV clones that contain wt CP or mutant CP(T42W) fused to the green fluorescent protein (GFP) (TMV-CP:GFP, TMV-CP(T42W):GFP) and clones harboring TMV movement protein (MP):GFP were followed in nontransgenic and transgenic tobacco BY-2 protoplasts and Nicotiana tabaccum Xanthi-nn plants that express wt CP or CP(T42W). On nontransgenic and wt CP transgenic plants, TMV-CP:GFP produced expanding, highly fluorescent disk-shaped areas. On plants expressing CP(T42W), infection by TMV-CP:GFP or TMV-MP:GFP-CP produced infection sites of smaller size that were characterized by low fluorescence, reflecting reduced levels of virus spread and reduced accumulation of both CP:GFP and MP:GFP. TMV-CP(T42W):GFP failed to produce visible infection sites on nontransgenic plants, yet produced normal infection sites on MP-transgenic plants that produce MP. TMV infection of transgenic BY-CP(T42W) protoplasts resulted in very low levels of MP accumulation, whereas on BY-CP protoplasts (containing wt CP), infection produced higher levels of MP than in nontransgenic BY-2 cells. The results suggest that wt CP has a positive effect on the production of MP, whereas the CP(T42W) has a negative effect on MP accumulation and/or function. This effect results in very high levels of resistance to TMV infection in plants containing CP(T42W). This report shows that the CP of a plant virus regulates production of the MP, and that a mutant CP interferes with MP accumulation and cell-to-cell movement of infection.

Therapeutic and reproductive cloning: a critique.

PubMed

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.

Cloning, Sequencing, and Role in Virulence of Two Phospholipases (A1 and C) from Mesophilic Aeromonas sp. Serogroup O:34

PubMed Central

Merino, Susana; Aguilar, Alicia; Nogueras, Maria Mercedes; Regue, Miguel; Swift, Simon; Tomás, Juan M.

1999-01-01

Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5α. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants of A. hydrophila AH-3 defective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic. A. hydrophila AH-3 plc mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic Aeromonas sp. serogroup O:34 infection process. PMID:10417167

Cloning and analysis of the positively acting regulatory gene amdR from Aspergillus nidulans.

PubMed Central

Andrianopoulos, A; Hynes, M J

1988-01-01

The positively acting regulatory gene amdR of Aspergillus nidulans coordinately regulates the expression of four unlinked structural genes involved in acetamide (amdS), omega amino acid (gatA and gabA), and lactam (lamA) catabolism. By the use of DNA-mediated transformation of A. nidulans, the amdR regulatory gene was cloned from a genomic cosmid library. Southern blot analysis of DNA from various loss-of-function amdR mutants revealed the presence of four detectable DNA rearrangements, including a deletion, an insertion, and a translocation. No detectable DNA rearrangements were found in several constitutive amdRc mutants. Analysis of the fate of amdR-bearing plasmids in transformants showed that 10 to 20% of the transformation events were homologous integrations or gene conversions, and this phenomenon was exploited in developing a strategy by which amdRc and amdR- alleles can be readily cloned and analyzed. Examination of the transcription of amdR by Northern blot (RNA blot) analysis revealed the presence of two mRNAs (2.7 and 1.8 kilobases) which were constitutively synthesized at a very low level. In addition, amdR transcription did not appear to depend on the presence of a functional amdR product nor was it altered in amdRc mutants. The dosage effects of multiple copies of amdR in transformants were examined, and it was shown that such transformants exhibited stronger growth than did the wild type on acetamide and pyrrolidinone media, indicating increased expression of the amdS and lamA genes, respectively. These results were used to formulate a model for amdR-mediated regulation of gene expression in which the low constitutive level of amdR product sets the upper limits of basal and induced transcription of the structural genes. Multiple copies of 5' sequences from the amdS gene can result in reduced growth on substrates whose utilization is dependent on amdR-controlled genes. This has been attributed to titration of limiting amdR gene product. Strong

Transposon mutagenesis in Bifidobacterium breve: construction and characterization of a Tn5 transposon mutant library for Bifidobacterium breve UCC2003.

PubMed

Ruiz, Lorena; Motherway, Mary O'Connell; Lanigan, Noreen; van Sinderen, Douwe

2013-01-01

Bifidobacteria are claimed to contribute positively to human health through a range of beneficial or probiotic activities, including amelioration of gastrointestinal and metabolic disorders, and therefore this particular group of gastrointestinal commensals has enjoyed increasing industrial and scientific attention in recent years. However, the molecular mechanisms underlying these probiotic mechanisms are still largely unknown, mainly due to the fact that molecular tools for bifidobacteria are rather poorly developed, with many strains lacking genetic accessibility. In this work, we describe the generation of transposon insertion mutants in two bifidobacterial strains, B. breve UCC2003 and B. breve NCFB2258. We also report the creation of the first transposon mutant library in a bifidobacterial strain, employing B. breve UCC2003 and a Tn5-based transposome strategy. The library was found to be composed of clones containing single transposon insertions which appear to be randomly distributed along the genome. The usefulness of the library to perform phenotypic screenings was confirmed through identification and analysis of mutants defective in D-galactose, D-lactose or pullulan utilization abilities.

Artificial cloning of domestic animals.

PubMed

Keefer, Carol L

2015-07-21

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

[Scientific ethics of human cloning].

PubMed

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.

Genomic clones for human cholinesterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kott, M.; Venta, P.J.; Larsen, J.

1987-05-01

A human genomic library was prepared from peripheral white blood cells from a single donor by inserting an MboI partial digest into BamHI poly-linker sites of EMBL3. This library was screened using an oligolabeled human cholinesterase cDNA probe over 700 bp long. The latter probe was obtained from a human basal ganglia cDNA library. Of approximately 2 million clones screened with high stringency conditions several positive clones were identified; two have been plaque purified. One of these clones has been partially mapped using restriction enzymes known to cut within the coded region of the cDNA for human serum cholinesterase. Hybridizationmore » of the fragments and their sizes are as expected if the genomic clone is cholinesterase. Sequencing of the DNA fragments in M13 is in progress to verify the identify of the clone and the location of introns.« less

The ethics of human reproductive cloning.

PubMed

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

NASA Astrophysics Data System (ADS)

Durt, Thomas; Fiurášek, Jaromír; Cerf, Nicolas J.

2005-11-01

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

Analysis of Yellow Striped Mutants of Zea mays Reveals Novel Loci Contributing to Iron Deficiency Chlorosis

PubMed Central

Chan-Rodriguez, David; Walker, Elsbeth L.

2018-01-01

The micronutrient iron (Fe) is essential for photosynthesis, respiration, and many other processes, but it is only sparingly soluble in aqueous solution, making adequate acquisition by plants a serious challenge. Fe is a limiting factor for plant growth on approximately 30% of the world’s arable lands. Moreover, Fe deficiency in humans is a global health issue, affecting 1.62 billion people, or about 25% of the world’s population. It is imperative that we gain a better understanding of the mechanisms that plants use to regulate iron homeostasis, since these will be important targets for future biofortification and crop improvement strategies. Grasses and non-grasses have evolved independent mechanisms for primary iron uptake from the soil. The grasses, which include most of the world’s staple grains, have evolved a distinct ‘chelation’ mechanism to acquire iron from the soil. Strong iron chelators called phytosiderophores (PSs) are synthesized by grasses and secreted into the rhizosphere where they bind and solubilize Fe(III). The Fe(III)-PS complex is then taken up into root cells via transporters specific for the Fe(III)-PS complex. In this study, 31 novel, uncharacterized striped maize mutants available through the Maize Genetics Cooperation Stock Center (MGCSC) were analyzed to determine whether their mutant phenotypes are caused by decreased iron. Many of these proved to be either pale yellow or white striped mutants. Complementation tests were performed by crossing the MGCSC mutants to ys1 and ys3 reference mutants. This allowed assignment of 10 ys1 alleles and 4 ys3 alleles among the novel mutants. In addition, four ys∗ mutant lines were identified that are not allelic to either ys1 or ys3. Three of these were characterized as being non-allelic to each other and as having low iron in leaves. These represent new genes involved in iron acquisition by maize, and future cloning of these genes may reveal novel aspects of the grass iron acquisition

Identification of putative methanol dehydrogenase (moxF) structural genes in methylotrophs and cloning of moxF genes from methylococcus capsulatus bath and Methylomonas albus BG8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stephens, R.L.; Haygood, M.G.; Lidstrom, M.E.

An open-reading-frame fragment of a Methylobacterium sp. strain AM1 gene (moxF) encoding a portion of the methanol dehydrogenase structural protein has been used as a hybridization probe to detect similar sequences in a variety of methylotrophic bacteria. This hybridization was used to isolate clones containing putative moxF genes from two obligate methanotrophic bacteria, Methylococcus capsulatus Bath and Methylomonas albus BG8. The identity of these genes was confirmed in two ways. A T7 expression vector was used to produce methanol dehydrogenase protein in Escherichia coli from the cloned genes,a and in each case the protein was identified by immunoblotting with antiserummore » against the Methylomonas albus methanol dehydrogenase. In addition, a moxF mutant of Methylobacterium strain AM1 was complemented to a methanol-positive phenotype that partially restored methanol dehydrogenase activity, using broad-host-range plasmids containing the moxF genes from each methanotroph. The partial complementation of a moxF mutant in a facultative serine pathway methanol utilizer by moxF genes from type I and type X obligate methane utilizers suggests broad functional conservation of the methanol oxidation system among gram-negative methylotrophs.« less

Novel mutants of Erwinia carotovora subsp. carotovora defective in the production of plant cell wall degrading enzymes generated by Mu transpososome-mediated insertion mutagenesis.

PubMed

Laasik, Eve; Ojarand, Merli; Pajunen, Maria; Savilahti, Harri; Mäe, Andres

2005-02-01

As in Erwinia carotovora subsp. carotovora the regulation details of the main virulence factors, encoding extracellular enzymes that degrade the plant cell wall, is only rudimentally understood, we performed a genetic screen to identify novel candidate genes involved in the process. Initially, we used Mu transpososome-mediated mutagenesis approach to generate a comprehensive transposon insertion mutant library of ca. 10000 clones and screened the clones for the loss of extracellular enzyme production. Extracellular enzymes production was abolished by mutations in the chromosomal helEcc, trkAEcc yheLEcc, glsEcc, igaAEcc and cysQEcc genes. The findings reported here demonstrate that we have isolated six new representatives that belong to the pool of genes modulating the production of virulence factors in E. carotovora.

Concurrent MPL515 and JAK2V617F mutations in myelofibrosis: chronology of clonal emergence and changes in mutant allele burden over time.

PubMed

Lasho, Terra L; Pardanani, Animesh; McClure, Rebecca F; Mesa, Ruben A; Levine, Ross L; Gilliland, D Gary; Tefferi, Ayalew

2006-12-01

MPLW515L/K and JAK2V617F can co-exist in myelofibrosis with myeloid metaplasia (MMM). The chronology of clonal emergence was studied in three such cases using serially stored bone marrow. At diagnosis, a major MPL515 mutant clone was accompanied by a minor JAK2V617F clone in all three instances. At 25 time points over a period of 4-8 years, allele burden fluctuated but remained high for MPLW515L/K and low for JAK2V617F. We conclude that MPLW515L/K and JAK2V617F are both early events in MMM and allele burden, rather than the mere presence of these mutations, might be relevant to phenotypic variation in myeloproliferative disorders.

Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase.

PubMed Central

Yen, K M; Karl, M R; Blatt, L M; Simon, M J; Winter, R B; Fausset, P R; Lu, H S; Harcourt, A A; Chen, K K

1991-01-01

Pseudomonas mendocina KR1 metabolizes toluene as a carbon source by a previously unknown pathway. The initial step of the pathway is hydroxylation of toluene to form p-cresol by a multicomponent toluene-4-monooxygenase (T4MO) system. The T4MO enzyme system has broad substrate specificity and provides a new opportunity for biodegradation of toxic compounds and bioconversions. Its known activities include conversion of a variety of phenyl compounds into the phenolic derivatives and the complete degradation of trichloroethylene. We have cloned and characterized a gene cluster from KR1 that determines the offO activity. To clone the T4MO genes, KR1 DNA libraries were constructed in Escherichia coli HB101 by using a broad-host-range vector and transferred to a KR1 mutant able to grow on p-cresol but not on toluene. An insert consisting of two SacI fragments of identical size (10.2 kb) was shown to complement the mutant for growth on toluene. One of the SacI fragments, when cloned into the E. coli vector pUC19, was found to direct the synthesis of indigo dye. The indigo-forming property was correlated with the presence of T4MO activity. The T4MO genes were mapped to a 3.6-kb region, and the direction of transcription was determined. DNA sequencing and N-terminal amino acid determination identified a five-gene cluster, tmoABCDE, within this region. Expression of this cluster carrying a single mutation in each gene demonstrated that each of the five genes is essential for T4MO activity. Other evidence presented indicated that none of the tmo genes was involved in the regulation of the tmo gene cluster, in the control of substrate transport for the T4MO system, or in major processing of the products of the tmo genes. It was tentatively concluded that the tmoABCDE genes encode structural polypeptides of the T4MO enzyme system. One of the tmo genes was tentatively identified as a ferredoxin gene. Images PMID:1885512

Human reproductive cloning: a conflict of liberties.

PubMed

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.

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-05-15

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

Construction of a novel porcine circovirus type 2 infectious clone as a basis for the development of a PCV2 iDNA vaccine.

PubMed

Wang, Wei-Cheng; Zeng, Zhi-Yong; Tang, De-Yuan; Liang, Hai-Ying; Liu, Zhao; Dai, Zhen-Jiang

2015-08-01

Porcine circovirus-associated disease is a highly contagious disease that has significant economic consequences. The disease is prevalent in many countries and regions. To generate a genetic marker strain of PCV2, a Sal I restriction enzyme site was inserted into the PCV2 clone as a genetic marker by applying iDNA infectious clone technology. The iDNA represents plasmids that encode the full-length DNA genome of PCV2 assembled in a pcDNA3.1-based vectors. The mutant PCV2 was rescued by transfecting an infectious clone into PK-15 cells and was characterised by an immunoperoxidase monolayer assay (IPMA). The viral genome could be differentiated from the wild-type parent by PCR and restriction fragment length polymorphism (PCR-RFLP). Kunming mice were inoculated with the PCV2 infectious clone or rescued virus via intranasal and intraperitoneal routes. Seroconversion to PCV2-specific antibody appeared in the majority of mice from the two inoculated groups at 7 days postinoculation (DPI), and the specific antibody level was steady for at least 42 days. Viraemia, beginning at 7 DPI and lasting 4 weeks, was detected in the majority of the pigs from the two inoculated groups. The animal experiments revealed that the PCV2 infectious clone and rescued virus both could replicate in mice and induce mice to generate anti-PCV2 antibodies. The infectious clones of PCV2 will be useful for further research investigating a potential tractable iDNA vaccine by reverse genetics technology for attenuated virulance. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification and Map-Based Cloning of the Light-Induced Lesion Mimic Mutant 1 (LIL1) Gene in Rice.

PubMed

Zhou, Qian; Zhang, Zhifei; Liu, Tiantian; Gao, Bida; Xiong, Xingyao

2017-01-01

The hypersensitive response (HR) is a mechanism by which plants prevent the spread of pathogen. Despite extensive study, the molecular mechanisms underlying HR remain poorly understood. Lesion mimic mutants (LMMs), such as LIL1 that was identified in an ethylmethane sulfonate mutagenized population of Indica rice ( Oryza sativa L. ssp. Indica ) 93-11, can be used to study the HR. Under natural field conditions, the leaves of LIL1 mutant plants exhibited light-induced, small, rust-red lesions that first appeared at the leaf tips and subsequently expanded throughout the entire leaf blade to the leaf sheath. Histochemical staining indicated that LIL1 lesions displayed an abnormal accumulation of reactive oxygen species (ROS) and resulted from programmed cell death (PCD). The LIL1 mutants also displayed increased expression of defense-related genes and enhanced resistance to rice blast fungus ( Magnaporthe grisea ). Genetic analysis showed that mutation of LIL1 created a semi-dominant allele. Using 1,758 individuals in the F 2 population, LIL1 was mapped in a 222.3 kb region on the long arm of chromosome 7. That contains 12 predicted open reading frames (ORFs). Sequence analysis of these 12 candidate genes revealed a G to A base substitution in the fourth exon of LOC_Os07g30510, a putative cysteine-rich receptor-like kinase (CRK), which led to an amino acid change (Val 429 to Ile) in the LIL1 protein. Comparison of the transcript accumulation of the 12 candidate genes between LIL1 and 93-11 revealed that LOC_Os07g30510 was up-regulated significantly in LIL1 . Overexpression of the LOC_Os07g30510 gene from LIL1 induced a LIL1 -like lesion phenotype in Nipponbare. Thus, LIL1 is a novel LMM in rice that will facilitate the further study of the molecular mechanisms of HR and the rice blast resistance.

Structural analysis of α1,3-linked galactose-containing oligosaccharides in Schizosaccharomyces pombe mutants harboring single and multiple α-galactosyltransferase genes disruptions.

PubMed

Ohashi, Takao; Nakakita, Shin-ichi; Sumiyoshi, Wataru; Yamada, Naotaka; Ikeda, Yuka; Tanaka, Naotaka; Takegawa, Kaoru

2011-03-01

In the fission yeast Schizosaccharomyces pombe, galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases in the lumen of the Golgi apparatus. In S. pombe, the major in vitro α1,2-galactosyltransferase activity has been purified, the gma12(+) gene has been cloned, and three α-galactosyltransferase genes (gmh1(+)-gmh3(+)) have also been partially characterized. In this study, we found three additional uncharacterized genes with homology to gmh1(+) (gmh4(+)-gmh6(+)) in the fission yeast genome sequence. All possible single disruption mutants and the septuple disruption strain were constructed and characterized. The electrophoretic mobility of acid phosphatase prepared from gma12Δ, gmh2Δ, gmh3Δ and gmh6Δ mutants was higher than that from wild type, indicating that Gma12p, Gmh2p, Gmh3p and Gmh6p are required for the galactosylation of N-linked oligosaccharides. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides from each single mutant showed that Gma12p, Gmh2p and Gmh6p are involved in galactosylation of O-linked oligosaccharides. The septuple mutant exhibited similar drug and temperature sensitivity as a gms1Δ mutant that is incapable of galactosylation. Oligosaccharide structural analysis based on HPLC and methylation analysis revealed that the septuple mutant still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating that an unknown α1,3-galactosyltransferase activity was still present in the septuple mutant.

Artificial cloning of domestic animals

PubMed Central

Keefer, Carol L.

2015-01-01

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. PMID:26195770

Catalytic site of human protein-glucosylgalactosylhydroxylysine glucosidase: Three crucial carboxyl residues were determined by cloning and site-directed mutagenesis.

PubMed

Hamazaki, Hideaki; Hamazaki, Michiko Horikawa

2016-01-15

Protein-glucosylgalactosylhydroxylysine glucosidase (PGGHG; EC3.2.1.107) cleaves glucose from disaccharide unit (Glc-α1,2-Gal) linked to hydroxylysine residues of collagen. In the present paper we first show that PGGHG is the product of ATHL1 gene as follows. (1) PGGHG was purified from chick embryos and digested with trypsin. LC-MS/MS analysis suggested the tryptic-peptides were from the ATHL1 gene product. (2) Chick embryo ATHL1 cDNA was cloned to a cloning and expression vector and two plasmid clones with different ATHL1 CDS insert were obtained. (3) Each plasmid DNA was transformed into Escherichia coli cells for expression and two isoforms of chicken PGGHG were obtained. (4) Both isoforms effectively released glucose from type IV collagen. Next, we searched for carboxyl residues crucial for catalytic activity as follows; human ATHL1 cDNA was cloned into a cloning and expression vector and 18 mutants were obtained by site-directed mutagenesis for 15 carboxyl residues conserved in ATHL1 of jawed vertebrates. The expression analysis indicated that substitutions of Asp301, Glu430 and Glu574 with sterically conservative (D301N, E430Q, E574Q) or functionally conservative (D301E, E430D, E574D) residues led to the complete elimination of enzyme activity. These findings lead us to the conclusion that PGGHG is encoded by ATHL1 and three carboxyl residues (corresponding to Asp301, Glu430 and Glu574 of human PGGHG) might be involved in the catalytic site of PGGHG. Copyright © 2015 Elsevier Inc. All rights reserved.

To Clone or Not To Clone: Method Analysis for Retrieving Consensus Sequences In Ancient DNA Samples

PubMed Central

Winters, Misa; Barta, Jodi Lynn; Monroe, Cara; Kemp, Brian M.

2011-01-01

The challenges associated with the retrieval and authentication of ancient DNA (aDNA) evidence are principally due to post-mortem damage which makes ancient samples particularly prone to contamination from “modern” DNA sources. The necessity for authentication of results has led many aDNA researchers to adopt methods considered to be “gold standards” in the field, including cloning aDNA amplicons as opposed to directly sequencing them. However, no standardized protocol has emerged regarding the necessary number of clones to sequence, how a consensus sequence is most appropriately derived, or how results should be reported in the literature. In addition, there has been no systematic demonstration of the degree to which direct sequences are affected by damage or whether direct sequencing would provide disparate results from a consensus of clones. To address this issue, a comparative study was designed to examine both cloned and direct sequences amplified from ∼3,500 year-old ancient northern fur seal DNA extracts. Majority rules and the Consensus Confidence Program were used to generate consensus sequences for each individual from the cloned sequences, which exhibited damage at 31 of 139 base pairs across all clones. In no instance did the consensus of clones differ from the direct sequence. This study demonstrates that, when appropriate, cloning need not be the default method, but instead, should be used as a measure of authentication on a case-by-case basis, especially when this practice adds time and cost to studies where it may be superfluous. PMID:21738625

The First Human Cloned Embryo.

ERIC Educational Resources Information Center

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)

Cloning of endangered mammalian species: any progress?

PubMed

Loi, Pasqualino; Galli, Cesare; Ptak, Grazyna

2007-05-01

Attempts through somatic cell nuclear transfer to expand wild populations that have shrunk to critical numbers is a logical extension of the successful cloning of mammals. However, although the first mammal was cloned 10 years ago, nuclear reprogramming remains phenomenological, with abnormal gene expression and epigenetic deregulation being associated with the cloning process. In addition, although cloning of wild animals using host oocytes from different species has been successful, little is known about the implication of partial or total mitochondrial DNA heteroplasmy in cloned embryos, fetuses and offspring. Finally, there is a need for suitable foster mothers for inter-intra specific cloned embryos. Considering these issues, the limited success achieved in cloning endangered animals is not surprising. However, optimism comes from the rapid gain in the understanding of the molecular clues underlying nuclear reprogramming. If it is possible to achieve a controlled reversal of the differentiated state of a cell then it is probable that other issues that impair the cloning of endangered animals, such as the inter-intra species oocyte or womb donor, will be overcome in the medium term.

Species-specific challenges in dog cloning.

PubMed

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.

Possible association between mutant frequency in peripheral lymphocytes and domestic radon concentrations.

PubMed

Bridges, B A; Cole, J; Arlett, C F; Green, M H; Waugh, A P; Beare, D; Henshaw, D L; Last, R D

1991-05-18

To investigate whether previously found geographical correlations between leukaemia incidence and exposure to radon are reflected in a detectable mutagenic effect on individuals, the frequency of mutations in the hypoxanthine guanine phosphoribosyl transferase gene (hprt) in peripheral blood T lymphocytes was measured in subjects with known domestic radon concentrations. These concentrations were measured in December, 1989, in houses in Street, Somerset, UK, by passive alpha-track radon detectors. 20 non-smoking subjects aged 36-55 years were selected from the patient list at the local health centre on the basis of the radon concentrations in their homes--the range selected varied by a factor of ten. Blood samples for preparation of T lymphocytes were taken in July, 1990. There was a significant association between the log mutant frequency and radon concentration (t = 3.47, p less than 0.01). A second analysis of a further set of radon measurements (October, 1990, to January, 1991), in both living rooms and bedrooms, and repeated mutant frequency determinations also showed a significant relation, which remained significant even after exclusion of the highest frequency and adjustment for subject's age and cloning efficiency. These data must be regarded as preliminary and further more extensive studies should be done to determine whether the observed association is causal.

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

PubMed Central

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

2014-01-01

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

Knowledge and attitudes toward human cloning in Israel.

PubMed

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.

Construction of a mutagenesis cartridge for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuhn, R.J.; Tada, H.; Ypma-Wong, M.F.

1988-01-01

By following a strategy of genetic analysis of poliovirus, the authors have constructed a synthetic mutagenesis cartridge spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type I (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed them to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alterationmore » of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. They suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).« less

Animal Cloning and Food Safety

MedlinePlus

... concluded that meat and milk from clones of cattle, swine (pigs), and goats, and the offspring of ... natural mating the composition of food products from cattle, swine, and goat clones, or the offspring of ...

Reproductive cloning combined with genetic modification.

PubMed

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.

Long-term Culture and Cloning of Primary Human Bronchial Basal Cells that Maintain Multipotent Differentiation Capacity and CFTR Channel Function.

PubMed

Peters-Hall, Jennifer Ruth; Coquelin, Melissa L; Torres, Michael J; LaRanger, Ryan; Alabi, Busola Ruth; Sho, Sei; Calva-Moreno, Jose Francisco; Thomas, Philip J; Shay, Jerry William

2018-05-03

While primary cystic fibrosis (CF) and non-CF human bronchial epithelial basal cells (HBECs) accurately represent in vivo phenotypes, one barrier to their wider use has been a limited ability to clone and expand cells in sufficient numbers to produce rare genotypes using genome editing tools. Recently, conditional reprogramming of cells (CRC) with a ROCK inhibitor and culture on an irradiated fibroblast feeder layer resulted in extension of the lifespan of HBECs, but differentiation capacity and CF transmembrane conductance regulator (CFTR) function decreased as a function of passage. This report details modifications to the standard HBEC CRC protocol (Mod CRC), including the use of bronchial epithelial growth medium instead of F-medium and 2% oxygen instead of 21% oxygen, that extend HBEC lifespan while preserving multipotent differentiation capacity and CFTR function. Critically, Mod CRC conditions support clonal growth of primary HBECs from a single cell and the resulting clonal HBEC population maintains multipotent differentiation capacity, including CFTR function, permitting gene editing of these cells. As a proof of concept, CRISPR/Cas9 genome editing and cloning was used to introduce insertions/deletions in CFTR exon 11. Mod CRC conditions overcome many barriers to the expanded use of HBECs for basic research and drug screens. Importantly, Mod CRC conditions support the creation of isogenic cell lines in which CFTR is mutant or wild-type in the same genetic background with no history of CF to enable determination of the primary defects of mutant CFTR.

Are Cloned Quantum States Macroscopic?

NASA Astrophysics Data System (ADS)

Fröwis, F.; Dür, W.

2012-10-01

We study quantum states produced by optimal phase covariant quantum cloners. We argue that cloned quantum superpositions are not macroscopic superpositions in the spirit of Schrödinger’s cat, despite their large particle number. This is indicated by calculating several measures for macroscopic superpositions from the literature, as well as by investigating the distinguishability of the two superposed cloned states. The latter rapidly diminishes when considering imperfect detectors or noisy states and does not increase with the system size. In contrast, we find that cloned quantum states themselves are macroscopic, in the sense of both proposed measures and their usefulness in quantum metrology with an optimal scaling in system size. We investigate the applicability of cloned states for parameter estimation in the presence of different kinds of noise.

Human cloning and 'posthuman' society.

PubMed

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.

Serial bull cloning by somatic cell nuclear transfer.

PubMed

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

2004-06-01

Although the list of species successfully cloned continues to grow, serial cloning has not been reported in species other than the mouse. Here we describe two live births of second-generation clones of a bull. Clones of the first and second generations appear healthy and have normal telomere lengths. Our attempts to produce the third generation of clones were unsuccessful.

Russell body inducing threshold depends on the variable domain sequences of individual human IgG clones and the cellular protein homeostasis.

PubMed

Stoops, Janelle; Byrd, Samantha; Hasegawa, Haruki

2012-10-01

Russell bodies are intracellular aggregates of immunoglobulins. Although the mechanism of Russell body biogenesis has been extensively studied by using truncated mutant heavy chains, the importance of the variable domain sequences in this process and in immunoglobulin biosynthesis remains largely unknown. Using a panel of structurally and functionally normal human immunoglobulin Gs, we show that individual immunoglobulin G clones possess distinctive Russell body inducing propensities that can surface differently under normal and abnormal cellular conditions. Russell body inducing predisposition unique to each immunoglobulin G clone was corroborated by the intrinsic physicochemical properties encoded in the heavy chain variable domain/light chain variable domain sequence combinations that define each immunoglobulin G clone. While the sequence based intrinsic factors predispose certain immunoglobulin G clones to be more prone to induce Russell bodies, extrinsic factors such as stressful cell culture conditions also play roles in unmasking Russell body propensity from immunoglobulin G clones that are normally refractory to developing Russell bodies. By taking advantage of heterologous expression systems, we dissected the roles of individual subunit chains in Russell body formation and examined the effect of non-cognate subunit chain pair co-expression on Russell body forming propensity. The results suggest that the properties embedded in the variable domain of individual light chain clones and their compatibility with the partnering heavy chain variable domain sequences underscore the efficiency of immunoglobulin G biosynthesis, the threshold for Russell body induction, and the level of immunoglobulin G secretion. We propose that an interplay between the unique properties encoded in variable domain sequences and the state of protein homeostasis determines whether an immunoglobulin G expressing cell will develop the Russell body phenotype in a dynamic cellular setting

The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: Molecular cloning and functional expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yun-Ling; Li, Li; Wu, Keqiang

1995-07-03

The biosynthesis of gibberellins (GAs) after GA{sub 12}-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidasemore » gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA{sub 53} to GA{sub 44} and GA{sub 19} to GA{sub 20}. The Arabidopsis GA 20-oxidase shares 55% identity and >80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA locus of Arabidopsis. The ga5 semidwarf mutant contains a G {yields} A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Arabidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA{sub 4} treatment, suggesting end-product repression in the GA biosynthetic pathway. 28 refs., 6

Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence-activated cell sorting.

PubMed

Wilke, Sonja; Krausze, Joern; Gossen, Manfred; Groebe, Lothar; Jäger, Volker; Gherardi, Ermanno; van den Heuvel, Joop; Büssow, Konrad

2010-06-01

Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N-linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence-activated cell sorting (FACS) and site-specific gene excision to establish high-yield glycoprotein expression for structural studies with stable clones derived from the well-established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single-chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome-associated membrane protein 3 (LAMP3d). In both cases, stable GFP-expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.

The globally disseminated M1T1 clone of Group A Streptococcus evades autophagy for intracellular replication

PubMed Central

Barnett, Timothy C.; Liebl, David; Seymour, Lisa M.; Gillen, Christine M.; Lim, Jin Yan; LaRock, Christopher N.; Davies, Mark R.; Schulz, Benjamin L.; Nizet, Victor; Teasdale, Rohan D.; Walker, Mark J.

2014-01-01

SUMMARY Autophagy is reported to be an important innate immune defence against the intracellular bacterial pathogen Group A Streptococcus (GAS). However, the GAS strains examined to-date belong to serotypes infrequently associated with human disease. We find that the globally disseminated serotype M1T1 clone of GAS can evade autophagy and replicate efficiently in the cytosol of infected cells. Cytosolic M1T1 GAS (strain 5448), but not M6 GAS (strain JRS4), avoids ubiquitylation and recognition by the host autophagy marker LC3 and ubiquitin-LC3 adaptor proteins NDP52, p62 and NBR1. Expression of SpeB, a streptococcal cysteine protease, is critical for this process, as an isogenic M1T1 ΔspeB mutant is targeted to autophagy and attenuated for intracellular replication. SpeB degrades p62, NDP52 and NBR1 in vitro and within the host cell cytosol. These results uncover a proteolytic mechanism utilized by GAS to escape the host autophagy pathway which may underpin the success of the M1T1 clone. PMID:24331465

Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu.

PubMed Central

He, S Y; Lindeberg, M; Chatterjee, A K; Collmer, A

1991-01-01

The out genes of the enterobacterial plant pathogen Erwinia chrysanthemi are responsible for the efficient extracellular secretion of multiple plant cell wall-degrading enzymes, including four isozymes of pectate lyase, exo-poly-alpha-D-galacturonosidase, pectin methylesterase, and cellulase. Out- mutants of Er. chrysanthemi are unable to export any of these proteins beyond the periplasm and are severely reduced in virulence. We have cloned out genes from Er. chrysanthemi in the stable, low-copy-number cosmid pCPP19 by complementing several transposon-induced mutations. The cloned out genes were clustered in a 12-kilobase chromosomal DNA region, complemented all existing out mutations in Er. chrysanthemi EC16, and enabled Escherichia coli strains to efficiently secrete the extracellular pectic enzymes produced from cloned Er. chrysanthemi genes, while retaining the periplasmic marker protein beta-lactamase. DNA sequencing of a 2.4-kilobase EcoRI fragment within the out cluster revealed four genes arranged colinearly and sharing substantial similarity with the Klebsiella pneumoniae genes pulH, pulI, pulJ, and pulK, which are necessary for pullulanase secretion. However, K. pneumoniae cells harboring the cloned Er. chrysanthemi pelE gene were unable to secrete the Erwinia pectate lyase. Furthermore, the Er. chrysanthemi Out system was unable to secrete an extracellular pectate lyase encoded by a gene from a closely related plant pathogen. Erwinia carotovora ssp. carotovora. The results suggest that these enterobacteria secrete polysaccharidases by a conserved mechanism whose protein-recognition capacities have diverged. Images PMID:1992458

Cloned Erwinia chrysanthemi out genes enable Escherichia coli to selectively secrete a diverse family of heterologous proteins to its milieu.

PubMed

He, S Y; Lindeberg, M; Chatterjee, A K; Collmer, A

1991-02-01

The out genes of the enterobacterial plant pathogen Erwinia chrysanthemi are responsible for the efficient extracellular secretion of multiple plant cell wall-degrading enzymes, including four isozymes of pectate lyase, exo-poly-alpha-D-galacturonosidase, pectin methylesterase, and cellulase. Out- mutants of Er. chrysanthemi are unable to export any of these proteins beyond the periplasm and are severely reduced in virulence. We have cloned out genes from Er. chrysanthemi in the stable, low-copy-number cosmid pCPP19 by complementing several transposon-induced mutations. The cloned out genes were clustered in a 12-kilobase chromosomal DNA region, complemented all existing out mutations in Er. chrysanthemi EC16, and enabled Escherichia coli strains to efficiently secrete the extracellular pectic enzymes produced from cloned Er. chrysanthemi genes, while retaining the periplasmic marker protein beta-lactamase. DNA sequencing of a 2.4-kilobase EcoRI fragment within the out cluster revealed four genes arranged colinearly and sharing substantial similarity with the Klebsiella pneumoniae genes pulH, pulI, pulJ, and pulK, which are necessary for pullulanase secretion. However, K. pneumoniae cells harboring the cloned Er. chrysanthemi pelE gene were unable to secrete the Erwinia pectate lyase. Furthermore, the Er. chrysanthemi Out system was unable to secrete an extracellular pectate lyase encoded by a gene from a closely related plant pathogen. Erwinia carotovora ssp. carotovora. The results suggest that these enterobacteria secrete polysaccharidases by a conserved mechanism whose protein-recognition capacities have diverged.

U.S. consumers attitudes toward farm animal cloning.

PubMed

Brooks, Kathleen R; Lusk, Jayson L

2011-10-01

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

Local circulating clones of Staphylococcus aureus in Ecuador.

PubMed

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.

Cloning and Characterization of the Scalloped Region of Drosophila Melanogaster

PubMed Central

Campbell, S. D.; Duttaroy, A.; Katzen, A. L.; Chovnick, A.

1991-01-01

Viable mutants of the scalloped gene (sd) of Drosophila melanogaster exhibit defects that can include gapping of the wing margin and ectopic bristle formation on the wing. Lethal sd alleles characterized in the present study now implicate this gene in a genetic function essential for normal development. In order to further characterize the developmental role of this gene, we have undertaken to clone and characterize the region where sd maps. A P[ry(+)] transposon insertion at 13F associated with sd([ry+2216]) served as the starting point for a 42-kb chromosomal walk. Molecular lesions associated with viable and lethal sd alleles were characterized by genomic hybridization analysis as a means of defining the extent of the gene. DNA rearrangements associated with 11 viable sd alleles map to a 2-kb interval which appears to be a ``hot spot'' for P element activity. Four of five recessive lethal sd mutations were mapped by denaturing gradient gel electrophoresis to a region 12-14 kb away from the region of viable lesions. In a sd(+) genotype, at least two structurally related and developmentally regulated transcripts hybridize to the genomic region where several sd lethal alleles have been localized. A viable mutation, sd(58), used for comparison in the transcript analysis, makes at least two slightly smaller transcripts that also hybridize to this region. Preliminary analysis of cDNA clones has identified three structurally related transcripts that hybridize to this genomic region. The 5' end of these transcripts extends into the 2-kb genomic region wherein DNA rearrangements were seen in the P element rearrangements. We favor the view that the transcripts represented by these cDNA clones are products of the sd gene. If this is true, the sd gene would include genomic sequences extending over at least 14 kb of the described chromosomal walk, and would appear to be subject to alternative splicing. PMID:1706292

Further yearly analyses of spontaneous pink mutant events in the stamen hairs of tradescantia clone BNL 4430 cultivated in the NSC growth chamber.

PubMed

Ichikawa, S; Wushur, S

2001-06-01

In order to confirm the results obtained in the previous 1-year-term (December 12, 1998, through December 10, 1999) scorings and analyses of spontaneous pink mutant events (PMEs) in the stamen hairs of Tradescantia clone BNL 4430 cultivated in a nutrient solution circulating (NSC) growth chamber, similar scorings and analyses were continued for another 52-week period from December 11, 1999, through December 8, 2000. The environmental conditions were not changed, except for a minor modification in the method of supplying the nutrient solution used. During the scoring period, 732,128 stamen hairs with an average cell number of 24.90 cells were observed, and 2,368 PMEs were detected. The overall spontaneous somatic mutation frequency was 1.35 +/- 0.03 PMEs per 10(4) hair-cell divisions, which was significantly lower than the value of 1.56 +/- 0.03 determined in the previous 52-week period, and the frequencies were lower during April through September than in other months, the period showing lower frequencies lasting 1-month longer than in the previous year. The present results reconfirmed the occurrence of a clear seasonal variation in the spontaneous mutation frequency in the NSC growth chamber, and the lower overall frequency, probably related to the minor modification in supplying the nutrient solution, is helpful for conducting mutagenicity tests at low levels, offering a lower background level. The analyses of the sectoring patterns of all these PMEs showed that the most of the 203 cases of multiple (two to five) pink sectors observed in the same stamen hairs (scored as 253 PMEs for calculating mutation frequency) were the results of events involving somatic recombinations occurred in single cells or cell lineages, rather than those of two or more independent somatic mutations occurred in different cells, agreeing with our previous study, and the significance of somatic recombinations in causing single PMEs was also reconfirmed.

The fate of mitochondrial loci in rho minus mutants induced by ultraviolet irradiation of Saccharomyces cerevisiae: effects of different post-irradiation treatments.

PubMed

Heude, M; Moustacchi, E

1979-09-01

Three main features regarding the loss of mitochondrial genetic markers among rho- mutants induced by ultraviolet irradiation are reported: (a) the frequency of loss of six loci examined increases with UV dose; (b) preferential loss of one region of the mitochondrial genome observed in spontaneous rho- mutants is enhanced by UV; and (c) the loss of each marker results from large deletions. Marker loss in rho- mutants was also investigated under conditions that modulate rho- induction. Liquid holding of irradiated exponential or stationary phase cells, as well as a split-dose regime applied to stationary phase cells, results in rho- mutants in which the loss of markers is correlated with rho- induction: the more sensitive the cells are to rho- induction, the more frequent are the marker losses among rho- clones derived from these cells. This correlation is not found in exponential-phase cells submitted to a split-dose treatment, suggesting that a different mechanism is involved in the latter case. It is known that UV-induced pyrimidine dimers are not excised in a controlled manner in mitochondrial DNA. However, our studies indicate that an accurate repair mechanism (of the recombinational type ?) can lead to the restoration of mitochondrial genetic information in growing cells.

Metabolomic phenotyping of a cloned pig model

PubMed Central

2011-01-01

Background Pigs are widely used as models for human physiological changes in intervention studies, because of the close resemblance between human and porcine physiology and the high degree of experimental control when using an animal model. Cloned animals have, in principle, identical genotypes and possibly also phenotypes and this offer an extra level of experimental control which could possibly make them a desirable tool for intervention studies. Therefore, in the present study, we address how phenotype and phenotypic variation is affected by cloning, through comparison of cloned pigs and normal outbred pigs. Results The metabolic phenotype of cloned pigs (n = 5) was for the first time elucidated by nuclear magnetic resonance (NMR)-based metabolomic analysis of multiple bio-fluids including plasma, bile and urine. The metabolic phenotype of the cloned pigs was compared with normal outbred pigs (n = 6) by multivariate data analysis, which revealed differences in the metabolic phenotypes. Plasma lactate was higher for cloned vs control pigs, while multiple metabolites were altered in the bile. However a lower inter-individual variability for cloned pigs compared with control pigs could not be established. Conclusions From the present study we conclude that cloned and normal outbred pigs are phenotypically different. However, it cannot be concluded that the use of cloned animals will reduce the inter-individual variation in intervention studies, though this is based on a limited number of animals. PMID:21859467

Human embryo cloning prohibited in Hong Kong.

PubMed

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.

Distribution of quantum Fisher information in asymmetric cloning machines

PubMed Central

Xiao, Xing; Yao, Yao; Zhou, Lei-Ming; Wang, Xiaoguang

2014-01-01

An unknown quantum state cannot be copied and broadcast freely due to the no-cloning theorem. Approximate cloning schemes have been proposed to achieve the optimal cloning characterized by the maximal fidelity between the original and its copies. Here, from the perspective of quantum Fisher information (QFI), we investigate the distribution of QFI in asymmetric cloning machines which produce two nonidentical copies. As one might expect, improving the QFI of one copy results in decreasing the QFI of the other copy. It is perhaps also unsurprising that asymmetric phase-covariant cloning outperforms universal cloning in distributing QFI since a priori information of the input state has been utilized. However, interesting results appear when we compare the distributabilities of fidelity (which quantifies the full information of quantum states), and QFI (which only captures the information of relevant parameters) in asymmetric cloning machines. Unlike the results of fidelity, where the distributability of symmetric cloning is always optimal for any d-dimensional cloning, we find that any asymmetric cloning outperforms symmetric cloning on the distribution of QFI for d ≤ 18, whereas some but not all asymmetric cloning strategies could be worse than symmetric ones when d > 18. PMID:25484234

Human cloning: Eastern Mediterranean Region perspective.

PubMed

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.

Mutation of a vitelline membrane protein, BmEP80, is responsible for the silkworm "Ming" lethal egg mutant.

PubMed

Chen, Anli; Gao, Peng; Zhao, Qiaoling; Tang, Shunming; Shen, Xingjia; Zhang, Guozheng; Qiu, Zhiyong; Xia, Dingguo; Huang, Yongping; Xu, Yunmin; He, Ningjia

2013-02-25

The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive "Ming" lethal egg mutants (l-e(m)) lose water and become concaved around 1h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-e(m) mutant to 360 kb on chromosome 10 using 2287 F(2) individuals. Using expression analysis and RNA interference, the best l-e(m) candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~1.9 kb region from the 3' untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a >100 kb DNA fragment in the l-e(m) mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-e(m) mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-e(m) mutant. These results indicate that BmEP80 is responsible for the l-e(m) mutation. Copyright © 2012 Elsevier B.V. All rights reserved.

Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

PubMed

Burgess, D; Penton, A; Dunsmuir, P; Dooner, H

1997-02-01

Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

Applications of quantum cloning

NASA Astrophysics Data System (ADS)

Pomarico, E.; Sanguinetti, B.; Sekatski, P.; Zbinden, H.; Gisin, N.

2011-10-01

Quantum Cloning Machines (QCMs) allow for the copying of information, within the limits imposed by quantum mechanics. These devices are particularly interesting in the high-gain regime, i.e., when one input qubit generates a state of many output qubits. In this regime, they allow for the study of certain aspects of the quantum to classical transition. The understanding of these aspects is the root of the two recent applications that we will review in this paper: the first one is the Quantum Cloning Radiometer, a device which is able to produce an absolute measure of spectral radiance. This device exploits the fact that in the quantum regime information can be copied with only finite fidelity, whereas when a state becomes macroscopic, this fidelity gradually increases to 1. Measuring the fidelity of the cloning operation then allows to precisely determine the absolute spectral radiance of the input optical source. We will then discuss whether a Quantum Cloning Machine could be used to produce a state visible by the naked human eye, and the possibility of a Bell Experiment with humans playing the role of detectors.

Rescue of a Porcine Anellovirus (Torque Teno Sus Virus 2) from Cloned Genomic DNA in Pigs

PubMed Central

Huang, Yao-Wei; Patterson, Abby R.; Opriessnig, Tanja; Dryman, Barbara A.; Gallei, Andreas; Harrall, Kylie K.; Vaughn, Eric M.; Roof, Michael B.

2012-01-01

Anelloviruses are a group of single-stranded circular DNA viruses infecting humans and other animal species. Animal models combined with reverse genetic systems of anellovirus have not been developed. We report here the construction and initial characterization of full-length DNA clones of a porcine anellovirus, torque teno sus virus 2 (TTSuV2), in vitro and in vivo. We first demonstrated that five cell lines, including PK-15 cells, are free of TTSuV1 or TTSuV2 contamination, as determined by a real-time PCR and an immunofluorescence assay (IFA) using anti-TTSuV antibodies. Recombinant plasmids harboring monomeric or tandem-dimerized genomic DNA of TTSuV2 from the United States and Germany were constructed. Circular TTSuV2 genomic DNA with or without introduced genetic markers and tandem-dimerized TTSuV2 plasmids were transfected into PK-15 cells, respectively. Splicing of viral mRNAs was identified in transfected cells. Expression of TTSuV2-specific open reading frame 1 (ORF1) in cell nuclei, especially in nucleoli, was detected by IFA. However, evidence of productive TTSuV2 infection was not observed in 12 different cell lines transfected with the TTSuV2 DNA clones. Transfection with circular DNA from a TTSuV2 deletion mutant did not produce ORF1 protein, suggesting that the observed ORF1 expression is driven by TTSuV2 DNA replication in cells. Pigs inoculated with either the tandem-dimerized clones or circular genomic DNA of U.S. TTSuV2 developed viremia, and the introduced genetic markers were retained in viral DNA recovered from the sera of infected pigs. The availability of an infectious DNA clone of TTSuV2 will facilitate future study of porcine anellovirus pathogenesis and biology. PMID:22491450

Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC Mutants in In Vitro and In Vivo Systems

PubMed Central

Lewis, David A.; Stevens, Marla K.; Latimer, Jo L.; Ward, Christine K.; Deng, Kaiping; Blick, Robert; Lumbley, Sheryl R.; Ison, Catherine A.; Hansen, Eric J.

2001-01-01

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtC genes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously described H. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900–3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid. PMID:11500438

Whole genome comparison of donor and cloned dogs

PubMed Central

Kim, Hak-Min; Cho, Yun Sung; Kim, Hyunmin; Jho, Sungwoong; Son, Bongjun; Choi, Joung Yoon; Kim, Sangsoo; Lee, Byeong Chun; Bhak, Jong; Jang, Goo

2013-01-01

Cloning is a process that produces genetically identical organisms. However, the genomic degree of genetic resemblance in clones needs to be determined. In this report, the genomes of a cloned dog and its donor were compared. Compared with a human monozygotic twin, the genome of the cloned dog showed little difference from the genome of the nuclear donor dog in terms of single nucleotide variations, chromosomal instability, and telomere lengths. These findings suggest that cloning by somatic cell nuclear transfer produced an almost identical genome. The whole genome sequence data of donor and cloned dogs can provide a resource for further investigations on epigenetic contributions in phenotypic differences. PMID:24141358

The ura5 gene of the ascomycete Sordaria macrospora: molecular cloning, characterization and expression in Escherichia coli.

PubMed

Le Chevanton, L; Leblon, G

1989-04-15

We cloned the ura5 gene coding for the orotate phosphoribosyl transferase from the ascomycete Sordaria macrospora by heterologous probing of a Sordaria genomic DNA library with the corresponding Podospora anserina sequence. The Sordaria gene was expressed in an Escherichia coli pyrE mutant strain defective for the same enzyme, and expression was shown to be promoted by plasmid sequences. The nucleotide sequence of the 1246-bp DNA fragment encompassing the region of homology with the Podospora gene has been determined. This sequence contains an open reading frame of 699 nucleotides. The deduced amino acid sequence shows 72% similarity with the corresponding Podospora protein.

"Goodbye Dolly?" The ethics of human cloning.

PubMed Central

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

Therapeutic cloning research and ethical oversight.

PubMed

Spriggs, M

2003-08-01

Cloning Trevor, a story about therapeutic cloning research, appeared in the June issue of The Atlantic Monthly. The story gives a human face to the people whom therapeutic cloning could benefit. It presents an argument for government funding and it puts the usual calls for a moratorium on embryonic stem cell research to allow for more debate, in a less favourable light. The story also highlights some problems with ethical oversight.

Ethical issues regarding human cloning: a nursing perspective.

PubMed

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].

PubMed

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.

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed Central

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-01-01

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes. Images PMID:1533935

Cloning cattle: the methods in the madness.

PubMed

Oback, Björn; Wells, David N

2007-01-01

Somatic cell nuclear transfer (SCNT) is much more widely and efficiently practiced in cattle than in any other species, making this arguably the most important mammal cloned to date. While the initial objective behind cattle cloning was commercially driven--in particular to multiply genetically superior animals with desired phenotypic traits and to produce genetically modified animals-researchers have now started to use bovine SCNT as a tool to address diverse questions in developmental and cell biology. In this paper, we review current cattle cloning methodologies and their potential technical or biological pitfalls at any step of the procedure. In doing so, we focus on one methodological parameter, namely donor cell selection. We emphasize the impact of epigenetic and genetic differences between embryonic, germ, and somatic donor cell types on cloning efficiency. Lastly, we discuss adult phenotypes and fitness of cloned cattle and their offspring and illustrate some of the more imminent commercial cattle cloning applications.

Cloning-free CRISPR

PubMed Central

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

2015-01-01

Summary 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 target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis. PMID:26527385

Tritium suicide selection of mammalian cell mutants defective in the transport of neutral amino acids. [Mouse lymphocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Finkelstein, M.C.; Slayman, C.W.; Adelberg, E.A.

Mouse lymphocytic cells of the established line GF-14 were allowed to accumulate intracellular /sup 3/H-labeled aminoisobutyric acid (AIB), frozen and stored over liquid N/sub 2/. After internal radiation had reduced survival to 1 in 10/sup 4/, survivors were plated and tested for their ability to transport AIB. Out of 200 clones tested, two (designated GF-17 and GF-18) were found to have reductions to 13 to 35% of the parent in the rate of transport of AIB, L-alanine, L-proline, and L-serine; GF-18 also showed significant reductions in the rate of transport of L-glutamate and DL-cysteine. Little or no change was observedmore » for 10 other amino acids or for thymidine. Kinetic analyses revealed that the mutants were not altered in K/sub m/ for AIB uptake, but had V/sub max/ values approximately 20% the value of the parent strain, GF-14, suggesting that either the number of AIB transport sites or the turnover rate of the sites has been reduced in the two mutants.« less

Capturing the metabolomic diversity of KRAS mutants in non-small-cell lung cancer cells

PubMed Central

Marabese, Mirko; Broggini, Massimo; Pastorelli, Roberta

2014-01-01

In non-small-cell lung cancer (NSCLC), one-fifth of patients have KRAS mutations, which are considered a negative predictive factor to first-line therapy. Evidence is emerging that not all KRAS mutations have the same biological activities and possible remodeling of cell metabolism by KRAS activation might complicate the scenario. An open question is whether different KRAS mutations at codon-12 affect cellular metabolism differently with possible implications for different responses to cancer treatments. We applied an explorative mass spectrometry-based untargeted metabolomics strategy to characterize the largest possible number of metabolites that might distinguish isogenic NSCLC cells overexpressing mutated forms of KRAS at codon-12 (G12C, G12D, G12V) and the wild-type. The glutamine deprivation assay and real-time PCR were used to confirm the involvement of some of the metabolic pathways highlighted. Cell clones indicated distinct metabolomic profiles in KRAS wild-type and mutants. Clones harboring different KRAS mutations at codon-12 also had different metabolic remodeling, such as a different redox buffering system and different glutamine-dependency not driven by the transcriptional state of enzymes involved in glutaminolysis. These findings indicate that KRAS mutations at codon-12 are associated with different metabolomic profiles that might affect the responses to cancer treatments. PMID:24952473

Cloning Mice.

PubMed

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.

No-cloning of quantum steering

NASA Astrophysics Data System (ADS)

Chiu, Ching-Yi; Lambert, Neill; Liao, Teh-Lu; Nori, Franco; Li, Che-Ming

2016-06-01

Einstein-Podolsky-Rosen (EPR) steering allows two parties to verify their entanglement, even if one party’s measurements are untrusted. This concept has not only provided new insights into the nature of non-local spatial correlations in quantum mechanics, but also serves as a resource for one-sided device-independent quantum information tasks. Here, we investigate how EPR steering behaves when one-half of a maximally entangled pair of qudits (multidimensional quantum systems) is cloned by a universal cloning machine. We find that EPR steering, as verified by a criterion based on the mutual information between qudits, can only be found in one of the copy subsystems but not both. We prove that this is also true for the single-system analogue of EPR steering. We find that this restriction, which we term ‘no-cloning of quantum steering’, elucidates the physical reason why steering can be used to secure sources and channels against cloning-based attacks when implementing quantum communication and quantum computation protocols.

Construction of an infectious plasmid clone of Muscovy duck parvovirus by TA cloning and creation of a partially attenuated strain.

PubMed

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.

Thiopurine S-methyltransferase deficiency: two nucleotide transitions define the most prevalent mutant allele associated with loss of catalytic activity in Caucasians.

PubMed

Tai, H L; Krynetski, E Y; Yates, C R; Loennechen, T; Fessing, M Y; Krynetskaia, N F; Evans, W E

1996-04-01

The autosomal recessive trait of thiopurine S-methytransferase (TPMT) deficiency is associated with severe hematopoietic toxicity when patients are treated with standard doses of mercaptopurine, azathioprine, or thioguanine. To define the molecular mechanism of this genetic polymorphism, we cloned and characterized the cDNA of a TPMT-deficient patient, which revealed a novel mutant allele (TPMT*3) containing two nucleotide transitions (G460-->A and A719-->G) producing amino acid changes at codons 154 (Ala-->Thr) and 240 (Tyr--> Cys), differing from the rare mutant TPMT allele we previously identified (i.e., TPMT*2 with only G238-->C). Site-directed mutagenesis and heterologous expression established that either TPMT*3 mutation alone leads to a reduction in catalytic activity (G460-->A, ninefold reduction; A719-->G, 1.4-fold reduction), while the presence of both mutations leads to complete loss of activity. Using mutation specific PCR-RFLP analysis, the TPMT*3 allele was detected in genomic DNA from approximately 75 percent of unrelated white subjects with heterozygous phenotypes, indicating that TPMT*3 is the most prevalent mutant allele associated with TPMT-deficiency in Caucasians.

Thiopurine S-methyltransferase deficiency: two nucleotide transitions define the most prevalent mutant allele associated with loss of catalytic activity in Caucasians.

PubMed Central

Tai, H. L.; Krynetski, E. Y.; Yates, C. R.; Loennechen, T.; Fessing, M. Y.; Krynetskaia, N. F.; Evans, W. E.

1996-01-01

The autosomal recessive trait of thiopurine S-methytransferase (TPMT) deficiency is associated with severe hematopoietic toxicity when patients are treated with standard doses of mercaptopurine, azathioprine, or thioguanine. To define the molecular mechanism of this genetic polymorphism, we cloned and characterized the cDNA of a TPMT-deficient patient, which revealed a novel mutant allele (TPMT*3) containing two nucleotide transitions (G460-->A and A719-->G) producing amino acid changes at codons 154 (Ala-->Thr) and 240 (Tyr--> Cys), differing from the rare mutant TPMT allele we previously identified (i.e., TPMT*2 with only G238-->C). Site-directed mutagenesis and heterologous expression established that either TPMT*3 mutation alone leads to a reduction in catalytic activity (G460-->A, ninefold reduction; A719-->G, 1.4-fold reduction), while the presence of both mutations leads to complete loss of activity. Using mutation specific PCR-RFLP analysis, the TPMT*3 allele was detected in genomic DNA from approximately 75 percent of unrelated white subjects with heterozygous phenotypes, indicating that TPMT*3 is the most prevalent mutant allele associated with TPMT-deficiency in Caucasians. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:8644731

Characterization of the snowy cotyledon 1 mutant of Arabidopsis thaliana: the impact of chloroplast elongation factor G on chloroplast development and plant vitality.

PubMed

Albrecht, Verónica; Ingenfeld, Anke; Apel, Klaus

2006-03-01

During seedling development chloroplast formation marks the transition from heterotrophic to autotrophic growth. The development and activity of chloroplasts may differ in cotyledons that initially serve as a storage organ and true leaves whose primary function is photosynthesis. A genetic screen was used for the identification of genes that affect selectively chloroplast function in cotyledons of Arabidopsis thaliana. Several mutants exhibiting pale cotyledons and green true leaves were isolated and dubbed snowy cotyledon (sco). One of the mutants, sco1, was characterized in more detail. The mutated gene was identified using map-based cloning. The mutant contains a point mutation in a gene encoding the chloroplast elongation factor G, leading to an amino acid exchange within the predicted 70S ribosome-binding domain. The mutation results in a delay in the onset of germination. At this early developmental stage embryos still contain undifferentiated proplastids, whose proper function seems necessary for seed germination. In light-grown sco1 seedlings the greening of cotyledons is severely impaired, whereas the following true leaves develop normally as in wild-type plants. Despite this apparent similarity of chloroplast development in true leaves of mutant and wild-type plants various aspects of mature plant development are also affected by the sco1 mutation such as the onset of flowering, the growth rate, and seed production. The onset of senescence in the mutant and the wild-type plants occurs, however, at the same time, suggesting that in the mutant this particular developmental step does not seem to suffer from reduced protein translation efficiency in chloroplasts.

A Suppressor of the Menadione-Hypersensitive Phenotype of a Xanthomonas campestris pv. phaseoli oxyR Mutant Reveals a Novel Mechanism of Toxicity and the Protective Role of Alkyl Hydroperoxide Reductase

PubMed Central

Vattanaviboon, Paiboon; Whangsuk, Wirongrong; Mongkolsuk, Skorn

2003-01-01

We isolated menadione-resistant mutants of Xanthomonas campestris pv. phaseoli oxyR (oxyRXp). The oxyRR2Xp mutant was hyperresistant to the superoxide generators menadione and plumbagin and was moderately resistant to H2O2 and tert-butyl hydroperoxide. Analysis of enzymes involved in oxidative-stress protection in the oxyRR2Xp mutant revealed a >10-fold increase in AhpC and AhpF levels, while the levels of superoxide dismutase (SOD), catalase, and the organic hydroperoxide resistance protein (Ohr) were not significantly altered. Inactivation of ahpC in the oxyRR2Xp mutant resulted in increased sensitivity to menadione killing. Moreover, high levels of expression of cloned ahpC and ahpF in the oxyRXp mutant complemented the menadione hypersensitivity phenotype. High levels of other oxidant-scavenging enzymes such as catalase and SOD did not protect the cells from menadione toxicity. These data strongly suggest that the toxicity of superoxide generators could be mediated via organic peroxide production and that alkyl hydroperoxide reductase has an important novel function in the protection against the toxicity of these compounds in X. campestris. PMID:12591894

PDGFRA-mutant syndrome.

PubMed

Ricci, Riccardo; Martini, Maurizio; Cenci, Tonia; Carbone, Arnaldo; Lanza, Paola; Biondi, Alberto; Rindi, Guido; Cassano, Alessandra; Larghi, Alberto; Persiani, Roberto; Larocca, Luigi M

2015-07-01

Germline PDGFRA mutations cause multiple heterogeneous gastrointestinal mesenchymal tumors. In its familial form this disease, which was formerly termed intestinal neurofibromatosis/neurofibromatosis 3b (INF/NF3b), has been included among familial gastrointestinal stromal tumors (GISTs) because of its genotype, described when GIST was the only known PDGFRA-mutant gastrointestinal tumor. Shortly afterwards, however, inflammatory fibroid polyps also revealed PDGFRA mutations. Subsequently, gastrointestinal CD34+ 'fibrous tumors' of uncertain classification were described in a germline PDGFRA-mutant context. Our aim was to characterize the syndrome produced by germline PDGFRA mutations and establish diagnostic criteria and management strategies for this hitherto puzzling disease. We studied a kindred displaying multiple gastrointestinal mesenchymal tumors, comparing it with published families/individuals with possible analogous conditions. We identified a novel inherited PDGFRA mutation (P653L), constituting the third reported example of familial PDGFRA mutation. In adult mutants we detected inflammatory fibroid polyps, gastric GISTs and gastrointestinal fibrous tumors of uncertain nosology. We demonstrate that the syndrome formerly defined as INF/NF3b (exemplified by the family reported herein) is simplistically considered a form of familial GIST, because inflammatory fibroid polyps often prevail. Fibrous tumors appear variants of inflammatory fibroid polyps. 'INF/NF3b' and 'familial GIST' are misleading terms which we propose changing to 'PDGFRA-mutant syndrome'. In this condition, unlike KIT-dependent familial GIST syndromes, if present, GISTs are stomach-restricted and diffuse Cajal cell hyperplasia is not observed. This restriction of GISTs to the stomach in PDGFRA-mutant syndrome: (i) focuses oncological concern on gastric masses, as inflammatory fibroid polyps are benign; (ii) supports a selective role of gastric environment for PDGFRA mutations to elicit GISTs

Cloning of a methanol-inducible moxF promoter and its analysis in moxB mutants of Methylobacterium extorquens AM1rif.

PubMed Central

Morris, C J; Lidstrom, M E

1992-01-01

In Methylobacterium extorquens AM1, gene encoding methanol dehydrogenase polypeptides are transcriptionally regulated in response to C1 compounds, including methanol (M. E. Lidstrom and D. I. Stirling, Annu. Rev. Microbiol. 44:27-57, 1990). In order to study this regulation, a transcriptional fusion has been constructed between a beta-galactosidase reporter gene and a 1.55-kb XhoI-SalI fragment of M. extorquens AM1rif DNA encoding the N terminus of the methanol dehydrogenase large subunit (moxF) and 1,289 bp of upstream DNA. The fusion exhibited orientation-specific promoter activity in M. extorquens AM1rif but was expressed constitutively when the transcriptional fusion was located on the plasmid. However, correct regulation was restored when the construction was inserted in the M. extorquens AM1rif chromosome. This DNA fragment was shown to contain both the moxFJGI promoter and the sequences necessary in cis for its transcriptional regulation by methanol. Transcription from this promoter was studied in the M. extorquens AM1rif moxB mutant strains UV4rif and UV25rif, which have a pleiotropic phenotype with regard to the components of methanol oxidation. In these mutants, beta-galactosidase activity from the fusion was reduced to a level equal to that of the vector background when the fusion was present in both plasmid and chromosomal locations. Since both constitutive and methanol-inducible promoter activities were lost in the mutants, moxB appears to be required for transcription of the genes encoding the methanol dehydrogenase polypeptides. Images PMID:1624436

Cloning, Sequencing, and Characterization of the cgmB Gene of Sinorhizobium meliloti Involved in Cyclic β-Glucan Biosynthesis

PubMed Central

Wang, Ping; Ingram-Smith, Cheryl; Hadley, Jill A.; Miller, Karen J.

1999-01-01

Periplasmic cyclic β-glucans of Rhizobium species provide important functions during plant infection and hypo-osmotic adaptation. In Sinorhizobium meliloti (also known as Rhizobium meliloti), these molecules are highly modified with phosphoglycerol and succinyl substituents. We have previously identified an S. meliloti Tn5 insertion mutant, S9, which is specifically impaired in its ability to transfer phosphoglycerol substituents to the cyclic β-glucan backbone (M. W. Breedveld, J. A. Hadley, and K. J. Miller, J. Bacteriol. 177:6346–6351, 1995). In the present study, we have cloned, sequenced, and characterized this mutation at the molecular level. By using the Tn5 flanking sequences (amplified by inverse PCR) as a probe, an S. meliloti genomic library was screened, and two overlapping cosmid clones which functionally complement S9 were isolated. A 3.1-kb HindIII-EcoRI fragment found in both cosmids was shown to fully complement mutant S9. Furthermore, when a plasmid containing this 3.1-kb fragment was used to transform Rhizobium leguminosarum bv. trifolii TA-1JH, a strain which normally synthesizes only neutral cyclic β-glucans, anionic glucans containing phosphoglycerol substituents were produced, consistent with the functional expression of an S. meliloti phosphoglycerol transferase gene. Sequence analysis revealed the presence of two major, overlapping open reading frames within the 3.1-kb fragment. Primer extension analysis revealed that one of these open reading frames, ORF1, was transcribed and its transcription was osmotically regulated. This novel locus of S. meliloti is designated the cgm (cyclic glucan modification) locus, and the product encoded by ORF1 is referred to as CgmB. PMID:10419956

Characterization of a Null Allelic Mutant of the Rice NAL1 Gene Reveals Its Role in Regulating Cell Division

PubMed Central

Jiang, Dan; Fang, Jingjing; Lou, Lamei; Zhao, Jinfeng; Yuan, Shoujiang; Yin, Liang; Sun, Wei; Peng, Lixiang; Guo, Baotai; Li, Xueyong

2015-01-01

Leaf morphology is closely associated with cell division. In rice, mutations in Narrow leaf 1 (NAL1) show narrow leaf phenotypes. Previous studies have shown that NAL1 plays a role in regulating vein patterning and increasing grain yield in indica cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of NARROW LEAF1 (NAL1), nal1-2 and nal1-3, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that nal1-2 is a null allelic mutant of NAL1 since both the whole promoter and a 404-bp fragment in the first exon of NAL1 were deleted, and that a 6-bp fragment was deleted in the mutant nal1-3. We demonstrated that NAL1 functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that NAL1 affects cell cycle regulation. Heterogenous expression of NAL1 in fission yeast (Schizosaccharomyces pombe) further supported that NAL1 affects cell division. These results suggest that NAL1 controls leaf width and plant height through its effects on cell division. PMID:25658704

Therapeutic cloning in the mouse

PubMed Central

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

Energy Values of Nine Populus Clones

Treesearch

Terry F. Strong

1980-01-01

Compares calorific values for components of nine Populus clones. The components include stem wood, stem bark, and branches. Also compares calorific values for clones of balsam poplar and black cottonwood parentages.

Benefits and problems with cloning animals.

PubMed Central

Smith, L C; Bordignon, V; Babkine, M; Fecteau, G; Keefer, C

2000-01-01

Animal cloning is becoming a useful technique for producing transgenic farm animals and is likely to be used to produce clones from valuable adults. Other applications will also undoubtedly be discovered in the near future, such as for preserving endangered breeds and species. Although cloning promises great advantages for commerce and research alike, its outcome is not always certain due to high pregnancy losses and high morbidity and mortality during the neonatal period. Research into the mechanisms involved in the reprogramming of the nucleus is being conducted throughout the world in an attempt to better understand the molecular and cellular mechanisms involved in correcting these problems. Although the cause of these anomalies remains mostly unknown, similar phenotypes have been observed in calves derived through in vitro fertilization, suggesting that culture conditions are involved in these phenomena. In the meantime, veterinarians and theriogenologists have an important role to play in improving the efficiency of cloning by finding treatments to assure normal gestation to term and to develop preventative and curative care for cloned neonates. Images Figure 1. PMID:11143925

Human cloning: category, dignity, and the role of bioethics.

PubMed

Shuster, Evelyne

2003-10-01

Human cloning has been simultaneously a running joke for massive worldwide publicity of fringe groups like the Raelians, and the core issue of an international movement at the United Nations in support of a treaty to ban the use of cloning techniques to produce a child (so called reproductive cloning). Yet, even though debates on human cloning have greatly increased since the birth of Dolly, the clone sheep, in 1997, we continue to wonder whether cloning is after all any different from other methods of medically assisted reproduction, and what exactly makes cloning an 'affront to the dignity of humans.' Categories we adopt matter mightily as they inform but can also misinform and lead to mistaken and unproductive decisions. And thus bioethicists have a responsibility to ensure that the proper categories are used in the cloning debates and denounce those who try to win the ethical debate through well-crafted labels rather than well-reasoned argumentations. But it is as important for bioethicists to take a position on broad issues such as human cloning and species altering interventions. One 'natural question' would be, for example, should there be an international treaty to ban human reproductive cloning?

[Cloning and law in Hungary].

PubMed

Julesz, Máté

2015-03-01

Reproductive human cloning is prohibited in Hungary, as in many other countries. Therapeutic human cloning is not prohibited, just like in many other countries. Stem cell therapy is also allowed. Article III, paragraph (3) of the Hungarian basic law (constitution) strictly forbids total human cloning. Article 1 of the Additional Protocol to the Oviedo Convention, on the Prohibition of Cloning Human Beings (1998) stipulates that any intervention seeking to create a human being genetically identical to another human being, whether living or dead, is prohibited. In Hungary, according to Article 174 of the Criminal Code, total human cloning constitutes a crime. Article 180, paragraph (3) of the Hungarian Act on Health declares that embryos shall not be brought about for research purposes; research shall be conducted only on embryos brought about for reproductive purposes when this is authorized by the persons entitled to decide upon its disposal, or when the embryo is damaged. Article 180, paragraph (5) of the Hungarian Act on Health stipulates that multiple individuals who genetically conform to one another shall not be brought about. According to Article 181, paragraph (1) of the Hungarian Act on Health, an embryo used for research shall be kept alive for not longer than 14 days, not counting the time it was frozen for storage and the time period of research.

Emotional reactions to human reproductive cloning.

PubMed

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/

Specific Detection of Naturally Occurring Hepatitis C Virus Mutants with Resistance to Telaprevir and Boceprevir (Protease Inhibitors) among Treatment-Naïve Infected Individuals

PubMed Central

Fonseca-Coronado, Salvador; Escobar-Gutiérrez, Alejandro; Ruiz-Tovar, Karina; Cruz-Rivera, Mayra Yolanda; Rivera-Osorio, Pilar; Vazquez-Pichardo, Mauricio; Carpio-Pedroza, Juan Carlos; Ruíz-Pacheco, Juan Alberto; Cazares, Fernando

2012-01-01

The use of telaprevir and boceprevir, both protease inhibitors (PI), as part of the specifically targeted antiviral therapy for hepatitis C (STAT-C) has significantly improved sustained virologic response (SVR) rates. However, different clinical studies have also identified several mutations associated with viral resistance to both PIs. In the absence of selective pressure, drug-resistant hepatitis C virus (HCV) mutants are generally present at low frequency, making mutation detection challenging. Here, we describe a mismatch amplification mutation assay (MAMA) PCR method for the specific detection of naturally occurring drug-resistant HCV mutants. MAMA PCR successfully identified the corresponding HCV variants, while conventional methods such as direct sequencing, endpoint limiting dilution (EPLD), and bacterial cloning were not sensitive enough to detect circulating drug-resistant mutants in clinical specimens. Ultradeep pyrosequencing was used to confirm the presence of the corresponding HCV mutants. In treatment-naïve patients, the frequency of all resistant variants was below 1%. Deep amplicon sequencing allowed a detailed analysis of the structure of the viral population among these patients, showing that the evolution of the NS3 is limited to a rather small sequence space. Monitoring of HCV drug resistance before and during treatment is likely to provide important information for management of patients undergoing anti-HCV therapy. PMID:22116161

Structure prediction and activity analysis of human heme oxygenase-1 and its mutant.

PubMed

Xia, Zhen-Wei; Zhou, Wen-Pu; Cui, Wen-Jun; Zhang, Xue-Hong; Shen, Qing-Xiang; Li, Yun-Zhu; Yu, Shan-Chang

2004-08-15

To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (delta hHO-1) structures, to clone and express them and analyze their activities. Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physical-chemical changes between wild and mutant hHO-1. hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E. coli DH5alpha. Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured. rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices. Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative. The mutated enzyme kept binding activity to heme. Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed. Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively. The activity of delta hHO-1 was reduced 91.21% after mutation compared with whHO-1. Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. delta hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. delta hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia.

Structure prediction and activity analysis of human heme oxygenase-1 and its mutant

PubMed Central

Xia, Zhen-Wei; Zhou, Wen-Pu; Cui, Wen-Jun; Zhang, Xue-Hong; Shen, Qing-Xiang; Li, Yun-Zhu; Yu, Shan-Chang

2004-01-01

AIM: To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (△hHO-1) structures, to clone and express them and analyze their activities. METHODS: Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physical-chemical changes between wild and mutant hHO-1. hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E. coli DH5α . Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured. RESULTS: rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices. Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative. The mutated enzyme kept binding activity to heme. Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed. Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively. The activity of △hHO-1 was reduced 91.21% after mutation compared with whHO-1. CONCLUSION: Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. △hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. △hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia. PMID:15285018

Cloning Mice and Men: Prohibiting the Use of iPS Cells for Human Reproductive Cloning

PubMed Central

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

2014-01-01

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. PMID:20085739

The effect of high-fat diet on the composition of the gut microbiota in cloned and non-cloned pigs of lean and obese phenotype

PubMed Central

Pedersen, Rebecca; Andersen, Anders Daniel; Hermann-Bank, Marie Louise; Stagsted, Jan; Boye, Mette

2013-01-01

The aim of this study was to investigate the effect of high-far-high-energy diet on cloned and non-cloned domestic pigs of both lean and obese phenotype and to evaluate if the lean cloned pigs had a lower inter-individual variation as compared with non-cloned pigs. The microbiota of colon and terminal ileum was investigated in cloned and non-cloned pigs that received a high-far-high-energy diet with either restricted or ad libitum access to feed, resulting in lean and obese phenotypes, respectively. The fecal microbiota of lean pigs was investigated by terminal restriction fragment length polymorphism (T-RFLP). The intestinal microbiota of lean and obese cloned and non-cloned pigs was analyzed by quantitative real time PCR and a novel high-throughput qPCR platform (Fluidigm). Principal component analysis (PCA) of the T-RFLP profiles revealed that lean cloned and non-cloned pigs had a different overall composition of their gut microbiota. The colon of lean cloned pigs contained relatively more bacteria belonging to the phylum Firmicutes and less from the phylum Bacteroidetes than obese cloned pigs as estimated by qPCR. Fluidigm qPCR results revealed differences in specific bacterial groups in the gut microbiota of both lean and obese pigs. Our results suggest that high-far-high-energy diet is associated with changes in the gut microbiota even in the absence of obesity. Overall, the cloned pigs had a different gut microbiota from that of non-cloned pigs. To our knowledge this is the first study to investigate the gut microbiota of cloned domestic pigs of lean and obese phenotype. PMID:23974297

The effect of high-fat diet on the composition of the gut microbiota in cloned and non-cloned pigs of lean and obese phenotype.

PubMed

Pedersen, Rebecca; Andersen, Anders Daniel; Hermann-Bank, Marie Louise; Stagsted, Jan; Boye, Mette

2013-01-01

The aim of this study was to investigate the effect of high-far-high-energy diet on cloned and non-cloned domestic pigs of both lean and obese phenotype and to evaluate if the lean cloned pigs had a lower inter-individual variation as compared with non-cloned pigs. The microbiota of colon and terminal ileum was investigated in cloned and non-cloned pigs that received a high-far-high-energy diet with either restricted or ad libitum access to feed, resulting in lean and obese phenotypes, respectively. The fecal microbiota of lean pigs was investigated by terminal restriction fragment length polymorphism (T-RFLP). The intestinal microbiota of lean and obese cloned and non-cloned pigs was analyzed by quantitative real time PCR and a novel high-throughput qPCR platform (Fluidigm). Principal component analysis (PCA) of the T-RFLP profiles revealed that lean cloned and non-cloned pigs had a different overall composition of their gut microbiota. The colon of lean cloned pigs contained relatively more bacteria belonging to the phylum Firmicutes and less from the phylum Bacteroidetes than obese cloned pigs as estimated by qPCR. Fluidigm qPCR results revealed differences in specific bacterial groups in the gut microbiota of both lean and obese pigs. Our results suggest that high-far-high-energy diet is associated with changes in the gut microbiota even in the absence of obesity. Overall, the cloned pigs had a different gut microbiota from that of non-cloned pigs. To our knowledge this is the first study to investigate the gut microbiota of cloned domestic pigs of lean and obese phenotype.

Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

PubMed Central

Yang, Hee-Jeong; Bogomolnaya, Lydia M.; Elfenbein, Johanna R.; Endicott-Yazdani, Tiana; Reynolds, M. Megan; Porwollik, Steffen; Cheng, Pui; Xia, Xiao-Qin

2016-01-01

Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection. PMID:26857572

Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene

PubMed Central

Sheng, Yanmin; Wang, Yingdian; Capell, Teresa; Shi, Lianxuan; Ni, Xiuzhen; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

2015-01-01

The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity. PMID:26030746

[Therapeutic cloning. Biology, perspectives and alternatives].

PubMed

Maddox-Hyttel, Poul

2003-02-24

Certain diseases are caused by or cause irreversible loss of cells and may in the future be treated by cell-based therapies where spare cells are introduced into the body. Therapeutic cloning constitutes a scientifically and ethically challenging route to the generation of autologous patient specific spare cells: Stem cells for subsequent differentiation and transplantation are isolated from one week old embryos, which are produced by cloning by nuclear transfer from normal cells retrieved from a patient. Research in therapeutic cloning should be pursued in line with alternative strategies for obtaining stem cells. Finally, the molecular biology of cloning by nuclear transfer may hold the key to understanding trans-differentiation, which ultimately may allow for de-differentiation and subsequent re-differentiation of adult somatic cells for therapeutic purposes.

Quantum cloning disturbed by thermal Davies environment

NASA Astrophysics Data System (ADS)

Dajka, Jerzy; Łuczka, Jerzy

2016-06-01

A network of quantum gates designed to implement universal quantum cloning machine is studied. We analyze how thermal environment coupled to auxiliary qubits, `blank paper' and `toner' required at the preparation stage of copying, modifies an output fidelity of the cloner. Thermal environment is described in terms of the Markovian Davies theory. We show that such a cloning machine is not universal any more but its output is independent of at least a part of parameters of the environment. As a case study, we consider cloning of states in a six-state cryptography's protocol. We also briefly discuss cloning of arbitrary input states.

Trichostatin A (TSA) improves the development of rabbit-rabbit intraspecies cloned embryos, but not rabbit-human interspecies cloned embryos.

PubMed

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.

Reproductive cloning in humans and therapeutic cloning in primates: is the ethical debate catching up with the recent scientific advances?

PubMed

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

PubMed Central

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

Aging of Cloned Animals: A Mini-Review.

PubMed

Burgstaller, Jörg Patrick; Brem, Gottfried

2017-01-01

The number of species for which somatic cell nuclear transfer (SCNT) protocols are established is still increasing. Due to the high number of cloned farm, companion, and sport animals, the topic of animal cloning never ceases to be of public interest. Numerous studies cover the health status of SCNT-derived animals, but very few cover the effects of SCNT on aging. However, only cloned animals that reach the full extent of the species-specific lifespan, doing so with only the normal age-related afflictions and diseases, would prove that SCNT can produce completely healthy offspring. Here, we review the available literature and own data to answer the question whether the aging process of cloned animals is qualitatively different from normal animals. We focus on 4 main factors that were proposed to influence aging in these animals: epigenetic (dys)regulation, accumulation of damaged macromolecules, shortened telomeres, and (nuclear donor-derived) age-related DNA damage. We find that at least some cloned animals can reach the species-specific maximum age with a performance that matches that of normal animals. However, for most species, only anecdotal evidence of cloned animals reaching high age is available. We therefore encourage reports on the aging of cloned animals to make further analysis on the performance of SCNT possible. © 2016 S. Karger AG, Basel.

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

PubMed Central

2005-01-01

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. PMID:15601595

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

PubMed

Pailhoux, Eric; Vigier, Bernard; Schibler, Laurent; Cribiu, Edmond P; Cotinot, Corinne; Vaiman, Daniel

2005-01-01

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.

High-dimensional quantum cloning and applications to quantum hacking

PubMed Central

Bouchard, Frédéric; Fickler, Robert; Boyd, Robert W.; Karimi, Ebrahim

2017-01-01

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. PMID:28168219

High-dimensional quantum cloning and applications to quantum hacking.

PubMed

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.

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

PubMed

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 mice and men: prohibiting the use of iPS cells for human reproductive cloning.

PubMed

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: can it be good for us? An overview of cloning technology and its moral implications.

PubMed

FitzGerald, K

2001-01-01

Adequate answers to moral questions about cloning require a working knowledge of the science and technology involved, both present and anticipated. This essay presents an overview of the current state of somatic cell nuclear transfer technology (SCNT), the type of cloning that now permits whole organism reproduction from adult DNA. This essay explains the basic science and technology of SCNT and explores its potential uses. Next, this essay notes remaining scientific obstacles and unanswered moral questions that must be resolved before SCNT can be used for human reproduction. Attention is given to aspects related to cloning for therapeutic and research purposes.

Memory B cells, but not long-lived plasma cells, possess antigen specificities for viral escape mutants

PubMed Central

Purtha, Whitney E.; Tedder, Thomas F.; Johnson, Syd

2011-01-01

Memory B cells (MBCs) and long-lived plasma cells (LLPCs) persist after clearance of infection, yet the specific and nonredundant role MBCs play in subsequent protection is unclear. After resolution of West Nile virus infection in mice, we demonstrate that LLPCs were specific for a single dominant neutralizing epitope, such that immune serum poorly inhibited a variant virus that encoded a mutation at this critical epitope. In contrast, a large fraction of MBC produced antibody that recognized both wild-type (WT) and mutant viral epitopes. Accordingly, antibody produced by the polyclonal pool of MBC neutralized WT and variant viruses equivalently. Remarkably, we also identified MBC clones that recognized the mutant epitope better than the WT protein, despite never having been exposed to the variant virus. The ability of MBCs to respond to variant viruses in vivo was confirmed by experiments in which MBCs were adoptively transferred or depleted before secondary challenge. Our data demonstrate that class-switched MBC can respond to variants of the original pathogen that escape neutralization of antibody produced by LLPC without a requirement for accumulating additional somatic mutations. PMID:22162833

Endangered wolves cloned from adult somatic cells.

PubMed

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

PubMed Central

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.

PubMed

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].

PubMed

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.

Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance.

PubMed

Arezi, Bahram; McKinney, Nancy; Hansen, Connie; Cayouette, Michelle; Fox, Jeffrey; Chen, Keith; Lapira, Jennifer; Hamilton, Sarah; Hogrefe, Holly

2014-01-01

Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd ) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.

Telomeres and the ethics of human cloning.

PubMed

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.

Mutant botrocetin-2 inhibits von Willebrand factor-induced platelet agglutination.

PubMed

Matsui, T; Hori, A; Hamako, J; Matsushita, F; Ozeki, Y; Sakurai, Y; Hayakawa, M; Matsumoto, M; Fujimura, Y

2017-03-01

Essentials Botrocetin-2 (Bot2) binds to von Willebrand factor (VWF) and induces platelet agglutination. We identified Bot2 residues that are required for binding to VWF and glycoprotein (GP) Ib. We produced a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Mutant Bot2 could be used as a potential anti-thrombotic reagent to block VWF-GPIb interaction. Background Botrocetin-2 (Bot2) is a botrocetin-like protein composed of α and β subunits that have been cloned from the snake Bothrops jararaca. Bot2 binds specifically to von Willebrand factor (VWF), and the complex induces glycoprotein (GP) Ib-dependent platelet agglutination. Objectives To exploit Bot2's VWF-binding capacity in order to attempt to create a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Methods and Results Several point mutations were introduced into Bot2 cDNA, and the recombinant protein (recombinant Bot2 [rBot2]) was purified on an anti-botrocetin column. The mutant rBot2 with either Ala at Asp70 in the β subunit (Aspβ70Ala), or Argβ115Ala and Lysβ117Ala, showed reduced platelet agglutination-inducing activity. rBot2 with Aspβ70Ala showed little binding activity towards immobilized VWF on an ELISA plate, whereas rBot2 with Argβ115Ala/Lysβ117Ala showed reduced binding activity towards GPIb (glycocalicin) after forming a complex with VWF. rBot2 point-mutated to oppositely charged Glu at both Argβ115 and Lysβ117 showed normal binding activity towards VWF but no platelet-agglutinating activity. Furthermore, this doubly mutated protein inhibited ristocetin-induced or high shear stress-induced platelet aggregation, and restrained thrombus formation under flow conditions. Conclusions Asp70 in the β subunit of botrocetin is important for VWF binding, and Arg115 and Lys117 in the β subunit are essential for interaction with GPIb. Doubly mutated rBot2, with Argβ115Glu and Lysβ117Glu, repels GPIb and might have potential as an antithrombotic reagent that

Probabilistic Metrology Attains Macroscopic Cloning of Quantum Clocks

NASA Astrophysics Data System (ADS)

Gendra, B.; Calsamiglia, J.; Muñoz-Tapia, R.; Bagan, E.; Chiribella, G.

2014-12-01

It has recently been shown that probabilistic protocols based on postselection boost the performances of the replication of quantum clocks and phase estimation. Here we demonstrate that the improvements in these two tasks have to match exactly in the macroscopic limit where the number of clones grows to infinity, preserving the equivalence between asymptotic cloning and state estimation for arbitrary values of the success probability. Remarkably, the cloning fidelity depends critically on the number of rationally independent eigenvalues of the clock Hamiltonian. We also prove that probabilistic metrology can simulate cloning in the macroscopic limit for arbitrary sets of states when the performance of the simulation is measured by testing small groups of clones.

[The discrete horror of cloning].

PubMed

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.

ERIC Educational Resources Information Center

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)

A comprehensive list of cloned human DNA sequences

PubMed Central

Schmidtke, Jörg; Cooper, David N.

1987-01-01

A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:3575113

A comprehensive list of cloned human DNA sequences

PubMed Central

Schmidtke, Jörg; Cooper, David N.

1990-01-01

A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:2333227

A comprehensive list of cloned human DNA sequences

PubMed Central

Schmidtke, Jörg; Cooper, David N.

1988-01-01

A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:3368330

A comprehensive list of cloned human DNA sequences

PubMed Central

Schmidtke, Jörg; Cooper, David N.

1989-01-01

A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:2654889

VH gene expression and regulation in the mutant Alicia rabbit. Rescue of VHa2 allotype expression.

PubMed

Chen, H T; Alexander, C B; Young-Cooper, G O; Mage, R G

1993-04-01

Rabbits of the Alicia strain, derived from rabbits expressing the VHa2 allotype, have a mutation in the H chain locus that has a cis effect upon the expression of VHa2 and VHa- genes. A small deletion at the most J-proximal (3') end of the VH locus leads to low expression of all the genes on the entire chromosome in heterozygous ali mutants and altered relative expression of VH genes in homozygotes. To study VH gene expression and regulation, we used the polymerase chain reaction to amplify the VH genes expressed in spleens of young and adult wild-type and mutant Alicia rabbits. The cDNA from reverse transcription of splenic mRNA was amplified and polymerase chain reaction libraries were constructed and screened with oligonucleotides from framework regions 1 and 3, as well as JH. Thirty-three VH-positive clones were sequenced and analyzed. We found that in mutant Alicia rabbits, products of the first functional VH gene (VH4a2), (or VH4a2-like genes) were expressed in 2- to 8-wk-olds. Expression of both the VHx and VHy types of VHa- genes was also elevated but the relative proportions of VHx and VHy, especially VHx, decreased whereas the relative levels of expression of VH4a2 or VH4a2-like genes increased with age. Our results suggest that the appearance of sequences resembling that of the VH1a2, which is deleted in the mutant ali rabbits, could be caused by alterations of the sequences of the rearranged VH4a2 genes by gene conversions and/or rearrangement of upstream VH1a2-like genes later in development.

[Telomere lengthening by trichostatin A treatment in cloned pigs].

PubMed

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 (P<0.05), indicating that nuclear reprogramming did not restore cellular age of donor cells after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA), an inhibitor of histone deacetylase, has proven to enhance the efficiency of nuclear reprogramming in several species. In order to test whether TSA also can effectively enhance reprogramming of telomeres, TSA (40 nmol/L) was used to treat porcine cloned embryos at 1-cell stage 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%, P<0.05). Notably, the telomere length of cloned porcine blastocysts was also significantly elongated (P<0.05). Although TSA did not improve the cloning efficiency (1.3% vs. 1.7%, TSA vs. control), the telomere lengths of cloned pig-lets were significantly longer compared with those of the control group and the donor fibroblasts (P<0.05). In conclusion, telomeres have not been effectively restored by SCNT in pigs but TSA can effectively lengthen the telomere lengths of cloned pigs.

Preservation and Reproduction of Microminipigs by Cloning Technology.

PubMed

Enya, Satoko; Kawarasaki, Tatsuo; Otake, Masayoshi; Kangawa, Akihisa; Uenishi, Hirohide; Mikawa, Satoshi; Nishimura, Takashi; Kuwahawa, Yasushi; Shibata, Masatoshi

Microminipigs have been maintained in small populations of closed colonies, involving risks of inbreeding depression and genetic drift. In order to avoid these risks, we assessed the applicability of cloning technology. Male and female clones were produced from a stock of cryopreserved somatic cells, obtaining offspring by means of natural mating. Phenotypic and genotypic characteristics of original microminipigs, clones and their offspring were analyzed and recorded. Clones presented characteristics similar to those of the cell-stock data. Although the body weight of clones tended to be heavier than that of the cell-stock data, body weights of their offspring were similar to those of previous reports. Thus, cloned microminipigs have the potential to be a valuable genetic resource for reproduction and breeding. Our proposed methodology might be useful to provide a large number of animals with adequate quality from a limited population with sufficient genetic diversity. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

[Product safety analysis of somatic cell cloned bovine].

PubMed

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.

Ultra-low background DNA cloning system.

PubMed

Goto, Kenta; Nagano, Yukio

2013-01-01

Yeast-based in vivo cloning is useful for cloning DNA fragments into plasmid vectors and is based on the ability of yeast to recombine the DNA fragments by homologous recombination. Although this method is efficient, it produces some by-products. We have developed an "ultra-low background DNA cloning system" on the basis of yeast-based in vivo cloning, by almost completely eliminating the generation of by-products and applying the method to commonly used Escherichia coli vectors, particularly those lacking yeast replication origins and carrying an ampicillin resistance gene (Amp(r)). First, we constructed a conversion cassette containing the DNA sequences in the following order: an Amp(r) 5' UTR (untranslated region) and coding region, an autonomous replication sequence and a centromere sequence from yeast, a TRP1 yeast selectable marker, and an Amp(r) 3' UTR. This cassette allowed conversion of the Amp(r)-containing vector into the yeast/E. coli shuttle vector through use of the Amp(r) sequence by homologous recombination. Furthermore, simultaneous transformation of the desired DNA fragment into yeast allowed cloning of this DNA fragment into the same vector. We rescued the plasmid vectors from all yeast transformants, and by-products containing the E. coli replication origin disappeared. Next, the rescued vectors were transformed into E. coli and the by-products containing the yeast replication origin disappeared. Thus, our method used yeast- and E. coli-specific "origins of replication" to eliminate the generation of by-products. Finally, we successfully cloned the DNA fragment into the vector with almost 100% efficiency.

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

PubMed

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.

Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia.

PubMed

Kaye, C; Crawford, N M; Malmberg, R L

1997-04-01

We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.

DNA hypomethylation of individual sequences in aborted cloned bovine fetuses.

PubMed

Chen, Tao; Jiang, Yan; Zhang, Yan-Ling; Liu, Jing-He; Hou, Yi; Schatten, Heide; Chen, Da-Yuan; Sun, Qing-Yuan

2005-09-01

Cloned bovines have a much higher abortion rate than those derived in vivo. Available evidence indicates that inappropriate epigenetic reprogramming of donor nuclei is the primary cause of cloning failure. To gain a better understanding of the DNA methylation changes associated with the high abortion rate of cloned bovines, we examined the DNA methylation status of a repeated sequence (satellite I) and the promoter regions of two single-copy genes (interleukin 3/cytokeratin) in aborted cloned fetuses, aborted fetuses derived from artificial insemination (AI), cloned adults and AI adults by bisulfite sequencing and restriction enzyme analysis. Two of four aborted cloned fetuses show very low methylation levels in the two single-copy gene promoter regions. One of the two fetuses also showed undermethylated status in the satellite I sequence. The other two aborted cloned fetuses have similar methylation levels to those of aborted AI fetuses. However, no difference in methylation was observed between cloned adults and AI adults. Our results demonstrate for the first time the undermethylated status of individual sequences in aborted cloned fetuses. These findings suggest that aberrant DNA methylation may contribute to the developmental failure of cloned bovine fetuses.

Role of the Bacillus methanolicus citrate synthase II gene, citY, in regulating the secretion of glutamate in L-lysine-secreting mutants.

PubMed

Brautaset, Trygve; Williams, Mark D; Dillingham, Richard D; Kaufmann, Christine; Bennaars, Assumpta; Crabbe, Edward; Flickinger, Michael C

2003-07-01

The thermotolerant, restrictive methylotroph Bacillus methanolicus MGA3 (ATCC 53907) can secrete 55 g of glutamate per liter (maximum yield, 0.36 g/g) at 50 degrees C with methanol as a carbon source and a source of ammonia in fed-batch bioreactors. A homoserine dehydrogenase mutant, 13A52-8A66, secreting up to 35 g of L-lysine per liter in fed-batch fermentations had minimal 2-oxoglutarate dehydrogenase activity [7.3 nmol min(-1) (mg of protein)(-1)], threefold-increased pyruvate carboxylase activity [535 nmol min(-1) (mg of protein)(-1)], and elevated citrate synthase (CS) activity [292 nmol min(-1) (mg of protein)(-1)] and simultaneously secreted glutamate (20 to 30 g per liter) and L-lysine. The flow of carbon from oxaloacetate is split between transamination to aspartate and formation of citrate. To investigate the regulation of this branch point, the B. methanolicus gene citY encoding a CSII protein with activity at 50 degrees C was cloned from 13A52-8A66 into a CS-deficient Escherichia coli K2-1-4 strain. A citY-deficient B. methanolicus mutant, NCS-L-7, was also isolated from the parent strain of 13A52-8A66 by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, followed by selection with monofluoroacetate disks on glutamate plates. Characterization of these strains confirmed that citY in strain 13A52-8A66 was not altered and that B. methanolicus possessed several forms of CS. Analysis of citY cloned from NCS-L-7 showed that the reduced CS activity resulted from a frameshift mutation. The level of glutamate secreted by NCS-L-7 was reduced sevenfold and the ratio of L-lysine to glutamate secreted was increased 4.5-fold compared to the wild type in fed-batch cultures with glutamate feeding. This indicates that glutamate secretion in L-lysine-overproducing mutants can be altered in favor of increased L-lysine secretion by regulating in vivo CS activity.

Role of the Bacillus methanolicus Citrate Synthase II Gene, citY, in Regulating the Secretion of Glutamate in l-Lysine-Secreting Mutants

PubMed Central

Brautaset, Trygve; Williams, Mark D.; Dillingham, Richard D.; Kaufmann, Christine; Bennaars, Assumpta; Crabbe, Edward; Flickinger, Michael C.

2003-01-01

The thermotolerant, restrictive methylotroph Bacillus methanolicus MGA3 (ATCC 53907) can secrete 55 g of glutamate per liter (maximum yield, 0.36 g/g) at 50°C with methanol as a carbon source and a source of ammonia in fed-batch bioreactors. A homoserine dehydrogenase mutant, 13A52-8A66, secreting up to 35 g of l-lysine per liter in fed-batch fermentations had minimal 2-oxoglutarate dehydrogenase activity [7.3 nmol min−1 (mg of protein)−1], threefold-increased pyruvate carboxylase activity [535 nmol min−1 (mg of protein)−1], and elevated citrate synthase (CS) activity [292 nmol min−1 (mg of protein)−1] and simultaneously secreted glutamate (20 to 30 g per liter) and l-lysine. The flow of carbon from oxaloacetate is split between transamination to aspartate and formation of citrate. To investigate the regulation of this branch point, the B. methanolicus gene citY encoding a CSII protein with activity at 50°C was cloned from 13A52-8A66 into a CS-deficient Escherichia coli K2-1-4 strain. A citY-deficient B. methanolicus mutant, NCS-L-7, was also isolated from the parent strain of 13A52-8A66 by N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis, followed by selection with monofluoroacetate disks on glutamate plates. Characterization of these strains confirmed that citY in strain 13A52-8A66 was not altered and that B. methanolicus possessed several forms of CS. Analysis of citY cloned from NCS-L-7 showed that the reduced CS activity resulted from a frameshift mutation. The level of glutamate secreted by NCS-L-7 was reduced sevenfold and the ratio of l-lysine to glutamate secreted was increased 4.5-fold compared to the wild type in fed-batch cultures with glutamate feeding. This indicates that glutamate secretion in l-lysine-overproducing mutants can be altered in favor of increased l-lysine secretion by regulating in vivo CS activity. PMID:12839772

Precocious Metamorphosis in the Juvenile Hormone–Deficient Mutant of the Silkworm, Bombyx mori

PubMed Central

Daimon, Takaaki; Kozaki, Toshinori; Niwa, Ryusuke; Kobayashi, Isao; Furuta, Kenjiro; Namiki, Toshiki; Uchino, Keiro; Banno, Yutaka; Katsuma, Susumu; Tamura, Toshiki; Mita, Kazuei; Sezutsu, Hideki; Nakayama, Masayoshi; Itoyama, Kyo; Shimada, Toru; Shinoda, Tetsuro

2012-01-01

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several “moltinism” mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval–larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval–pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH–deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis. PMID:22412378

Marked decrease in specific activity contributes to disease phenotype in two human glucose 6-phosphate dehydrogenase mutants, G6PD(Union) and G6PD(Andalus).

PubMed

Wang, Xiao-Tao; Lam, Veronica M S; Engel, Paul C

2005-09-01

Clones overexpressing clinical glucose 6-phosphate dehydrogenase (G6PD) mutants Union (c.1360C>T/p.Arg454Cys) and Andalus (c.1361G>A/p.Arg454His), have been constructed. These abolish a salt bridge between Arg454 and Asp 286. One mutant is reportedly a Class II clinical variant and the other a Class I. Kinetic studies of the purified proteins reveal that, for both mutants, kcat is about 10-fold decreased, thus giving a 90% decrease in the WHO assay, and also presumably under physiological conditions. In contrast with unfavourable changes in Vmax for both mutants, Km values for both G6P and NADP+ are decreased approximately 5-fold. Measurements with alternative substrates confirm that G6PD Union, like the wild-type enzyme, follows a rapid-equilibrium random-order mechanism, allowing calculation of enzyme-substrate dissociation constants from initial-rate parameters. The mutations result in several-fold tighter binding of glucose 6-phosphate to the free enzyme. Binding, however, is clearly less productive than with normal enzyme. G6PD mutations are thought to cause haemolytic anaemia by compromising enzyme stability. Both these mutants indeed show somewhat decreased thermostability. However, at 37 degrees C and with NADP+, the stability differences are only moderate. Decreased catalytic efficiency clearly contributes to the disease phenotype of these two mutants, entirely accounting for reported decrease in leukocyte G6PD levels, though not for still lower levels in erythrocytes. Neither the kinetic nor the stability effects appear to justify the different clinical classification of these mutations.

Cloning and characterization of new bioluminescent proteins

NASA Astrophysics Data System (ADS)

Szent-Gyorgyi, Christopher; Ballou, Byron T.; Dagnal, Erich; Bryan, Bruce

1999-07-01

Over the past two years Prolume has undertaken a comprehensive program to clone luciferases and associated 'green fluorescent proteins' (GFPs) from marine animals that use coelenterazine as the luciferin. To data we have cloned several bioluminescent proteins, including two novel copepod luciferases and two anthozoan GFPs. These four proteins have sequences that differ greatly form previously cloned analogous proteins; the sequence diversity apparently is due to independent evolutionary origins and unusual evolutionary constraints. Thus coelenterazine-based bioluminescent systems may also manifest a variety of useful properties. We discuss form this taxonomic perspective the initial biochemical and spectral characterization of our cloned proteins. Emphasis is placed on the anthozoan luciferase-GFP systems, whose efficient resonance energy transfer has elicited much current interest.

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

PubMed

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.

Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hao, Z.; Chen, S.; Wilson, D.B.

1999-11-01

An Mn{sup 2+} and Cd{sup 2+} uptake gene, mntA, was cloned from Lactobacillus plantarum ATCC 14917 into Escherichia coli. Its expression conferred on E. coli cells increased Cd{sup 2+} sensitivity as well as energy-dependent Cd{sup 2+} uptake activity. Both transcription and translation of mntA were induced by Mn{sup 2+} starvation in L. plantarum, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd{sup 2+} uptake systems have been identified in L. plantarum: one is a high-affinity Mn{sup 2+} and Cd{sup 2+} uptake system that is expressed in Mn{sup 2+}-starved cells, and the other is a nonsaturable Cd{sup 2+} uptake systemmore » that is expressed in Cd{sup 2+}-sufficient cells. MntA was not detected in an Mn{sup 2+}-dependent mutant of L. plantarum which had lost high-affinity Mn{sup 2+} and Cd{sup 2+} uptake activity. The results suggest that mntA is the gene encoding the high-affinity Mn{sup 2+} and Cd{sup 2+} transporter. On the basis of its predicted amino acid sequence, MntA belongs to the family of P-type cation-translocating ATPases. The topology and potential Mn{sup 2+}- and Cd{sup 2+}-binding sites of MntA are discussed. A second clone containing a low-affinity Cd{sup 2+} transport system was also isolated.« less

Photosynthesis and leaf water relations in four American sycamore clones

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Z.; Land, S.B. Jr.

1995-11-01

Photosynthesis, transpiration, stomatal conductance, and xylem pressure potential were studied to examine clonal variation and clone-by-season interactions in rooted cuttings of four sycamore clones (Platanus occidentalis L.). These physiological parameters were measured during June through November of the second and third growing seasons in the field. Stomatal conductance, xylem pressure potential, and photosynthesis were higher in June-July than in August-November. The four clones did not differ significantly in yearly average photosynthetic rates, but clone 11 tended to have higher rates early in each growing season (June-July) than did the other three clones. Dry periods during August-September of the second seasonmore » and during October of the third season apparently caused clone 11 to close its stomata more than clone 17, as indicated by significant clone-by-season interactions for reductions in stomatal conductance and transpiration late in the morning. Clone 17 was generally able to maintain high xylem pressure potential, stomatal conductance, and transpiration throughout the growing season, probably because of its large root system. 36 refs., 2 figs., 5 tabs.« less

Human research cloning, embryos, and embryo-like artifacts.

PubMed

Hyun, Insoo; Jung, Kyu Won

2006-01-01

Research suggests that cloning is incapable of producing a viable embryo when it is used on primate eggs. In fact, the entity created may not qualify as an embryo at all. If the results stand, cloning avoids the moral objections typically lodged against it, and cloning is itself an "alternative source" of stem cells.

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

PubMed Central

Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-01-01

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion® Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure. PMID:26633465

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.

PubMed

Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-12-01

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

Cloning animals by somatic cell nuclear transfer--biological factors.

PubMed

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

2003-11-13

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 species, this review will be focused on somatic cell cloning of cattle.

Photonic quantum simulator for unbiased phase covariant cloning

NASA Astrophysics Data System (ADS)

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.

Transmitter responsiveness in two newly isolated clones of neuroblastoma X glioma hybrid.

PubMed

Ogura, A; Amano, T

1983-01-10

Mouse neuroblastoma clone N1E-115 cells and rat glioma clone C6 cells were hybridized and two new clones were isolated. One clone, designated NG115-301, possessed weak electric excitability to an applied current pulse, while another clone, NG115-401, generated an action potential in response to the pulse. The former clone responded to serotonin and catecholamines with slow hyperpolarizations, while the latter clone responded to catecholamines with transient depolarizations. Both clones did not respond to acetylcholine. These types of responses have not been reported in any available clones. These clones may enrich the repertoire of cell clones useful for the characterization of transmitter reception mechanisms in the nervous system.

Optimization of embryo culture conditions for increasing efficiency of cloning in buffalo (Bubalus bubalis) and generation of transgenic embryos via cloning.

PubMed

Wadhwa, Neerja; Kunj, Neetu; Tiwari, Shuchita; Saraiya, Megha; Majumdar, Subeer S

2009-09-01

Cloning in bovine species is marred by low efficiency of blastocyst formation. Any increase in the efficiency of blastocyst formation upon nuclear transfer will greatly enhance the efficiency of cloning. In the present study, the effect of various media, protein sources, and growth factors on the development of cloned buffalo embryos was evaluated. Among various combinations tested, culture of cloned embryos in TCM-199 media on the feeder layer of Buffalo Oviductal Epithelial Cells (BOEC) in the presence of bovine serum albumin-free fatty acid (BSA-FFA) and leukemia inhibitory factor (LIF) provided most suitable environment for efficient development of cloned blastocysts. Under these conditions, we achieved a blastocyst formation rate of 43%, which is better than those reported previously. Because preimplantation embryonic development, in vivo, occurs in an environment of oviductal cells, the blastocysts generated by this method may presumably be more suitable for implantation and further development. Additionally, we generated green blastocysts from enucleated oocytes by transfer of nuclei from cells transfected with EGFP transgene, showing possibility of transgenesis via cloning in this species. To our knowledge, this is the first report regarding the production of transgenic cloned buffalo embryos and their developmental competence with respect to various media, cocultures, and supplements.

Kasugamycin-dependent mutants of Escherichia coli.

PubMed Central

Dabbs, E R

1978-01-01

Kasugamycin-dependent mutants have been isolated from Escherichia coli B. They were obtained through mutagenesis with ethyl methane sulfonate or nitrosoguanidine in conjunction with an antibiotic underlay technique. In the case of nitrosoguanidine, dependent mutants were obtained at a frequency of about 3% of survivors growing up in the selection. In the case of ethyl methane sulfonate, the corresponding value was 1%. Nineteen mutants showing a kasugamycin-dependent phenotype were studied. In terms of response to various temperatures and antibiotic concentrations, they were very heterogeneous, although most fell into two general classes. Genetic analysis indicated that in at least some cases, the kasugamycin-dependent phenotype was the product of two mutations. Two-dimensional gel electropherograms revealed alterations in the ribosomal proteins of seven mutants. One mutant had an alteration in protein S13, and one had an alteration in protein L14. Three showed changes in protein S9. Each of two mutants had changes in two proteins, S18 and L11. Three of these mutants additionally had protein S18 occurring in a partly altered, partly unaltered form. Images PMID:363701

Sequential quantum cloning under real-life conditions

NASA Astrophysics Data System (ADS)

Saberi, Hamed; Mardoukhi, Yousof

2012-05-01

We consider a sequential implementation of the optimal quantum cloning machine of Gisin and Massar and propose optimization protocols for experimental realization of such a quantum cloner subject to the real-life restrictions. We demonstrate how exploiting the matrix-product state (MPS) formalism and the ensuing variational optimization techniques reveals the intriguing algebraic structure of the Gisin-Massar output of the cloning procedure and brings about significant improvements to the optimality of the sequential cloning prescription of Delgado [Phys. Rev. Lett.PRLTAO0031-900710.1103/PhysRevLett.98.150502 98, 150502 (2007)]. Our numerical results show that the orthodox paradigm of optimal quantum cloning can in practice be realized in a much more economical manner by utilizing a considerably lesser amount of informational and numerical resources than hitherto estimated. Instead of the previously predicted linear scaling of the required ancilla dimension D with the number of qubits n, our recipe allows a realization of such a sequential cloning setup with an experimentally manageable ancilla of dimension at most D=3 up to n=15 qubits. We also address satisfactorily the possibility of providing an optimal range of sequential ancilla-qubit interactions for optimal cloning of arbitrary states under realistic experimental circumstances when only a restricted class of such bipartite interactions can be engineered in practice.

Post-Death Cloning of Endangered Jeju Black Cattle (Korean Native Cattle): Fertility and Serum Chemistry in a Cloned Bull and Cow and Their Offspring

PubMed Central

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

2013-01-01

Abstract 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. PMID:23955237

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

PubMed

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.

Characterization of an activation-tagged mutant uncovers a role of GLABRA2 in anthocyanin biosynthesis in Arabidopsis

DOE PAGES

Wang, Xiaoyu; Wang, Xianling; Hu, Qingnan; ...

2015-06-17

In Arabidopsis, anthocyanin biosynthesis is controlled by a MYB-bHLH-WD40 (MBW) transcriptional activator complex. The MBW complex activates the transcription of late biosynthesis genes in the flavonoid pathway, leading to the production of anthocyanins. A similar MBW complex regulates epidermal cell fate by activating the transcription of GLABRA2 (GL2), a homeodomain transcription factor required for trichome formation in shoots and non-hair cell formation in roots. Here we provide experimental evidence to show that GL2 also plays a role in regulating anthocyanin biosynthesis in Arabidopsis. From an activation-tagged mutagenized population of Arabidopsis plants, we isolated a dominant, gain-of-function mutant with reduced anthocyanins.more » Molecular cloning revealed that this phenotype is caused by an elevated expression of GL2, thus the mutant was named gl2-1D. Consistent with the view that GL2 acts as a negative regulator of anthocyanin biosynthesis, gl2-1D seedlings accumulated less whereas gl2-3 seedlings accumulated more anthocyanins in response to sucrose. Gene expression analysis indicated that expression of late, but not early, biosynthesis genes in the flavonoid pathway was dramatically reduced in gl2-1D but elevated in gl2-3 mutants. Further analysis showed that expression of some MBW component genes involved in the regulation of late biosynthesis genes was reduced in gl2-1D but elevated in gl2-3 mutants, and chromatin immunoprecipitation results indicated that some MBW component genes are targets of GL2. We also showed that GL2 functions as a transcriptional repressor. Altogether, these results indicate that GL2 negatively regulates anthocyanin biosynthesis in Arabidopsis by directly repressing the expression of some MBW component genes.« less

Characterization of an activation-tagged mutant uncovers a role of GLABRA2 in anthocyanin biosynthesis in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xiaoyu; Wang, Xianling; Hu, Qingnan

In Arabidopsis, anthocyanin biosynthesis is controlled by a MYB-bHLH-WD40 (MBW) transcriptional activator complex. The MBW complex activates the transcription of late biosynthesis genes in the flavonoid pathway, leading to the production of anthocyanins. A similar MBW complex regulates epidermal cell fate by activating the transcription of GLABRA2 (GL2), a homeodomain transcription factor required for trichome formation in shoots and non-hair cell formation in roots. Here we provide experimental evidence to show that GL2 also plays a role in regulating anthocyanin biosynthesis in Arabidopsis. From an activation-tagged mutagenized population of Arabidopsis plants, we isolated a dominant, gain-of-function mutant with reduced anthocyanins.more » Molecular cloning revealed that this phenotype is caused by an elevated expression of GL2, thus the mutant was named gl2-1D. Consistent with the view that GL2 acts as a negative regulator of anthocyanin biosynthesis, gl2-1D seedlings accumulated less whereas gl2-3 seedlings accumulated more anthocyanins in response to sucrose. Gene expression analysis indicated that expression of late, but not early, biosynthesis genes in the flavonoid pathway was dramatically reduced in gl2-1D but elevated in gl2-3 mutants. Further analysis showed that expression of some MBW component genes involved in the regulation of late biosynthesis genes was reduced in gl2-1D but elevated in gl2-3 mutants, and chromatin immunoprecipitation results indicated that some MBW component genes are targets of GL2. We also showed that GL2 functions as a transcriptional repressor. Altogether, these results indicate that GL2 negatively regulates anthocyanin biosynthesis in Arabidopsis by directly repressing the expression of some MBW component genes.« less

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

ERIC Educational Resources Information Center

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)

Mapped Clone and Functional Analysis of Leaf-Color Gene Ygl7 in a Rice Hybrid (Oryza sativa L. ssp. indica)

PubMed Central

Deng, Xiao-juan; Zhang, Hai-qing; Wang, Yue; He, Feng; Liu, Jin-ling; Xiao, Xiao; Shu, Zhi-feng; Li, Wei; Wang, Guo-huai; Wang, Guo-liang

2014-01-01

Leaf-color is an effective marker to identify the hybridization of rice. Leaf-color related genes function in chloroplast development and the photosynthetic pigment biosynthesis of higher plants. The ygl7 (yellow-green leaf 7) is a mutant with spontaneous yellow-green leaf phenotype across the whole lifespan but with no change to its yield traits. We cloned gene Ygl7 (Os03g59640) which encodes a magnesium-chelatase ChlD protein. Expression of ygl7 turns green-leaves to yellow, whereas RNAi-mediated silence of Ygl7 causes a lethal phenotype of the transgenic plants. This indicates the importance of the gene for rice plant. On the other hand, it corroborates that ygl7 is a non-null mutants. The content of photosynthetic pigment is lower in Ygl7 than the wild type, but its light efficiency was comparatively high. All these results indicated that the mutational YGL7 protein does not cause a complete loss of original function but instead acts as a new protein performing a new function. This new function partially includes its preceding function and possesses an additional feature to promote photosynthesis. Chl1, Ygl98, and Ygl3 are three alleles of the OsChlD gene that have been documented previously. However, mutational sites of OsChlD mutant gene and their encoded protein products were different in the three mutants. The three mutants have suppressed grain output. In our experiment, plant materials of three mutants (ygl7, chl1, and ygl98) all exhibited mutational leaf-color during the whole growth period. This result was somewhat different from previous studies. We used ygl7 as female crossed with chl1 and ygl98, respectively. Both the F1 and F2 generation display yellow-green leaf phenotype with their chlorophyll and carotenoid content falling between the values of their parents. Moreover, we noted an important phenomenon: ygl7-NIL's leaf-color is yellow, not yellowy-green, and this is also true of all back-crossed offspring with ygl7. PMID:24932524

Mapped clone and functional analysis of leaf-color gene Ygl7 in a rice hybrid (Oryza sativa L. ssp. indica).

PubMed

Deng, Xiao-juan; Zhang, Hai-qing; Wang, Yue; He, Feng; Liu, Jin-ling; Xiao, Xiao; Shu, Zhi-feng; Li, Wei; Wang, Guo-huai; Wang, Guo-liang

2014-01-01

Leaf-color is an effective marker to identify the hybridization of rice. Leaf-color related genes function in chloroplast development and the photosynthetic pigment biosynthesis of higher plants. The ygl7 (yellow-green leaf 7) is a mutant with spontaneous yellow-green leaf phenotype across the whole lifespan but with no change to its yield traits. We cloned gene Ygl7 (Os03g59640) which encodes a magnesium-chelatase ChlD protein. Expression of ygl7 turns green-leaves to yellow, whereas RNAi-mediated silence of Ygl7 causes a lethal phenotype of the transgenic plants. This indicates the importance of the gene for rice plant. On the other hand, it corroborates that ygl7 is a non-null mutants. The content of photosynthetic pigment is lower in Ygl7 than the wild type, but its light efficiency was comparatively high. All these results indicated that the mutational YGL7 protein does not cause a complete loss of original function but instead acts as a new protein performing a new function. This new function partially includes its preceding function and possesses an additional feature to promote photosynthesis. Chl1, Ygl98, and Ygl3 are three alleles of the OsChlD gene that have been documented previously. However, mutational sites of OsChlD mutant gene and their encoded protein products were different in the three mutants. The three mutants have suppressed grain output. In our experiment, plant materials of three mutants (ygl7, chl1, and ygl98) all exhibited mutational leaf-color during the whole growth period. This result was somewhat different from previous studies. We used ygl7 as female crossed with chl1 and ygl98, respectively. Both the F1 and F2 generation display yellow-green leaf phenotype with their chlorophyll and carotenoid content falling between the values of their parents. Moreover, we noted an important phenomenon: ygl7-NIL's leaf-color is yellow, not yellowy-green, and this is also true of all back-crossed offspring with ygl7.

Factors influencing the commercialisation of cloning in the pork industry.

PubMed

Pratt, S L; Sherrer, E S; Reeves, D E; Stice, S L

2006-01-01

Production of cloned pigs using somatic cell nuclear transfer (SCNT) is a repeatable and predictable procedure and multiple labs around the world have generated cloned pigs and genetically modified cloned pigs. Due to the integrated nature of the pork production industry, pork producers are the most likely to benefit and are in the best position to introduce cloning in to production systems. Cloning can be used to amplify superior genetics or be used in conjunction with genetic modifications to produce animals with superior economic traits. Though unproven, cloning could add value by reducing pig-to-pig variability in economically significant traits such as growth rate, feed efficiency, and carcass characteristics. However, cloning efficiencies using SCNT are low, but predictable. The inefficiencies are due to the intrusive nature of the procedure, the quality of oocytes and/or the somatic cells used in the procedure, the quality of the nuclear transfer embryos transferred into recipients, pregnancy rates of the recipients, and neonatal survival of the clones. Furthermore, in commercial animal agriculture, clones produced must be able to grow and thrive under normal management conditions, which include attainment of puberty and subsequent capability to reproduce. To integrate SCNT into the pork industry, inefficiencies at each step of the procedure must be overcome. In addition, it is likely that non-surgical embryo transfer will be required to deliver cloned embryos, and/or additional methods to generate high health clones will need to be developed. This review will focus on the state-of-the-art for SCNT in pigs and the steps required for practical implementation of pig cloning in animal agriculture.

Transcriptome Analysis of a Premature Leaf Senescence Mutant of Common Wheat (Triticum aestivum L.)

PubMed Central

Xia, Chuan; Zhang, Lichao; Dong, Chunhao; Liu, Xu; Kong, Xiuying

2018-01-01

Leaf senescence is an important agronomic trait that affects both crop yield and quality. In this study, we characterized a premature leaf senescence mutant of wheat (Triticum aestivum L.) obtained by ethylmethane sulfonate (EMS) mutagenesis, named m68. Genetic analysis showed that the leaf senescence phenotype of m68 is controlled by a single recessive nuclear gene. We compared the transcriptome of wheat leaves between the wild type (WT) and the m68 mutant at four time points. Differentially expressed gene (DEG) analysis revealed many genes that were closely related to senescence genes. Gene Ontology (GO) enrichment analysis suggested that transcription factors and protein transport genes might function in the beginning of leaf senescence, while genes that were associated with chlorophyll and carbon metabolism might function in the later stage. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the genes that are involved in plant hormone signal transduction were significantly enriched. Through expression pattern clustering of DEGs, we identified 1012 genes that were induced during senescence, and we found that the WRKY family and zinc finger transcription factors might be more important than other transcription factors in the early stage of leaf senescence. These results will not only support further gene cloning and functional analysis of m68, but also facilitate the study of leaf senescence in wheat. PMID:29534430

Myths about Cloning

MedlinePlus

... to have health problems all their lives. Myth: Cow clones make human pharmaceuticals in their milk. Myth: ... actual animal. For example, if they’re Holstein cows, the pattern of their spots, or the shape ...

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

PubMed

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.

Ethical dimensions of therapeutic human cloning.

PubMed

Reiss, Michael J

2002-09-11

Therapeutic human cloning has the potential significantly to reduce human suffering and enhance human happiness. This is the main ethical argument in its favour. The main ethical arguments against it centre on questions to do with the moral status of the human embryo. A subsidiary set of arguments arises from the connections between therapeutic human cloning and reproductive cloning. Most of the ethical questions concerning the status of the human embryo have long been examined in the context of abortion, though they are being re-examined in the context of genetic screening and embryo research. A consensus on such matters seems extremely unlikely to result in the near future. The current role of ethicists may not, therefore, be so much to attempt to produce a definitive answer to the question of the status of the human embryo at the very early developmental stages at which therapeutic human cloning would take place, but more to help clarify arguments and indicate the implications of particular approaches. That is what this paper seeks to do.

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

PubMed Central

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.

PubMed

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.

Counterfactual quantum cloning without transmitting any physical particles

NASA Astrophysics Data System (ADS)

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.

pPCV, a versatile vector for cloning PCR products.

PubMed

Janner, Christiane R; Brito, Ana Lívia P; Moraes, Lidia Maria P; Reis, Viviane Cb; Torres, Fernando Ag

2013-01-01

The efficiency of PCR product cloning depends on the nature of the DNA polymerase employed because amplicons may have blunt-ends or 3' adenosines overhangs. Therefore, for amplicon cloning, available commercial vectors are either blunt-ended or have a single 3' overhanging thymidine. The aim of this work was to offer in a single vector the ability to clone both types of PCR products. For that purpose, a minimal polylinker was designed to include restriction sites for EcoRV and XcmI which enable direct cloning of amplicons bearing blunt-ends or A-overhangs, respectively, still offering blue/white selection. When tested, the resulting vector, pPCV, presented high efficiency cloning of both types of amplicons.

Towards an understanding of British public attitudes concerning human cloning.

PubMed

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

[Offspring quality and its related factors of different Brachionus calyciflorus clones].

PubMed

Dong, Lili; Xi, Yilong; Zhang, Lei

2006-12-01

This paper studied the neonate starvation-endurance duration of four Brachionus calyciflorus clones (Clone A, B, C and D) with different biochemical-genetic characteristics at 15 degrees C, 20 degrees C, 25 degrees C and 30 degrees C, and the relationships of this duration with the temperature and the body- and egg volumes of B. calyciflorus. The results showed that at 15 degrees C, the neonates of Clone B had the shortest starvation-endurance duration (45.67 h); at 20 degrees C and 25 degrees C, the neonates' starvation-endurance duration of Clone C was the longest, being 61.33 h and 72.01 h, respectively; while at 30 degrees C, this duration of Clone A was the longest (40.11 h). The neonates' starvation-endurance duration of Clone A was the longest at 15 degrees C, those of Clone B and C were the shortest at 30 degrees C, while that of Clone D decreased with raising temperature. The neonates' starvation-endurance duration of all the four clones was negatively correlated with temperature. There was a negative correlation between this duration of Clone A and its egg volume, and the reverse was true for Clone C. The neonates' starvation-endurance duration of Clone B and D was positively correlated with the body volume of rotifer mother.

Whole organ, venation and epidermal cell morphological variations are correlated in the leaves of Arabidopsis mutants.

PubMed

Pérez-Pérez, José Manuel; Rubio-Díaz, Silvia; Dhondt, Stijn; Hernández-Romero, Diana; Sánchez-Soriano, Joaquín; Beemster, Gerrit T S; Ponce, María Rosa; Micol, José Luis

2011-12-01

Despite the large number of genes known to affect leaf shape or size, we still have a relatively poor understanding of how leaf morphology is established. For example, little is known about how cell division and cell expansion are controlled and coordinated within a growing leaf to eventually develop into a laminar organ of a definite size. To obtain a global perspective of the cellular basis of variations in leaf morphology at the organ, tissue and cell levels, we studied a collection of 111 non-allelic mutants with abnormally shaped and/or sized leaves, which broadly represent the mutational variations in Arabidopsis thaliana leaf morphology not associated with lethality. We used image-processing techniques on these mutants to quantify morphological parameters running the gamut from the palisade mesophyll and epidermal cells to the venation, whole leaf and rosette levels. We found positive correlations between epidermal cell size and leaf area, which is consistent with long-standing Avery's hypothesis that the epidermis drives leaf growth. In addition, venation parameters were positively correlated with leaf area, suggesting that leaf growth and vein patterning share some genetic controls. Positional cloning of the genes affected by the studied mutations will eventually establish functional links between genotypes, molecular functions, cellular parameters and leaf phenotypes. © 2011 Blackwell Publishing Ltd.

Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient γ -ECS and glutathione synthetase mutants impaired in cadmium-induced sulfate assimilation

PubMed Central

Jobe, Timothy O.; Sung, Dong-Yul; Akmakjian, Garo; Pham, Allis; Komives, Elizabeth A.; Mendoza-Cózatl, David G.; Schroeder, Julian I.

2015-01-01

Summary Plants exposed to heavy metals rapidly induce changes in gene expression that activate and enhance detoxification mechanisms, including toxic-metal chelation and the scavenging of reactive oxygen species. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. Microarray studies identified a high-affinity sulfate transporter (SULTR1;2) among the most robust and rapid cadmium-inducible transcripts. The SULTR1;2 promoter (2.2 kb) was fused with the firefly luciferase reporter gene to quantitatively report the transcriptional response of plants exposed to cadmium. Stably transformed luciferase reporter lines were ethyl methanesulfonate (EMS) mutagenized, and stable M2 seedlings were screened for an abnormal luciferase response during exposure to cadmium. The screen identified non-allelic mutant lines that fell into one of three categories: (i) super response to cadmium (SRC) mutants; (ii) constitutive response to cadmium (CRC) mutants; or (iii) non-response and reduced response to cadmium (NRC) mutants. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. The nrc1 mutation was mapped to the γ-glutamylcysteine synthetase gene and the nrc2 mutation was identified as the first viable recessive mutant allele in the glutathione synthetase gene. Moreover, genetic, HPLC mass spectrometry, and gene expression analysis of the nrc1 and nrc2 mutants, revealed that intracellular glutathione depletion alone would be insufficient to induce gene expression of sulfate uptake and assimilation mechanisms. Our results modify the glutathione-depletion driven model for sulfate assimilation gene induction during cadmium stress, and suggest that an enhanced oxidative state and depletion of upstream thiols, in addition to glutathione

Mutant fatty acid desaturase

DOEpatents

Shanklin, John; Cahoon, Edgar B.

2004-02-03

The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

Differences in virulence of pneumolysin and autolysin mutants constructed by insertion duplication mutagenesis and in-frame deletion in Streptococcus pneumoniae

PubMed Central

2014-01-01

Background Insertion duplication mutagenesis (IDM) and in-frame deletion (IFD) are common techniques for studying gene function, and have been applied to pneumolysin (ply), a virulence gene in Streptococcus pneumoniae (D39). Discrepancies in virulence between the two techniques were observed in both the previous and present studies. This phenomenon was also observed during mutation analysis of autolysin (lytA). Results Our data showed that target gene restoration (TGR) occurred in IDM mutants, even in the presence of antibiotics, while the IFD mutants were stable. In PCR result, TGR occurred later in IDM-ply and -lytA mutants cultured in non-supplemented medium (4–5 h) compared with those grown in medium supplemented with erythromycin (erm)/chloramphenicol (cat) (3–4 h), but plateaued faster. Real-time PCR for detecting TGR had been performed. When compared with 8-h culture, TGR detection increased from Day 1 and Day 2 of IDM mutant’s culture. erm-sensitive clones from IDM mutant were found. Southern blot hybridization and Western blotting also confirmed the phenomenon of TGR. The median survival of mice following intraperitoneal (IP) injection with a 3-h culture of IDM-mutants was significantly longer than that with an 8-h culture, irrespective of antibiotic usage. The median survival time of mice following IP injection of a 3-h culture versus an 8-h culture of IDM-ply in the absence of antibiotics was 10 days versus 2 days (p = 0.031), respectively, while in the presence of erm, the median survival was 5 days versus 2.5 days (p = 0.037), respectively. For an IDM-lytA mutant, the corresponding values were 8.5 days versus 2 days (p = 0.019), respectively, for non-supplemented medium, and 2.5 versus 2 days (p = 0.021), respectively, in the presence of cat. A comparable survival rate was observed between WT D39 and an 8-h IDM culture. Conclusion TGR in IDM mutants should be monitored to avoid inconsistent results, and misinterpretation

Chaperone-mediated autophagy degrades mutant p53

PubMed Central

Vakifahmetoglu-Norberg, Helin; Kim, Minsu; Xia, Hong-guang; Iwanicki, Marcin P.; Ofengeim, Dimitry; Coloff, Jonathan L.; Pan, Lifeng; Ince, Tan A.; Kroemer, Guido; Brugge, Joan S.; Yuan, Junying

2013-01-01

Missense mutations in the gene TP53, which encodes p53, one of the most important tumor suppressors, are common in human cancers. Accumulated mutant p53 proteins are known to actively contribute to tumor development and metastasis. Thus, promoting the removal of mutant p53 proteins in cancer cells may have therapeutic significance. Here we investigated the mechanisms that govern the turnover of mutant p53 in nonproliferating tumor cells using a combination of pharmacological and genetic approaches. We show that suppression of macroautophagy by multiple means promotes the degradation of mutant p53 through chaperone-mediated autophagy in a lysosome-dependent fashion. In addition, depletion of mutant p53 expression due to macroautophagy inhibition sensitizes the death of dormant cancer cells under nonproliferating conditions. Taken together, our results delineate a novel strategy for killing tumor cells that depend on mutant p53 expression by the activation of chaperone-mediated autophagy and potential pharmacological means to reduce the levels of accumulated mutant p53 without the restriction of mutant p53 conformation in quiescent tumor cells. PMID:23913924

Isolation, Oxygen Sensitivity, and Virulence of NADH Oxidase Mutants of the Anaerobic Spirochete Brachyspira (Serpulina) hyodysenteriae, Etiologic Agent of Swine Dysentery

PubMed Central

Stanton, Thad B.; Rosey, Everett L.; Kennedy, Michael J.; Jensen, Neil S.; Bosworth, Brad T.

1999-01-01

Brachyspira (Serpulina) hyodysenteriae, the etiologic agent of swine dysentery, uses the enzyme NADH oxidase to consume oxygen. To investigate possible roles for NADH oxidase in the growth and virulence of this anaerobic spirochete, mutant strains deficient in oxidase activity were isolated and characterized. The cloned NADH oxidase gene (nox; GenBank accession no. U19610) on plasmid pER218 was inactivated by replacing 321 bp of coding sequence with either a gene for chloramphenicol resistance (cat) or a gene for kanamycin resistance (kan). The resulting plasmids, respectively, pCmΔNOX and pKmΔNOX, were used to transform wild-type B. hyodysenteriae B204 cells and generate the antibiotic-resistant strains Nox-Cm and Nox-Km. PCR and Southern hybridization analyses indicated that the chromosomal wild-type nox genes in these strains had been replaced, through allelic exchange, by the inactivated nox gene containing cat or kan. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis revealed that both nox mutant cell lysates were missing the 48-kDa Nox protein. Soluble NADH oxidase activity levels in cell lysates of Nox-Cm and Nox-Km were reduced 92 to 96% compared to the activity level in parent strain B204. In an aerotolerance test, cells of both nox mutants were at least 100-fold more sensitive to oxygen exposure than were cells of the wild-type parent strain B204. In swine experimental infections, both nox mutants were less virulent than strain B204 in that fewer animals were colonized by the mutant cells and infected animals displayed mild, transient signs of disease, with no deaths. These results provide evidence that NADH oxidase serves to protect B. hyodysenteriae cells against oxygen toxicity and that the enzyme, in that role, contributes to the pathogenic ability of the spirochete. PMID:10543819

Systematic cloning of an ORFeome using the Gateway system.

PubMed

Matsuyama, Akihisa; Yoshida, Minoru

2009-01-01

With the completion of the genome projects, there are increasing demands on the experimental systems that enable to exploit the entire set of protein-coding open reading frames (ORFs), viz. ORFeome, en masse. Systematic proteomic studies based on cloned ORFeomes are called "reverse proteomics," and have been launched in many organisms in recent years. Cloning of an ORFeome is such an attractive way for comprehensive understanding of biological phenomena, but is a challenging and daunting task. However, recent advances in techniques for DNA cloning using site-specific recombination and for high-throughput experimental techniques have made it feasible to clone an ORFeome with the minimum of exertion. The Gateway system is one of such the approaches, employing the recombination reaction of the bacteriophage lambda. Combining traditional DNA manipulation methods with modern technique of the recombination-based cloning system, it is possible to clone an ORFeome of an organism on an individual level.

Cloning and heterologous expression of chlorophyll a synthase in Rhodobacter sphaeroides.

PubMed

Ipekoğlu, Emre M; Göçmen, Koray; Öz, Mehmet T; Gürgan, Muazzez; Yücel, Meral

2017-03-01

Rhodobacter sphaeroides is a purple non-sulfur bacterium which photoheterotrophically produces hydrogen from organic acids under anaerobic conditions. A gene coding for putative chlorophyll a synthase (chlG) from cyanobacterium Prochlorococcus marinus was amplified by nested polymerase chain reaction and cloned into an inducible-expression plasmid which was subsequently transferred to R. sphaeroides for heterologous expression. Induced expression of chlG in R. sphaeroides led to changes in light absorption spectrum within 400-700 nm. The hydrogen production capacity of the mutant strain was evaluated on hydrogen production medium with 15 mM malate and 2 mM glutamate. Hydrogen yield and productivity were increased by 13.6 and 22.6%, respectively, compared to the wild type strain. The results demonstrated the feasibility of genetic engineering to combine chlorophyll and bacteriochlorophyll biosynthetic pathways which utilize common intermediates. Heterologous expression of key enzymes from biosynthetic pathways of various pigments is proposed here as a general strategy to improve absorption spectra and yield of photosynthesis and hydrogen gas production in bacteria. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cloning and expression of two chitin deacetylase genes of Saccharomyces cerevisiae.

PubMed

Mishra, C; Semino, C E; McCreath, K J; de la Vega, H; Jones, B J; Specht, C A; Robbins, P W

1997-03-30

Chitin deacetylase (EC 3.5.1.41), which hydrolyses the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, has been demonstrated in crude extracts from sporulating Saccharomyces cerevisiae. Two S. cerevisiae open reading frames (ORFs), identified by the Yeast Genome Project, have protein sequence homology to a chitin deacetylase from Mucor rouxii. Northern blot hybridizations show each ORF was transcribed in diploid cells after transfer to sporulation medium and prior to formation of asci. Each ORF was cloned in a vector under transcriptional control of the GAL 1, 10 promoter and introduced back into haploid strains of S. cerevisiae. Chitin deacetylase activity was detected by in vitro assays from vegetative cells grown in galactose. Chemical analysis of these cells also demonstrated the synthesis of chitosam in vivo. Both recombinant chitin deacetylases showed similar qualitative and quantitative activities toward chitooligosaccharides in vitro. A diploid strain deleted to both ORFs, when sporulated, did not show deacetylase activity. The mutant spores were hypersensitive to lytic enzymes (Glusulase or Zymolyase).

Cloning of Plasmodium falciparum by single-cell sorting

PubMed Central

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

Cloning of Plasmodium falciparum by single-cell sorting.

PubMed

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.

Transposon-containing DNA cloning vector and uses thereof

DOEpatents

Berg, C.M.; Berg, D.E.; Wang, G.

1997-07-08

The present invention discloses a rapid method of restriction mapping, sequencing or localizing genetic features in a segment of deoxyribonucleic acid (DNA) that is up to 42 kb in size. The method in part comprises cloning of the DNA segment in a specialized cloning vector and then isolating nested deletions in either direction in vivo by intramolecular transposition into the cloned DNA. A plasmid has been prepared and disclosed. 4 figs.

Transposon-containing DNA cloning vector and uses thereof

DOEpatents

Berg, Claire M.; Berg, Douglas E.; Wang, Gan

1997-01-01

The present invention discloses a rapid method of restriction mapping, sequencing or localizing genetic features in a segment of deoxyribonucleic acid (DNA) that is up to 42 kb in size. The method in part comprises cloning of the DNA segment in a specialized cloning vector and then isolating nested deletions in either direction in vivo by intramolecular transposition into the cloned DNA. A plasmid has been prepared and disclosed.

A strategy for clone selection under different production conditions.

PubMed

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).

In vivo and in vitro cloning and phenotype characterization of tellurite resistance determinant conferred by plasmid pTE53 of a clinical isolate of Escherichia coli.

PubMed

Burian, J; Tu, N; Kl'ucár, L; Guller, L; Lloyd-Jones, G; Stuchlík, S; Fejdi, P; Siekel, P; Turna, J

1998-01-01

A determinant encoding resistance against potassium tellurite (Te(r)) was discovered in a clinical isolate of Escherichia coli strain KL53. The strain formed typical black colonies on solid LB medium with tellurite. The determinant was located on a large conjugative plasmid designated pTE53. Electron-dense particles were observed in cells harboring pTE53 by electron microscopy. X-Ray identification analysis identified these deposits as elemental tellurium and X-ray diffraction analysis showed patterns typical of crystalline structures. Comparison with JCPDS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals. In common with other characterized Te(r) determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tellurite did not contribute to the resistance mechanism. Tellurite accumulation rates for E. coli strain AB1157 harboring pTE53 were twice higher than for the plasmid-free host strain. In addition, no efflux mechanism was detected. The potassium tellurite resistance determinant of plasmid pTE53 was cloned using both in vitro and in vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46. Cloning of the functional Te(r) determinant into high-copy cloning vectors pTZ19R and mini-Mu derivatives pBEf and pJT2 was not successful. During in vivo cloning experiments, clones with unusual "white colony" phenotypes were found on solid LB with tellurite. All these clones were Mucts62 lysogens. Their tellurite resistance levels were in the same order as the wild type strains. Clones with the "white" phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing strain. Transformation of a "white" mutant with a recombinant pACYC184 based Te(r) plasmid did not change the phenotype. However, when one clone was cured from Mucts62 the "white" phenotype reverted to the wild-type "black" phenotype. It was suggested

Proper reprogramming of imprinted and non-imprinted genes in cloned cattle gametogenesis.

PubMed

Kaneda, Masahiro; Watanabe, Shinya; Akagi, Satoshi; Inaba, Yasushi; Geshi, Masaya; Nagai, Takashi

2017-11-01

Epigenetic abnormalities in cloned animals are caused by incomplete reprogramming of the donor nucleus during the nuclear transfer step (first reprogramming). However, during the second reprogramming step that occurs only in the germline cells, epigenetic errors not corrected during the first step are repaired. Consequently, epigenetic abnormalities in the somatic cells of cloned animals should be erased in their spermatozoa or oocytes. This is supported by the fact that offspring from cloned animals do not exhibit defects at birth or during postnatal development. To test this hypothesis in cloned cattle, we compared the DNA methylation level of two imprinted genes (H19 and PEG3) and three non-imprinted genes (XIST, OCT4 and NANOG) and two repetitive elements (Satellite I and Satellite II) in blood and sperm DNAs from cloned and non-cloned bulls. We found no differences between cloned and non-cloned bulls. We also analyzed the DNA methylation levels of four repetitive elements (Satellite I, Satellite II, Alpha-satellite and Art2) in oocytes recovered from cloned and non-cloned cows. Again, no significant differences were observed between clones and non-clones. These results suggested that imprinted and non-imprinted genes and repetitive elements were properly reprogramed during gametogenesis in cloned cattle; therefore, they contributed to the soundness of cloned cattle offspring. © 2017 Japanese Society of Animal Science.

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

PubMed Central

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; 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. PMID:29091919

High-throughput cloning and expression library creation for functional proteomics.

PubMed

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

2013-05-01

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 particularly 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 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(TM) 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). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An efficient transgenic system by TA cloning vectors and RNAi for C. elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gengyo-Ando, Keiko; CREST, JST, 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012; Yoshina, Sawako

2006-11-03

In the nematode, transgenic analyses have been performed by microinjection of DNA from various sources into the syncytium gonad. To expedite these transgenic analyses, we solved two potential problems in this work. First, we constructed an efficient TA-cloning vector system which is useful for any promoter. By amplifying the genomic DNA fragments which contain regulatory sequences with or without the coding region, we could easily construct plasmids expressing fluorescent protein fusion without considering restriction sites. We could dissect motor neurons with three colors in a single animal. Second, we used feeding RNAi to isolate transgenic strains which express lag-2::venus fusionmore » gene. We found that the fusion protein is toxic when ectopically expressed in embryos but is functional to rescue a loss of function mutant in the lag-2 gene. Thus, the transgenic system described here should be useful to examine the protein function in the nematode.« less

Cloning of DOG1, a quantitative trait locus controlling seed dormancy in Arabidopsis.

PubMed

Bentsink, Leónie; Jowett, Jemma; Hanhart, Corrie J; Koornneef, Maarten

2006-11-07

Genetic variation for seed dormancy in nature is a typical quantitative trait controlled by multiple loci on which environmental factors have a strong effect. Finding the genes underlying dormancy quantitative trait loci is a major scientific challenge, which also has relevance for agriculture and ecology. In this study we describe the identification of the DELAY OF GERMINATION 1 (DOG1) gene previously identified as a quantitative trait locus involved in the control of seed dormancy. This gene was isolated by a combination of positional cloning and mutant analysis and is absolutely required for the induction of seed dormancy. DOG1 is a member of a small gene family of unknown molecular function, with five members in Arabidopsis. The functional natural allelic variation present in Arabidopsis is caused by polymorphisms in the cis-regulatory region of the DOG1 gene and results in considerable expression differences between the DOG1 alleles of the accessions analyzed.

Cloning of hydrogenase genes and fine structure analysis of an operon essential for H/sub 2/ metabolism in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sankar, P.; Lee, J.H.; Shanmugam, K.T.

1985-04-01

Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2more » (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome.« less

Superior triacylglycerol (TAG) accumulation in starchless mutants of Scenedesmus obliquus: (I) mutant generation and characterization

PubMed Central

2014-01-01

Background Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities. Results Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogen-replete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type. Conclusions The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under

Pig cloning by microinjection of fetal fibroblast nuclei.

PubMed

Onishi, A; Iwamoto, M; Akita, T; Mikawa, S; Takeda, K; Awata, T; Hanada, H; Perry, A C

2000-08-18

Pig cloning will have a marked impact on the optimization of meat production and xenotransplantation. To clone pigs from differentiated cells, we microinjected the nuclei of porcine (Sus scrofa) fetal fibroblasts into enucleated oocytes, and development was induced by electroactivation. The transfer of 110 cloned embryos to four surrogate mothers produced an apparently normal female piglet. The clonal provenance of the piglet was indicated by her coat color and confirmed by DNA microsatellite analysis.

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

PubMed

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.

Problem-Solving Test: Tryptophan Operon Mutants

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2010-01-01

This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

(New hosts and vectors for genome cloning)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

The main goal of our project remains the development of new bacterial hosts and vectors for the stable propagation of human DNA clones in E. coli. During the past six months of our current budget period, we have (1) continued to develop new hosts that permit the stable maintenance of unstable features of human DNA, and (2) developed a series of vectors for (a) cloning large DNA inserts, (b) assessing the frequency of human sequences that are lethal to the growth of E. coli, and (c) assessing the stability of human sequences cloned in M13 for large-scale sequencing projects.

Cloning animals by somatic cell nuclear transfer – biological factors

PubMed Central

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

Phenotypic effects of somatic cell cloning in the mouse.

PubMed

Ogura, A; Inoue, K; Ogonuki, N; Lee, J; Kohda, T; Ishino, F

2002-01-01

Although a variety of phenotypes and epigenetic alterations have been reported in animals cloned from somatic cells, the exact nature and consequences of cloning remain unclear. We cloned mice using fresh or short-term cultures of donor cells (cumulus cells, immature Sertoli cells, and fetal or adult fibroblast cells) with defined genetic backgrounds, and then compared the phenotypic and epigenetic characteristics of the cloned mice with those of fertilization-derived control mice. Irrespective of the nucleus-donor cell type, about 50% of the reconstructed embryos developed to the morula/blastocyst stage, but about 90% of these clones showed arrested development between days 5 and 8, shortly after implantation. Most of the clones were alive at term, readily recovered respiration, and did not show any malformations or overgrowths. However, their placentas were two- to threefold larger than those of the controls, due to hyperplasia of the basal (or spongiotrophoblast) layer. Although there was significant suppression of a subset of both imprinted and non-imprinted placental genes, fetal gene suppression was minimal. The seven imprinted genes that we examined were all expressed correctly from the parental alleles. These findings were consistent for every cell type from the midgestation through term stages. Therefore, cloning by nuclear transfer does not perturb the parent-specific imprinting memory that is established during gametogenesis, and the phenotypic and epigenetic effects of cloning are restricted to placental development at the midgestation and term stages. Twelve male mice that were born in a normal manner following nuclear transfer with immature Sertoli cells (B6D2F1 genetic background) were subjected to long-term observation. They died earlier than the genotype-matched controls (50% survival point: 550 days vs. 1028 days, respectively), most probably due to severe pneumonia, which indicates that unexpected phenotypes can appear as a result of the long

Cloning and characterization of mouse ACF7, a novel member of the dystonin subfamily of actin binding proteins.

PubMed

Bernier, G; Mathieu, M; De Repentigny, Y; Vidal, S M; Kothary, R

1996-11-15

We have recently cloned the gene responsible for the mouse neurological disorder dystonia musculorum. The predicted product of this gene, dystonin (Dst), is a neural isoform of bullous pemphigoid antigen 1 (Bpag1) with an N-terminal actin binding domain. Here we report on the cloning and characterization of mouse ACF7. Sequence analysis revealed extended homology of mACF7 with both the actin binding domain (ABD) and the Bpag1 portions of dystonin. Moreover, mACF7 and Dst display similar isoform diversity and encode similar sized transcripts in the nervous system. Phylogenetic analysis of mACF7 and dystonin ABD sequences suggests a recent evolutionary origin and that these proteins form a separate novel subfamily within the beta-spectrin superfamily of actin binding proteins. Given the implication of several actin binding proteins in genetic disorders, it is important to know the pattern of mACF7 expression. mACF7 transcripts are detected principally in lung, brain, spinal cord, skeletal and cardiac muscle, and skin. Intriguingly, mACF7 expression in lung is strongly induced just before birth and is restricted to type II alveolar cells. To determine whether spontaneous mutants that may be defective in mACF7 exist, we have mapped the mACF7 gene to mouse chromosome 4.

Cloning of genes required for hypersensitivity and pathogenicity in Pseudomonas syringae pv. aptata.

PubMed

Minardi, P

1995-01-01

A genomic library of Pseudomonas syringae pv. aptata strain NCPPB 2664, which causes bacterial blight of sugar beet, lettuce and other plants, was constructed in the cosmid vector pCPP31. The 13.4 kb EcoRI fragment of the cosmid pHIR11, containing the hrp (hypersensitive response and pathogenicity) gene cluster of the closely related bacterium Pseudomonas syringae pv. syringae strain 61, was used as a probe to identify a homologous hrp gene cluster in P. syringae pv. aptata. Thirty of 2500 cosmid clones, screened by colony hybridization, gave a strong hybridization signal with the probe, but none of these conferred to the non-pathogenic bacterium, Pseudomonas fluorescens, the ability to elicit the hypersensitive response (HR) in tobacco. Southern blot analysis of EcoRI-digested genomic DNA of P. syringae pv. aptata showed hybridizing bands of 12 kb and 4.4 kb. Only a 12 kb fragment hybridized in digests of the cosmids. Cosmid clone pCPP1069 was mutagenized with Tn10-minitet and marker-exchanged into the genome of P. syringae pv. aptata. Three resulting prototrophic mutant strains failed to elicit the HR in tobacco and to cause disease in lettuce. The DNA flanking the Tn10-minitet insertions from mutated derivatives of pCPP1069 hybridized with the 10.6 kb Bg/II fragment of pHIR11. These results indicate that P. syringae pv. aptata harbours hrp genes that are similar to, but arranged differently from, homologous hrp genes of P. syringae pv. syringae.

Information-theoretic limitations on approximate quantum cloning and broadcasting

NASA Astrophysics Data System (ADS)

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.

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

PubMed

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.

Cloning and adoption: a reply to Levy and Lotz.

PubMed

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.

Biotechnology. Perseverance leads to cloned pig in Japan.

PubMed

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].

PubMed

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.

Therapeutic cloning: from consequences to contradiction.

PubMed

Coors, Marilyn

2002-06-01

The British Parliament legalized therapeutic cloning in December 2000 despite opposition from the European Union. The watershed event in Parliament's move was the active and unprecedented government support for the generation and destruction of human embryonic life merely as a means of medical advancement. This article contends that the utilitarian analysis of this procedure is necessary to identify the real world risks of therapeutic cloning but insufficient to identify the breach of defensible ethical limits that this procedure represents. A value-oriented approach to Kantian ethics demonstrates that the utilitarian endorsement of therapeutic cloning entails a contradiction of the necessity of human vulnerability and a faulty valuation of the human embryo. The concern is that a narrow utilitarian focus ultimately commodifies human embryonic life and preferences outcomes as the sole determinant of moral value.

Dogs cloned from adult somatic cells.

PubMed

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.

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

PubMed

Bower, S; Perkins, J; Yocum, R R; Serror, P; Sorokin, A; Rahaim, P; Howitt, C L; Prasad, N; Ehrlich, S D; Pero, J

1995-05-01

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli.

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

PubMed Central

Bower, S; Perkins, J; Yocum, R R; Serror, P; Sorokin, A; Rahaim, P; Howitt, C L; Prasad, N; Ehrlich, S D; Pero, J

1995-01-01

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli. PMID:7730294

Preneoplastic lesion growth driven by the death of adjacent normal stem cells

PubMed Central

Chao, Dennis L.; Eck, J. Thomas; Brash, Douglas E.; Maley, Carlo C.; Luebeck, E. Georg

2008-01-01

Clonal expansion of premalignant lesions is an important step in the progression to cancer. This process is commonly considered to be a consequence of sustaining a proliferative mutation. Here, we investigate whether the growth trajectory of clones can be better described by a model in which clone growth does not depend on a proliferative advantage. We developed a simple computer model of clonal expansion in an epithelium in which mutant clones can only colonize space left unoccupied by the death of adjacent normal stem cells. In this model, competition for space occurs along the frontier between mutant and normal territories, and both the shapes and the growth rates of lesions are governed by the differences between mutant and normal cells' replication or apoptosis rates. The behavior of this model of clonal expansion along a mutant clone's frontier, when apoptosis of both normal and mutant cells is included, matches the growth of UVB-induced p53-mutant clones in mouse dorsal epidermis better than a standard exponential growth model that does not include tissue architecture. The model predicts precancer cell mutation and death rates that agree with biological observations. These results support the hypothesis that clonal expansion of premalignant lesions can be driven by agents, such as ionizing or nonionizing radiation, that cause cell killing but do not directly stimulate cell replication. PMID:18815380

Mutant power: using mutant allele collections for yeast functional genomics.

PubMed

Norman, Kaitlyn L; Kumar, Anuj

2016-03-01

The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Mode of action of the qcr9 and cat3 mutations in restoring the ability of Saccharomyces cerevisiae tps1 mutants to grow on glucose.

PubMed

Blázquez, M A; Gancedo, C

1995-12-20

Mutations in the TPS1 gene, which encodes trehalose-6-P synthase, cause a glucose-negative phenotype in Saccharomyces cerevisiae. Antimycin A or disruption of the QCR9 gene, which encodes one subunit of the cytochrome bc1 complex, restore the ability to grow in glucose-containing media. Under these conditions the cell excreted a large amount of glycerol, corresponding to about 20% of the glucose taken up. Suppression appears to be achieved by diversion of accumulated glycolytic intermediates to the production of glycerol, thereby providing NAD+ and phosphate for the glyceraldehyde-3-P dehydrogenase reaction. Analysis of the mutation sci1-1, which also suppresses the glucose-negative phenotype of tps1 mutants, showed that glucose transport was decreased in sci1-1 mutants. The gene SCI1 was cloned and its nucleotide sequence revealed it to be identical to CAT3/SNF4. The suppression mediated by sci1-1 is attributable to a decrease in glycolytic flux.

High-Throughput Cloning and Expression Library Creation for Functional Proteomics

PubMed Central

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

Misfolded rhodopsin mutants display variable aggregation properties.

PubMed

Gragg, Megan; Park, Paul S-H

2018-06-08

The largest class of rhodopsin mutations causing autosomal dominant retinitis pigmentosa (adRP) is mutations that lead to misfolding and aggregation of the receptor. The misfolding mutants have been characterized biochemically, and categorized as either partial or complete misfolding mutants. This classification is incomplete and does not provide sufficient information to fully understand the disease pathogenesis and evaluate therapeutic strategies. A Förster resonance energy transfer (FRET) method was utilized to directly assess the aggregation properties of misfolding rhodopsin mutants within the cell. Partial (P23H and P267L) and complete (G188R, H211P, and P267R) misfolding mutants were characterized to reveal variability in aggregation properties. The complete misfolding mutants all behaved similarly, forming aggregates when expressed alone, minimally interacting with the wild-type receptor when coexpressed, and were unresponsive to treatment with the pharmacological chaperone 9-cis retinal. In contrast, variability was observed between the partial misfolding mutants. In the opsin form, the P23H mutant behaved similarly as the complete misfolding mutants. In contrast, the opsin form of the P267L mutant existed as both aggregates and oligomers when expressed alone and formed mostly oligomers with the wild-type receptor when coexpressed. The partial misfolding mutants both reacted similarly to the pharmacological chaperone 9-cis retinal, displaying improved folding and oligomerization when expressed alone but aggregating with wild-type receptor when coexpressed. The observed differences in aggregation properties and effect of 9-cis retinal predict different outcomes in disease pathophysiology and suggest that retinoid-based chaperones will be ineffective or even detrimental. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of PblB mediating galactose-specific adhesion in a successful Streptococcus pneumoniae clone

PubMed Central

Hsieh, Yu-Chia; Lin, Tzu-Lung; Lin, Che-Ming; Wang, Jin-Town

2015-01-01

The pneumococcal genome is variable and there are minimal data on the influence of the accessory genome on phenotype. Pneumococcal serotype 14 sequence type (ST) 46 had been the most prevalent clone causing pneumonia in children in Taiwan. A microarray was constructed using the genomic DNA of a clinical strain (NTUH-P15) of serotype 14 ST46. Using DNA hybridization, genomic variations in NTUH-P15 were compared to those of 3 control strains. Microarray analysis identified 7 genomic regions that had significant increases in hybridization signals in the NTUH-P15 strain compared to control strains. One of these regions encoded PblB, a phage-encoded virulence factor implicated (in Streptococcus mitis) in infective endocarditis. The isogenic pblB mutant decreased adherence to A549 human lung epithelial cell compared to wild-type NTUH-P15 strain (P = 0.01). Complementation with pblB restored the adherence. PblB is predicted to contain a galactose-binding domain-like region. Preincubation of NTUH-P15 with D-galactose resulted in decreases of adherence to A549 cell in a dose-dependent manner. Challenge of mice with NTUH-P15, isogenic pblB mutant and pblB complementation strains determined that PblB was required for bacterial persistence in the nasopharynx and lung. PblB, as an adhesin mediating the galactose-specific adhesion activity of pneumococci, promote pneumococcal clonal success. PMID:26193794

Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, Melloni N.; Dunning, Jonathan P; Wiley, Ronald G

2007-01-01

We report on a behavioral screening test battery that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and employed a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open field mutants (one displaying hyper-locomotion, the other hypo-locomotion), four tail suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsivenessmore » to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning and memory mutant (displaying reduced response to the conditioned stimulus) These findings highlight the utility of a set of behavioral tasks used in a high throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.« less

Fundamental resource-allocating model in colleges and universities based on Immune Clone Algorithms

NASA Astrophysics Data System (ADS)

Ye, Mengdie

2017-05-01

In this thesis we will seek the combination of antibodies and antigens converted from the optimal course arrangement and make an analogy with Immune Clone Algorithms. According to the character of the Algorithms, we apply clone, clone gene and clone selection to arrange courses. Clone operator can combine evolutionary search and random search, global search and local search. By cloning and clone mutating candidate solutions, we can find the global optimal solution quickly.

Cloning Expeditions: Risky but Rewarding

PubMed Central

2013-01-01

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

Analysis of an epigenetic argument against human reproductive cloning.

PubMed

Nordgren, Anders

2006-08-01

Human reproductive cloning is a much disputed ethical issue. This technology is often condemned as being contrary to human dignity. However, there are also risk arguments. An ethical argument that is often put forward by scientists but seldom developed in more detail focuses on health risks in animal cloning. There is a high risk that animal clones exhibit abnormalities and these are increasingly believed to be due to errors in epigenetic reprogramming. The argument is that human reproductive cloning should not be carried out because human clones are also likely to exhibit abnormalities due to inappropriate epigenetic reprogramming. Different versions of this epigenetic argument are analysed, a categorical version and a non-categorical. The non-categorical version is suggested to be more well-considered. With regard to policy making on human reproductive cloning, the categorical version can be used to prescribe a permanent ban, while the non-categorical version can be used to prescribe a temporary ban. The implications of the precautionary principle--as interpreted in the European Union--are investigated. The conclusion is that it seems possible to support a temporary ban by reference to this principle.

Probabilistic quantum cloning of a subset of linearly dependent states

NASA Astrophysics Data System (ADS)

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.

Twelve years before the quantum no-cloning theorem

NASA Astrophysics Data System (ADS)

Ortigoso, Juan

2018-03-01

The celebrated quantum no-cloning theorem establishes the impossibility of making a perfect copy of an unknown quantum state. The discovery of this important theorem for the field of quantum information is currently dated 1982. I show here that an article published in 1970 [J. L. Park, Found. Phys. 1, 23-33 (1970)] contained an explicit mathematical proof of the impossibility of cloning quantum states. I analyze Park's demonstration in the light of published explanations concerning the genesis of the better-known papers on no-cloning.

The cleft lip and palate defects in Dancer mutant mice result from gain of function of the Tbx10 gene

PubMed Central

Bush, Jeffrey O.; Lan, Yu; Jiang, Rulang

2004-01-01

Cleft lip and palate (CL/P) is a common disfiguring birth defect with complex, poorly understood etiology. Mice carrying a spontaneous mutation, Dancer (Dc), exhibit CL/P in homozygotes and show significantly increased susceptibility to CL/P in heterozygotes [Deol, M. S. & Lane, P. W. (1966) J. Embryol. Exp. Morphol. 16, 543–558 and Trasler, D. G., Kemp, D. & Trasler, T. A. (1984) Teratology 29, 101–104], providing an animal model for understanding the molecular pathogenesis of CL/P. We genetically mapped Dc to within a 1-cM region near the centromere of chromosome 19. In situ hybridization analysis showed that one positional candidate gene, Tbx10, is ectopically expressed in Dc mutant embryos. Positional cloning of the Dc locus revealed an insertion of a 3.3-kb sequence containing the 5′ region of the p23 gene into the first intron of Tbx10, which causes ectopic expression of a p23-Tbx10 chimeric transcript encoding a protein product identical to a normal variant of the Tbx10 protein. Furthermore, we show that ectopic expression of Tbx10 in transgenic mice recapitulates the Dc mutant phenotype, indicating that CL/Pin Dc mutant mice results from the p23 insertion-induced ectopic Tbx10 expression. These results identify gain of function of a T-box transcription factor gene as a mechanism underlying CL/P pathogenesis. PMID:15118109

[Cloning of cDNA for RNA polymerase subunit from the fission yeast Schizosaccharomyces pombe by heterospecific complementation in Saccharomyces cerevisiae].

PubMed

Shpakovskiĭ, G V; Lebedenko, E N; Thuriaux, P

1997-02-01

The rpb10 cDNA of the fission yeast Schizosaccharomyces pombe, encoding one of the five small subunits common to all three nuclear DNA-dependent RNA polymerases, was isolated from an expression cDNA library by two independent approaches: PCR-based screening and direct suppression by means of heterospecific complementation of a temperature-sensitive mutant defective in the corresponding gene of Saccharomyces cerevisiae. The cloned Sz. pombe cDNA encodes a protein Rpb10 of 71 amino acids with an M of 8,275 Da, sharing 51 amino acids (71% identity) with the subunit ABC10 beta of RNA polymerases I-III from S. cerevisiae. All eukaryotic members of this protein family have the same general organization featuring two highly conserved motifs (RCFT/SCGK and RYCCRRM) around an atypical zinc finger and an additional invariant HVDLIEK motif toward the C-terminal end. The last motif is only characteristics for homologs from eukaryotes. In keeping with this remarkable structural conservation, the Sz. pombe cDNA also fully complemented a S. cerevisiae deletion mutant lacking subunit ABC10 beta (null allele rpb10-delta 1::HIS3).

Molecular cloning and analysis of the ergopeptine assembly system in the ergot fungus Claviceps purpurea.

PubMed

Correia, Telmo; Grammel, Nicolas; Ortel, Ingo; Keller, Ullrich; Tudzynski, Paul

2003-12-01

Claviceps purpurea produces the pharmacological important ergopeptines, a class of cyclol-structured alkaloid peptides containing D-lysergic acid. These compounds are assembled from D-lysergic acid and three different amino acids by the nonribosomal peptide synthetase enzymes LPS1 and LPS2. Cloning of alkaloid biosynthesis genes from C. purpurea has revealed a gene cluster including two NRPS genes, cpps 1 and cpps 2. Protein sequence data had assigned earlier cpps1 to encode the trimodular LPS1 assembling the tripeptide portion of ergopeptines. Here, we show by transcriptional analysis, targeted inactivation, analysis of disruption mutants, and heterologous expression that cpps 2 encodes the monomodular LPS2 responsible for D-lysergic acid activation and incorporation into the ergopeptine backbone. The presence of two distinct NRPS subunits catalyzing formation of ergot peptides is the first example of a fungal NRPS system consisting of different NRPS subunits.

Isolation and characterization of cDNA clones for human erythrocyte beta-spectrin.

PubMed Central

Prchal, J T; Morley, B J; Yoon, S H; Coetzer, T L; Palek, J; Conboy, J G; Kan, Y W

1987-01-01

Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical alpha (Mr 240,000) and beta (Mr 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. We report here the isolation and characterization of a human erythroid-specific beta-spectrin cDNA clone that encodes parts of the beta-9 through beta-12 repeat segments. This cDNA was used as a hybridization probe to assign the beta-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte beta-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the beta-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities. Images PMID:3478706

Involvement of the pituitary-specific transcription factor pit-1 in somatolactotrope cell growth and death: an approach using dominant-negative pit-1 mutants.

PubMed

Pellegrini, Isabelle; Roche, Cathy; Quentien, Marie-Helene; Ferrand, Mireille; Gunz, Ginette; Thirion, Sylvie; Bagnis, Claude; Enjalbert, Alain; Franc, Jean-Louis

2006-12-01

The anterior pituitary-specific transcription factor Pit-1 was initially identified and cloned as a transactivator of the prolactin (PRL) and GH genes and later as a regulator of the TSHb gene. It was found to be a major developmental regulator, because natural Pit-1 gene mutations cause a dwarf phenotype in mice and cause combined pituitary hormone deficiency associated with pituitary hypoplasia in humans. To further investigate the growth-promoting effects of Pit-1, we used a strategy based on the use of dominant-negative Pit-1 mutants as an alternative means of inactivating endogenous Pit-1 functions. R271W, a Pit-1 mutant identified in one allele in patients with severe combined pituitary hormone deficiency, and Pit-1Delta1-123, a deletion mutant in which only the DNA binding domain of Pit-1 is conserved, were generated, and their ability to abolish the effects of the endogenous native Pit-1 in the differentiated proliferating somatolactotrope GH4C1 cell line was investigated. Enforced expression of the dominant-negative mutants in GH4C1 cells using recombinant lentiviral vectors decreased the levels of expression of known Pit-1 target genes such as PRL and GH, abolished the hormone release, and reduced cell viability by decreasing the growth rate and inducing apoptosis via a caspase-independent pathway. These results show for the first time that the growth-promoting effects of Pit-1 are at least partly due to the fact that this transcription factor prevents apoptotic cell death.

DNA cloning: A personal view after 40 years

PubMed Central

Cohen, Stanley N.

2013-01-01

In November 1973, my colleagues A. C. Y. Chang, H. W. Boyer, R. B. Helling, and I reported in PNAS that individual genes can be cloned and isolated by enzymatically cleaving DNA molecules into fragments, linking the fragments to an autonomously replicating plasmid, and introducing the resulting recombinant DNA molecules into bacteria. A few months later, Chang and I reported that genes from unrelated bacterial species can be combined and propagated using the same approach and that interspecies recombinant DNA molecules can produce a biologically functional protein in a foreign host. Soon afterward, Boyer’s laboratory and mine published our collaborative discovery that even genes from animal cells can be cloned in bacteria. These three PNAS papers quickly led to the use of DNA cloning methods in multiple areas of the biological and chemical sciences. They also resulted in a highly public controversy about the potential hazards of laboratory manipulation of genetic material, a decision by Stanford University and the University of California to seek patents on the technology that Boyer and I had invented, and the application of DNA cloning methods for commercial purposes. In the 40 years that have passed since publication of our findings, use of DNA cloning has produced insights about the workings of genes and cells in health and disease and has altered the nature of the biotechnology and biopharmaceutical industries. Here, I provide a personal perspective of the events that led to, and followed, our report of DNA cloning. PMID:24043817

Experimental Eavesdropping Based on Optimal Quantum Cloning

NASA Astrophysics Data System (ADS)

Bartkiewicz, Karol; Lemr, Karel; Černoch, Antonín; Soubusta, Jan; Miranowicz, Adam

2013-04-01

The security of quantum cryptography is guaranteed by the no-cloning theorem, which implies that an eavesdropper copying transmitted qubits in unknown states causes their disturbance. Nevertheless, in real cryptographic systems some level of disturbance has to be allowed to cover, e.g., transmission losses. An eavesdropper can attack such systems by replacing a noisy channel by a better one and by performing approximate cloning of transmitted qubits which disturb them but below the noise level assumed by legitimate users. We experimentally demonstrate such symmetric individual eavesdropping on the quantum key distribution protocols of Bennett and Brassard (BB84) and the trine-state spherical code of Renes (R04) with two-level probes prepared using a recently developed photonic multifunctional quantum cloner [Lemr et al., Phys. Rev. A 85, 050307(R) (2012)PLRAAN1050-2947]. We demonstrated that our optimal cloning device with high-success rate makes the eavesdropping possible by hiding it in usual transmission losses. We believe that this experiment can stimulate the quest for other operational applications of quantum cloning.

Entangled cloning of stabilizer codes and free fermions

NASA Astrophysics Data System (ADS)

Hsieh, Timothy H.

2016-10-01

Though the no-cloning theorem [Wooters and Zurek, Nature (London) 299, 802 (1982), 10.1038/299802a0] prohibits exact replication of arbitrary quantum states, there are many instances in quantum information processing and entanglement measurement in which a weaker form of cloning may be useful. Here, I provide a construction for generating an "entangled clone" for a particular but rather expansive and rich class of states. Given a stabilizer code or free fermion Hamiltonian, this construction generates an exact entangled clone of the original ground state, in the sense that the entanglement between the original and the exact copy can be tuned to be arbitrarily small but finite, or large, and the relation between the original and the copy can also be modified to some extent. For example, this Rapid Communication focuses on generating time-reversed copies of stabilizer codes and particle-hole transformed ground states of free fermion systems, although untransformed clones can also be generated. The protocol leverages entanglement to simulate a transformed copy of the Hamiltonian without having to physically implement it and can potentially be realized in superconducting qubits or ultracold atomic systems.

Human reproductive cloning and reasons for deprivation.

PubMed

Jensen, D A

2008-08-01

Human reproductive cloning provides the possibility of genetically related children for persons for whom present technologies are ineffective. I argue that the desire for genetically related children is not, by itself, a sufficient reason to engage in human reproductive cloning. I show this by arguing that the value underlying the desire for genetically related children implies a tension between the parent and the future child. This tension stems from an instance of a deprivation and violates a general principle of reasons for deprivation. Alternative considerations, such as a right to procreative autonomy, do not appear helpful in making the case for human reproductive cloning merely on the basis of the desire for genetically related children.

Transformation of Schwanniomyces occidentalis with an ADE2 gene cloned from S. occidentalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klein, R.D.; Favreau, M.A.

1988-12-01

We have developed an efficient transformation system for the industrial yeast Schwanniomyces occidentalis (formerly Schwanniomyces castellii). The transformation system is based on ade2 mutants of S. occidentalis deficient for phosphoribosylaminoimidazole carboxylase that were generated by mutagenesis. As a selectable marker, we isolated and characterized the S. occidentalis ADE2 gene by complementation in an ade2 strain of Saccharomyces cerevisiae. S. occidentalis was transformed with the recombinant plasmid pADE, consisting of a 4.5-kilobase-pair (kbp) DNA fragment from S. occidentalis containing the ADE2 gene inserted into the S. cerevisiae expression vector pYcDE8 by a modification of the spheroplasting procedure of Beggs. Intact plasmidsmore » were recovered in Escherichia coli from whole-cell lysates of ADE+ transformants, indicating that plasmids were replicating autonomously. High-molecular-mass species of pADE2 were found by Southern hybridization analysis of intact genomic DNA preparations. The shift to higher molecular mass of these plasmids during electrophoresis in the presence ethidium bromide after exposure to shortwave UV suggests that they exist in a supercoiled form in the transformed host. Subclones of the 4.5-kbp insert indicated that ADE2-complementing activity and sequences conferring autonomous replication in S. occidentalis were located within a 2.7-kbp EcoRI-SphI fragment. Plasmids containing this region cloned into the bacterial vector pUC19 complemented ade2 mutants of S. occidentalis with efficiencies identical to those of the original plasmid pADE.« less

Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts.

PubMed

Kang, Tae-Jin; Han, So-Chon; Kim, Mi-Young; Kim, Young-Sook; Yang, Moon-Sik

2004-11-01

Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-labile toxin. LTK63 is devoid of any toxic activity, but still retains its mucosal adjuvanticity. The LTK63 was cloned into chloroplast targeting vector and transformed to tobacco chloroplasts by particle bombardment. PCR and Southern blot analyses confirmed stable homologous recombination of the LTK63 gene into the chloroplast genome. The amount of LTK63 protein detected in tobacco chloroplasts was approximately 3.7% of the total soluble protein. The GM1-ganglioside binding assay confirmed that chloroplast-synthesized LTB of LTK63 binds to the intestinal membrane GM1-ganglioside receptor. Thus, the expression of LTK63 in chloroplasts provides a potential route toward the development of a plant-based edible vaccine for high expression system and environmentally friendly approach.

Purification of FKBP-70, a novel immunophilin from Saccharomyces cerevisiae, and cloning of its structural gene, FPR3.

PubMed

Manning-Krieg, U C; Henríquez, R; Cammas, F; Graff, P; Gavériaux, S; Movva, N R

1994-09-19

A novel protein, belonging to the yeast family of FKBPs (FK-binding proteins), FKBP-70, was isolated from Saccharomyces cerevisiae by its interaction with the immunosuppressive agent FK-520. Its structural gene, FPR3, was cloned and the protein expressed and purified from Escherichia coli. This third member of the FKBP family in yeast is homologous to the other FKBPs at its carboxy terminus, showing conserved ligand binding and proline isomerase regions. It is, however, a longer acidic protein with several potential nuclear targeting sequences and a region of homology to nucleolins. Yeast strains deleted for FPR3, as well as a triple deletion mutant of this family of genes, FPR1, FPR2 and FPR3, are viable under normal conditions of growth, indicating that the FPR genes are not essential for life.

The Three Major Spanish Clones of Penicillin-Resistant Streptococcus pneumoniae Are the Most Common Clones Recovered in Recent Cases of Meningitis in Spain

PubMed Central

Enright, Mark C.; Fenoll, Asunción; Griffiths, David; Spratt, Brian G.

1999-01-01

One hundred six isolates of Streptococcus pneumoniae recovered in Spain from patients with meningitis in 1997 and 1998 were characterized by multilocus sequence typing. A heterogeneous collection of genotypes was associated with meningitis in Spain: 65 different sequence types were resolved and, even at a genetic distance of 0.43, there were 37 distinct lineages. Thirty-eight percent of the isolates, including all isolates of serotypes 6B, 9V, 14, and 23F, were resistant to penicillin, and 24% of the isolates were members of the three major Spanish penicillin-resistant or multidrug-resistant clones of serotypes 6B, 9V, and 23F or serotype variants of these clones. These three clones (MICs, 1 to 2 μg of penicillin/ml) were the most common clones associated with pneumococcal meningitis in Spain during 1997 and 1998. Only two of the other clones associated with meningitis were penicillin resistant (MICs, 0.12 to 0.5 μg/ml). One of the two most prevalent penicillin-susceptible clones causing meningitis (serotype 3) has not been detected outside of Spain, whereas the other (serotype 18C) has been recovered from patients with meningitis in the United Kingdom, The Netherlands, and Denmark. The prevalence of meningitis caused by isolates of the three major Spanish penicillin-resistant or multiply antibiotic-resistant clones, which are now globally distributed, is disturbing and clearly establishes their ability to cause life-threatening disease. PMID:10488179

Characterization of a Spontaneous Nonmagnetic Mutant of Magnetospirillum gryphiswaldense Reveals a Large Deletion Comprising a Putative Magnetosome Island

PubMed Central

Schübbe, Sabrina; Kube, Michael; Scheffel, André; Wawer, Cathrin; Heyen, Udo; Meyerdierks, Anke; Madkour, Mohamed H.; Mayer, Frank; Reinhardt, Richard; Schüler, Dirk

2003-01-01

Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosome-associated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria. PMID:13129949

Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan.

PubMed

Gómez-Manzo, Saúl; Marcial-Quino, Jaime; Vanoye-Carlo, America; Serrano-Posada, Hugo; González-Valdez, Abigail; Martínez-Rosas, Víctor; Hernández-Ochoa, Beatriz; Sierra-Palacios, Edgar; Castillo-Rodríguez, Rosa Angélica; Cuevas-Cruz, Miguel; Rodríguez-Bustamante, Eduardo; Arreguin-Espinosa, Roberto

2016-05-21

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site.

Escherichia coli mutants impaired in maltodextrin transport.

PubMed

Wandersman, C; Schwartz, M; Ferenci, T

1979-10-01

Wild-type Escherichia coli K-12 was found to grow equally well on maltose and on maltodextrins containing up to seven glucose residues. Three classes of mutants unable to grow on maltodextrins, but still able to grow on maltose, were investigated in detail. The first class, already known, was composed of phage lambda-resistant mutants, which lack the outer membrane protein coded by gene lamB. These mutants grow on maltose and maltotriose but not at all on maltotetraose and longer maltodextrins which cannot cross the outer membrane. A second class of mutants were affected in malE, the structural gene of the periplasmic maltose binding protein. The maltose binding proteins isolated from the new mutants were altered in their substrate binding properties, but not in a way that could account for the mutant phenotypes. Rather, the results of growth experiments and transport studies suggest that these malE mutants are impaired in their ability to transport maltodextrins across the outer membrane. This implies that the maltose binding protein (in wild-type strains) cooperates with the lambda receptor in permeation through the outer membrane. The last class of mutants described in this paper were affected in malG, or perhaps in an as yet undetected gene close to malG. They were defective in the transfer of maltodextrins from the periplasmic space to the cytoplasm but only slightly affected in the transport of maltose.

Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis.

PubMed

Osanai-Futahashi, M; Tatematsu, K-I; Futahashi, R; Narukawa, J; Takasu, Y; Kayukawa, T; Shinoda, T; Ishige, T; Yajima, S; Tamura, T; Yamamoto, K; Sezutsu, H

2016-02-01

Ommochromes are major insect pigments involved in coloration of compound eyes, eggs, epidermis and wings. In the silkworm Bombyx mori, adult compound eyes and eggs contain a mixture of the ommochrome pigments such as ommin and xanthommatin. Here, we identified the gene involved in ommochrome biosynthesis by positional cloning of B. mori egg and eye color mutant pink-eyed white egg (pe). The recessive homozygote of pe has bright red eyes and white or pale pink eggs instead of a normal dark coloration due to the decrease of dark ommochrome pigments. By genetic linkage analysis, we narrowed down the pe-linked region to ~258 kb, containing 17 predicted genes. RNA sequencing analyses showed that the expression of one candidate gene, the ortholog of Drosophila haem peroxidase cardinal, coincided with egg pigmentation timing, similar to other ommochrome-related genes such as Bm-scarlet and Bm-re. In two pe strains, a common missense mutation was found within a conserved motif of B. mori cardinal homolog (Bm-cardinal). RNA interference-mediated knockdown and transcription activator-like effector nuclease (TALEN)-mediated knockout of the Bm-cardinal gene produced the same phenotype as pe in terms of egg, adult eye and larval epidermis coloration. A complementation test of the pe mutant with the TALEN-mediated Bm-cardinal-deficient strain showed that the mutant phenotype could not be rescued, indicating that Bm-cardinal is responsible for pe. Moreover, knockdown of the cardinal homolog in Tribolium castaneum also induced red compound eyes. Our results indicate that cardinal plays a major role in ommochrome synthesis of holometabolous insects.

Imaginal Disc Abnormalities in Lethal Mutants of Drosophila

PubMed Central

Shearn, Allen; Rice, Thomas; Garen, Alan; Gehring, Walter

1971-01-01

Late lethal mutants of Drosophila melanogaster, dying after the larval stage of development, were isolated. The homozygous mutant larvae were examined for abnormal imaginal disc morphology, and the discs were injected into normal larval hosts to test their capacities to differentiate into adult structures. In about half of the mutants analyzed, disc abnormalities were found. Included among the abnormalities were missing discs, small discs incapable of differentiating, morphologically normal discs with limited capacities for differentiation, and discs with homeotic transformations. In some mutants all discs were affected, and in others only certain discs. The most extreme abnormal phenotype is a class of “discless” mutants. The viability of these mutant larvae indicates that the discs are essential only for the development of an adult and not of a larva. The late lethals are therefore a major source of mutants for studying the genetic control of disc formation. Images PMID:5002822

Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology

PubMed Central

Kouprina, Natalay; Larionov, Vladimir

2016-01-01

Transformation-associated recombination (TAR) cloning represents a unique tool for isolation and manipulation of large DNA molecules. The technique exploits a high level of homologous recombination in the yeast Sacharomyces cerevisiae. So far, TAR cloning is the only method available to selectively recover chromosomal segments up to 300 kb in length from complex and simple genomes. In addition, TAR cloning allows the assembly and cloning of entire microbe genomes up to several Mb as well as engineering of large metabolic pathways. In this review, we summarize applications of TAR cloning for functional/structural genomics and synthetic biology. PMID:27116033

Absence of mutation at the 5'-upstream promoter region of the TPM4 gene from cardiac mutant axolotl (Ambystoma mexicanum).

PubMed

Denz, Christopher R; Zhang, Chi; Jia, Pingping; Du, Jianfeng; Huang, Xupei; Dube, Syamalima; Thomas, Anish; Poiesz, Bernard J; Dube, Dipak K

2011-09-01

Tropomyosins are a family of actin-binding proteins that show cell-specific diversity by a combination of multiple genes and alternative RNA splicing. Of the 4 different tropomyosin genes, TPM4 plays a pivotal role in myofibrillogenesis as well as cardiac contractility in amphibians. In this study, we amplified and sequenced the upstream regulatory region of the TPM4 gene from both normal and mutant axolotl hearts. To identify the cis-elements that are essential for the expression of the TPM4, we created various deletion mutants of the TPM4 promoter DNA, inserted the deleted segments into PGL3 vector, and performed promoter-reporter assay using luciferase as the reporter gene. Comparison of sequences of the promoter region of the TPM4 gene from normal and mutant axolotl revealed no mutations in the promoter sequence of the mutant TPM4 gene. CArG box elements that are generally involved in controlling the expression of several other muscle-specific gene promoters were not found in the upstream regulatory region of the TPM4 gene. In deletion experiments, loss of activity of the reporter gene was noted upon deletion which was then restored upon further deletion suggesting the presence of both positive and negative cis-elements in the upstream regulatory region of the TPM4 gene. We believe that this is the first axolotl promoter that has ever been cloned and studied with clear evidence that it functions in mammalian cell lines. Although striated muscle-specific cis-acting elements are absent from the promoter region of TPM4 gene, our results suggest the presence of positive and negative cis-elements in the promoter region, which in conjunction with positive and negative trans-elements may be involved in regulating the expression of TPM4 gene in a tissue-specific manner.

Japan. Human cloning ban allows some research.

PubMed

Normile, D

2000-12-08

TOKYO--Japanese legislators last week approved a ban on human cloning that leaves room for the use of certain techniques in basic research. The action comes at the same time officials in two other countries--China and France--aired similar proposals that would prohibit so-called reproductive cloning while recognizing the possible importance of the technology in combating disease and improving human health.

Thermally induced disintegration of the oligomeric structure of alphaB-crystallin mutant F28S is associated with diminished chaperone activity.

PubMed

Kelley, Patrick B; Abraham, Edathara C

2003-10-01

alphaB-crystallin, a member of the small heat-shock protein (hsp) family of proteins, is able to function as a molecular chaperone by protecting other proteins from stress-induced aggregation by recognizing and binding to partially unfolded species of damaged proteins. The present work has investigated the role of phenylalanine-28 (F28) of the 22RLFDQFF28 region of alphaB-crystallin in maintaining chaperone function and oligomeric structure under physiological condition and under thermal stress. Bovine alphaB-crystallin was cloned for the first time and the cDNA sequence revealed greater than 90% homology to that of human, rat and mouse alphaB-crystallins. F28 was mutated to a serine followed by expression of the mutant F28S and the wild-type alphaB (alphaB-wt) in E. coli and subsequent purification of the protein by size-exclusion chromatography. Secondary and tertiary structure analyses showed some structural changes in the mutant. Chaperone activity and oligomeric size of the mutant was unchanged at 37 degrees C whereas at 58 degrees C the chaperone activity was significantly decreased and the oligomeric size ranged from low molecular weight to high molecular weight showing disintegration of the oligomeric structure. The data support the idea that the participation of large oligomeric structure rather than smaller units is required to have optimal chaperone activity and the hydrophobic F28 residue is needed for maintaining the native oligomeric structure under thermal stress.

Transformation of apple (Malus × domestica) using mutants of apple acetolactate synthase as a selectable marker and analysis of the T-DNA integration sites.

PubMed

Yao, Jia-Long; Tomes, Sumathi; Gleave, Andrew P

2013-05-01

Apple acetolactate synthase mutants were generated by site-specific mutagenesis and successfully used as selection marker in tobacco and apple transformation. T-DNA/Apple genome junctions were analysed using genome-walking PCR and sequencing. An Agrobacterium-mediated genetic transformation system was developed for apple (Malus × domestica), using mutants of apple acetolactate synthase (ALS) as a selectable marker. Four apple ALS mutants were generated by site-specific mutagenesis and subsequently cloned under the transcriptional control of the CaMV 35S promoter and ocs 3' terminator, in a pART27-derived plant transformation vector. Three of the four mutations were found to confer resistance to the herbicide Glean(®), containing the active agent chlorsulfuron, in tobacco (Nicotiana tabacum) transformation. In apple transformation, leaf explants infected with Agrobacterium tumefaciens EHA105 containing one of the three ALS mutants resulted in the production of shoots on medium containing 2-8 μg L(-1) Glean(®), whilst uninfected wild-type explants failed to regenerate shoots or survive on medium containing 1 and 3 μg L(-1) Glean(®), respectively. Glean(®)-resistant, regenerated shoots were further multiplied and rooted on medium containing 10 μg L(-1) Glean(®). The T-DNA and apple genome-DNA junctions from eight rooted transgenic apple plants were analysed using genome-walking PCR amplification and sequencing. This analysis confirmed T-DNA integration into the apple genome, identified the genome integration sites and revealed the extent of any vector backbone integration, T-DNA rearrangements and deletions of apple genome DNA at the sites of integration.

Mice cloned from skin cells.

PubMed

Li, Jinsong; Greco, Valentina; Guasch, Géraldine; Fuchs, Elaine; Mombaerts, Peter

2007-02-20

Adult stem cells represent unique populations of undifferentiated cells with self-renewal capacity. In many tissues, stem cells divide less often than their progeny. It has been widely speculated, but largely untested, that their undifferentiated and quiescent state may make stem cells more efficient as donors for cloning by nuclear transfer (NT). Here, we report the use of nuclei from hair follicle stem cells and other skin keratinocytes as NT donors. When keratinocyte stem cells (KSCs) were used as NT donors, 19 liveborn mice were obtained, 9 of which survived to adulthood. Embryonic keratinocytes and cumulus cells also gave rise to cloned mice. Although cloning efficiencies were similar (<6% per transferred blastocyst), success rates were consistently higher for males than for females. Adult keratinocyte stem cells were better NT donors than so-called transit amplifying (TA) keratinocytes in both sexes (1.6% vs. 0% in females and 5.4% vs. 2.8% in males). Our findings reveal skin as a source of readily accessible stem cells, the nuclei of which can be reprogrammed to the pluripotent state by exposure to the cytoplasm of unfertilized oocytes.

Feedback inhibition by thiols outranks glutathione depletion: a luciferase-based screen reveals glutathione-deficient γ-ECS and glutathione synthetase mutants impaired in cadmium-induced sulfate assimilation.

PubMed

Jobe, Timothy O; Sung, Dong-Yul; Akmakjian, Garo; Pham, Allis; Komives, Elizabeth A; Mendoza-Cózatl, David G; Schroeder, Julian I

2012-06-01

Plants exposed to heavy metals rapidly induce changes in gene expression that activate and enhance detoxification mechanisms, including toxic-metal chelation and the scavenging of reactive oxygen species. However, the mechanisms mediating toxic heavy metal-induced gene expression remain largely unknown. To genetically elucidate cadmium-specific transcriptional responses in Arabidopsis, we designed a genetic screen based on the activation of a cadmium-inducible reporter gene. Microarray studies identified a high-affinity sulfate transporter (SULTR1;2) among the most robust and rapid cadmium-inducible transcripts. The SULTR1;2 promoter (2.2 kb) was fused with the firefly luciferase reporter gene to quantitatively report the transcriptional response of plants exposed to cadmium. Stably transformed luciferase reporter lines were ethyl methanesulfonate (EMS) mutagenized, and stable M(2) seedlings were screened for an abnormal luciferase response during exposure to cadmium. The screen identified non-allelic mutant lines that fell into one of three categories: (i) super response to cadmium (SRC) mutants; (ii) constitutive response to cadmium (CRC) mutants; or (iii) non-response and reduced response to cadmium (NRC) mutants. Two nrc mutants, nrc1 and nrc2, were mapped, cloned and further characterized. The nrc1 mutation was mapped to the γ-glutamylcysteine synthetase gene and the nrc2 mutation was identified as the first viable recessive mutant allele in the glutathione synthetase gene. Moreover, genetic, HPLC mass spectrometry, and gene expression analysis of the nrc1 and nrc2 mutants, revealed that intracellular glutathione depletion alone would be insufficient to induce gene expression of sulfate uptake and assimilation mechanisms. Our results modify the glutathione-depletion driven model for sulfate assimilation gene induction during cadmium stress, and suggest that an enhanced oxidative state and depletion of upstream thiols, in addition to glutathione depletion

Photonic Programmable Tele-Cloning Network.

PubMed

Li, Wei; Chen, Ming-Cheng

2016-06-29