Formation of functional asialoglycoprotein receptor after transfection with cDNAs encoding the receptor proteins.

PubMed Central

McPhaul, M; Berg, P

1986-01-01

The rat asialoglycoprotein receptor (ASGP-R) has been expressed in cultured rat hepatoma cells (HTC cells) after transfection with cloned cDNAs. Fluorescence-activated cell sorting of transfected cells was used to identify the functional cDNA clones and to isolate cells expressing the ASGP-R. Simultaneous or sequential transfections with two cloned cDNAs that encode related but distinctive polypeptide chains were needed to obtain ASGP-R activity; transfection with either cDNA alone failed to produce detectable ASGP-R. The affinity of transduced ASGP-R for asialo orosomucoid is less than that of the native rat ASGP-R, and the number of surface receptors in clones expressing ASGP-R is about one-fifth that found on rat hepatocytes. Images PMID:3466162

Identification of Prostate Cancer-Specific microDNAs

DTIC Science & Technology

2016-02-01

circular DNA by rolling circle amplification (RCA) and then amplified DNA fragments were subject to deep sequencing. Deep sequencing of the...demonstrate the existence of microDNAs in prostate cancer. We adopted multiple displacement amplification (MDA) with random 2 primers for enriched...prostate cancer cells through multiple displacement amplification and next generation sequencing. R e la ti v e c e ll g ro w th ( % ) 0 20

Fluorescence biosensor for inorganic pyrophosphatase activity.

PubMed

Zhang, Ying; Guo, Yajuan; Zhao, Mengmeng; Lin, Cuiying; Lin, Zhenyu; Luo, Fang; Chen, Guonan

2017-02-01

A highly sensitive and selective fluorescence biosensor for inorganic pyrophosphatase (PPase) activity has been developed based on special click ligation trigger hyperbranched rolling circle amplification (CLT-HRCA). Pyrophosphate ion (PPi) can coordinate with Cu 2+ to form stable PPi/Cu 2+ complex and Cu 2+ in the complex cannot be reduced to Cu + . The addition of PPase causes the hydrolysis of PPi into orthophosphate (Pi) and therefore induces the releasing of Cu 2+ from the stable PPi/Cu 2+ complex, and the free Cu 2+ is easily reduced to Cu + by sodium ascorbate. Then Cu + catalyzes the cyclization reaction between the specially designed 5'-azide and 3'-alkyne tagged padlock probes through Cu + catalyzed azide-alkyne cycloaddition (CuAAC), which in turn initiates the hyperbranched rolling circle amplification (HRCA). Given that the CLT-HRCA products contain large amounts of double-stranded DNAs (dsDNAs), the addition of SYBR Green I resulted in the enhanced fluorescence signal. There was a linear relationship between the enhanced fluorescence intensity and the logarithm PPase activity ranging from 0.05 to 25 mU with a detection limit of 0.02 mU. Such proposed biosensor has been successfully applied to screen the potential PPase inhibitors and has accessed the related inhibit ability with high efficiency.

A label-free and high-efficient GO-based aptasensor for cancer cells based on cyclic enzymatic signal amplification.

PubMed

Xiao, Kunyi; Liu, Juan; Chen, Hui; Zhang, Song; Kong, Jilie

2017-05-15

A label-free and high-efficient graphene oxide (GO)-based aptasensor was developed for the detection of low quantity cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA). In the absence of target cells, hairpin aptamer probes (HAPs) and dye-labeled linker DNAs stably coexisted in solution, and the fluorescence was quenched by the GO-based FÖrster resonance energy transfer (FRET) process. In the presence of target cells, the specific binding of HAPs with the target cells triggered a conformational alternation, which resulted in linker DNA complementary pairing and cleavage by nicking endonuclease-strand scission cycles. Consequently, more cleaved fragments of linker DNAs with more the terminal labeled dyes could show the enhanced fluorescence because these cleaved DNA fragments hardly combine with GOs and prevent the FRET process. Fluorescence analysis demonstrated that this GO-based aptasensor exhibited selective and sensitive response to the presence of target CCRF-CEM cells in the concentration range from 50 to 10 5 cells. The detection limit of this method was 25 cells, which was approximately 20 times lower than the detection limit of normal fluorescence aptasensors without amplification. With high sensitivity and specificity, it provided a simple and cost-effective approach for early cancer diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Variations in 5S rDNAs in diploid and tetraploid offspring of red crucian carp × common carp.

PubMed

Ye, Lihai; Zhang, Chun; Tang, Xiaojun; Chen, Yiyi; Liu, Shaojun

2017-08-08

The allotetraploid hybrid fish (4nAT) that was created in a previous study through an intergeneric cross between red crucian carp (Carassius auratus red var., ♀) and common carp (Cyprinus carpio L., ♂) provided an excellent platform to investigate the effect of hybridization and polyploidization on the evolution of 5S rDNA. The 5S rDNAs of paternal common carp were made up of a coding sequence (CDS) and a non-transcribed spacer (NTS) unit, and while the 5S rDNAs of maternal red crucian carp contained a CDS and a NTS unit, they also contained a variable number of interposed regions (IPRs). The CDSs of the 5S rDNAs in both parental fishes were conserved, while their NTS units seemed to have been subjected to rapid evolution. The diploid hybrid 2nF 1 inherited all the types of 5S rDNAs in both progenitors and there were no signs of homeologous recombination in the 5S rDNAs of 2nF 1 by sequencing of PCR products. We obtained two segments of 5S rDNA with a total length of 16,457 bp from allotetraploid offspring 4nAT through bacterial artificial chromosome (BAC) sequencing. Using this sequence together with the 5S rDNA sequences amplified from the genomic DNA of 4nAT, we deduced that the 5S rDNAs of 4nAT might be inherited from the maternal progenitor red crucian carp. Additionally, the IPRs in the 5S rDNAs of 4nAT contained A-repeats and TA-repeats, which was not the case for the IPRs in the 5S rDNAs of 2nF 1 . We also detected two signals of a 200-bp fragment of 5S rDNA in the chromosomes of parental progenitors and hybrid progenies by fluorescence in situ hybridization (FISH). We deduced that during the evolution of 5S rDNAs in different ploidy hybrid fishes, interlocus gene conversion events and tandem repeat insertion events might occurred in the process of polyploidization. This study provided new insights into the relationship among the evolution of 5S rDNAs, hybridization and polyploidization, which were significant in clarifying the genome evolution of polyploid fish.

Mitochondrial Recombination Reveals Mito-Mito Epistasis in Yeast.

PubMed

Wolters, John F; Charron, Guillaume; Gaspary, Alec; Landry, Christian R; Fiumera, Anthony C; Fiumera, Heather L

2018-05-01

Genetic variation in mitochondrial DNA (mtDNA) provides adaptive potential although the underlying genetic architecture of fitness components within mtDNAs is not known. To dissect functional variation within mtDNAs, we first identified naturally occurring mtDNAs that conferred high or low fitness in Saccharomyces cerevisiae by comparing growth in strains containing identical nuclear genotypes but different mtDNAs. During respiratory growth under temperature and oxidative stress conditions, mitotype effects were largely independent of nuclear genotypes even in the presence of mito-nuclear interactions. Recombinant mtDNAs were generated to determine fitness components within high- and low-fitness mtDNAs. Based on phenotypic distributions of isogenic strains containing recombinant mtDNAs, we found that multiple loci contributed to mitotype fitness differences. These mitochondrial loci interacted in epistatic, nonadditive ways in certain environmental conditions. Mito-mito epistasis ( i.e. , nonadditive interactions between mitochondrial loci) influenced fitness in progeny from four different crosses, suggesting that mito-mito epistasis is a widespread phenomenon in yeast and other systems with recombining mtDNAs. Furthermore, we found that interruption of coadapted mito-mito interactions produced recombinant mtDNAs with lower fitness. Our results demonstrate that mito-mito epistasis results in functional variation through mitochondrial recombination in fungi, providing modes for adaptive evolution and the generation of mito-mito incompatibilities. Copyright © 2018 by the Genetics Society of America.

Cohesin Can Remain Associated with Chromosomes during DNA Replication.

PubMed

Rhodes, James D P; Haarhuis, Judith H I; Grimm, Jonathan B; Rowland, Benjamin D; Lavis, Luke D; Nasmyth, Kim A

2017-09-19

To ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication. We developed a non-fluorescent HaloTag ligand to discriminate the fluorescence recovery signal from labeling of newly synthesized Halo-tagged Scc1 protein (pulse-chase or pcFRAP). In cells where cohesin turnover is inactivated by deletion of WAPL, Scc1 can remain associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to un-replicated DNA. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

A gold@polydopamine core-shell nanoprobe for long-term intracellular detection of microRNAs in differentiating stem cells.

PubMed

Choi, Chun Kit K; Li, Jinming; Wei, Kongchang; Xu, Yang J; Ho, Lok Wai C; Zhu, Meiling; To, Kenneth K W; Choi, Chung Hang J; Bian, Liming

2015-06-17

The capability of monitoring the differentiation process in living stem cells is crucial to the understanding of stem cell biology and the practical application of stem-cell-based therapies, yet conventional methods for the analysis of biomarkers related to differentiation require a large number of cells as well as cell lysis. Such requirements lead to the unavoidable loss of cell sources and preclude real-time monitoring of cellular events. In this work, we report the detection of microRNAs (miRNAs) in living human mesenchymal stem cells (hMSCs) by using polydopamine-coated gold nanoparticles (Au@PDA NPs). The PDA shell facilitates the immobilization of fluorescently labeled hairpin DNA strands (hpDNAs) that can recognize specific miRNA targets. The gold core and PDA shell quench the fluorescence of the immobilized hpDNAs, and subsequent binding of the hpDNAs to the target miRNAs leads to their dissociation from Au@PDA NPs and the recovery of fluorescence signals. Remarkably, these Au@PDA-hpDNA nanoprobes can naturally enter stem cells, which are known for their poor transfection efficiency, without the aid of transfection agents. Upon cellular uptake of these nanoprobes, we observe intense and time-dependent fluorescence responses from two important osteogenic marker miRNAs, namely, miR-29b and miR-31, only in hMSCs undergoing osteogenic differentiation and living primary osteoblasts but not in undifferentiated hMSCs and 3T3 fibroblasts. Strikingly, our nanoprobes can afford long-term tracking of miRNAs (5 days) in the differentiating hMSCs without the need of continuously replenishing cell culture medium with fresh nanoprobes. Our results demonstrate the capability of our Au@PDA-hpDNA nanoprobes for monitoring the differentiation status of hMSCs (i.e., differentiating versus undifferentiated) via the detection of specific miRNAs in living stem cells. Our nanoprobes show great promise in the investigation of the long-term dynamics of stem cell differentiation, identification and isolation of specific cell types, and high-throughput drug screening.

Analysis of T-DNA integration and generative segregation in transgenic winter triticale (x Triticosecale Wittmack)

PubMed Central

2012-01-01

Background While the genetic transformation of the major cereal crops has become relatively routine, to date only a few reports were published on transgenic triticale, and robust data on T-DNA integration and segregation have not been available in this species. Results Here, we present a comprehensive analysis of stable transgenic winter triticale cv. Bogo carrying the selectable marker gene HYGROMYCIN PHOSPHOTRANSFERASE (HPT) and a synthetic green fluorescent protein gene (gfp). Progeny of four independent transgenic plants were comprehensively investigated with regard to the number of integrated T-DNA copies, the number of plant genomic integration loci, the integrity and functionality of individual T-DNA copies, as well as the segregation of transgenes in T1 and T2 generations, which also enabled us to identify homozygous transgenic lines. The truncation of some integrated T-DNAs at their left end along with the occurrence of independent segregation of multiple T-DNAs unintendedly resulted in a single-copy segregant that is selectable marker-free and homozygous for the gfp gene. The heritable expression of gfp driven by the maize UBI-1 promoter was demonstrated by confocal laser scanning microscopy. Conclusions The used transformation method is a valuable tool for the genetic engineering of triticale. Here we show that comprehensive molecular analyses are required for the correct interpretation of phenotypic data collected from the transgenic plants. PMID:23006412

Analysis of T-DNA integration and generative segregation in transgenic winter triticale (x Triticosecale Wittmack).

PubMed

Hensel, Goetz; Oleszczuk, Sylwia; Daghma, Diaa Eldin S; Zimny, Janusz; Melzer, Michael; Kumlehn, Jochen

2012-09-25

While the genetic transformation of the major cereal crops has become relatively routine, to date only a few reports were published on transgenic triticale, and robust data on T-DNA integration and segregation have not been available in this species. Here, we present a comprehensive analysis of stable transgenic winter triticale cv. Bogo carrying the selectable marker gene HYGROMYCIN PHOSPHOTRANSFERASE (HPT) and a synthetic green fluorescent protein gene (gfp). Progeny of four independent transgenic plants were comprehensively investigated with regard to the number of integrated T-DNA copies, the number of plant genomic integration loci, the integrity and functionality of individual T-DNA copies, as well as the segregation of transgenes in T1 and T2 generations, which also enabled us to identify homozygous transgenic lines. The truncation of some integrated T-DNAs at their left end along with the occurrence of independent segregation of multiple T-DNAs unintendedly resulted in a single-copy segregant that is selectable marker-free and homozygous for the gfp gene. The heritable expression of gfp driven by the maize UBI-1 promoter was demonstrated by confocal laser scanning microscopy. The used transformation method is a valuable tool for the genetic engineering of triticale. Here we show that comprehensive molecular analyses are required for the correct interpretation of phenotypic data collected from the transgenic plants.

Characterization of Bleomycin-Mediated Cleavage of a Hairpin DNA Library

PubMed Central

Segerman, Zachary J.; Roy, Basab; Hecht, Sidney M.

2013-01-01

A study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal free BLM. The ability of Fe(II)•BLM to effect cleavage on both the 3' and 5'-arms of the hairpin DNAs was characterized. The strongly bound DNAs were found to be efficient substrates for Fe•BLM A5-mediated hairpin DNA cleavage. Surprisingly, the most prevalent site of BLM-mediated cleavage was found to be the 5′-AT-3′ dinucleotide sequence. This dinucleotide sequence, and other sequences generally not cleaved well by BLM when examined using arbitrarily chosen DNA substrates, were apparent when examining the library of ten hairpin DNAs. In total, 132 sites of DNA cleavage were produced by exposure of the hairpin DNA library to Fe•BLM A5. The existence of multiple sites of cleavage on both the 3′- and 5′-arms of the hairpin DNAs suggested that some of these might be double-strand cleavage events. Accordingly, an assay was developed with which to test the propensity of the hairpin DNAs to undergo double-strand DNA damage. One hairpin DNA was characterized using this method, and gave results consistent with earlier reports of double-strand DNA cleavage, but with a sequence selectivity different from those reported previously. PMID:23834496

Rapid detection of Wuchereria bancrofti and Brugia malayi in mosquito vectors (Diptera: Culicidae) using a real-time fluorescence resonance energy transfer multiplex PCR and melting curve analysis.

PubMed

Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Maleewong, Wanchai

2009-01-01

We developed a single-step real-time fluorescence resonance energy transfer (FRET) multiplex polymerase chain reaction (PCR) merged with melting curve analysis for the detection of Wuchereria bancrofti and Brugia malayi DNA in blood-fed mosquitoes. Real-time FRET multiplex PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two families of repeated DNA elements: the 188 bp SspI repeated sequence, specific to W. bancrofti, and the 153-bp HhaI repeated sequence, specific to the genus Brugia and two pairs of specific fluorophore-labeled probes. Both W. bancrofti and B. malayi can be differentially detected in infected vectors by this process through their different fluorescence channel and melting temperatures. The assay could distinguish both human filarial DNAs in infected vectors from the DNAs of Dirofilaria immitis- and Plasmodium falciparum-infected human red blood cells and noninfected mosquitoes and human leukocytes. The technique showed 100% sensitivity and specificity and offers a rapid and reliable procedure for differentially identifying lymphatic filariasis. The introduced real-time FRET multiplex PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The test can be used to screen mosquito vectors in endemic areas and therefore should be a useful diagnostic tool for the evaluation of infection rate of the mosquito populations and for xenomonitoring in the community after eradication programs such as the Global Program to Eliminate Lymphatic Filariasis.

Identification and characterization of a subtelomeric satellite DNA in Callitrichini monkeys.

PubMed

Araújo, Naiara Pereira; de Lima, Leonardo Gomes; Dias, Guilherme Borges; Kuhn, Gustavo Campos Silva; de Melo, Alan Lane; Yonenaga-Yassuda, Yatiyo; Stanyon, Roscoe; Svartman, Marta

2017-08-01

Repetitive DNAs are abundant fast-evolving components of eukaryotic genomes, which often possess important structural and functional roles. Despite their ubiquity, repetitive DNAs are poorly studied when compared with the genic fraction of genomes. Here, we took advantage of the availability of the sequenced genome of the common marmoset Callithrix jacchus to assess its satellite DNAs (satDNAs) and their distribution in Callitrichini. After clustering analysis of all reads and comparisons by similarity, we identified a satDNA composed by 171 bp motifs, named MarmoSAT, which composes 1.09% of the C. jacchus genome. Fluorescent in situ hybridization on chromosomes of species from the genera Callithrix, Mico and Callimico showed that MarmoSAT had a subtelomeric location. In addition to the common monomeric, we found that MarmoSAT was also organized in higher-order repeats of 338 bp in Callimico goeldii. Our phylogenetic analyses showed that MarmoSAT repeats from C. jacchus lack chromosome-specific features, suggesting exchange events among subterminal regions of non-homologous chromosomes. MarmoSAT is transcribed in several tissues of C. jacchus, with the highest transcription levels in spleen, thymus and heart. The transcription profile and subtelomeric location suggest that MarmoSAT may be involved in the regulation of telomerase and modulation of telomeric chromatin. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Male specific genes from dioecious white campion identified by fluorescent differential display.

PubMed

Scutt, Charles P; Jenkins, Tom; Furuya, Masaki; Gilmartin, Philip M

2002-05-01

Fluorescent differential display (FDD) has been used to screen for cDNAs that are differentially up-regulated in male flowers of the dioecious plant Silene latifolia in which an X/Y chromosome system of sex determination operates. To adapt FDD to the cloning of large numbers of differential cDNAs, a novel method of confirming the differential expression of these has been devised. FDD gels were Southern electro-blotted and probed with mixtures of individual cDNA clones derived from different FDD product ligation reactions. These Southern blots were then stripped and re-probed with further mixtures of individual cloned FDD products to identify the maximum number of recombinant clones carrying the true differential amplification products. Of 135 differential bands identified by FDD, 56 differential amplification products were confirmed; these represent 23 unique differentially expressed genes as determined by virtual Northern analysis and two genes expressed at or below the level of detection by virtual Northern analysis. These two low expressed genes show bands of hybridization on genomic Southern blots that are specific to male plants, indicating that they are derived from, or closely related to, Y chromosome genes.

Chromosomal study of lettuce and its allied species (Lactuca spp., Asteraceae) by means of karyotype analysis and fluorescence in situ hybridization.

PubMed

Matoba, Hideyuki; Mizutani, Takayuki; Nagano, Katsuya; Hoshi, Yoshikazu; Uchiyama, Hiroshi

2007-12-01

In this study, in addition to the karyotype analysis, the chromosomal distributions of 5 S and 18 S rDNAs, and the Arabidopsis-type (T3AG3) telomeric sequences were detected by means of fluorescence in situ hybridization (FISH) to promote the information of chromosomal organization and evolution in the cultivated lettuce and its wild relatives, L. sativa, L. serriola, L. saligna and L. virosa. The karyotype analysis revealed the dissimilarity between L. virosa and the remaining species. In all four Lactuca species studied, one 5 S rDNA and two 18 S rDNA loci were detected. The simultaneous FISH of 5 S and 18 S rDNAs revealed that both rDNA loci of L. sativa, L. serriola and L. saligna were identical, however, that of L. virosa was different from the other species. These analyses indicate the closer relationships between L. sativa/L. serriola and L. saligna rather than L. virosa. Arabidopsis-type telomeric sequences were detected at both ends of their chromatids of all chromosomes not in the other regions. This observation suggests the lack of telomere-mediated chromosomal rearrangements among the Lactuca chromosomes.

mtDNA lineage analysis of mouse L-cell lines reveals the accumulation of multiple mtDNA mutants and intermolecular recombination

PubMed Central

Fan, Weiwei; Lin, Chun Shi; Potluri, Prasanth; Procaccio, Vincent; Wallace, Douglas C.

2012-01-01

The role of mitochondrial DNA (mtDNA) mutations and mtDNA recombination in cancer cell proliferation and developmental biology remains controversial. While analyzing the mtDNAs of several mouse L cell lines, we discovered that every cell line harbored multiple mtDNA mutants. These included four missense mutations, two frameshift mutations, and one tRNA homopolymer expansion. The LA9 cell lines lacked wild-type mtDNAs but harbored a heteroplasmic mixture of mtDNAs, each with a different combination of these variants. We isolated each of the mtDNAs in a separate cybrid cell line. This permitted determination of the linkage phase of each mtDNA and its physiological characteristics. All of the polypeptide mutations inhibited their oxidative phosphorylation (OXPHOS) complexes. However, they also increased mitochondrial reactive oxygen species (ROS) production, and the level of ROS production was proportional to the cellular proliferation rate. By comparing the mtDNA haplotypes of the different cell lines, we were able to reconstruct the mtDNA mutational history of the L–L929 cell line. This revealed that every heteroplasmic L-cell line harbored a mtDNA that had been generated by intracellular mtDNA homologous recombination. Therefore, deleterious mtDNA mutations that increase ROS production can provide a proliferative advantage to cancer or stem cells, and optimal combinations of mutant loci can be generated through recombination. PMID:22345519

Multiplex and high-throughput DNA detection using surface plasmon mediated fluorescence

NASA Astrophysics Data System (ADS)

Mei, Zhong

The overall objective of this research project was to develop a user-friendly and sensitive biosensor for nucleic acid aptamers with multiplexing and high-throughput capability. The sensing was based on the fluorescence signals emitted by the fluorophores coupling with plamonic nanoparticle (gold nanorod) deposited on a patterned substrate. Gold nanorods (GNRs) were synthesized using a binary mixture of hexadecyltrimethylammonium bromide (CTAB) and sodium oleate (NaOL) in seed mediated growth method. Polytetrafluoroethylene (PTFE) printed glass slides were selectively coated with a gold thin-film to define hydrophilic areas for GNR deposition. Due to the wettablity contrast, GNR solution dropped on the slide was induced to assemble exclusively in the hydrophilic spots. By controlling temperature and humidity of the evaporation process, vertically-standing GNR arrays were achieved on the pattered slide. Fluorescence was conjugated to GNR surface via DNA double strand with tunable length. Theoretical simulation predicted a flat layer ( 30 nm thick) of uniform "hot spots" presented on the GNR tips, which could modify the nearby fluorescence. Experimentally, the vertical GNR arrays yielded metallic enhanced fluorescence (MEF) effect, which was dependent on the spectrum overlap and GNR-fluorophore distance. Specifically, the maximum enhancement of Quasar 670 and Alexa 750 was observed when it was coupled with GNR664 (plasmonic wavelength 664 nm) and GNR778 respectively at a distance of 16 nm, while the carboxyfluorescein (FAM) was at maximal intensity when attached to gold nanosphere520. This offers an opportunity for multiplexed DNA sensing. Based on this, we developed a novel GNR mediated fluorescence biosensor for DNA detection. Fluorescence labeled haipin-DNA probes were introduced to designated spots of GNR array with the matching LSPR wavelengths on the substrate. The fluorescence was quenched originally because of Forster resonance energy transfer (FRET) effect. Upon hybridization with their complimentary target DNAs, hairpin structures were opened and the fluorescence enhancement from each GNR sensing spot was measured by fluorescence scanning. We demonstrated multiple DNA sequences were simultaneously detected at a picomolar level with high-throughput capability using the ordered GNR array biochip.

Application of a multi-channel microfluidic chip on the simultaneous detection of DNAs by using microbead-quantum dots.

PubMed

Le, Ngoc Tam; Kim, Jong Sung

2014-12-01

Several researches have shown that cancer is caused by genetic mutations especially in genes involved in cell growth and regulation. Ras family members are frequently found in their mutated, oncogenic forms in human tumors. Mutant RAS proteins are constitutively active, owing to reduce intrinsic GTPase activity and insensitivity to GTPase-activating protein (GAPs). In total, activating mutations in the RAS genes occur in approximately 20% of all human cancers, mainly in codon 12, 13 or 61. Activating mutations in the NRAS gene not only result in the reduction of intrinsic GTPase activity but also in the induction of resistance against molecules inducing such activity. In this paper, we reported a rapid, simple and portable method for detecting the mutant types of NRAS genes codon 12 and 61 simultaneously by using bead-quantum dots (QDs) based multi-channel microfluidic chip. Probe DNAs are conjugated to bead-QDs and packed in the pillars of channels in the microfluidic chip. After injection of target DNAs and intercalating dyes, the fluorescence quenching of QDs by intercalating dye was observed due to FRET phenomena. The platform can be effortlessly applied in other biological and clinical areas.

PCR cloning and characterization of multiple ADP-glucose pyrophosphorylase cDNAs from tomato

NASA Technical Reports Server (NTRS)

Chen, B. Y.; Janes, H. W.; Gianfagna, T.

1998-01-01

Four ADP-glucose pyrophosphorylase (AGP) cDNAs were cloned from tomato fruit and leaves by the PCR techniques. Three of them (agp S1, agp S2, and agp S3) encode the large subunit of AGP, the fourth one (agp B) encodes the small subunit. The deduced amino acid sequences of the cDNAs show very high identities (96-98%) to the corresponding potato AGP isoforms, although there are major differences in tissue expression profiles. All four tomato AGP transcripts were detected in fruit and leaves; the predominant ones in fruit are agp B and agp S1, whereas in leaves they are agp B and agp S3. Genomic southern analysis suggests that the four AGP transcripts are encoded by distinct genes.

Highly Sensitive Colorimetric Cancer Cell Detection Based on Dual Signal Amplification.

PubMed

Yu, Tao; Dai, Pan-Pan; Xu, Jing-Juan; Chen, Hong-Yuan

2016-02-01

Facile and efficient detection of cancer cells at their preclinical stages is one of the central challenges in cancer diagnostics. A direct, rapid, highly sensitive and specific biosensor for detection of cancer biomarkers is desirable in early diagnosis and prognosis of cancer. In this work, we developed, for the first time, an easy and intuitive dispersion-dominated colorimetric strategy for cancer cell detection based on combining multi-DNA released from an aptamer scaffold with cyclic enzymatic amplification, which was triggered by aptamer DNA conformational switch and demonstrated by non-cross-linking gold nanoparticles (Au NPs) aggregation. First, five kinds of messenger DNAs (mDNAs) were aligned on the cancer cell aptamers modified on magnetic beads (MBs) to form mDNAs-Apt-MBs biocompatible nanosensors. In the presence of target cells, the aptamer would bind to the receptors on the cell membranes, and mDNAs would be released, resulting in the first amplification that one biological binding event would cause the release of multiple kinds of mDNAs simultaneously. After magnetic separation, the released mDNAs were introduced into the cyclic enzymatic amplification to cleave more single strand DNA (ssDNA) fragments. Instead of modification of Au NPs, these fragments and mDNAs could be adsorbed on the surface of Au NPs to prevent particle aggregation and ensure the stability and color of solution in high salt environments. The linear response for HL-60 cells in a concentration range from 10 to 10(4) cells was obtained with a detection limit of four cells in buffer solution. Moreover, the feasibility of the proposed strategy was demonstrated in a diluted serum sample. This dual signal amplification method can be extended to other types of cancer cells, which has potential application in point-of-care cancer diagnosis.

Contrasting the Chromosomal Organization of Repetitive DNAs in Two Gryllidae Crickets with Highly Divergent Karyotypes

PubMed Central

Palacios-Gimenez, Octavio M.; Carvalho, Carlos Roberto; Ferrari Soares, Fernanda Aparecida; Cabral-de-Mello, Diogo C.

2015-01-01

A large percentage of eukaryotic genomes consist of repetitive DNA that plays an important role in the organization, size and evolution. In the case of crickets, chromosomal variability has been found using classical cytogenetics, but almost no information concerning the organization of their repetitive DNAs is available. To better understand the chromosomal organization and diversification of repetitive DNAs in crickets, we studied the chromosomes of two Gryllidae species with highly divergent karyotypes, i.e., 2n(♂) = 29,X0 (Gryllus assimilis) and 2n = 9, neo-X1X2Y (Eneoptera surinamensis). The analyses were performed using classical cytogenetic techniques, repetitive DNA mapping and genome-size estimation. Conserved characteristics were observed, such as the occurrence of a small number of clusters of rDNAs and U snDNAs, in contrast to the multiple clusters/dispersal of the H3 histone genes. The positions of U2 snDNA and 18S rDNA are also conserved, being intermingled within the largest autosome. The distribution and base-pair composition of the heterochromatin and repetitive DNA pools of these organisms differed, suggesting reorganization. Although the microsatellite arrays had a similar distribution pattern, being dispersed along entire chromosomes, as has been observed in some grasshopper species, a band-like pattern was also observed in the E. surinamensis chromosomes, putatively due to their amplification and clustering. In addition to these differences, the genome of E. surinamensis is approximately 2.5 times larger than that of G. assimilis, which we hypothesize is due to the amplification of repetitive DNAs. Finally, we discuss the possible involvement of repetitive DNAs in the differentiation of the neo-sex chromosomes of E. surinamensis, as has been reported in other eukaryotic groups. This study provided an opportunity to explore the evolutionary dynamics of repetitive DNAs in two non-model species and will contribute to the understanding of chromosomal evolution in a group about which little chromosomal and genomic information is known. PMID:26630487

Contrasting the Chromosomal Organization of Repetitive DNAs in Two Gryllidae Crickets with Highly Divergent Karyotypes.

PubMed

Palacios-Gimenez, Octavio M; Carvalho, Carlos Roberto; Ferrari Soares, Fernanda Aparecida; Cabral-de-Mello, Diogo C

2015-01-01

A large percentage of eukaryotic genomes consist of repetitive DNA that plays an important role in the organization, size and evolution. In the case of crickets, chromosomal variability has been found using classical cytogenetics, but almost no information concerning the organization of their repetitive DNAs is available. To better understand the chromosomal organization and diversification of repetitive DNAs in crickets, we studied the chromosomes of two Gryllidae species with highly divergent karyotypes, i.e., 2n(♂) = 29,X0 (Gryllus assimilis) and 2n = 9, neo-X1X2Y (Eneoptera surinamensis). The analyses were performed using classical cytogenetic techniques, repetitive DNA mapping and genome-size estimation. Conserved characteristics were observed, such as the occurrence of a small number of clusters of rDNAs and U snDNAs, in contrast to the multiple clusters/dispersal of the H3 histone genes. The positions of U2 snDNA and 18S rDNA are also conserved, being intermingled within the largest autosome. The distribution and base-pair composition of the heterochromatin and repetitive DNA pools of these organisms differed, suggesting reorganization. Although the microsatellite arrays had a similar distribution pattern, being dispersed along entire chromosomes, as has been observed in some grasshopper species, a band-like pattern was also observed in the E. surinamensis chromosomes, putatively due to their amplification and clustering. In addition to these differences, the genome of E. surinamensis is approximately 2.5 times larger than that of G. assimilis, which we hypothesize is due to the amplification of repetitive DNAs. Finally, we discuss the possible involvement of repetitive DNAs in the differentiation of the neo-sex chromosomes of E. surinamensis, as has been reported in other eukaryotic groups. This study provided an opportunity to explore the evolutionary dynamics of repetitive DNAs in two non-model species and will contribute to the understanding of chromosomal evolution in a group about which little chromosomal and genomic information is known.

Reversible conformational switching of i-motif DNA studied by fluorescence spectroscopy.

PubMed

Choi, Jungkweon; Majima, Tetsuro

2013-01-01

Non-B DNAs, which can form unique structures other than double helix of B-DNA, have attracted considerable attention from scientists in various fields including biology, chemistry and physics etc. Among them, i-motif DNA, which is formed from cytosine (C)-rich sequences found in telomeric DNA and the promoter region of oncogenes, has been extensively investigated as a signpost and controller for the oncogene expression at the transcription level and as a promising material in nanotechnology. Fluorescence techniques such as fluorescence resonance energy transfer (FRET) and the fluorescence quenching are important for studying DNA and in particular for the visualization of reversible conformational switching of i-motif DNA that is triggered by the protonation. Here, we review the latest studies on the conformational dynamics of i-motif DNA as well as the application of FRET and fluorescence quenching techniques to the visualization of reversible conformational switching of i-motif DNA in nano-biotechnology. © 2013 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2013 The American Society of Photobiology.

Fluorescence studies with DNA probes: dynamic aspects of DNA structure and DNA-protein interactions

NASA Astrophysics Data System (ADS)

Millar, David P.; Carver, Theodore E.

1994-08-01

Time-resolved fluorescence measurements of optical probes incorporated at specific sites in DNA provides a new approach to studies of DNA structure and DNA:protein interactions. This approach can be used to study complex multi-state behavior, such as the folding of DNA into alternative higher order structures or the transfer of DNA between multiple binding sites on a protein. In this study, fluorescence anisotropy decay of an internal dansyl probe attached to 17/27-mer oligonucleotides was used to monitor the distribution of DNA 3' termini bound at either the polymerase of 3' to 5' exonuclease sites of the Klenow fragment of DNA polymerase I. Partitioning of the primer terminus between the two active sites of the enzyme resulted in a heterogeneous probe environment, reflected in the associative behavior of the fluorescence anisotropy decay. Analysis of the anisotropy decay with a two state model of solvent-exposed and protein-associated dansyl probes was used to determine the fraction of DNA bound at each site. We examined complexes of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus caused a 3-fold increase in the equilibrium partitioning of DNA into the exonuclease site, while two or more consecutive G:G mismatches caused the DNA to bind exclusively at the exonuclease site, with a partitioning constant at least 250- fold greater than that of the corresponding matched DNA sequence. Internal single mismatches located up to four bases from the primer terminus produced larger effects than the same mismatch at the primer terminus. These results provide insight into the recognition mechanisms that enable DNA polymerases to proofread misincorporated bases during DNA replication.

Caught in the act: the lifetime of synaptic intermediates during the search for homology on DNA

PubMed Central

Mani, Adam; Braslavsky, Ido; Arbel-Goren, Rinat; Stavans, Joel

2010-01-01

Homologous recombination plays pivotal roles in DNA repair and in the generation of genetic diversity. To locate homologous target sequences at which strand exchange can occur within a timescale that a cell’s biology demands, a single-stranded DNA-recombinase complex must search among a large number of sequences on a genome by forming synapses with chromosomal segments of DNA. A key element in the search is the time it takes for the two sequences of DNA to be compared, i.e. the synapse lifetime. Here, we visualize for the first time fluorescently tagged individual synapses formed by RecA, a prokaryotic recombinase, and measure their lifetime as a function of synapse length and differences in sequence between the participating DNAs. Surprisingly, lifetimes can be ∼10 s long when the DNAs are fully heterologous, and much longer for partial homology, consistently with ensemble FRET measurements. Synapse lifetime increases rapidly as the length of a region of full homology at either the 3′- or 5′-ends of the invading single-stranded DNA increases above 30 bases. A few mismatches can reduce dramatically the lifetime of synapses formed with nearly homologous DNAs. These results suggest the need for facilitated homology search mechanisms to locate homology successfully within the timescales observed in vivo. PMID:20044347

High-throughput analysis of the satellitome illuminates satellite DNA evolution

NASA Astrophysics Data System (ADS)

Ruiz-Ruano, Francisco J.; López-León, María Dolores; Cabrero, Josefa; Camacho, Juan Pedro M.

2016-07-01

Satellite DNA (satDNA) is a major component yet the great unknown of eukaryote genomes and clearly underrepresented in genome sequencing projects. Here we show the high-throughput analysis of satellite DNA content in the migratory locust by means of the bioinformatic analysis of Illumina reads with the RepeatExplorer and RepeatMasker programs. This unveiled 62 satDNA families and we propose the term “satellitome” for the whole collection of different satDNA families in a genome. The finding that satDNAs were present in many contigs of the migratory locust draft genome indicates that they show many genomic locations invisible by fluorescent in situ hybridization (FISH). The cytological pattern of five satellites showing common descent (belonging to the SF3 superfamily) suggests that non-clustered satDNAs can become into clustered through local amplification at any of the many genomic loci resulting from previous dissemination of short satDNA arrays. The fact that all kinds of satDNA (micro- mini- and satellites) can show the non-clustered and clustered states suggests that all these elements are mostly similar, except for repeat length. Finally, the presence of VNTRs in bacteria, showing similar properties to non-clustered satDNAs in eukaryotes, suggests that this kind of tandem repeats show common properties in all living beings.

Pyrrolo-dC modified duplex DNA as a novel probe for the sensitive assay of base excision repair enzyme activity.

PubMed

Lee, Chang Yeol; Park, Ki Soo; Park, Hyun Gyu

2017-12-15

We develop a novel approach to determine formamidopyrimidine DNA glycosylase (Fpg) activity by taking advantage of the unique fluorescence property of pyrrolo-dC (PdC) positioned opposite to 8-oxoguanine (8-oxoG) in duplex DNA. In its initial state, PdC in duplex DNA undergoes the efficient stacking and collisional quenching interactions, showing the low fluorescence signal. In contrast, the presence of Fpg, which specifically removes 8-oxoG and incises resulting apurinic (AP) site, transforms duplex DNA into single-stranded (ss) DNAs. As a result, the intrinsic fluorescence signal of PdC in ssDNA is recovered to exhibit the significantly enhanced fluorescence signal. Based on this Fpg-dependent fluorescence response of PdC, we could reliably determine Fpg activity down to 1.25U/ml with a linear response from 0 to 50U/ml. In addition, the diagnostic capability of this strategy was successfully demonstrated by reliably assaying Fpg activity in human blood serum, showing its great potential in the practical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Engineered Plants as Biosensors

DTIC Science & Technology

2003-05-28

GFP fluorescence was detectable in the lower leaves and especially in the roots of one transgenic plant compared to negative and positive control...mgfp5-er gene, lane 5 contains cDNA from a 35s-mgfp5-er transgenic plant , lanes 6-10 contain cDNAs from gn1/gfp plants. RNA extraction was performed 7...contains transgenic plant sprayed with water (negative control). Lanes 5-12 are independent gn1/gfp transgenic events sprayed with 5 mM BTH. Lanes

Two Successive Reactions on a DNA Template: A Strategy for Improving Background and Specificity in Nucleic Acid Detection

PubMed Central

Franzini, Raphael M.

2015-01-01

We report a new strategy for template-mediated fluorogenic chemistry that results in enhanced performance for the fluorescence detection of nucleic acids. In this approach, two successive templated reactions are required to induce a fluorescence signal, rather than only one. These novel fluorescein-labeled oligonucleotide probes, termed 2-STAR probes, contain two quencher groups tethered by separate reductively cleavable linkers. When a 2-STAR quenched probe binds adjacent to either two successive mono triphenyl-phosphine (TPP)-DNAs or a dual TPP-DNA, the two quenchers are released, resulting in a fluorescence signal. Because of the requirement for two consecutive reactions, 2-STAR probes display an unprecedented level of sequence-specificity for template-mediated probe designs. At the same time, background emission generated by off-template reactions or incomplete quenching is among the lowest of any fluorogenic reactive probes for the detection of DNA or RNA. PMID:21294182

An aptamer-based bio-barcode assay with isothermal recombinase polymerase amplification for cytochrome-c detection and anti-cancer drug screening.

PubMed

Loo, Jacky F C; Lau, P M; Ho, H P; Kong, S K

2013-10-15

Based on a recently reported ultra-sensitive bio-barcode (BBC) assay, we have developed an aptamer-based bio-barcode (ABC) alternative to detect a cell death marker cytochrome-c (Cyto-c) and its subsequent application to screen anti-cancer drugs. Aptamer is a short single-stranded DNA selected from a synthetic DNA library by virtue of its high binding affinity and specificity to its target based on its unique 3D structure from the nucleotide sequence after folding. In the BBC assay, an antigen (Ag) in analytes is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Abs). Gold nanoparticles (NPs) with another recognition Ab against the same target and hundreds of identical DNA molecules of known sequence are subsequently added to allow the formation of sandwich structures ([MMP-Ab1]-Ag-[Ab2-NP-DNA]). After isolating the sandwiches by a magnetic field, the DNAs hybridized to their complementary DNAs covalently bound on the NPs are released from the sandwiches after heating. Acting as an Ag identification tag, these bio-barcode DNAs with known DNA sequence are then amplified by polymerase chain reaction (PCR) and detected by fluorescence. In our ABC assay, we employed a Cyto-c-specific aptamer to substitute both the recognition Ab and barcode DNAs on the NPs in the BBC assay; and a novel isothermal recombinase polymerase amplification for the time-consuming PCR. The detection limit of our ABC assay for the Cyto-c was found to be 10 ng/mL and this new assay can be completed within 3h. Several potential anti-cancer drugs have been tested in vitro for their efficacy to kill liver cancer with or without multi-drug resistance. © 2013 Elsevier B.V. All rights reserved.

Picosecond-resolved FRET on non-amplified DNA for identifying individuals genetically susceptible to type-1 diabetes

NASA Astrophysics Data System (ADS)

Nardo, Luca; Tosi, Giovanna; Bondani, Maria; Accolla, Roberto; Andreoni, Alessandra

2012-06-01

By tens-of-picosecond resolved fluorescence detection we study Förster resonance energy transfer between a donor and a black-hole-quencher bound at the 5'- and 3'-positions of an oligonucleotide probe matching the highly polymorphic region between codons 51 and 58 of the human leukocyte antigen DQB1 0201 allele, conferring susceptibility to type-1 diabetes. The probe is annealed with non-amplified genomic DNAs carrying either the 0201 sequence or other DQB1 allelic variants. We detect the longest-lived donor fluorescence in the case of hybridization with the 0201 allele and definitely faster and distinct decays for the other allelic variants, some of which are single-nucleotide polymorphic.

[Identification of genes that are specifically/preferentially expressed in developing cotton fibers by mRNA fluorescence differential display (FDD)].

PubMed

Sun, Jie; Li, Yuan-Li; Wang, Ruo-Hai; Xia, Gui-Xian

2004-01-01

Fluorescence differential display (FDD) technique was used to identify genes that are specifically or preferentially expressed in different developmental stages of cotton fiber cells. One hundred and nine differentially displayed cDNA fragments were isolated using 9, 21 and 27 DPA (days postanthesis) fibers as experimental materials. By a combination of two rounds of reverse Northern hybridization and Northern blot analyses, a number of such cDNA fragments were proved to represent fiber-specific/preferential genes. Sequencing determination and database searching indicated that most of these genes are novel. This work is an important step towards cloning the full-length cDNAs and characterizing the cellular functions of aforementioned genes in fiber development.

Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

PubMed Central

Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L.; Tomkinson, Alan E.; Tainer, John A.; Ellenberger, Tom

2015-01-01

Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation. PMID:26130724

Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining.

PubMed

Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L; Tomkinson, Alan E; Tainer, John A; Ellenberger, Tom

2015-08-18

Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Multiplexed resequencing analysis to identify rare variants in pooled DNA with barcode indexing using next-generation sequencer.

PubMed

Mitsui, Jun; Fukuda, Yoko; Azuma, Kyo; Tozaki, Hirokazu; Ishiura, Hiroyuki; Takahashi, Yuji; Goto, Jun; Tsuji, Shoji

2010-07-01

We have recently found that multiple rare variants of the glucocerebrosidase gene (GBA) confer a robust risk for Parkinson disease, supporting the 'common disease-multiple rare variants' hypothesis. To develop an efficient method of identifying rare variants in a large number of samples, we applied multiplexed resequencing using a next-generation sequencer to identification of rare variants of GBA. Sixteen sets of pooled DNAs from six pooled DNA samples were prepared. Each set of pooled DNAs was subjected to polymerase chain reaction to amplify the target gene (GBA) covering 6.5 kb, pooled into one tube with barcode indexing, and then subjected to extensive sequence analysis using the SOLiD System. Individual samples were also subjected to direct nucleotide sequence analysis. With the optimization of data processing, we were able to extract all the variants from 96 samples with acceptable rates of false-positive single-nucleotide variants.

Hierarchical assembly of viral nanotemplates with encoded microparticles via nucleic acid hybridization.

PubMed

Tan, Wui Siew; Lewis, Christina L; Horelik, Nicholas E; Pregibon, Daniel C; Doyle, Patrick S; Yi, Hyunmin

2008-11-04

We demonstrate hierarchical assembly of tobacco mosaic virus (TMV)-based nanotemplates with hydrogel-based encoded microparticles via nucleic acid hybridization. TMV nanotemplates possess a highly defined structure and a genetically engineered high density thiol functionality. The encoded microparticles are produced in a high throughput microfluidic device via stop-flow lithography (SFL) and consist of spatially discrete regions containing encoded identity information, an internal control, and capture DNAs. For the hybridization-based assembly, partially disassembled TMVs were programmed with linker DNAs that contain sequences complementary to both the virus 5' end and a selected capture DNA. Fluorescence microscopy, atomic force microscopy (AFM), and confocal microscopy results clearly indicate facile assembly of TMV nanotemplates onto microparticles with high spatial and sequence selectivity. We anticipate that our hybridization-based assembly strategy could be employed to create multifunctional viral-synthetic hybrid materials in a rapid and high-throughput manner. Additionally, we believe that these viral-synthetic hybrid microparticles may find broad applications in high capacity, multiplexed target sensing.

High throughput protein production screening

DOEpatents

Beernink, Peter T [Walnut Creek, CA; Coleman, Matthew A [Oakland, CA; Segelke, Brent W [San Ramon, CA

2009-09-08

Methods, compositions, and kits for the cell-free production and analysis of proteins are provided. The invention allows for the production of proteins from prokaryotic sequences or eukaryotic sequences, including human cDNAs using PCR and IVT methods and detecting the proteins through fluorescence or immunoblot techniques. This invention can be used to identify optimized PCR and WT conditions, codon usages and mutations. The methods are readily automated and can be used for high throughput analysis of protein expression levels, interactions, and functional states.

Inkjet Gene Printing: A Novel Approach to Achieve Gene Modified Cells for Tissue Engineering

DTIC Science & Technology

2008-12-01

and pIRES-VEGF-GFP (BD Biosciences, Bedford, MA) encoding the cDNAs of jellyfish Aequorea victoria green fluorescent protein, driven by the...prepared from rat-tail Type I collagen gels using a previously reported protocol(Xu et al. 2005). Briefly, rat- tail Type I collagen (BD Biosciences...aliquots of the mixture were dispersed onto coverslips and cured in an incubator for 3–5 h. Once the gel set, the collagen bio-paper was ready for

Large-scale identification and characterization of alternative splicing variants of human gene transcripts using 56 419 completely sequenced and manually annotated full-length cDNAs

PubMed Central

Takeda, Jun-ichi; Suzuki, Yutaka; Nakao, Mitsuteru; Barrero, Roberto A.; Koyanagi, Kanako O.; Jin, Lihua; Motono, Chie; Hata, Hiroko; Isogai, Takao; Nagai, Keiichi; Otsuki, Tetsuji; Kuryshev, Vladimir; Shionyu, Masafumi; Yura, Kei; Go, Mitiko; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Wiemann, Stefan; Nomura, Nobuo; Sugano, Sumio; Gojobori, Takashi; Imanishi, Tadashi

2006-01-01

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56 419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37 670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants. PMID:16914452

Cloning of human cDNAs for Apg-1 and Apg-2, members of the Hsp110 family, and chromosomal assignment of their genes.

PubMed

Nonoguchi, K; Itoh, K; Xue, J H; Tokuchi, H; Nishiyama, H; Kaneko, Y; Tatsumi, K; Okuno, H; Tomiwa, K; Fujita, J

1999-09-03

In mice, the Hsp110/SSE family is composed of the heat shock protein (Hsp)110/105, Apg-1 and Apg-2. In humans, however, only the Hsp110/105 homolog has been identified as a member, and two cDNAs, Hsp70RY and HS24/p52, potentially encoding proteins structurally similar to, but smaller than, mouse Apg-2 have been reported. To clarify the membership of Hsp110 family in humans, we isolated Apg-1 and Apg-2 cDNAs from a human testis cDNA library. The human Apg-1 was 100% and 91.8% identical in length and amino acid (aa) sequence, respectively, to mouse Apg-1. Human Apg-2 was one aa shorter than and 95.5% identical in sequence to mouse Apg-2. In ECV304, human endothelial cells Apg-1 but not Apg-2 transcripts were induced in 2 h by a temperature shift from 32 degrees C to 39 degrees C. As found in mice, the response was stronger than that to a 37-42 degrees C shift. The human Apg-1 and Apg-2 genes were mapped to the chromosomal loci 4q28 and 5q23.3-q31.1, respectively, by fluorescence in-situ hybridization. We isolated cDNA and genomic clones encompassing the region critical for the difference between Apg-2 and HS24/p52. Although the primer sets used were derived from the sequences common to both cDNAs, all cDNA and genomic clones corresponded to Apg-2. Using a similar approach, the relationship between Apg-2 and Hsp70RY was assessed, and no clone corresponding to Hsp70RY was obtained. These results demonstrated that the Hsp110 family consists of at least three members, Apg-1, Apg-2 and Hsp110 in humans as well as in mice. The significance of HS24/p52 and Hsp70RY cDNAs previously reported remains to be determined.

Functional DNA-Containing Nanomaterials: Cellular Applications in Biosensing, Imaging, and Targeted Therapy

PubMed Central

2015-01-01

Conspectus DNA performs a vital function as a carrier of genetic code, but in the field of nanotechnology, DNA molecules can catalyze chemical reactions in the cell, that is, DNAzymes, or bind with target-specific ligands, that is, aptamers. These functional DNAs with different modifications have been developed for sensing, imaging, and therapeutic systems. Thus, functional DNAs hold great promise for future applications in nanotechnology and bioanalysis. However, these functional DNAs face challenges, especially in the field of biomedicine. For example, functional DNAs typically require the use of cationic transfection reagents to realize cellular uptake. Such reagents enter the cells, increasing the difficulty of performing bioassays in vivo and potentially damaging the cell’s nucleus. To address this obstacle, nanomaterials, such as metallic, carbon, silica, or magnetic materials, have been utilized as DNA carriers or assistants. In this Account, we describe selected examples of functional DNA-containing nanomaterials and their applications from our recent research and those of others. As models, we have chosen to highlight DNA/nanomaterial complexes consisting of gold nanoparticles, graphene oxides, and aptamer–micelles, and we illustrate the potential of such complexes in biosensing, imaging, and medical diagnostics. Under proper conditions, multiple ligand–receptor interactions, decreased steric hindrance, and increased surface roughness can be achieved from a high density of DNA that is bound to the surface of nanomaterials, resulting in a higher affinity for complementary DNA and other targets. In addition, this high density of DNA causes a high local salt concentration and negative charge density, which can prevent DNA degradation. For example, DNAzymes assembled on gold nanoparticles can effectively catalyze chemical reactions even in living cells. And it has been confirmed that DNA–nanomaterial complexes can enter cells more easily than free single-stranded DNA. Nanomaterials can be designed and synthesized in needed sizes and shapes, and they possess unique chemical and physical properties, which make them useful as DNA carriers or assistants, excellent signal reporters, transducers, and amplifiers. When nanomaterials are combined with functional DNAs to create novel assay platforms, highly sensitive biosensing and high-resolution imaging result. For example, gold nanoparticles and graphene oxides can quench fluorescence efficiently to achieve low background and effectively increase the signal-to-background ratio. Meanwhile, gold nanoparticles themselves can be colorimetric reporters because of their different optical absorptions between monodispersion and aggregation. DNA self-assembled nanomaterials contain several properties of both DNA and nanomaterials. Compared with DNA–nanomaterial complexes, DNA self-assembled nanomaterials more closely resemble living beings, and therefore they have lower cytotoxicity at high concentrations. Functional DNA self-assemblies also have high density of DNA for multivalent reaction and three-dimensional nanostructures for cell uptake. Now and in the future, we envision the use of DNA bases in making designer molecules for many challenging applications confronting chemists. With the further development of artificial DNA bases using smart organic synthesis, DNA macromolecules based on elegant molecular assembly approaches are expected to achieve great diversity, additional versatility, and advanced functions. PMID:24780000

Functional DNA-containing nanomaterials: cellular applications in biosensing, imaging, and targeted therapy.

PubMed

Liang, Hao; Zhang, Xiao-Bing; Lv, Yifan; Gong, Liang; Wang, Ruowen; Zhu, Xiaoyan; Yang, Ronghua; Tan, Weihong

2014-06-17

CONSPECTUS: DNA performs a vital function as a carrier of genetic code, but in the field of nanotechnology, DNA molecules can catalyze chemical reactions in the cell, that is, DNAzymes, or bind with target-specific ligands, that is, aptamers. These functional DNAs with different modifications have been developed for sensing, imaging, and therapeutic systems. Thus, functional DNAs hold great promise for future applications in nanotechnology and bioanalysis. However, these functional DNAs face challenges, especially in the field of biomedicine. For example, functional DNAs typically require the use of cationic transfection reagents to realize cellular uptake. Such reagents enter the cells, increasing the difficulty of performing bioassays in vivo and potentially damaging the cell's nucleus. To address this obstacle, nanomaterials, such as metallic, carbon, silica, or magnetic materials, have been utilized as DNA carriers or assistants. In this Account, we describe selected examples of functional DNA-containing nanomaterials and their applications from our recent research and those of others. As models, we have chosen to highlight DNA/nanomaterial complexes consisting of gold nanoparticles, graphene oxides, and aptamer-micelles, and we illustrate the potential of such complexes in biosensing, imaging, and medical diagnostics. Under proper conditions, multiple ligand-receptor interactions, decreased steric hindrance, and increased surface roughness can be achieved from a high density of DNA that is bound to the surface of nanomaterials, resulting in a higher affinity for complementary DNA and other targets. In addition, this high density of DNA causes a high local salt concentration and negative charge density, which can prevent DNA degradation. For example, DNAzymes assembled on gold nanoparticles can effectively catalyze chemical reactions even in living cells. And it has been confirmed that DNA-nanomaterial complexes can enter cells more easily than free single-stranded DNA. Nanomaterials can be designed and synthesized in needed sizes and shapes, and they possess unique chemical and physical properties, which make them useful as DNA carriers or assistants, excellent signal reporters, transducers, and amplifiers. When nanomaterials are combined with functional DNAs to create novel assay platforms, highly sensitive biosensing and high-resolution imaging result. For example, gold nanoparticles and graphene oxides can quench fluorescence efficiently to achieve low background and effectively increase the signal-to-background ratio. Meanwhile, gold nanoparticles themselves can be colorimetric reporters because of their different optical absorptions between monodispersion and aggregation. DNA self-assembled nanomaterials contain several properties of both DNA and nanomaterials. Compared with DNA-nanomaterial complexes, DNA self-assembled nanomaterials more closely resemble living beings, and therefore they have lower cytotoxicity at high concentrations. Functional DNA self-assemblies also have high density of DNA for multivalent reaction and three-dimensional nanostructures for cell uptake. Now and in the future, we envision the use of DNA bases in making designer molecules for many challenging applications confronting chemists. With the further development of artificial DNA bases using smart organic synthesis, DNA macromolecules based on elegant molecular assembly approaches are expected to achieve great diversity, additional versatility, and advanced functions.

Color transitions in coral's fluorescent proteins by site-directed mutagenesis

PubMed Central

Gurskaya, Nadya G; Savitsky, Alexander P; Yanushevich, Yurii G; Lukyanov, Sergey A; Lukyanov, Konstantin A

2001-01-01

Background Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (~ 485 nm), green (~ 505 nm), yellow (~ 540 nm), and red (>580 nm) emitters. Results Here we applied site-directed mutagenesis in order to investigate the structural background of color variety and possibility of shifting between different types of fluorescence. First, a blue-shifted mutant of cyan amFP486 was generated. Second, it was established that cyan and green emitters can be modified so as to produce an intermediate spectrum of fluorescence. Third, the relationship between green and yellow fluorescence was inspected on closely homologous green zFP506 and yellow zFP538 proteins. The following transitions of colors were performed: yellow to green; yellow to dual color (green and yellow); and green to yellow. Fourth, we generated a mutant of cyan emitter dsFP483 that demonstrated dual color (cyan and red) fluorescence. Conclusions Several amino acid substitutions were found to strongly affect fluorescence maxima. Some positions primarily found by sequence comparison were proved to be crucial for fluorescence of particular color. These results are the first step towards predicting the color of natural GFP-like proteins corresponding to newly identified cDNAs from corals. PMID:11459517

Olfactory Proteins Mediating Chemical Communication in the Navel Orangeworm Moth, Amyelois transitella

PubMed Central

Leal, Walter S.; Ishida, Yuko; Pelletier, Julien; Xu, Wei; Rayo, Josep; Xu, Xianzhong; Ames, James B.

2009-01-01

Background The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control — like pheromone-based approaches — are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins. Methodology By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components. Conclusion We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH. PMID:19789654

A light-up probe targeting for Bcl-2 2345 G-quadruplex DNA with carbazole TO

NASA Astrophysics Data System (ADS)

Gu, Yingchun; Lin, Dayong; Tang, Yalin; Fei, Xuening; Wang, Cuihong; Zhang, Baolian; Zhou, Jianguo

2018-02-01

As its significant role, the selective recognition of G-quadruplex with specific structures and functions is important in biological and medicinal chemistry. Carbazole derivatives have been reported as a kind of fluorescent probe with many excellent optical properties. In the present study, the fluorescence of the dye (carbazole TO) increased almost 70 fold in the presence of bcl-2 2345 G4 compared to that alone in aqueous buffer condition with almost no fluorescence and 10-30 fold than those in the presence of other DNAs. The binding study results by activity inhibition of G4/Hemin peroxidase experiment, NMR titration and molecular docking simulation showed the high affinity and selectivity to bcl-2 2345 G4 arises from its end-stacking interaction with G-quartet. It is said that a facile approach with excellent sensitive, good selectivity and quick response for bcl-2 2345 G-quadruplex was developed and may be used for antitumor recognition or antitumor agents.

[Development of a mouse cell line containing stably integrated copies of pMCLacI/Neo plasmid: a model for studying mutations in vitro].

PubMed

Lu, Y; Li, H; Fu, J

2000-04-01

To establish a suitable model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells. The NIH3T3 cells were transfected with the linearized pMCLacI/Neo DNAs by liposome-mediated transfection, and grew in the presence of G418. One drug resistant cell clone was selected to proliferate and to be analyzed with Southern blot and RT-PCR analyses on its genomic DNAs. (1) Multiple copies of pMCLacI/Neo plasmid DNA were intactly integrated in the genomic DNAs of the cell clone. (2) One of lac I target genes in the integrated plasmid could be transcribed in the NIH3T3 cells while the other could not. (3) The pMCLacI/Neo plasmid DNA could be efficiently rescued from the genomic DNAs of the cell clone with the average rescue efficiency of 410 cfu/microg DNA. The NIH3T3 cell line containing copies of a stably integrated pMCLacI/Neo has been established. The two lacI target genes in the cell line could imitate the functional states of expressed and non-expressed genes in mammalian cells respectively. The cell line will be a useful model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells.

Fluorescence of size-expanded DNA bases: reporting on DNA sequence and structure with an unnatural genetic set.

PubMed

Krueger, Andrew T; Kool, Eric T

2008-03-26

We recently described the synthesis and helix assembly properties of expanded DNA (xDNA), which contains base pairs 2.4 A larger than natural DNA pairs. This designed genetic set is under study with the goals of mimicking the functions of the natural DNA-based genetic system and of developing useful research tools. Here, we study the fluorescence properties of the four expanded bases of xDNA (xA, xC, xG, xT) and evaluate how their emission varies with changes in oligomer length, composition, and hybridization. Experiments were carried out with short oligomers of xDNA nucleosides conjugated to a DNA oligonucleotide, and we investigated the effects of hybridizing these fluorescent oligomers to short complementary DNAs with varied bases opposite the xDNA bases. As monomer nucleosides, the xDNA bases absorb light in two bands: one at approximately 260 nm (similar to DNA) and one at longer wavelength ( approximately 330 nm). All are efficient violet-blue fluorophores with emission maxima at approximately 380-410 nm and quantum yields (Phifl) of 0.30-0.52. Short homo-oligomers of the xDNA bases (length 1-4 monomers) showed moderate self-quenching except xC, which showed enhancement of Phifl with increasing length. Interestingly, multimers of xA emitted at longer wavelengths (520 nm) as an apparent excimer. Hybridization of an oligonucleotide to the DNA adjacent to the xDNA bases (with the xDNA portion overhanging) resulted in no change in fluorescence. However, addition of one, two, or more DNA bases in these duplexes opposite the xDNA portion resulted in a number of significant fluorescence responses, including wavelength shifts, enhancements, or quenching. The strongest responses were the enhancement of (xG)n emission by hybridization of one or more adenines opposite them, and the quenching of (xT)n and (xC)n emission by guanines opposite. The data suggest multiple ways in which the xDNA bases, both alone and in oligomers, may be useful as tools in biophysical analysis and biotechnological applications.

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss.

PubMed

Weld, R; Heinemann, J; Eady, C

2001-03-01

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.

Identification of Prostate Cancer-Specific microDNAs

DTIC Science & Technology

2014-12-01

displacement amplification (MDA). 2 adopted multiple displacement amplification (MDA) with random primers for enriched circular DNA by rolling circle ... amplification (RCA) (Fig. 1) and then amplified DNA fragments were subject to deep sequencing. Sequence NO of Reads seq 1 184 seq 2 133 seq 3 2407 seq...prostate cancer cells through multiple displacement amplification .  Clone #7 is the top candidate which has been cloned in an expression vector and it

Molecular cloning of peroxidase cDNAs from dehydration-treated fibrous roots of sweetpotato and their differential expression in response to stress.

PubMed

Kim, Yun-Hee; Yang, Kyoung-Sil; Kim, Cha Young; Ryu, Sun-Hwa; Song, Wan-Keun; Kwon, Suk-Yoon; Lee, Haeng-Soon; Bang, Jae-Wook; Kwak, Sang-Soo

2008-03-31

Three peroxidase (POD) cDNAs were isolated from dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas) plant via the screening of a cDNA library, and their expressions were assessed to characterize functions of each POD in relation to environmental stress. Three PODs were divided into two groups, designated the basic PODs (swpb4, swpb5) and the anionic PODs (swpa7), on the basis of the pI values of mature proteins. Fluorescence microscope analysis indicated that three PODs are secreted into the extracellular space. RTPCR analysis revealed that POD genes have diverse expression patterns in a variety of plant tissues. Swpb4 was abundantly expressed in stem tissues, whereas the expression levels of swpb5 and swpa7 transcripts were high in fibrous and thick pigmented roots. Swpb4 and swpa7 showed abundant expression levels in suspension cultured cells. Three POD genes responded differently in the leaf and fibrous roots in response to a variety of stresses including dehydration, temperature stress, stress-associated chemicals, and pathogenic bacteria.

A convenient method of attaching fluorescent dyes on single-walled carbon nanotubes pre-wrapped with DNA molecules.

PubMed

Tomura, Akihiro; Umemura, Kazuo

2018-04-15

We demonstrated the attachment of different kinds of dyes, Uranine, Rhodamime 800 (R800), and Indocyanine green (ICG), to single-walled carbon nanotubes pre-wrapped with single-stranded DNAs (ssDNA-SWCNTs). A new but simple method was employed, in which a dye solution was added to ssDNA-SWCNTs that had been prepared beforehand in the conventional way. Resulting conjugates of dyes, DNA, and SWCNTs were precisely evaluated by ultraviolet to near-infrared fluorescence/absorbance spectrometry and atomic force microscopy. In particular, simultaneous measurements of fluorescence and absorbance spectroscopy enabled us to find differences in the behaviors of the dyes on SWCNT surfaces. As a result, the fluorescence/absorbance spectra of dyes showed significant changes upon adsorption on SWCNTs. The fluorescence/absorbance peaks of Uranine, R800, and ICG were quenched by 41.3/2.8%, 72.3/48.9%, and 88.3/45.0%, respectively, in the presence of 11.5 μg/mL SWCNTs. We concluded firstly that by pre-wrapping SWCNTs with ssDNA, stable hybrids with these components were obtained even if the dyes used were relatively hydrophobic and secondly that Uranine retained light absorption on the surface of SWCNT while R800 and ICG did not. Copyright © 2018 Elsevier Inc. All rights reserved.

Mapping neurofibromatosis 1 homologous loci by fluorescence in situ hybridization

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

Viskochil, D.; Breidenbach, H.H.; Cawthon, R.

Neurofibromatosis 1 maps to chromosome band 17q11.2 and the NF1 gene is comprised of 59 exons that span approximately 335 kb of genomic DNA. In order to further analyze the structure of NF1 from exons 2 through 27b, we isolated a number of cosmid and bacteriophage P-1 genomic clones using NF1-exon probes under high-stringency hybridization conditions. Using tagged, intron-based primers and DNA from various clones as a template, we PCR-amplified and sequenced individual NF1 exons. The exon sequences in PCR products from several genomic clones differed from the exon sequence derived from cloned NF1 cDNAs. Clones with variant sequences weremore » mapped by fluorescence in situ hybridization under high-stringency conditions. Three clones mapped to chromosome band 15q11.2, one mapped to 14q11.2, one mapped to both 2q14.1-14.3 and 14q11.2, one mapped to 2q33-34, and one mapped to both 18q11.2 and 21q21. Even though some PCR-product sequences retained proper splice junctions and open reading frames, we have yet to identify cDNAs that correspond to the variant exon sequences. We are now sequencing clones that map to NF1-homologous loci in order to develop discriminating primer pairs for the exclusive amplification of NF1-specific sequences in our efforts to develop a comprehensive NF1 mutation screen using genomic DNA as template. The role of NF1-homologous sequences may play in neurofibromatosis 1 is not clear.« less

Label-Free Sensitive Detection of DNA Methyltransferase by Target-Induced Hyperbranched Amplification with Zero Background Signal.

PubMed

Zhang, Yan; Wang, Xin-Yan; Zhang, Qianyi; Zhang, Chun-Yang

2017-11-21

DNA methyltransferases (MTases) may specifically recognize the short palindromic sequences and transfer a methyl group from S-adenosyl-l-methionine to target cytosine/adenine. The aberrant DNA methylation is linked to the abnormal DNA MTase activity, and some DNA MTases have become promising targets of anticancer/antimicrobial drugs. However, the reported DNA MTase assays often involve laborious operation, expensive instruments, and radio-labeled substrates. Here, we develop a simple and label-free fluorescent method to sensitively detect DNA adenine methyltransferase (Dam) on the basis of terminal deoxynucleotidyl transferase (TdT)-activated Endonuclease IV (Endo IV)-assisted hyperbranched amplification. We design a hairpin probe with a palindromic sequence in the stem as the substrate and a NH 2 -modified 3' end for the prevention of nonspecific amplification. The substrate may be methylated by Dam and subsequently cleaved by DpnI, producing three single-stranded DNAs, two of which with 3'-OH termini may be amplified by hyperbranched amplification to generate a distinct fluorescence signal. Because high exactitude of TdT enables the amplification only in the presence of free 3'-OH termini and Endo IV only hydrolyzes the intact apurinic/apyrimidinic sites in double-stranded DNAs, zero background signal can be achieved. This method exhibits excellent selectivity and high sensitivity with a limit of detection of 0.003 U/mL for pure Dam and 9.61 × 10 -6 mg/mL for Dam in E. coli cells. Moreover, it can be used to screen the Dam inhibitors, holding great potentials in disease diagnosis and drug development.

The chloroplast genome sequence of the green alga Leptosira terrestris: multiple losses of the inverted repeat and extensive genome rearrangements within the Trebouxiophyceae

PubMed Central

de Cambiaire, Jean-Charles; Otis, Christian; Turmel, Monique; Lemieux, Claude

2007-01-01

Background In the Chlorophyta – the green algal phylum comprising the classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae and Chlorophyceae – the chloroplast genome displays a highly variable architecture. While chlorophycean chloroplast DNAs (cpDNAs) deviate considerably from the ancestral pattern described for the prasinophyte Nephroselmis olivacea, the degree of remodelling sustained by the two ulvophyte cpDNAs completely sequenced to date is intermediate relative to those observed for chlorophycean and trebouxiophyte cpDNAs. Chlorella vulgaris (Chlorellales) is currently the only photosynthetic trebouxiophyte whose complete cpDNA sequence has been reported. To gain insights into the evolutionary trends of the chloroplast genome in the Trebouxiophyceae, we sequenced cpDNA from the filamentous alga Leptosira terrestris (Ctenocladales). Results The 195,081-bp Leptosira chloroplast genome resembles the 150,613-bp Chlorella genome in lacking a large inverted repeat (IR) but differs greatly in gene order. Six of the conserved genes present in Chlorella cpDNA are missing from the Leptosira gene repertoire. The 106 conserved genes, four introns and 11 free standing open reading frames (ORFs) account for 48.3% of the genome sequence. This is the lowest gene density yet observed among chlorophyte cpDNAs. Contrary to the situation in Chlorella but similar to that in the chlorophycean Scenedesmus obliquus, the gene distribution is highly biased over the two DNA strands in Leptosira. Nine genes, compared to only three in Chlorella, have significantly expanded coding regions relative to their homologues in ancestral-type green algal cpDNAs. As observed in chlorophycean genomes, the rpoB gene is fragmented into two ORFs. Short repeats account for 5.1% of the Leptosira genome sequence and are present mainly in intergenic regions. Conclusion Our results highlight the great plasticity of the chloroplast genome in the Trebouxiophyceae and indicate that the IR was lost on at least two separate occasions. The intriguing similarities of the derived features exhibited by Leptosira cpDNA and its chlorophycean counterparts suggest that the same evolutionary forces shaped the IR-lacking chloroplast genomes in these two algal lineages. PMID:17610731

Comparative chromosomal localization of 45S and 5S rDNAs and implications for genome evolution in Cucumis.

PubMed

Zhang, Zhen-Tao; Yang, Shu-Qiong; Li, Zi-Ang; Zhang, Yun-Xia; Wang, Yun-Zhu; Cheng, Chun-Yan; Li, Ji; Chen, Jin-Feng; Lou, Qun-Feng

2016-07-01

Ribosomal DNAs are useful cytogenetic markers for chromosome analysis. Studies investigating site numbers and distributions of rDNAs have provided important information for elucidating genome organization and chromosomal relationships of many species by fluorescence in situ hybridization. But relevant studies are scarce for species of the genus Cucumis, especially in wild species. In the present study, FISH was conducted to investigate the organization of 45S and 5S rDNA among 20 Cucumis accessions, including cultivars and wild accessions. Our results showed that the number of 45S rDNA sites varied from one to five pairs in different accessions, and most of these sites are located at the terminal regions of chromosomes. Interestingly, up to five pairs of 45S rDNA sites were observed in C. sativus var. sativus, the species which has the lowest chromosome number, i.e., 2n = 14. Only one pair of 5S rDNA sites was detected in all accessions, except for C. heptadactylus, C. sp, and C. spp that had two pairs of 5S rDNA sites. The distributions of 5S rDNA sites showed more variation than 45S rDNA sites. The phylogenetic analysis in this study showed that 45S and 5S rDNA have contrasting evolutionary patterns. We find that 5S rDNA has a polyploidization-related tendency towards the terminal location from an interstitial location but maintains a conserved site number, whereas the 45S rDNA showed a trend of increasing site number but a relatively conserved location.

Signal sequence and keyword trap in silico for selection of full-length human cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries.

PubMed

Otsuki, Tetsuji; Ota, Toshio; Nishikawa, Tetsuo; Hayashi, Koji; Suzuki, Yutaka; Yamamoto, Jun-ichi; Wakamatsu, Ai; Kimura, Kouichi; Sakamoto, Katsuhiko; Hatano, Naoto; Kawai, Yuri; Ishii, Shizuko; Saito, Kaoru; Kojima, Shin-ichi; Sugiyama, Tomoyasu; Ono, Tetsuyoshi; Okano, Kazunori; Yoshikawa, Yoko; Aotsuka, Satoshi; Sasaki, Naokazu; Hattori, Atsushi; Okumura, Koji; Nagai, Keiichi; Sugano, Sumio; Isogai, Takao

2005-01-01

We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5'-end single-pass sequences, cDNAs having the signal sequence were selected by PSORT ('signal sequence trap'). We also applied 'secretion or membrane protein-related keyword trap' based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165(80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by 'keyword trap' preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

Soft fruit traceability in food matrices using real-time PCR.

PubMed

Palmieri, Luisa; Bozza, Elisa; Giongo, Lara

2009-02-01

Food product authentication provides a means of monitoring and identifying products for consumer protection and regulatory compliance. There is a scarcity of analytical methods for confirming the identity of fruit pulp in products containing Soft Fruit. In the present work we have developed a very sensible qualitative and quantitative method to determine the presence of berry DNAs in different food matrices. To our knowledge, this is the first study that shows the applicability, to Soft Fruit traceability, of melting curve analysis and multiplexed fluorescent probes, in a Real-Time PCR platform. This methodology aims to protect the consumer from label misrepresentation.

Solvent-modified ultrafast decay dynamics in conjugated polymer/dye labeled single stranded DNA

NASA Astrophysics Data System (ADS)

Kim, Inhong; Kang, Mijeong; Woo, Han Young; Oh, Jin-Woo; Kyhm, Kwangseuk

2015-07-01

We have investigated that organic solvent (DMSO, dimethyl sulfoxide) modifies energy transfer efficiency between conjugated polymers (donors) and fluorescein-labeled single stranded DNAs (acceptors). In a mixture of buffer and organic solvent, fluorescence of the acceptors is significantly enhanced compared to that of pure water solution. This result can be attributed to change of the donor-acceptor environment such as decreased hydrophobicity of polymers, screening effect of organic solvent molecules, resulting in an enhanced energy transfer efficiency. Time-resolved fluorescence decay of the donors and the acceptors was modelled by considering the competition between the energy harvesting Foerster resonance energy transfer and the energy-wasting quenching. This enables to quantity that the Foerster distance (R0 = 43.3 Å) and resonance energy transfer efficiency (EFRET = 58.7 %) of pure buffer solution become R0 = 38.6 Å and EFRET = 48.0 % when 80% DMSO/buffer mixture is added.

Separating homeologs by phasing in the tetraploid wheat transcriptome.

PubMed

Krasileva, Ksenia V; Buffalo, Vince; Bailey, Paul; Pearce, Stephen; Ayling, Sarah; Tabbita, Facundo; Soria, Marcelo; Wang, Shichen; Akhunov, Eduard; Uauy, Cristobal; Dubcovsky, Jorge

2013-06-25

The high level of identity among duplicated homoeologous genomes in tetraploid pasta wheat presents substantial challenges for de novo transcriptome assembly. To solve this problem, we develop a specialized bioinformatics workflow that optimizes transcriptome assembly and separation of merged homoeologs. To evaluate our strategy, we sequence and assemble the transcriptome of one of the diploid ancestors of pasta wheat, and compare both assemblies with a benchmark set of 13,472 full-length, non-redundant bread wheat cDNAs. A total of 489 million 100 bp paired-end reads from tetraploid wheat assemble in 140,118 contigs, including 96% of the benchmark cDNAs. We used a comparative genomics approach to annotate 66,633 open reading frames. The multiple k-mer assembly strategy increases the proportion of cDNAs assembled full-length in a single contig by 22% relative to the best single k-mer size. Homoeologs are separated using a post-assembly pipeline that includes polymorphism identification, phasing of SNPs, read sorting, and re-assembly of phased reads. Using a reference set of genes, we determine that 98.7% of SNPs analyzed are correctly separated by phasing. Our study shows that de novo transcriptome assembly of tetraploid wheat benefit from multiple k-mer assembly strategies more than diploid wheat. Our results also demonstrate that phasing approaches originally designed for heterozygous diploid organisms can be used to separate the close homoeologous genomes of tetraploid wheat. The predicted tetraploid wheat proteome and gene models provide a valuable tool for the wheat research community and for those interested in comparative genomic studies.

Separating homeologs by phasing in the tetraploid wheat transcriptome

PubMed Central

2013-01-01

Background The high level of identity among duplicated homoeologous genomes in tetraploid pasta wheat presents substantial challenges for de novo transcriptome assembly. To solve this problem, we develop a specialized bioinformatics workflow that optimizes transcriptome assembly and separation of merged homoeologs. To evaluate our strategy, we sequence and assemble the transcriptome of one of the diploid ancestors of pasta wheat, and compare both assemblies with a benchmark set of 13,472 full-length, non-redundant bread wheat cDNAs. Results A total of 489 million 100 bp paired-end reads from tetraploid wheat assemble in 140,118 contigs, including 96% of the benchmark cDNAs. We used a comparative genomics approach to annotate 66,633 open reading frames. The multiple k-mer assembly strategy increases the proportion of cDNAs assembled full-length in a single contig by 22% relative to the best single k-mer size. Homoeologs are separated using a post-assembly pipeline that includes polymorphism identification, phasing of SNPs, read sorting, and re-assembly of phased reads. Using a reference set of genes, we determine that 98.7% of SNPs analyzed are correctly separated by phasing. Conclusions Our study shows that de novo transcriptome assembly of tetraploid wheat benefit from multiple k-mer assembly strategies more than diploid wheat. Our results also demonstrate that phasing approaches originally designed for heterozygous diploid organisms can be used to separate the close homoeologous genomes of tetraploid wheat. The predicted tetraploid wheat proteome and gene models provide a valuable tool for the wheat research community and for those interested in comparative genomic studies. PMID:23800085

Transcription of telomeric DNA leads to high levels of homologous recombination and t-loops.

PubMed

Kar, Anirban; Willcox, Smaranda; Griffith, Jack D

2016-11-02

The formation of DNA loops at chromosome ends (t-loops) and the transcription of telomeres producing G-rich RNA (TERRA) represent two central features of telomeres. To explore a possible link between them we employed artificial human telomeres containing long arrays of TTAGGG repeats flanked by the T7 or T3 promoters. Transcription of these DNAs generates a high frequency of t-loops within individual molecules and homologous recombination events between different DNAs at their telomeric sequences. T-loop formation does not require a single strand overhang, arguing that both terminal strands insert into the preceding duplex. The loops are very stable and some RNase H resistant TERRA remains at the t-loop, likely adding to their stability. Transcription of DNAs containing TTAGTG or TGAGTG repeats showed greatly reduced loop formation. While in the cell multiple pathways may lead to t-loop formation, the pathway revealed here does not depend on the shelterins but rather on the unique character of telomeric DNA when it is opened for transcription. Hence, telomeric sequences may have evolved to facilitate their ability to loop back on themselves. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Free energy landscape and transition pathways from Watson–Crick to Hoogsteen base pairing in free duplex DNA

PubMed Central

Yang, Changwon; Kim, Eunae; Pak, Youngshang

2015-01-01

Houghton (HG) base pairing plays a central role in the DNA binding of proteins and small ligands. Probing detailed transition mechanism from Watson–Crick (WC) to HG base pair (bp) formation in duplex DNAs is of fundamental importance in terms of revealing intrinsic functions of double helical DNAs beyond their sequence determined functions. We investigated a free energy landscape of a free B-DNA with an adenosine–thymine (A–T) rich sequence to probe its conformational transition pathways from WC to HG base pairing. The free energy landscape was computed with a state-of-art two-dimensional umbrella molecular dynamics simulation at the all-atom level. The present simulation showed that in an isolated duplex DNA, the spontaneous transition from WC to HG bp takes place via multiple pathways. Notably, base flipping into the major and minor grooves was found to play an important role in forming these multiple transition pathways. This finding suggests that naked B-DNA under normal conditions has an inherent ability to form HG bps via spontaneous base opening events. PMID:26250116

Experimental single-strain mobilomics reveals events that shape pathogen emergence

DOE PAGES

Schoeniger, Joseph S.; Hudson, Corey M.; Bent, Zachary W.; ...

2016-07-04

Virulence and resistance genes carried on mobile DNAs such as genomic islands (GIs) and plasmids promote bacterial pathogen emergence. An early step in the mobilization of GIs is their excision, which produces both a circular form of the GI and a deletion site in the chromosome; circular forms have also been described for some bacterial insertion sequences (ISs). We demonstrate that the recombinant sequence produced at the junction of such circles, and their corresponding deletion sites, can be detected sensitively in high throughput sequencing data, using new computational methods that enable empirical discovery of new mobile DNAs. Applied to themore » rich mobilome of a single strain (Kpn2146) of the emerging multidrug-resistant pathogen Klebsiella pneumoniae, our approach detected circular junctions for six GIs and seven IS types (several of the latter not previously known to circularize). Our methods further revealed differential biology of multiple mobile DNAs, imprecision of integrases and transposases, and differential activity among identical IS copies for IS26, ISKpn18 and ISKpn21. Exonuclease was used to enrich for circular dsDNA molecules, and internal calibration with the native Kpn2146 plasmids showed that not all molecules bearing GI and IS circular junctions were circular dsDNAs. Transposition events were also detected, revealing replicon preference (ISKpn18 preferring a conjugative IncA/C2 plasmid), local action (IS26), regional preferences, selection (against capsule synthesis), and left-right IS end swapping. Efficient discovery and global characterization of numerous mobile elements per experiment will allow detailed accounting of bacterial evolution, explaining the new gene combinations that arise in emerging pathogens.« less

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

Schoeniger, Joseph S.; Hudson, Corey M.; Bent, Zachary W.

Virulence and resistance genes carried on mobile DNAs such as genomic islands (GIs) and plasmids promote bacterial pathogen emergence. An early step in the mobilization of GIs is their excision, which produces both a circular form of the GI and a deletion site in the chromosome; circular forms have also been described for some bacterial insertion sequences (ISs). We demonstrate that the recombinant sequence produced at the junction of such circles, and their corresponding deletion sites, can be detected sensitively in high throughput sequencing data, using new computational methods that enable empirical discovery of new mobile DNAs. Applied to themore » rich mobilome of a single strain (Kpn2146) of the emerging multidrug-resistant pathogen Klebsiella pneumoniae, our approach detected circular junctions for six GIs and seven IS types (several of the latter not previously known to circularize). Our methods further revealed differential biology of multiple mobile DNAs, imprecision of integrases and transposases, and differential activity among identical IS copies for IS26, ISKpn18 and ISKpn21. Exonuclease was used to enrich for circular dsDNA molecules, and internal calibration with the native Kpn2146 plasmids showed that not all molecules bearing GI and IS circular junctions were circular dsDNAs. Transposition events were also detected, revealing replicon preference (ISKpn18 preferring a conjugative IncA/C2 plasmid), local action (IS26), regional preferences, selection (against capsule synthesis), and left-right IS end swapping. Efficient discovery and global characterization of numerous mobile elements per experiment will allow detailed accounting of bacterial evolution, explaining the new gene combinations that arise in emerging pathogens.« less

Excited State Proton Transfer of Natural Flavonoids and Their Chromophores in Duplex and Tetraplex DNAs

PubMed Central

2015-01-01

Fisetin (3,7,3′,4′-tetrahydroxyflavone) and quercetin (3,5,7,3′,4′-pentahydroxyflavone) are the bioactive plant flavonoids that are potentially useful therapeutic drugs for the treatment of a broad spectrum of diseases, including atherosclerosis, cardiovascular disease, obesity, hypertension, and cancer. 3-Hydroxyflavone (3HF) and 7-hydroxyflavone (7HF) are the synthetic chromophores of fisetin and quercetin. We have exploited dual luminescence properties of fisetin and quercetin along with 3-HF and 7HF to examine their efficacy of binding and compare their interactions with DNA, which is one of the macromolecular targets of flavonoids in physiological systems. Following the sequence of the human telomeric DNA 5′-d (CCCTAA-)n/(-TTAGGG)n-5′, two single-stranded DNA oligonucleotides, 5′-d(C3TA2)3C3-3′ and 5′-d(T2AG3)4-3′, and their duplex were used as receptors to study binding by the ligands quercetin, fisetin, and their chromophores. Circular dichroism, differential absorption, UV thermal melting, and size exclusion chromatographic studies indicated the formation of unusual DNA structures (such as C4 and G4 tetraplexes) for both the C- and G-rich single-stranded DNAs. Upon binding to DNA, dramatic changes were observed in the intrinsic fluorescence behavior of the flavonoids. Molecular docking studies were performed to describe the likely binding sites for the ligands. The spectroscopic studies on flavonoid–DNA interactions described herein demonstrate a powerful approach for examining their DNA binding through exploiting the highly sensitive intrinsic fluorescence properties of the flavonoids as their own “reporter” for their interactions with macromolecular targets. PMID:25393681

Dendritic silica nanomaterials (KCC-1) with fibrous pore structure possess high DNA adsorption capacity and effectively deliver genes in vitro.

PubMed

Huang, Xiaoxi; Tao, Zhimin; Praskavich, John C; Goswami, Anandarup; Al-Sharab, Jafar F; Minko, Tamara; Polshettiwar, Vivek; Asefa, Tewodros

2014-09-16

The pore size and pore structure of nanoporous materials can affect the materials' physical properties, as well as potential applications in different areas, including catalysis, drug delivery, and biomolecular therapeutics. KCC-1, one of the newest members of silica nanomaterials, possesses fibrous, large pore, dendritic pore networks with wide pore entrances, large pore size distribution, spacious pore volume and large surface area--structural features that are conducive for adsorption and release of large guest molecules and biomacromolecules (e.g., proteins and DNAs). Here, we report the results of our comparative studies of adsorption of salmon DNA in a series of KCC-1-based nanomaterials that are functionalized with different organoamine groups on different parts of their surfaces (channel walls, external surfaces or both). For comparison the results of our studies of adsorption of salmon DNA in similarly functionalized, MCM-41 mesoporous silica nanomaterials with cylindrical pores, some of the most studied silica nanomaterials for drug/gene delivery, are also included. Our results indicate that, despite their relatively lower specific surface area, the KCC-1-based nanomaterials show high adsorption capacity for DNA than the corresponding MCM-41-based nanomaterials, most likely because of KCC-1's large pores, wide pore mouths, fibrous pore network, and thereby more accessible and amenable structure for DNA molecules to diffuse through. Conversely, the MCM-41-based nanomaterials adsorb much less DNA, presumably because their outer surfaces/cylindrical channel pore entrances can get blocked by the DNA molecules, making the inner parts of the materials inaccessible. Moreover, experiments involving fluorescent dye-tagged DNAs suggest that the amine-grafted KCC-1 materials are better suited for delivering the DNAs adsorbed on their surfaces into cellular environments than their MCM-41 counterparts. Finally, cellular toxicity tests show that the KCC-1-based materials are biocompatible. On the basis of these results, the fibrous and porous KCC-1-based nanomaterials can be said to be more suitable to carry, transport, and deliver DNAs and genes than cylindrical porous nanomaterials such as MCM-41.

Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and non-label sensitive fluorescent detection of thrombin.

PubMed

Xu, Yunying; Zhou, Wenjiao; Zhou, Ming; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

2015-02-15

Based on a new signal amplification strategy by the toehold strand displacement-driven cyclic assembly of G-quadruplex DNA, the development of an enzyme-free and non-label aptamer sensing approach for sensitive fluorescent detection of thrombin is described. The target thrombin associates with the corresponding aptamer of the partial dsDNA probes and liberates single stranded initiation sequences, which trigger the toehold strand displacement assembly of two G-quadruplex containing hairpin DNAs. This toehold strand displacement reaction leads to the cyclic reuse of the initiation sequences and the production of DNA assemblies with numerous G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binds to these G-quadruplex structures and generates significantly amplified fluorescent signals to achieve highly sensitive detection of thrombin down to 5 pM. Besides, this method shows high selectivity towards the target thrombin against other control proteins. The developed thrombin sensing method herein avoids the modification of the probes and the involvement of any enzyme or nanomaterial labels for signal amplification. With the successful demonstration for thrombin detection, our approach can be easily adopted to monitor other target molecules in a simple, low-cost, sensitive and selective way by choosing appropriate aptamer/ligand pairs. Copyright © 2014 Elsevier B.V. All rights reserved.

Tumor Touch Imprints as Source for Whole Genome Analysis of Neuroblastoma Tumors

PubMed Central

Brunner, Clemens; Brunner-Herglotz, Bettina; Ziegler, Andrea; Frech, Christian; Amann, Gabriele; Ladenstein, Ruth; Ambros, Inge M.; Ambros, Peter F.

2016-01-01

Introduction Tumor touch imprints (TTIs) are routinely used for the molecular diagnosis of neuroblastomas by interphase fluorescence in-situ hybridization (I-FISH). However, in order to facilitate a comprehensive, up-to-date molecular diagnosis of neuroblastomas and to identify new markers to refine risk and therapy stratification methods, whole genome approaches are needed. We examined the applicability of an ultra-high density SNP array platform that identifies copy number changes of varying sizes down to a few exons for the detection of genomic changes in tumor DNA extracted from TTIs. Material and Methods DNAs were extracted from TTIs of 46 neuroblastoma and 4 other pediatric tumors. The DNAs were analyzed on the Cytoscan HD SNP array platform to evaluate numerical and structural genomic aberrations. The quality of the data obtained from TTIs was compared to that from randomly chosen fresh or fresh frozen solid tumors (n = 212) and I-FISH validation was performed. Results SNP array profiles were obtained from 48 (out of 50) TTI DNAs of which 47 showed genomic aberrations. The high marker density allowed for single gene analysis, e.g. loss of nine exons in the ATRX gene and the visualization of chromothripsis. Data quality was comparable to fresh or fresh frozen tumor SNP profiles. SNP array results were confirmed by I-FISH. Conclusion TTIs are an excellent source for SNP array processing with the advantage of simple handling, distribution and storage of tumor tissue on glass slides. The minimal amount of tumor tissue needed to analyze whole genomes makes TTIs an economic surrogate source in the molecular diagnostic work up of tumor samples. PMID:27560999

Crowding Induces Complex Ergodic Diffusion and Dynamic Elongation of Large DNA Molecules

PubMed Central

Chapman, Cole D.; Gorczyca, Stephanie; Robertson-Anderson, Rae M.

2015-01-01

Despite the ubiquity of molecular crowding in living cells, the effects of crowding on the dynamics of genome-sized DNA are poorly understood. Here, we track single, fluorescent-labeled large DNA molecules (11, 115 kbp) diffusing in dextran solutions that mimic intracellular crowding conditions (0–40%), and determine the effects of crowding on both DNA mobility and conformation. Both DNAs exhibit ergodic Brownian motion and comparable mobility reduction in all conditions; however, crowder size (10 vs. 500 kDa) plays a critical role in the underlying diffusive mechanisms and dependence on crowder concentration. Surprisingly, in 10-kDa dextran, crowder influence saturates at ∼20% with an ∼5× drop in DNA diffusion, in stark contrast to exponentially retarded mobility, coupled to weak anomalous subdiffusion, with increasing concentration of 500-kDa dextran. Both DNAs elongate into lower-entropy states (compared to random coil conformations) when crowded, with elongation states that are gamma distributed and fluctuate in time. However, the broadness of the distribution of states and the time-dependence and length scale of elongation length fluctuations depend on both DNA and crowder size with concentration having surprisingly little impact. Results collectively show that mobility reduction and coil elongation of large crowded DNAs are due to a complex interplay between entropic effects and crowder mobility. Although elongation and initial mobility retardation are driven by depletion interactions, subdiffusive dynamics, and the drastic exponential slowing of DNA, up to ∼300×, arise from the reduced mobility of larger crowders. Our results elucidate the highly important and widely debated effects of cellular crowding on genome-sized DNA. PMID:25762333

High-throughput analysis of the satellitome revealed enormous diversity of satellite DNAs in the neo-Y chromosome of the cricket Eneoptera surinamensis.

PubMed

Palacios-Gimenez, Octavio Manuel; Dias, Guilherme Borges; de Lima, Leonardo Gomes; Kuhn, Gustavo Campos E Silva; Ramos, Érica; Martins, Cesar; Cabral-de-Mello, Diogo Cavalcanti

2017-07-25

Satellite DNAs (satDNAs) constitute large portion of eukaryote genomes, comprising non-protein-coding sequences tandemly repeated. They are mostly found in heterochromatic regions of chromosomes such as around centromere or near telomeres, in intercalary heterochromatin, and often in non-recombining segments of sex chromosomes. We examined the satellitome in the cricket Eneoptera surinamensis (2n = 9, neo-X 1 X 2 Y, males) to characterize the molecular evolution of its neo-sex chromosomes. To achieve this, we analyzed illumina reads using graph-based clustering and complementary analyses. We found an unusually high number of 45 families of satDNAs, ranging from 4 bp to 517 bp, accounting for about 14% of the genome and showing different modular structures and high diversity of arrays. FISH mapping revealed that satDNAs are located mostly in C-positive pericentromeric regions of the chromosomes. SatDNAs enrichment was also observed in the neo-sex chromosomes in comparison to autosomes. Especially astonishing accumulation of satDNAs loci was found in the highly differentiated neo-Y, including 39 satDNAs over-represented in this chromosome, which is the greatest satDNAs diversity yet reported for sex chromosomes. Our results suggest possible involvement of satDNAs in genome increasing and in molecular differentiation of the neo-sex chromosomes in this species, contributing to the understanding of sex chromosome composition and evolution in Orthoptera.

Preparation and evaluation of nanoparticles loading plasmid DNAs inserted with siRNA fragments targeting c-Myc gene.

PubMed

Ma, Tao; Jiang, Jin-Ling; Liu, Ying; Ye, Zheng-Bao; Zhang, Jun

2014-09-01

c-Myc plays a key role in glioma cancer stem cell maintenance. A drug delivery system, nanoparticles loading plasmid DNAs inserted with siRNA fragments targeting c-Myc gene (NPs-c-Myc-siRNA-pDNAs), for the treatment of glioma, has not previously been reported. NPs-c-Myc-siRNA-pDNAs were prepared and evaluated in vitro. Three kinds of c-Myc-siRNA fragments were separately synthesized and linked with empty siRNA expression vectors in the mole ratio of 3:1 by T4 DNA ligase. The linked products were then separately transfected into Escherichia coli. DH5α followed by extraction with Endofree plasmid Mega kit (Qiagen, Hilden, Germany) obtained c-Myc-siRNA-pDNAs. Finally, the recombinant c-Myc-siRNA3-pDNAs, generating the highest transfection efficiency and the greatest apoptotic ability, were chosen for encapsulation into NPs by the double-emulsion solvent-evaporation procedure, followed by stability, transfection efficiency, as well as qualitative and quantitative apoptosis evaluation. NPs-c-Myc-siRNA3-pDNAs were obtained with spherical shape in uniform size below 150 nm, with the zeta potential about -18 mV, the encapsulation efficiency and loading capacity as 76.3 ± 5.4% and 1.91 ± 0.06%, respectively. The stability results showed that c-Myc-siRNA3-pDNAs remained structurally and functionally stable after encapsulated into NPs, and NPs could prevent the loaded c-Myc-siRNA3-pDNAs from DNase degradation. The transfection efficiency of NPs-c-Myc-siRNA3-pDNAs was proven to be positive. Furthermore, NPs-c-Myc-siRNA3-pDNAs produced significant apoptosis with the apoptotic rate at 24.77 ± 5.39% and early apoptosis cells observed. Methoxy-poly-(ethylene-glycol)-poly-(lactide-co-glycolide) nanoparticles (MPEG-PLGA-NPs) are potential delivery carriers for c-Myc-siRNA3-pDNAs.

Soft Fruit Traceability in Food Matrices using Real-Time PCR

PubMed Central

Palmieri, Luisa; Bozza, Elisa; Giongo, Lara

2009-01-01

Food product authentication provides a means of monitoring and identifying products for consumer protection and regulatory compliance. There is a scarcity of analytical methods for confirming the identity of fruit pulp in products containing Soft Fruit. In the present work we have developed a very sensible qualitative and quantitative method to determine the presence of berry DNAs in different food matrices. To our knowledge, this is the first study that shows the applicability, to Soft Fruit traceability, of melting curve analysis and multiplexed fluorescent probes, in a Real-Time PCR platform. This methodology aims to protect the consumer from label misrepresentation. PMID:22253987

Nature and distribution of feline sarcoma virus nucleotide sequences.

PubMed Central

Frankel, A E; Gilbert, J H; Porzig, K J; Scolnick, E M; Aaronson, S A

1979-01-01

The genomes of three independent isolates of feline sarcoma virus (FeSV) were compared by molecular hybridization techniques. Using complementary DNAs prepared from two strains, SM- and ST-FeSV, common complementary DNA'S were selected by sequential hybridization to FeSV and feline leukemia virus RNAs. These DNAs were shown to be highly related among the three independent sarcoma virus isolates. FeSV-specific complementary DNAs were prepared by selection for hybridization by the homologous FeSV RNA and against hybridization by fline leukemia virus RNA. Sarcoma virus-specific sequences of SM-FeSV were shown to differ from those of either ST- or GA-FeSV strains, whereas ST-FeSV-specific DNA shared extensive sequence homology with GA-FeSV. By molecular hybridization, each set of FeSV-specific sequences was demonstrated to be present in normal cat cellular DNA in approximately one copy per haploid genome and was conserved throughout Felidae. In contrast, FeSV-common sequences were present in multiple DNA copies and were found only in Mediterranean cats. The present results are consistent with the concept that each FeSV strain has arisen by a mechanism involving recombination between feline leukemia virus and cat cellular DNA sequences, the latter represented within the cat genome in a manner analogous to that of a cellular gene. PMID:225544

Isolation of genes negatively or positively co-expressed with human recombination activating gene 1 (RAG1) by differential display PCR (DD RT-PCR).

PubMed

Verkoczy, L K; Berinstein, N L

1998-10-01

Differential display PCR (DD RT-PCR) has been extensively used for analysis of differential gene expression, but continues to be hampered by technical limitations that impair its effectiveness. In order to isolate novel genes co-expressing with human RAG1, we have developed an effective, multi-tiered screening/purification approach which effectively complements the standard DD RT-PCR methodology. In 'primary' screens, standard DD RT-PCR was used, detecting 22 reproducible differentially expressed amplicons between clonally related cell variants with differential constitutive expression of RAG mRNAs. 'Secondary' screens used differential display (DD) amplicons as probes in low and high stringency northern blotting. Eight of 22 independent DD amplicons detected nine independent differentially expressed transcripts. 'Tertiary' screens used reconfirmed amplicons as probes in northern analysis of multiple RAG-and RAG+sources. Reconfirmed DD amplicons detected six independent RAG co-expressing transcripts. All DD amplicons reconfirmed by northern blot were a heterogeneous mixture of cDNAs, necessitating further purification to isolate single cDNAs prior to subcloning and sequencing. To effectively select the appropriate cDNAs from DD amplicons, we excised and eluted the cDNA(s) directly from regions of prior northern blots in which differentially expressed transcripts were detected. Sequences of six purified cDNA clones specifically detecting RAG co-expressing transcripts included matches to portions of the human RAG2 and BSAP regions and to four novel partial cDNAs (three with homologies to human ESTs). Overall, our results also suggest that even when using clonally related variants from the same cell line in addition to all appropriate internal controls previously reported, further screening and purification steps are still required in order to efficiently and specifically isolate differentially expressed genes by DD RT-PCR.

Human Gamma Satellite Insulator Sequences to Prevent Gene Silencing | NCI Technology Transfer Center | TTC

Cancer.gov

This invention describes the use of chromatin insulators, or gamma satellite DNA, to inhibit gene silencing in a cell, which may have a significant impact on gene therapy across multiple diseases where gene silencing is the cause. Experimental data has demonstrated these gamma satellite DNAs overcome gene position effects and ultimately inhibit gene silencing.

Free energy landscape and transition pathways from Watson-Crick to Hoogsteen base pairing in free duplex DNA.

PubMed

Yang, Changwon; Kim, Eunae; Pak, Youngshang

2015-09-18

Houghton (HG) base pairing plays a central role in the DNA binding of proteins and small ligands. Probing detailed transition mechanism from Watson-Crick (WC) to HG base pair (bp) formation in duplex DNAs is of fundamental importance in terms of revealing intrinsic functions of double helical DNAs beyond their sequence determined functions. We investigated a free energy landscape of a free B-DNA with an adenosine-thymine (A-T) rich sequence to probe its conformational transition pathways from WC to HG base pairing. The free energy landscape was computed with a state-of-art two-dimensional umbrella molecular dynamics simulation at the all-atom level. The present simulation showed that in an isolated duplex DNA, the spontaneous transition from WC to HG bp takes place via multiple pathways. Notably, base flipping into the major and minor grooves was found to play an important role in forming these multiple transition pathways. This finding suggests that naked B-DNA under normal conditions has an inherent ability to form HG bps via spontaneous base opening events. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Pathogenic role of mtDNA duplications in mitochondrial diseases associated with mtDNA deletions.

PubMed

Odoardi, Francesca; Rana, Michele; Broccolini, Aldobrando; Mirabella, Massimiliano; Modoni, Anna; D'Amico, Adele; Papacci, Manuela; Tonali, Pietro; Servidei, Serenella; Silvestri, Gabriella

2003-04-30

We estimated the frequency of multiple mtDNA rearrangements by Southern blot in 32 patients affected by mitochondrial disorders associated with single deletions in order to assess genotype-phenotype correlations and elucidate the pathogenic significance of mtDNA duplications. Muscle in situ hybridization studies were performed in patients showing mtDNA duplications at Southern blot. We found multiple rearrangements in 12/32 (37.5%) patients; in particular, mtDNA duplications were detected in 4/4 Kearns-Sayre syndrome (KSS), in 1 Pearson's syndrome, in 1/3 encephalomyopathies with progressive external ophthalmoplegia (PEO), and in 2/23 PEO. In situ studies documented an exclusive accumulation of deleted mtDNAs in cytochrome c oxidase negative fibers of patients with mtDNA duplications. The presence of mtDNA duplications significantly correlated with onset of symptoms before age 15 and occurrence of clinical multisystem involvement. Analysis of biochemical data documented a predominant reduction of complex III in patients without duplications compared to patients with mtDNA duplications. Our data indicate that multiple mtDNA rearrangements are detectable in a considerable proportion of patients with single deletions and that mtDNA duplications do not cause any oxidative impairment. They more likely play a pathogenic role in the determination of clinical expression of mitochondrial diseases associated with single mtDNA deletions, possibly generating deleted mtDNAs in embryonic tissues by homologous recombination. Copyright 2003 Wiley-Liss, Inc.

Rapid on-site/in-situ detection of heavy metal ions in environmental water using a structure-switching DNA optical biosensor.

PubMed

Long, Feng; Zhu, Anna; Shi, Hanchang; Wang, Hongchen; Liu, Jingquan

2013-01-01

A structure-switching DNA optical biosensor for rapid on-site/in situ detection of heavy metal ions is reported. Mercury ions (Hg²⁺), highly toxic and ubiquitous pollutants, were selected as model target. In this system, fluorescence-labeled DNA containing T-T mismatch structure was introduced to bind with DNA probes immobilized onto the sensor surface. In the presence of Hg²⁺, some of the fluorescence-labeled DNAs bind with Hg²⁺ to form T-Hg²⁺-T complexes through the folding of themselves into a hairpin structure and dehybridization from the sensor surface, which leads to decrease in fluorescence signal. The total analysis time for a single sample was less than 10 min with detection limit of 1.2 nM. The rapid on-site/in situ determination of Hg²⁺ was readily performed in natural water. This sensing strategy can be extended in principle to other metal ions by substituting the T-Hg²⁺-T complexes with other specificity structures that selectively bind to other analytes.

Two-color, 30 second microwave-accelerated Metal-Enhanced Fluorescence DNA assays: a new Rapid Catch and Signal (RCS) technology.

PubMed

Dragan, Anatoliy I; Golberg, Karina; Elbaz, Amit; Marks, Robert; Zhang, Yongxia; Geddes, Chris D

2011-03-07

For analyses of DNA fragment sequences in solution we introduce a 2-color DNA assay, utilizing a combination of the Metal-Enhanced Fluorescence (MEF) effect and microwave-accelerated DNA hybridization. The assay is based on a new "Catch and Signal" technology, i.e. the simultaneous specific recognition of two target DNA sequences in one well by complementary anchor-ssDNAs, attached to silver island films (SiFs). It is shown that fluorescent labels (Alexa 488 and Alexa 594), covalently attached to ssDNA fragments, play the role of biosensor recognition probes, demonstrating strong response upon DNA hybridization, locating fluorophores in close proximity to silver NPs, which is ideal for MEF. Subsequently the emission dramatically increases, while the excited state lifetime decreases. It is also shown that 30s microwave irradiation of wells, containing DNA molecules, considerably (~1000-fold) speeds up the highly selective hybridization of DNA fragments at ambient temperature. The 2-color "Catch and Signal" DNA assay platform can radically expedite quantitative analysis of genome DNA sequences, creating a simple and fast bio-medical platform for nucleic acid analysis. Copyright © 2010 Elsevier B.V. All rights reserved.

Fluorescent biosensors enabled by graphene and graphene oxide.

PubMed

Zhang, Huan; Zhang, Honglu; Aldalbahi, Ali; Zuo, Xiaolei; Fan, Chunhai; Mi, Xianqiang

2017-03-15

During the past few years, graphene and graphene oxide (GO) have attracted numerous attentions for the potential applications in various fields from energy technology, biosensing to biomedical diagnosis and therapy due to their various functionalization, high volume surface ratio, unique physical and electrical properties. Among which, graphene and graphene oxide based fluorescent biosensors enabled by their fluorescence-quenching properties have attracted great interests. The fluorescence of fluorophore or dye labeled on probes (such as molecular beacon, aptamer, DNAzymes and so on) was quenched after adsorbed on to the surface of graphene. While in the present of the targets, due to the strong interactions between probes and targets, the probes were detached from the surface of graphene, generating dramatic fluorescence, which could be used as signals for detection of the targets. This strategy was simple and economy, together with great programmable abilities of probes; we could realize detection of different kinds of species. In this review, we first briefly introduced the history of graphene and graphene oxide, and then summarized the fluorescent biosensors enabled by graphene and GO, with a detailed account of the design mechanism and comparison with other nanomaterials (e.g. carbon nanotubes and gold nanoparticles). Following that, different sensing platforms for detection of DNAs, ions, biomolecules and pathogens or cells as well as the cytotoxicity issue of graphene and GO based in vivo biosensing were further discussed. We hope that this review would do some help to researchers who are interested in graphene related biosening research work. Copyright © 2016 Elsevier B.V. All rights reserved.

Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing

PubMed Central

Kon, Tatsuya; Yoshikawa, Nobuyuki

2014-01-01

Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification. PMID:25426109

Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

PubMed

Cha, Jaehyun; Kwon, Inchan

2018-02-27

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Localization of a translocation breakpoint involved in Smith-Lemli-Opitz syndrome

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

Alley, T.L.; Gray, B.A.; Lee, S.

1994-09-01

Smith-Lemli-Opitz syndrome (SLOS) is a multiple congenital anomaly/mental retardation syndrome, with features including toe syndactyly, genital anomalies, unusual facies, and occasional organ malformations. The gene(s) for this autosomal recessive disorder has not been mapped. Recent biochemical studies suggest that the defect may involve the penultimate step in cholesterol synthesis, as patients have low serum cholesterol and increased 7-dehydrocholesterol (7-DHC) levels. However, the enzyme putatively involved (7-DHC reductase) has not been isolated. We identified an SLOS patient with a de novo balanced chromosome translocation [t(7;20)(q32.1;q13.2)], and we propose that the translocation interrupts one of the patient`s SLOS alleles. We are pursuingmore » positional cloning to identify the SLOS gene. Using fluorescence in situ hybridization (FISH), we recently identified a chromosome 7 yeast artificial chromosome (YAC) that spans the breakpoint and places it onto physical and genetic maps. We are in the process of narrowing this region via overlapping YACs and YAC subclones, from which we will isolate candidate cDNAs. Any candidate gene disrupted by the translocation and mutated on the other allele will be proven to be the SLOS gene. Functional analysis of an SLOS cDNA may also determine its relationship to cholesterol metabolism and the observed biochemical abnormalities.« less

Microarray-Based Comparative Genomic Hybridization Using Sex-Matched Reference DNA Provides Greater Sensitivity for Detection of Sex Chromosome Imbalances than Array-Comparative Genomic Hybridization with Sex-Mismatched Reference DNA

PubMed Central

Yatsenko, Svetlana A.; Shaw, Chad A.; Ou, Zhishuo; Pursley, Amber N.; Patel, Ankita; Bi, Weimin; Cheung, Sau Wai; Lupski, James R.; Chinault, A. Craig; Beaudet, Arthur L.

2009-01-01

In array-comparative genomic hybridization (array-CGH) experiments, the measurement of DNA copy number of sex chromosomal regions depends on the sex of the patient and the reference DNAs used. We evaluated the ability of bacterial artificial chromosomes/P1-derived artificial and oligonucleotide array-CGH analyses to detect constitutional sex chromosome imbalances using sex-mismatched reference DNAs. Twenty-two samples with imbalances involving either the X or Y chromosome, including deletions, duplications, triplications, derivative or isodicentric chromosomes, and aneuploidy, were analyzed. Although concordant results were obtained for approximately one-half of the samples when using sex-mismatched and sex-matched reference DNAs, array-CGH analyses with sex-mismatched reference DNAs did not detect genomic imbalances that were detected using sex-matched reference DNAs in 6 of 22 patients. Small duplications and deletions of the X chromosome were most difficult to detect in female and male patients, respectively, when sex-mismatched reference DNAs were used. Sex-matched reference DNAs in array-CGH analyses provides optimal sensitivity and enables an automated statistical evaluation for the detection of sex chromosome imbalances when compared with an experimental design using sex-mismatched reference DNAs. Using sex-mismatched reference DNAs in array-CGH analyses may generate false-negative, false-positive, and ambiguous results for sex chromosome-specific probes, thus masking potential pathogenic genomic imbalances. Therefore, to optimize both detection of clinically relevant sex chromosome imbalances and ensure proper experimental performance, we suggest that alternative internal controls be developed and used instead of using sex-mismatched reference DNAs. PMID:19324990

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

Pack, Chan-Gi, E-mail: changipack@amc.seoul.kr; Ahn, Sang-Gun

The cellular response to stress is primarily controlled in cells via transcriptional activation by heat shock factor 1 (HSF1). HSF1 is well-known to form homotrimers for activation upon heat shock and subsequently bind to target DNAs, such as heat-shock elements, by forming stress granules. A previous study demonstrated that nuclear HSF1 and HSF2 molecules in live cells interacted with target DNAs on the stress granules. However, the process underlying the binding interactions of HSF family in cells upon heat shock remains unclear. This study demonstrate for the first time that the interaction kinetics among nuclear HSF1, HSF2, and HSF4 uponmore » heat shock can be detected directly in live cells using dual color fluorescence cross-correlation spectroscopy (FCCS). FCCS analyses indicated that the binding between HSFs was dramatically changed by heat shock. Interestingly, the recovery kinetics of interaction between HSF1 molecules after heat shock could be represented by changes in the relative interaction amplitude and mobility. - Highlights: • The binding interactions among nuclear HSFs were successfully detected. • The binding kinetics between HSF1s during recovery was quantified. • HSF2 and HSF4 strongly formed hetero-complex, even before heat shock. • Nuclear HSF2 and HSF4 bound to HSF1 only after heat shock.« less

Comparative Analysis of Repetitive DNA between the Main Vectors of Chagas Disease: Triatoma infestans and Rhodnius prolixus.

PubMed

Pita, Sebastián; Mora, Pablo; Vela, Jesús; Palomeque, Teresa; Sánchez, Antonio; Panzera, Francisco; Lorite, Pedro

2018-04-24

Chagas disease or American trypanosomiasis affects six to seven million people worldwide, mostly in Latin America. This disease is transmitted by hematophagous insects known as "kissing bugs" (Hemiptera, Triatominae), with Triatoma infestans and Rhodnius prolixus being the two most important vector species. Despite the fact that both species present the same diploid chromosome number (2 n = 22), they have remarkable differences in their total DNA content, chromosome structure and genome organization. Variations in the DNA genome size are expected to be due to differences in the amount of repetitive DNA sequences. The T. infestans genome-wide analysis revealed the existence of 42 satellite DNA families. BLAST searches of these sequences against the R. prolixus genome assembly revealed that only four of these satellite DNA families are shared between both species, suggesting a great differentiation between the Triatoma and Rhodnius genomes. Fluorescence in situ hybridization (FISH) location of these repetitive DNAs in both species showed that they are dispersed on the euchromatic regions of all autosomes and the X chromosome. Regarding the Y chromosome, these common satellite DNAs are absent in T. infestans but they are present in the R. prolixus Y chromosome. These results support a different origin and/or evolution in the Y chromosome of both species.

A novel sensitive pathogen detection system based on Microbead Quantum Dot System.

PubMed

Wu, Tzong-Yuan; Su, Yi-Yu; Shu, Wei-Hsien; Mercado, Augustus T; Wang, Shi-Kwun; Hsu, Ling-Yi; Tsai, Yow-Fu; Chen, Chung-Yung

2016-04-15

A fast and accurate detection system for pathogens can provide immediate measurements for the identification of infectious agents. Therefore, the Microbead Quantum-dots Detection System (MQDS) was developed to identify and measure target DNAs of pathogenic microorganisms and eliminated the need of PCR amplifications. This nanomaterial-based technique can detect different microorganisms by flow cytometry measurements. In MQDS, pathogen specific DNA probes were designed to form a hairpin structure and conjugated on microbeads. In the presence of the complementary target DNA sequence, the probes will compete for binding with the reporter probes but will not interfere with the binding between the probe and internal control DNA. To monitor the binding process by flow cytometry, both the reporter probes and internal control probes were conjugated with Quantum dots that fluoresce at different emission wavelengths using the click reaction. When MQDS was used to detect the pathogens in environmental samples, a high correlation coefficient (R=0.994) for Legionella spp., with a detection limit of 0.1 ng of the extracted DNAs and 10 CFU/test, can be achieved. Thus, this newly developed technique can also be applied to detect other pathogens, particularly viruses and other genetic diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

A Conserved, Mg2+-Dependent Exonuclease Degrades Organelle DNA during Arabidopsis Pollen Development[C][W

PubMed Central

Matsushima, Ryo; Tang, Lay Yin; Zhang, Lingang; Yamada, Hiroshi; Twell, David; Sakamoto, Wataru

2011-01-01

In plant cells, mitochondria and plastids contain their own genomes derived from the ancestral bacteria endosymbiont. Despite their limited genetic capacity, these multicopy organelle genomes account for a substantial fraction of total cellular DNA, raising the question of whether organelle DNA quantity is controlled spatially or temporally. In this study, we genetically dissected the organelle DNA decrease in pollen, a phenomenon that appears to be common in most angiosperm species. By staining mature pollen grains with fluorescent DNA dye, we screened Arabidopsis thaliana for mutants in which extrachromosomal DNAs had accumulated. Such a recessive mutant, termed defective in pollen organelle DNA degradation1 (dpd1), showing elevated levels of DNAs in both plastids and mitochondria, was isolated and characterized. DPD1 encodes a protein belonging to the exonuclease family, whose homologs appear to be found in angiosperms. Indeed, DPD1 has Mg2+-dependent exonuclease activity when expressed as a fusion protein and when assayed in vitro and is highly active in developing pollen. Consistent with the dpd phenotype, DPD1 is dual-targeted to plastids and mitochondria. Therefore, we provide evidence of active organelle DNA degradation in the angiosperm male gametophyte, primarily independent of maternal inheritance; the biological function of organellar DNA degradation in pollen is currently unclear. PMID:21521697

Design and numerical analysis of highly sensitive Au-MoS2-graphene based hybrid surface plasmon resonance biosensor

NASA Astrophysics Data System (ADS)

Rahman, M. Saifur; Anower, Md. Shamim; Hasan, Md. Rabiul; Hossain, Md. Biplob; Haque, Md. Ismail

2017-08-01

We demonstrate a highly sensitive Au-MoS2-Graphene based hybrid surface plasmon resonance (SPR) biosensor for the detection of DNA hybridization. The performance parameters of the proposed sensor are investigated in terms of sensitivity, detection accuracy and quality factor at operating wavelength of 633 nm. We observed in the numerical study that sensitivity can be greatly increased by adding MoS2 layer in the middle of a Graphene-on-Au layer. It is shown that by using single layer of MoS2 in between gold and graphene layer, the proposed biosensor exhibits simultaneously high sensitivity of 87.8 deg/RIU, high detection accuracy of 1.28 and quality factor of 17.56 with gold layer thickness of 50 nm. This increased performance is due to the absorption ability and optical characteristics of graphene biomolecules and high fluorescence quenching ability of MoS2. On the basis of changing in SPR angle and minimum reflectance, the proposed sensor can sense nucleotides bonding happened between double-stranded DNA (dsDNA) helix structures. Therefore, this sensor can successfully detect the hybridization of target DNAs to the probe DNAs pre-immobilized on the Au-MoS2-Graphene hybrid with capability of distinguishing single-base mismatch.

Identification of Bacterial Plant Pathogens Using Multilocus Polymerase Chain Reaction/Electrospray Ionization-Mass Spectrometry

DTIC Science & Technology

2008-01-01

sandii FK-53; OLF#1 Oleander; USA 1 Xylophilus ampelinus FB-1178 Grape; S. Africa 1 Xylophilus ampelinus FJ-3; 60002 Grape; S. Africa 1 1160...campestris Xanthomonas campestris Xylella fastidiosa (6 strains) Xylella fastidiosa Xylophilus ampelinus (2 strains) Xylophilus ampelinus ...Rathayibacter iranicus Rathayibacter iranicus Xylophilus ampelinus Xylophilus ampelinus a Purified DNAs from multiple bacteria were mixed at equal

Organelle DNA variation and systematic relationships in the genus Zea: Teosinte

PubMed Central

Timothy, D. H.; Levings, C. S.; Pring, D. R.; Conde, M. F.; Kermicle, J. L.

1979-01-01

Chloroplast and mitochondrial DNAs from six races of annual teosinte (Guatemala, Huehuetenango, Balsas, Central Plateau, Chalco, and Nobogame), perennial teosinte, and maize were compared and grouped by restriction endonuclease fragment analyses. Three groups of chloroplast DNAs were detected: (i) perennial teosinte and Guatemala; (ii) Balsas and Huehuetenango; and (iii) all other teosintes. Four groups of mitochondrial DNAs were separated: (i) perennial teosinte; (ii) Guatemala; (iii) Nobogame; and (iv) all other teosintes. Separation of the teosinte and maize organelle DNAs into five groups (Guatemala; perennial teosinte; Balsas and Huehuetenango; Central Plateau and Chalco; Nobogame and maize) approximated the biosystematic relationships of the taxa. It was suggested that the evolutions of the chloroplast and mitochondrial DNAs may be independent of each other, that variation of organelle DNA within a species complex of an organism may be the common condition, and that the DNAs of the organelle and nuclear systems evolve in reasonable harmony. Images PMID:16592708

Development of an isothermal recombinase polymerase amplification assay for rapid detection of pseudorabies virus.

PubMed

Yang, Yang; Qin, Xiaodong; Zhang, Wei; Li, Zhiyong; Zhang, Shuaijun; Li, Yanmin; Zhang, Zhidong

2017-06-01

Recombinase polymerase amplification assays using real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the gD gene of pseudorabies virus (PRV). Both assays were performed at 39 °C within 20 min. The sensitivity of the real-time RPA assay and the RPA LFD assay was 100 copies per reaction and 160 copies per reaction, respectively. Both assays did not detect DNAs from other virus or PRV negative samples. Therefore, the developed RPA assays provide a rapid, simple, sensitive and specific alternative tool for detection of PRV. Copyright © 2017. Published by Elsevier Ltd.

The structures of non-CG-repeat Z-DNAs co-crystallized with the Z-DNA-binding domain, hZ alpha(ADAR1).

PubMed

Ha, Sung Chul; Choi, Jongkeun; Hwang, Hye-Yeon; Rich, Alexander; Kim, Yang-Gyun; Kim, Kyeong Kyu

2009-02-01

The Z-DNA conformation preferentially occurs at alternating purine-pyrimidine repeats, and is specifically recognized by Z alpha domains identified in several Z-DNA-binding proteins. The binding of Z alpha to foreign or chromosomal DNA in various sequence contexts is known to influence various biological functions, including the DNA-mediated innate immune response and transcriptional modulation of gene expression. For these reasons, understanding its binding mode and the conformational diversity of Z alpha bound Z-DNAs is of considerable importance. However, structural studies of Z alpha bound Z-DNA have been mostly limited to standard CG-repeat DNAs. Here, we have solved the crystal structures of three representative non-CG repeat DNAs, d(CACGTG)(2), d(CGTACG)(2) and d(CGGCCG)(2) complexed to hZ alpha(ADAR1) and compared those structures with that of hZ alpha(ADAR1)/d(CGCGCG)(2) and the Z alpha-free Z-DNAs. hZ alpha(ADAR1) bound to each of the three Z-DNAs showed a well conserved binding mode with very limited structural deviation irrespective of the DNA sequence, although varying numbers of residues were in contact with Z-DNA. Z-DNAs display less structural alterations in the Z alpha-bound state than in their free form, thereby suggesting that conformational diversities of Z-DNAs are restrained by the binding pocket of Z alpha. These data suggest that Z-DNAs are recognized by Z alpha through common conformational features regardless of the sequence and structural alterations.

Experimental single-strain mobilomics reveals events that shape pathogen emergence.

PubMed

Schoeniger, Joseph S; Hudson, Corey M; Bent, Zachary W; Sinha, Anupama; Williams, Kelly P

2016-08-19

Virulence genes on mobile DNAs such as genomic islands (GIs) and plasmids promote bacterial pathogen emergence. Excision is an early step in GI mobilization, producing a circular GI and a deletion site in the chromosome; circular forms are also known for some bacterial insertion sequences (ISs). The recombinant sequence at the junctions of such circles and deletions can be detected sensitively in high-throughput sequencing data, using new computational methods that enable empirical discovery of mobile DNAs. For the rich mobilome of a hospital Klebsiella pneumoniae strain, circularization junctions (CJs) were detected for six GIs and seven IS types. Our methods revealed differential biology of multiple mobile DNAs, imprecision of integrases and transposases, and differential activity among identical IS copies for IS26, ISKpn18 and ISKpn21 Using the resistance of circular dsDNA molecules to exonuclease, internally calibrated with the native plasmids, showed that not all molecules bearing GI CJs were circular. Transpositions were also detected, revealing replicon preference (ISKpn18 prefers a conjugative IncA/C2 plasmid), local action (IS26), regional preferences, selection (against capsule synthesis) and IS polarity inversion. Efficient discovery and global characterization of numerous mobile elements per experiment improves accounting for the new gene combinations that arise in emerging pathogens. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification of MHC class I sequences in Chinese-origin rhesus macaques

PubMed Central

Karl, Julie A.; Wiseman, Roger W.; Campbell, Kevin J.; Blasky, Alex J.; Hughes, Austin L.; Ferguson, Betsy; Read, Daniel S.

2010-01-01

The rhesus macaque (Macaca mulatta) is an excellent model for human disease and vaccine research. Two populations exhibiting distinctive morphological and physiological characteristics, Indian- and Chinese-origin rhesus macaques, are commonly used in research. Genetic analysis has focused on the Indian macaque population, but the accessibility of these animals for research is limited. Due to their greater availability, Chinese rhesus macaques are now being used more frequently, particularly in vaccine and biodefense studies, although relatively little is known about their immunogenetics. In this study, we discovered major histocompatibility complex (MHC) class I cDNAs in 12 Chinese rhesus macaques and detected 41 distinct Mamu-A and Mamu-B sequences. Twenty-seven of these class I cDNAs were novel, while six and eight of these sequences were previously reported in Chinese and Indian rhesus macaques, respectively. We then performed microsatellite analysis on DNA from these 12 animals, as well as an additional 18 animals, and developed sequence specific primer PCR (PCR-SSP) assays for eight cDNAs found in multiple animals. We also examined our cohort for potential admixture of Chinese and Indian origin animals using a recently developed panel of single nucleotide polymorphisms (SNPs). The discovery of 27 novel MHC class I sequences in this analysis underscores the genetic diversity of Chinese rhesus macaques and contributes reagents that will be valuable for studying cellular immunology in this population. PMID:18097659

Cell cycle effect on the activity of deoxynucleoside analogue metabolising enzymes

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

Fyrberg, Anna; Albertioni, Freidoun; Lotfi, Kourosh

Deoxynucleoside analogues (dNAs) are cytotoxic towards both replicating and indolent malignancies. The impact of fluctuations in the metabolism of dNAs in relation to cell cycle could have strong implications regarding the activity of dNAs. Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) are important enzymes for phosphorylation/activation of dNAs. These drugs can be dephosphorylated/deactivated by 5'-nucleotidases (5'-NTs) and elevated activities of 5'-NTs and decreased dCK and/or dGK activities represent resistance mechanisms towards dNAs. The activities of dCK, dGK, and three 5'-NTs were investigated in four human leukemic cell lines in relationship to cell cycle progression and cytotoxicity of dNAs. Synchronization ofmore » cell cultures to arrest in G0/G1 by serum-deprivation was performed followed by serum-supplementation for cell cycle progression. The activities of dCK and dGK increased up to 3-fold in CEM, HL60, and MOLT-4 cells as they started to proliferate, while the activity of cytosolic nucleotidase I was reduced in proliferating cells. CEM, HL60, and MOLT-4 cells were also more sensitive to cladribine, cytarabine, 9-{beta}-D-arabinofuranosylguanine and clofarabine than K562 cells which demonstrated lower levels and less alteration of these enzymes and were least susceptible to the cytotoxic effects of most dNAs. The results suggest that, in the cell lines studied, the proliferation process is associated with a general shift in the direction of activation of dNAs by inducing activities of dCK/dGK and reducing the activity of cN-I which is favourable for the cytotoxic effects of cladribine, cytarabine and, 9-{beta}-D-arabinofuranosylguanine. These results emphasize the importance of cellular proliferation and dNA metabolism by both phosphorylation and dephosphorylation for susceptibility to dNAs. It underscores the need to understand the mechanisms of action and resistance to dNAs in order to increase efficacy of dNAs treatment by new rational.« less

Multiple Origins of a Mitochondrial Mutation Conferring Deafness

PubMed Central

Hutchin, T. P.; Cortopassi, G. A.

1997-01-01

A point mutation (1555G) in the smaller ribosomal subunit of the mitochondrial DNA (mtDNA) has been associated with maternally inherited traits of hypersensitivity to streptomycin and sensorineural deafness in a number of families from China, Japan, Israel, and Africa. To determine whether this distribution was the result of a single or multiple mutational events, we carried out genetic distance analysis and phylogenetic analysis of 10 independent mtDNA D-loop sequences from Africa and Asia. The mtDNA sequence diversity was high (2.21%). Phylogenetic analysis assigned 1555G-bearing haplotypes at very divergent points in the human mtDNA evolutionary tree, and the 1555G mutations occur in many cases on race-specific mtDNA haplotypes, both facts are inconsistent with a recent introgression of the mutation into these races. The simplest interpretation of the available data is that there have been multiple origins of the 1555G mutation. The genetic distance among mtDNAs bearing the pathogenic 1555G mutation is much larger than among mtDNAs bearing either evolutionarily neutral or weakly deleterious nucleotide substitutions (such as the 4336G mutation). These results are consistent with the view that pathogenic mtDNA haplotypes such as 1555G arise on disparate mtDNA lineages which because of negative natural selection leave relatively few related descendants. The co-existence of the same mutation with deafness in individuals with very different nuclear and mitochondrial genetic backgrounds confirms the pathogenicity of the 1555G mutation. PMID:9055086

Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification.

PubMed

Wang, Yi; Wang, Yan; Ma, Ai-Jing; Li, Dong-Xun; Luo, Li-Juan; Liu, Dong-Xin; Jin, Dong; Liu, Kai; Ye, Chang-Yun

2015-07-08

We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61-65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.

Synchronous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment based on a zwitterionic copper (II) metal-organic framework.

PubMed

Qiu, Gui-Hua; Weng, Zi-Hua; Hu, Pei-Pei; Duan, Wen-Jun; Xie, Bao-Ping; Sun, Bin; Tang, Xiao-Yan; Chen, Jin-Xiang

2018-04-01

From a three-dimensional (3D) metal-organic framework (MOF) of {[Cu(Cmdcp)(phen)(H 2 O)] 2 ·9H 2 O} n (1, H 3 CmdcpBr = N-carboxymethyl-(3,5-dicarboxyl)pyridinium bromide, phen = phenanthroline), a sensitive and selective fluorescence sensor has been developed for the simultaneous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded microRNA-like (miRNA-like) fragment. The results from molecular dynamics simulation confirmed that MOF 1 absorbs carboxyfluorescein (FAM)-tagged and 5(6)-carboxyrhodamine, triethylammonium salt (ROX)-tagged probe ss-DNA (probe DNA, P-DNA) by π … π stacking and hydrogen bonding, as well as additional electrostatic interactions to form a sensing platform of P-DNAs@1 with quenched FAM and ROX fluorescence. In the presence of targeted ebolavirus conserved RNA sequences or ebolavirus-encoded miRNA-like fragment, the fluorophore-labeled P-DNA hybridizes with the analyte to give a P-DNA@RNA duplex and released from MOF 1, triggering a fluorescence recovery. Simultaneous detection of two target RNAs has also been realized by single and synchronous fluorescence analysis. The formed sensing platform shows high sensitivity for ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment with detection limits at the picomolar level and high selectivity without cross-reaction between the two probes. MOF 1 thus shows the potential as an effective fluorescent sensing platform for the synchronous detection of two ebolavirus-related sequences, and offer improved diagnostic accuracy of Ebola virus disease. Copyright © 2017 Elsevier B.V. All rights reserved.

"Bright" and "dark" excited states of an alternating at oligomer characterized by femtosecond broadband spectroscopy.

PubMed

Kwok, Wai-Ming; Ma, Chensheng; Phillips, David Lee

2009-08-20

The nature and dynamics of DNA excited states is of pivotal importance in determining both DNA ultraviolet photostability and its vulnerability toward photodamage. The complexity regarding the involvement of "bright" and "dark" excited states, their molecular origin, and the roles played by these states in the course of electronic energy relaxation constitute an active and contentious area in current research of DNA excited states. As a case study, we report here a combined broadband femtosecond time-resolved fluorescence (TRF) and transient absorption (TA) study on a self-complementary d(AT)(10) oligomer and a reference system of an equal molar mixture of the constituent bases represented by adenosine and thymidine (A+T). Comparison of the spectral character and temporal evolution of the TRF and TA data for 267 nm excited d(AT)(10) and A+T provides evidence for a base-localized excitation feature for an early (< approximately 50 fs) "bright" S(LE) state and its ensuing evolution within approximately 3 ps into a approximately 72 ps "dark" S(E) exciplex in d(AT)(10). Combined analysis of the d(AT)(10) TRF and TA results suggests the presence of a weakly fluorescent transient S(G) state that acts as a gateway to mediate the excitation transfer and energy elimination. A distinct base conformation-dependent model involving an ultrafast approximately 0.3 ps conversion of the S(LE) to S(G) that then evolves by approximately 3 ps into the S(E) has been proposed to account for the collective deactivation character of d(AT)(10). This presents a novel excited-state picture that can unify the seemingly conflicting time-resolved results reported previously for related AT DNAs. The direct spectral and dynamical data provided here contributes important photophysical parameters for the description of the excited states of AT oligomers. The possible connection between the energy transfer giving the S(E) and the photostability vs photodamage of A/T DNAs is briefly discussed.

A fluorescent 3,7-bis-(naphthalen-1-ylethynylated)-2'-deoxyadenosine analogue reports thymidine in complementary DNA by a large emission Stokes shift.

PubMed

Yanagi, Masaki; Suzuki, Azusa; Hudson, Robert H E; Saito, Yoshio

2018-02-28

The new environmentally responsive fluorescent nucleosides, 3,7-bis-(naphthalen-1-ylethynyl)-8-aza-3,7-dideaza-2'-deoxyadenosine (3n7nzA, 1) and 7-(naphthalen-1-ylethynyl)-8-aza-3,7-dideaza-2'-deoxyadenosine (37nzA, 2), have been synthesized. Both 3n7nzA (1) and 37nzA (2) possess large π-conjugated systems which extend into both the minor and major grooves or the major groove alone, respectively. The nucleosides exhibited large solvatochromic shifts (3n7nzA: Δλ = 45 nm, 37nzA: Δλ = 78 nm) and were examined for their ability to fluorimetrically report hybridization events. When incorporated into ODN probes, the bis-substituted 3n7nzA (1) selectively recognized thymidine on target strands which was reported by a distinct change in its emission wavelength in the long wavelength region, whereas 37nzA (2) showed a preference for pairing to cytidine and a smaller wavelength shift. Thus, 3n7nzA (1) has the potential for use as a fluorescent probe for structural studies of DNAs/RNAs including the detection of single-base alterations in target DNA sequences.

Synthesis of viral DNA forms in Nicotiana plumbaginifolia protoplasts inoculated with cassava latent virus (CLV); evidence for the independent replication of one component of the CLV genome.

PubMed Central

Townsend, R; Watts, J; Stanley, J

1986-01-01

Totipotent leaf mesophyll protoplasts of Nicotiana plumbaginifolia, Viviani were inoculated with cassava latent virus (CLV) or with full length copies of CLV genomic DNAs 1 and 2 excised from replicative forms of M13 clones. Virus specific DNAs began to appear 48-72h after inoculation with virus or cloned DNAs, coincident with the onset of host cell division. Infected cells accumulated supercoiled forms of DNAs 1 and 2 as well as progeny single-stranded (ss) virion (+) sense DNAs representing each component of the genome. Both supercoiled and ss molecules were synthesised by cells inoculated with cloned DNA 1 alone but DNA 2 failed to replicate independently. Images PMID:3951986

High-performance electrochemical mercury aptasensor based on synergistic amplification of Pt nanotube arrays and Fe3O4/rGO nanoprobes.

PubMed

Luo, Jingyi; Jiang, Danfeng; Liu, Tao; Peng, Jingmeng; Chu, Zhenyu; Jin, Wanqin

2018-05-01

In this work, a novel sandwich-type aptasensor was designed for the ultrasensitive recognition of trace mercury ions in water. Numerous oriented platinum nanotube arrays (PtNAs) were in-situ crystallized on a flexible electrode as a sensing interface, while thionine labelled Fe 3 O 4 /rGO nanocomposites as signal amplifiers. Both PtNAs/CF and nanocomposites were synthesized by easy hydrothermal processes. With their large surface area, it was favorable for electrochemical performance and immobilization of capture DNAs (cDNA) and report DNAs (rDNA). Upon the existence of Hg 2+ , partial linker DNAs were tightly bound with cDNAs through thymine-Hg 2+ -thymine pairing (T-Hg 2+ -T). Then rDNAs attached Fe 3 O 4 /rGO nanoprobes were fixed on the electrode through the match of remaining linker DNAs and rDNAs. Under the optimal conditions, the Hg 2+ aptasensor showed a synergistic amplification performance with a wide linear range from 0.1nM to 100nM, as well as a low detection limit of 30pM. Moreover, the as-prepared aptasensor also exhibited reliable performance for assay in real lake water samples. Copyright © 2017. Published by Elsevier B.V.

Novel method for preparation of the template DNA and selection of primers to differentiate the material rice cultivars of rice wine by PCR.

PubMed

Ohtsubo, Ken'ichi; Suzuki, Keitaro; Haraguchi, Kazutomo; Nakamura, Sumiko

2008-04-24

As many rice wine brewers label the name of the cultivar of the material rice, authentication technology is necessary. The problems are (1) decomposition of DNAs during the fermentation, (2) contamination of DNAs from microorganisms, (3) co-existence of PCR inhibitors, such as polyphenols. The present authors improved the PCR method by (1) lyophilizing and pulverizing the rice wine to concentrate DNAs, (2) decomposition of starches and proteins so as not to inhibit DNA extraction by the use of heat-resistant amylase and proteinase K, (3) purification of the template DNA by the combination of CTAB method and fractional precipitation by 70% EtOH. To prevent the amplification of microorganism's DNAs during PCR, the present authors selected the suitable plant-specific primers. It became possible to prepare the template DNAs for PCR from the rice wine. The sequences of the amplified DNAs by PCR were ascertained to be same with those of material rice. Mislabeling of material rice cultivar was detected by PCR using the commercial rice wine. It became possible to extract and purify the template DNAs for PCR from the rice wine and to differentiate the material rice cultivars by the PCR using the rice wine as a sample.

Infectious mutants of cassava latent virus generated in vivo from intact recombinant DNA clones containing single copies of the genome.

PubMed Central

Stanley, J; Townsend, R

1986-01-01

Intact recombinant DNAs containing single copies of either component of the cassava latent virus genome can elicit infection when mechanically inoculated to host plants in the presence of the appropriate second component. Characterisation of infectious mutant progeny viruses, by analysis of virus-specific supercoiled DNA intermediates, indicates that most if not all of the cloning vector has been deleted, achieved at least in some cases by intermolecular recombination in vivo between DNAs 1 and 2. Significant rearrangements within the intergenic region of DNA 2, predominantly external to the common region, can be tolerated without loss of infectivity suggesting a somewhat passive role in virus multiplication for the sequences in question. Although packaging constraints might impose limits on the amount of DNA within geminate particles, isolation of an infectious coat protein mutant defective in virion production suggests that packaging is not essential for systemic spread of the viral DNA. Images PMID:2875435

Before In Vivo Imaging: Evaluation of Fluorescent Probes Using Fluorescence Microscopy, Multiplate Reader, and Cytotoxicity Assays.

PubMed

Zhang, Shaojuan

2016-01-01

Fluorescent probes are widely utilized for noninvasive fluorescence imaging. Continuing efforts have been made in developing novel fluorescent probes with improved fluorescence quantum yield, enhanced target-specificity, and lower cytotoxicity. Before such probes are administrated into a living system, it is essential to evaluate the subcellular uptake, targeting specificity, and cytotoxicity in vitro. In this chapter, we briefly outline common methods used to evaluate fluorescent probes using fluorescence microscopy, multiplate reader, and cytotoxicity assay.

DNA Hairpins Containing the Cytidine Analog Pyrrolo-dC: Structural, Thermodynamic, and Spectroscopic Studies

PubMed Central

Zhang, Xu; Wadkins, Randy M.

2009-01-01

Structures formed by single-strand DNA have become increasingly interesting because of their roles in a number of biological processes, particularly transcription and its regulation. Of particular importance is the fact that antitumor drugs such as Actinomycin D can selectively bind DNA hairpins over fully paired, double-strand DNA. A new fluorescent base analog, pyrrolo-deoxycytidine (PdC), can now be routinely incorporated into single-strand DNA. The fluorescence of PdC is particularly useful for studying the formation of single-strand DNA in regions of double-strand DNA. The fluorescence is quenched when PdC is paired with a complementary guanine residue, and thus is greatly enhanced upon formation of single-strand DNA. Hence, any process that results in melting or opening of DNA strands produces an increase in the fluorescence intensity of this base analog. In this study we measured the structural effects of incorporating PdC into DNA hairpins, and the effect of this incorporation on the binding of the hairpins by a fluorescent analog of the drug Actinomycin D. Two hairpin DNAs were used: one with PdC in the stem (basepaired) and one with PdC in the loop (unpaired). The thermal stability, 7-aminoactinomycin D binding, and three-dimensional structures of PdC incorporated into these DNA hairpins were all quite similar as compared to the hairpins containing an unmodified dC residue. Fluorescence lifetime measurements indicate that two lifetimes are present in PdC, and that the increase in fluorescence of the unpaired PdC residue compared to the basepaired PdC is due to an increase in the contribution of the longer lifetime to the average fluorescence lifetime. Our data indicate that PdC can be used effectively to differentiate paired and unpaired bases in DNA hairpin secondary structures, and should be similarly applicable for related structures such as cruciforms and quadruplexes. Further, our data indicate that PdC can act as a fluorescence resonance energy transfer donor for the fluorescent drug 7-aminoactinomycin D. PMID:19254547

DNA hairpins containing the cytidine analog pyrrolo-dC: structural, thermodynamic, and spectroscopic studies.

PubMed

Zhang, Xu; Wadkins, Randy M

2009-03-04

Structures formed by single-strand DNA have become increasingly interesting because of their roles in a number of biological processes, particularly transcription and its regulation. Of particular importance is the fact that antitumor drugs such as Actinomycin D can selectively bind DNA hairpins over fully paired, double-strand DNA. A new fluorescent base analog, pyrrolo-deoxycytidine (PdC), can now be routinely incorporated into single-strand DNA. The fluorescence of PdC is particularly useful for studying the formation of single-strand DNA in regions of double-strand DNA. The fluorescence is quenched when PdC is paired with a complementary guanine residue, and thus is greatly enhanced upon formation of single-strand DNA. Hence, any process that results in melting or opening of DNA strands produces an increase in the fluorescence intensity of this base analog. In this study we measured the structural effects of incorporating PdC into DNA hairpins, and the effect of this incorporation on the binding of the hairpins by a fluorescent analog of the drug Actinomycin D. Two hairpin DNAs were used: one with PdC in the stem (basepaired) and one with PdC in the loop (unpaired). The thermal stability, 7-aminoactinomycin D binding, and three-dimensional structures of PdC incorporated into these DNA hairpins were all quite similar as compared to the hairpins containing an unmodified dC residue. Fluorescence lifetime measurements indicate that two lifetimes are present in PdC, and that the increase in fluorescence of the unpaired PdC residue compared to the basepaired PdC is due to an increase in the contribution of the longer lifetime to the average fluorescence lifetime. Our data indicate that PdC can be used effectively to differentiate paired and unpaired bases in DNA hairpin secondary structures, and should be similarly applicable for related structures such as cruciforms and quadruplexes. Further, our data indicate that PdC can act as a fluorescence resonance energy transfer donor for the fluorescent drug 7-aminoactinomycin D.

Single-molecule fluorescence reveals the unwinding stepping mechanism of replicative helicase.

PubMed

Syed, Salman; Pandey, Manjula; Patel, Smita S; Ha, Taekjip

2014-03-27

Bacteriophage T7 gp4 serves as a model protein for replicative helicases that couples deoxythymidine triphosphate (dTTP) hydrolysis to directional movement and DNA strand separation. We employed single-molecule fluorescence resonance energy transfer methods to resolve steps during DNA unwinding by T7 helicase. We confirm that the unwinding rate of T7 helicase decreases with increasing base pair stability. For duplexes containing >35% guanine-cytosine (GC) base pairs, we observed stochastic pauses every 2-3 bp during unwinding. The dwells on each pause were distributed nonexponentially, consistent with two or three rounds of dTTP hydrolysis before each unwinding step. Moreover, we observed backward movements of the enzyme on GC-rich DNAs at low dTTP concentrations. Our data suggest a coupling ratio of 1:1 between base pairs unwound and dTTP hydrolysis, and they further support the concept that nucleic acid motors can have a hierarchy of different-sized steps or can accumulate elastic energy before transitioning to a subsequent phase. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Armored DNA in recombinant Baculoviruses as controls in molecular genetic assays.

PubMed

Freystetter, Andrea; Paar, Christian; Stekel, Herbert; Berg, Jörg

2017-10-01

The widespread use of molecular PCR-based assays in analytical and clinical laboratories brings about the need for test-specific, stable, and reliable external controls (EC) as well as standards and internal amplification controls (IC), in order to arrive at consistent test results. In addition, there is also a growing need to produce and provide stable, well-characterized molecular controls for quality assurance programs. In this study, we describe a novel approach to generate armored double-stranded DNA controls, which are encapsulated in baculovirus (BV) particles of the species Autographa californica multiple nucleopolyhedrovirus. We used the well-known BacPAK™ Baculovirus Expression System (Takara-Clontech), removed the polyhedrin promoter used for protein expression, and generated recombinant BV-armored DNAs. The obtained BV-armored DNAs were readily extracted by standard clinical DNA extraction methods, showed favorable linearity and performance in our clinical PCR assays, were resistant to DNase I digestion, and exhibited marked stability in human plasma and serum. BV-armored DNA ought to be used as ECs, quantification standards, and ICs in molecular assays, with the latter application allowing for the entire monitoring of clinical molecular assays for sample adequacy. BV-armored DNA may also be used to produce double-stranded DNA reference materials for, e.g., quality assurance programs. The ease to produce BV-armored DNA should make this approach feasible for a broad spectrum of molecular applications. Finally, as BV-armored DNAs are non-infectious to mammals, they may be even more conveniently shipped than clinical specimen.

An artificial intelligence approach fit for tRNA gene studies in the era of big sequence data.

PubMed

Iwasaki, Yuki; Abe, Takashi; Wada, Kennosuke; Wada, Yoshiko; Ikemura, Toshimichi

2017-09-12

Unsupervised data mining capable of extracting a wide range of knowledge from big data without prior knowledge or particular models is a timely application in the era of big sequence data accumulation in genome research. By handling oligonucleotide compositions as high-dimensional data, we have previously modified the conventional self-organizing map (SOM) for genome informatics and established BLSOM, which can analyze more than ten million sequences simultaneously. Here, we develop BLSOM specialized for tRNA genes (tDNAs) that can cluster (self-organize) more than one million microbial tDNAs according to their cognate amino acid solely depending on tetra- and pentanucleotide compositions. This unsupervised clustering can reveal combinatorial oligonucleotide motifs that are responsible for the amino acid-dependent clustering, as well as other functionally and structurally important consensus motifs, which have been evolutionarily conserved. BLSOM is also useful for identifying tDNAs as phylogenetic markers for special phylotypes. When we constructed BLSOM with 'species-unknown' tDNAs from metagenomic sequences plus 'species-known' microbial tDNAs, a large portion of metagenomic tDNAs self-organized with species-known tDNAs, yielding information on microbial communities in environmental samples. BLSOM can also enhance accuracy in the tDNA database obtained from big sequence data. This unsupervised data mining should become important for studying numerous functionally unclear RNAs obtained from a wide range of organisms.

Simultaneous fluorescence light-up and selective multicolor nucleobase recognition based on sequence-dependent strong binding of berberine to DNA abasic site.

PubMed

Wu, Fei; Shao, Yong; Ma, Kun; Cui, Qinghua; Liu, Guiying; Xu, Shujuan

2012-04-28

Label-free DNA nucleobase recognition by fluorescent small molecules has received much attention due to its simplicity in mutation identification and drug screening. However, sequence-dependent fluorescence light-up nucleobase recognition and multicolor emission with individual emission energy for individual nucleobases have been seldom realized. Herein, an abasic site (AP site) in a DNA duplex was employed as a binding field for berberine, one of isoquinoline alkaloids. Unlike weak binding of berberine to the fully matched DNAs without the AP site, strong binding of berberine to the AP site occurs and the berberine's fluorescence light-up behaviors are highly dependent on the target nucleobases opposite the AP site in which the targets thymine and cytosine produce dual emission bands, while the targets guanine and adenine only give a single emission band. Furthermore, more intense emissions are observed for the target pyrimidines than purines. The flanking bases of the AP site also produce some modifications of the berberine's emission behavior. The binding selectivity of berberine at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, excited-state lifetime, DNA melting and fluorescence quenching by ferrocyanide and sodium chloride. It is expected that the target pyrimidines cause berberine to be stacked well within DNA base pairs near the AP site, which results in a strong resonance coupling of the electronic transitions to the particular vibration mode to produce the dual emissions. The fluorescent signal-on and emission energy-modulated sensing for nucleobases based on this fluorophore is substantially advantageous over the previously used fluorophores. We expect that this approach will be developed as a practical device for differentiating pyrimidines from purines by positioning an AP site toward a target that is available for readout by this alkaloid probe. This journal is © The Royal Society of Chemistry 2012

The mitochondrial genome of the gymnosperm Cycas taitungensis contains a novel family of short interspersed elements, Bpu sequences, and abundant RNA editing sites.

PubMed

Chaw, Shu-Miaw; Shih, Arthur Chun-Chieh; Wang, Daryi; Wu, Yu-Wei; Liu, Shu-Mei; Chou, The-Yuan

2008-03-01

The mtDNA of Cycas taitungensis is a circular molecule of 414,903 bp, making it 2- to 6-fold larger than the known mtDNAs of charophytes and bryophytes, but similar to the average of 7 elucidated angiosperm mtDNAs. It is characterized by abundant RNA editing sites (1,084), more than twice the number found in the angiosperm mtDNAs. The A + T content of Cycas mtDNA is 53.1%, the lowest among known land plants. About 5% of the Cycas mtDNA is composed of a novel family of mobile elements, which we designated as "Bpu sequences." They share a consensus sequence of 36 bp with 2 terminal direct repeats (AAGG) and a recognition site for the Bpu 10I restriction endonuclease (CCTGAAGC). Comparison of the Cycas mtDNA with other plant mtDNAs revealed many new insights into the biology and evolution of land plant mtDNAs. For example, the noncoding sequences in mtDNAs have drastically expanded as land plants have evolved, with abrupt increases appearing in the bryophytes, and then in the seed plants. As a result, the genomic organizations of seed plant mtDNAs are much less compact than in other plants. Also, the Cycas mtDNA appears to have been exempted from the frequent gene loss observed in angiosperm mtDNAs. Similar to the angiosperms, the 3 Cycas genes nad1, nad2, and nad5 are disrupted by 5 group II intron squences, which have brought the genes into trans-splicing arrangements. The evolutionary origin and invasion/duplication mechanism of the Bpu sequences in Cycas mtDNA are hypothesized and discussed.

Expression of three mammalian cDNAs that interfere with RAS function in Saccharomyces cerevisiae.

PubMed Central

Colicelli, J; Nicolette, C; Birchmeier, C; Rodgers, L; Riggs, M; Wigler, M

1991-01-01

Saccharomyces cerevisiae strains expressing the activated RAS2Val19 gene or lacking both cAMP phosphodiesterase genes, PDE1 and PDE2, have impaired growth control and display an acute sensitivity to heat shock. We have isolated two classes of mammalian cDNAs from yeast expression libraries that suppress the heat shock-sensitive phenotype of RAS2Val19 strain. Members of the first class of cDNAs also suppress the heat shock-sensitive phenotype of pde1- pde2- strains and encode cAMP phosphodiesterases. Members of the second class fail to suppress the phenotype of pde1- pde2- strains and therefore are candidate cDNAs encoding proteins that interact with RAS proteins. We report the nucleotide sequence of three members of this class. Two of these cDNAs share considerable sequence similarity, but none are clearly similar to previously isolated genes. Images PMID:1849280

DNA Translator and Aligner: HyperCard utilities to aid phylogenetic analysis of molecules.

PubMed

Eernisse, D J

1992-04-01

DNA Translator and Aligner are molecular phylogenetics HyperCard stacks for Macintosh computers. They manipulate sequence data to provide graphical gene mapping, conversions, translations and manual multiple-sequence alignment editing. DNA Translator is able to convert documented GenBank or EMBL documented sequences into linearized, rescalable gene maps whose gene sequences are extractable by clicking on the corresponding map button or by selection from a scrolling list. Provided gene maps, complete with extractable sequences, consist of nine metazoan, one yeast, and one ciliate mitochondrial DNAs and three green plant chloroplast DNAs. Single or multiple sequences can be manipulated to aid in phylogenetic analysis. Sequences can be translated between nucleic acids and proteins in either direction with flexible support of alternate genetic codes and ambiguous nucleotide symbols. Multiple aligned sequence output from diverse sources can be converted to Nexus, Hennig86 or PHYLIP format for subsequent phylogenetic analysis. Input or output alignments can be examined with Aligner, a convenient accessory stack included in the DNA Translator package. Aligner is an editor for the manual alignment of up to 100 sequences that toggles between display of matched characters and normal unmatched sequences. DNA Translator also generates graphic displays of amino acid coding and codon usage frequency relative to all other, or only synonymous, codons for approximately 70 select organism-organelle combinations. Codon usage data is compatible with spreadsheet or UWGCG formats for incorporation of additional molecules of interest. The complete package is available via anonymous ftp and is free for non-commercial uses.

Fluorescence quenching studies of potential-dependent DNA reorientation dynamics at glassy carbon electrode surfaces.

PubMed

Li, Qin; Cui, Chenchen; Higgins, Daniel A; Li, Jun

2012-09-05

The potential-dependent reorientation dynamics of double-stranded DNA (ds-DNA) attached to planar glassy carbon electrode (GCE) surfaces were investigated. The orientation state of surface-bound ds-DNA was followed by monitoring the fluorescence from a 6-carboxyfluorescein (FAM6) fluorophore covalently linked to the distal end of the DNA. Positive potentials (i.e., +0.2 V vs open circuit potential, OCP) caused the ds-DNA to align parallel to the electrode surface, resulting in strong dipole-electrode quenching of FAM6 fluorescence. Switching of the GCE potential to negative values (i.e., -0.2 V vs OCP) caused the ds-DNA to reorient perpendicular to the electrode surface, with a concomitant increase in FAM6 fluorescence. In addition to the very fast (submilliseconds) dynamics of the initial reorientation process, slow (0.1-0.9 s) relaxation of FAM6 fluorescence to intermediate levels was also observed after potential switching. These dynamics have not been previously described in the literature. They are too slow to be explained by double layer charging, and chronoamperometry data showed no evidence of such effects. Both the amplitude and rate of the dynamics were found to depend upon buffer concentration, and ds-DNA length, demonstrating a dependence on the double layer field. The dynamics are concluded to arise from previously undetected complexities in the mechanism of potential-dependent ds-DNA reorientation. The possible origins of these dynamics are discussed. A better understanding of these dynamics will lead to improved models for potential-dependent ds-DNA reorientation at electrode surfaces and will facilitate the development of advanced electrochemical devices for detection of target DNAs.

Dynamics of bleomycin interaction with a strongly bound hairpin DNA substrate, and implications for cleavage of the bound DNA.

PubMed

Bozeman, Trevor C; Nanjunda, Rupesh; Tang, Chenhong; Liu, Yang; Segerman, Zachary J; Zaleski, Paul A; Wilson, W David; Hecht, Sidney M

2012-10-31

Recent studies involving DNAs bound strongly by bleomycins have documented that such DNAs are degraded by the antitumor antibiotic with characteristics different from those observed when studying the cleavage of randomly chosen DNAs in the presence of excess Fe·BLM. In the present study, surface plasmon resonance has been used to characterize the dynamics of BLM B(2) binding to a strongly bound hairpin DNA, to define the effects of Fe(3+), salt, and temperature on BLM-DNA interaction. One strong primary DNA binding site, and at least one much weaker site, were documented. In contrast, more than one strong cleavage site was found, an observation also made for two other hairpin DNAs. Evidence is presented for BLM equilibration between the stronger and weaker binding sites in a way that renders BLM unavailable to other, less strongly bound DNAs. Thus, enhanced binding to a given site does not necessarily result in increased DNA degradation at that site; i.e., for strongly bound DNAs, the facility of DNA cleavage must involve other parameters in addition to the intrinsic rate of C-4' H atom abstraction from DNA sugars.

Two divergent endo-beta-1,4-glucanase genes exhibit overlapping expression in ripening fruit and abscising flowers.

PubMed Central

Lashbrook, C C; Gonzalez-Bosch, C; Bennett, A B

1994-01-01

Two structurally divergent endo-beta-1,4-glucanase (EGase) cDNAs were cloned from tomato. Although both cDNAs (Cel1 and Cel2) encode potentially glycosylated, basic proteins of 51 to 53 kD and possess multiple amino acid domains conserved in both plant and microbial EGases, Cel1 and Cel2 exhibit only 50% amino acid identity at the overall sequence level. Amino acid sequence comparisons to other plant EGases indicate that tomato Cel1 is most similar to bean abscission zone EGase (68%), whereas Cel2 exhibits greatest sequence identity to avocado fruit EGase (57%). Sequence comparisons suggest the presence of at least two structurally divergent EGase families in plants. Unlike ripening avocado fruit and bean abscission zones in which a single EGase mRNA predominates, EGase expression in tomato reflects the overlapping accumulation of both Cel1 and Cel2 transcripts in ripening fruit and in plant organs undergoing cell separation. Cel1 mRNA contributes significantly to total EGase mRNA accumulation within plant organs undergoing cell separation (abscission zones and mature anthers), whereas Cel2 mRNA is most abundant in ripening fruit. The overlapping expression of divergent EGase genes within a single species may suggest that multiple activities are required for the cooperative disassembly of cell wall components during fruit ripening, floral abscission, and anther dehiscence. PMID:7994180

Time-resolved multicolor two-photon excitation fluorescence microscopy of cells and tissues

NASA Astrophysics Data System (ADS)

Zheng, Wei

2014-11-01

Multilabeling which maps the distribution of different targets is an indispensable technique in many biochemical and biophysical studies. Two-photon excitation fluorescence (TPEF) microscopy of endogenous fluorophores combining with conventional fluorescence labeling techniques such as genetically encoded fluorescent protein (FP) and fluorescent dyes staining could be a powerful tool for imaging living cells. However, the challenge is that the excitation and emission wavelength of these endogenous fluorophores and fluorescent labels are very different. A multi-color ultrafast source is required for the excitation of multiple fluorescence molecules. In this study, we developed a two-photon imaging system with excitations from the pump femtosecond laser and the selected supercontinuum generated from a photonic crystal fiber (PCF). Multiple endogenous fluorophores, fluorescent proteins and fluorescent dyes were excited in their optimal wavelengths simultaneously. A time- and spectral-resolved detection system was used to record the TPEF signals. This detection technique separated the TPEF signals from multiple sources in time and wavelength domains. Cellular organelles such as nucleus, mitochondria, microtubule and endoplasmic reticulum, were clearly revealed in the TPEF images. The simultaneous imaging of multiple fluorophores of cells will greatly aid the study of sub-cellular compartments and protein localization.

African-American mitochondrial DNAs often match mtDNAs found in multiple African ethnic groups

PubMed Central

Ely, Bert; Wilson, Jamie Lee; Jackson, Fatimah; Jackson, Bruce A

2006-01-01

Background Mitochondrial DNA (mtDNA) haplotypes have become popular tools for tracing maternal ancestry, and several companies offer this service to the general public. Numerous studies have demonstrated that human mtDNA haplotypes can be used with confidence to identify the continent where the haplotype originated. Ideally, mtDNA haplotypes could also be used to identify a particular country or ethnic group from which the maternal ancestor emanated. However, the geographic distribution of mtDNA haplotypes is greatly influenced by the movement of both individuals and population groups. Consequently, common mtDNA haplotypes are shared among multiple ethnic groups. We have studied the distribution of mtDNA haplotypes among West African ethnic groups to determine how often mtDNA haplotypes can be used to reconnect Americans of African descent to a country or ethnic group of a maternal African ancestor. The nucleotide sequence of the mtDNA hypervariable segment I (HVS-I) usually provides sufficient information to assign a particular mtDNA to the proper haplogroup, and it contains most of the variation that is available to distinguish a particular mtDNA haplotype from closely related haplotypes. In this study, samples of general African-American and specific Gullah/Geechee HVS-I haplotypes were compared with two databases of HVS-I haplotypes from sub-Saharan Africa, and the incidence of perfect matches recorded for each sample. Results When two independent African-American samples were analyzed, more than half of the sampled HVS-I mtDNA haplotypes exactly matched common haplotypes that were shared among multiple African ethnic groups. Another 40% did not match any sequence in the database, and fewer than 10% were an exact match to a sequence from a single African ethnic group. Differences in the regional distribution of haplotypes were observed in the African database, and the African-American haplotypes were more likely to match haplotypes found in ethnic groups from West or West Central Africa than those found in eastern or southern Africa. Fewer than 14% of the African-American mtDNA sequences matched sequences from only West Africa or only West Central Africa. Conclusion Our database of sub-Saharan mtDNA sequences includes the most common haplotypes that are shared among ethnic groups from multiple regions of Africa. These common haplotypes have been found in half of all sub-Saharan Africans. More than 60% of the remaining haplotypes differ from the common haplotypes at a single nucleotide position in the HVS-I region, and they are likely to occur at varying frequencies within sub-Saharan Africa. However, the finding that 40% of the African-American mtDNAs analyzed had no match in the database indicates that only a small fraction of the total number of African haplotypes has been identified. In addition, the finding that fewer than 10% of African-American mtDNAs matched mtDNA sequences from a single African region suggests that few African Americans might be able to trace their mtDNA lineages to a particular region of Africa, and even fewer will be able to trace their mtDNA to a single ethnic group. However, no firm conclusions should be made until a much larger database is available. It is clear, however, that when identical mtDNA haplotypes are shared among many ethnic groups from different parts of Africa, it is impossible to determine which single ethnic group was the source of a particular maternal ancestor based on the mtDNA sequence. PMID:17038170

Ultrasensitive FRET-based DNA sensor using PNA/DNA hybridization.

PubMed

Yang, Lan-Hee; Ahn, Dong June; Koo, Eunhae

2016-12-01

In the diagnosis of genetic diseases, rapid and highly sensitive DNA detection is crucial. Therefore, many strategies for detecting target DNA have been developed, including electrical, optical, and mechanical methods. Herein, a highly sensitive FRET based sensor was developed by using PNA (Peptide Nucleic Acid) probe and QD, in which red color QDs are hybridized with capture probes, reporter probes and target DNAs by EDC-NHS coupling. The hybridized probe with target DNA gives off fluorescent signal due to the energy transfer from QD to Cy5 dye in the reporter probe. Compared to the conventional DNA sensor using DNA probes, the DNA sensor using PNA probes shows higher FRET factor and efficiency due to the higher reactivity between PNA and target DNA. In addition, to elicit the effect of the distance between the donor and the acceptor, we have investigated two types of the reporter probes having Cy5 dyes attached at the different positions of the reporter probes. Results show that the shorter the distance between QDs and Cy5s, the stronger the signal intensity. Furthermore, based on the fluorescence microscopy images using microcapillary chips, the FRET signal is enhanced to be up to 276% times stronger than the signal obtained using the cuvette by the fluorescence spectrometer. These results suggest that the PNA probe system conjugated with QDs can be used as ultrasensitive DNA nanosensors. Copyright © 2016. Published by Elsevier B.V.

Generation of recombinant rotaviruses expressing fluorescent proteins using an optimized reverse genetics system.

PubMed

Komoto, Satoshi; Fukuda, Saori; Ide, Tomihiko; Ito, Naoto; Sugiyama, Makoto; Yoshikawa, Tetsushi; Murata, Takayuki; Taniguchi, Koki

2018-04-18

An entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids for NSP2 and NSP5 genes. Utilizing this highly efficient system, we then engineered infectious recombinant rotaviruses expressing bioluminescent (NanoLuc luciferase) and fluorescent (EGFP and mCherry) reporters. These recombinant rotaviruses expressing reporters remained genetically stable during serial passages. Our reverse genetics approach and recombinant rotaviruses carrying reporter genes will be great additions to the tool kit for studying the molecular virology of rotavirus, and for developing future next-generation vaccines and expression vectors. IMPORTANCE Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. In this paper, we describe a robust and simple reverse genetics system based on only rotavirus cDNAs, and its application for engineering infectious recombinant rotaviruses harboring bioluminescent (NanoLuc) and fluorescent (EGFP and mCherry) protein genes. This highly efficient reverse genetics system and recombinant RVAs expressing reporters could be powerful tools for the study of different aspects of rotavirus replication. Furthermore, they may be useful for next-generation vaccine production for this medically important virus. Copyright © 2018 American Society for Microbiology.

Electron microscopic studies of bacteriophage M13 DNA replication. [Escherichia coli

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

Allison, D.P.; Ganesan, A.T.; Olson, A.C.

Intracellular forms of M13 phage DNA isolated after infection of Escherichia coli with wild-type phage have been studied by electron microscopy and ultracentrifugation. The data indicate the involvement of rolling-circle intermediates in single-stranded DNA synthesis. In addition to single-stranded, circular DNA, we observed covalently closed and nicked replicative-form (RF) DNAs, dimer RF DNAs, concatenated RF DNAs, RF DNAs with single-stranded tails (sigma, rolling circles), and, occasionally, RF DNAs with theta structures. The tails in sigma molecules are always single stranded and are never longer than the DNA from mature phage; the proportion of sigma to other RF molecules does notmore » change significantly with time after infection. The origin of single-stranded DNA synthesis has been mapped by electron microscopy at a unique location on RF DNA by use of partial denaturation mapping and restriction endonuclease digestion. This location is between gene IV and gene II, and synthesis proceeds in a counterclockwise direction on the conventional genetic map.« less

Fluorescent DNA-templated silver nanoclusters

NASA Astrophysics Data System (ADS)

Lin, Ruoqian

Because of the ultra-small size and biocompatibility of silver nanoclusters, they have attracted much research interest for their applications in biolabeling. Among the many ways of synthesizing silver nanoclusters, DNA templated method is particularly attractive---the high tunability of DNA sequences provides another degree of freedom for controlling the chemical and photophysical properties. However, systematic studies about how DNA sequences and concentrations are controlling the photophysical properties are still lacking. The aim of this thesis is to investigate the binding mechanisms of silver clusters binding and single stranded DNAs. Here in this thesis, we report synthesis and characterization of DNA-templated silver nanoclusters and provide a systematic interrogation of the effects of DNA concentrations and sequences, including lengths and secondary structures. We performed a series of syntheses utilizing five different sequences to explore the optimal synthesis condition. By characterizing samples with UV-vis and fluorescence spectroscopy, we achieved the most proper reactants ratio and synthesis conditions. Two of them were chosen for further concentration dependence studies and sequence dependence studies. We found that cytosine-rich sequences are more likely to produce silver nanoclusters with stronger fluorescence signals; however, sequences with hairpin secondary structures are more capable in stabilizing silver nanoclusters. In addition, the fluorescence peak emission intensities and wavelengths of the DNA templated silver clusters have sequence dependent fingerprints. This potentially can be applied to sequence sensing in the future. However all the current conclusions are not warranted; there is still difficulty in formulating general rules in DNA strand design and silver nanocluster production. Further investigation of more sequences could solve these questions in the future.

Interaction Potency of Single-Walled Carbon Nanotubes with DNAs: A Novel Assay for Assessment of Hazard Risk

PubMed Central

Yao, Chunhe; Carlisi, Cristina; Li, Yuning; Chen, Da; Ding, Jianfu; Feng, Yong-Lai

2016-01-01

Increasing use of single-walled carbon nanotubes (SWCNTs) necessitates a novel method for hazard risk assessment. In this work, we investigated the interaction of several types of commercial SWCNTs with single-stranded (ss) and double-stranded (ds) DNA oligonucleotides (20-mer and 20 bp). Based on the results achieved, we proposed a novel assay that employed the DNA interaction potency to assess the hazard risk of SWCNTs. It was found that SWCNTs in different sizes or different batches of the same product number of SWCNTs showed dramatically different potency of interaction with DNAs. In addition, the same SWCNTs also exerted strikingly different interaction potency with ss- versus ds- DNAs. The interaction rates of SWCNTs with DNAs were investigated, which could be utilized as the indicator of potential hazard for acute exposure. Compared to solid SWCNTs, the SWCNTs dispersed in liquid medium (2% sodium cholate solution) exhibited dramatically different interaction potency with DNAs. This indicates that the exposure medium may greatly influence the subsequent toxicity and hazard risk produced by SWCNTs. Based on the findings of dose-dependences and time-dependences from the interactions between SWCNTs and DNAs, a new chemistry based assay for hazard risk assessment of nanomaterials including SWCNTs has been presented. PMID:27936089

Interaction Potency of Single-Walled Carbon Nanotubes with DNAs: A Novel Assay for Assessment of Hazard Risk.

PubMed

Yao, Chunhe; Carlisi, Cristina; Li, Yuning; Chen, Da; Ding, Jianfu; Feng, Yong-Lai

2016-01-01

Increasing use of single-walled carbon nanotubes (SWCNTs) necessitates a novel method for hazard risk assessment. In this work, we investigated the interaction of several types of commercial SWCNTs with single-stranded (ss) and double-stranded (ds) DNA oligonucleotides (20-mer and 20 bp). Based on the results achieved, we proposed a novel assay that employed the DNA interaction potency to assess the hazard risk of SWCNTs. It was found that SWCNTs in different sizes or different batches of the same product number of SWCNTs showed dramatically different potency of interaction with DNAs. In addition, the same SWCNTs also exerted strikingly different interaction potency with ss- versus ds- DNAs. The interaction rates of SWCNTs with DNAs were investigated, which could be utilized as the indicator of potential hazard for acute exposure. Compared to solid SWCNTs, the SWCNTs dispersed in liquid medium (2% sodium cholate solution) exhibited dramatically different interaction potency with DNAs. This indicates that the exposure medium may greatly influence the subsequent toxicity and hazard risk produced by SWCNTs. Based on the findings of dose-dependences and time-dependences from the interactions between SWCNTs and DNAs, a new chemistry based assay for hazard risk assessment of nanomaterials including SWCNTs has been presented.

Evolutionary dynamics and sites of illegitimate recombination revealed in the interspersion and sequence junctions of two nonhomologous satellite DNAs in cactophilic Drosophila species.

PubMed

Kuhn, G C S; Teo, C H; Schwarzacher, T; Heslop-Harrison, J S

2009-05-01

Satellite DNA (satDNA) is a major component of genomes but relatively little is known about the fine-scale organization of unrelated satDNAs residing at the same chromosome location, and the sequence structure and dynamics of satDNA junctions. We studied the organization and sequence junctions of two nonhomologous satDNAs, pBuM and DBC-150, in three species from the neotropical Drosophila buzzatii cluster (repleta group). In situ hybridization to microchromosomes, interphase nuclei and extended DNA fibers showed frequent interspersion of the two satellites in D. gouveai, D. antonietae and, to a lesser extent, D. seriema. We isolated by PCR six pBuM x DBC-150 junctions: four are exclusive to D. gouveai and two are exclusive to D. antonietae. The six junction breakpoints occur at different positions within monomers, suggesting independent origin. Four junctions showed abrupt transitions between the two satellites, whereas two junctions showed a distinct 10 bp tandem duplication before the junction. Unlike pBuM, DBC-150 junction repeats are more variable than randomly cloned monomers and showed diagnostic features in common to a 3-monomer higher-order repeat seen in the sister species D. serido. The high levels of interspersion between pBuM and DBC-150 repeats suggest extensive rearrangements between the two satellites, maybe favored by specific features of the microchromosomes. Our interpretation is that the junctions evolved by multiples events of illegitimate recombination between nonhomologous satDNA repeats, with subsequent rounds of unequal crossing-over expanding the copy number of some of the junctions.

Microarray-Based Analysis of Subnanogram Quantities of Microbial Community DNAs by Using Whole-Community Genome Amplification†

PubMed Central

Wu, Liyou; Liu, Xueduan; Schadt, Christopher W.; Zhou, Jizhong

2006-01-01

Microarray technology provides the opportunity to identify thousands of microbial genes or populations simultaneously, but low microbial biomass often prevents application of this technology to many natural microbial communities. We developed a whole-community genome amplification-assisted microarray detection approach based on multiple displacement amplification. The representativeness of amplification was evaluated using several types of microarrays and quantitative indexes. Representative detection of individual genes or genomes was obtained with 1 to 100 ng DNA from individual or mixed genomes, in equal or unequal abundance, and with 1 to 500 ng community DNAs from groundwater. Lower concentrations of DNA (as low as 10 fg) could be detected, but the lower template concentrations affected the representativeness of amplification. Robust quantitative detection was also observed by significant linear relationships between signal intensities and initial DNA concentrations ranging from (i) 0.04 to 125 ng (r2 = 0.65 to 0.99) for DNA from pure cultures as detected by whole-genome open reading frame arrays, (ii) 0.1 to 1,000 ng (r2 = 0.91) for genomic DNA using community genome arrays, and (iii) 0.01 to 250 ng (r2 = 0.96 to 0.98) for community DNAs from ethanol-amended groundwater using 50-mer functional gene arrays. This method allowed us to investigate the oligotrophic microbial communities in groundwater contaminated with uranium and other metals. The results indicated that microorganisms containing genes involved in contaminant degradation and immobilization are present in these communities, that their spatial distribution is heterogeneous, and that microbial diversity is greatly reduced in the highly contaminated environment. PMID:16820490

Investigating the Genetic Diversity, Population Differentiation and Population Dynamics of Cycas segmentifida (Cycadaceae) Endemic to Southwest China by Multiple Molecular Markers

PubMed Central

Feng, Xiuyan; Liu, Jian; Chiang, Yu-Chung; Gong, Xun

2017-01-01

Climate change, species dispersal ability and habitat fragmentation are major factors influencing species distribution and genetic diversity, especially for the range-restricted and threatened taxa. Here, using four sequences of chloroplast DNAs (cpDNAs), three nuclear genes (nDNAs) and 12 nuclear microsatellites (SSRs), we investigated the genetic diversity, genetic structure, divergence time and population dynamics of Cycas segmentifida D. Y. Wang and C. Y. Deng, a threatened cycad species endemic to Southwest China. High levels of genetic diversity and genetic differentiation were revealed in C. segmentifida. Haplotypes of networks showed two evolutionary units in C. segmentifida, with the exception of the nuclear gene GTP network. Meanwhile, the UPGMA tree, structure and PCoA analyses suggested that 14 populations of C. segmentifida were divided into two clades. There was significant effect of isolation by distance (IBD) in this species. However, this species did not display a significant phylogeographic structure. The divergence time estimation suggested that its haplotypes diverged during the Middle Pleistocene. Additionally, the population dynamics inferred from different DNA sequences analyses were discordant. Bottleneck analysis showed that populations of C. segmentifida did not experience any recent bottleneck effect, but rather pointed to a contraction of its effective population size over time. Furthermore, our results suggested that the population BM which held an intact population structure and occupied undisturbed habitat was at the Hardy–Weinberg equilibrium, implying that this population is a free-mating system. These genetic features provide important information for the sustainable management of C. segmentifida. PMID:28580005

A homogeneous, Anti-dsDNA antibody-based assay for multicolor detection of cancer stem cell transcription factors.

PubMed

Ma, Jiehua; Shi, Hai; Zhang, Meiling; Li, Chao; Xiang, Yang; Liu, Ping

2018-10-31

Cancer stem cells (CSCs) are responsible for maintaining tumor growth, metastasis and recurrence. The high expression of cancer stem cell transcription factors (Oct4, Sox2 and Nanog) is a valuable prognostic factor, suggesting a higher risk of tumor recurrence and metastasis. So, the development of a convenient and cost-effective method for multiplex assay of these transcription factors (TFs) is highly required. In this work, we have proposed a universal homogeneous assay for multicolor detection of these TFs based on anti-dsDNA antibody-decorated Fe 3 O 4 magnetite nanoparticles (aadMNPs). In the presence of analytes, the dye-labeled dsDNAs are bound by specific TFs, which will inhibit the interactions between the dsDNAs and aadMNPs, generating higher fluorescence that may provide signal readout for the immunosensing process. By using the proposed method, Oct4 can be determined in a linear range from 3 to 1200 ng/mL with a detection limit of 0.035 ng/mL. Furthermore, we have presented assays for the sensitive, selective and rapid detection of Oct4, Sox2 and Nanog in cell extract, as well as the analysis of binding affinity of the mutated binding sequences. This work may provide potential applications in clinical CSCs detections, and may open new opportunity for the study of nucleotide polymorphisms in TF binding sites. Copyright © 2018 Elsevier B.V. All rights reserved.

The Molecular Cytogenetic Characterization of Pistachio (Pistacia vera L.) Suggests the Arrest of Recombination in the Largest Heteropycnotic Pair HC1.

PubMed

Sola-Campoy, Pedro J; Robles, Francisca; Schwarzacher, Trude; Ruiz Rejón, Carmelo; de la Herrán, Roberto; Navajas-Pérez, Rafael

2015-01-01

This paper represents the first molecular cytogenetic characterization of the strictly dioecious pistachio tree (Pistacia vera L.). The karyotype was characterized by fluorescent in situ hybridization (FISH) with probes for 5S and 45S rDNAs, and the pistachio specific satellite DNAs PIVE-40, and PIVE-180, together with DAPI-staining. PIVE-180 has a monomeric unit of 176-178 bp and high sequence homology between family members; PIVE-40 has a 43 bp consensus monomeric unit, and is most likely arranged in higher order repeats (HORs) of two units. The P. vera genome is highly heterochromatic, and prominent DAPI positive blocks are detected in most chromosomes. Despite the difficulty in classifying chromosomes according to morphology, 10 out of 15 pairs (2n = 30) could be distinguished by their unique banding patterns using a combination of FISH probes. Significantly, the largest pair, designated HC1, is strongly heteropycnotic, shows differential condensation, and has massive enrichment in PIVE-40 repeats. There are two types of HC1 chromosomes (type-I and type-II) with differing PIVE-40 hybridization signal. Only type-I/II heterozygotes and type-I homozygotes individuals were found. We speculate that the differentiation between the two HC1 chromosomes is due to suppression of homologous recombination at meiosis, reinforced by the presence of PIVE-40 HORs and differences in PIVE-40 abundance. This would be compatible with a ZW sex-determination system in the pistachio tree.

The Molecular Cytogenetic Characterization of Pistachio (Pistacia vera L.) Suggests the Arrest of Recombination in the Largest Heteropycnotic Pair HC1

PubMed Central

Sola-Campoy, Pedro J.; Robles, Francisca; Schwarzacher, Trude; Ruiz Rejón, Carmelo; de la Herrán, Roberto; Navajas-Pérez, Rafael

2015-01-01

This paper represents the first molecular cytogenetic characterization of the strictly dioecious pistachio tree (Pistacia vera L.). The karyotype was characterized by fluorescent in situ hybridization (FISH) with probes for 5S and 45S rDNAs, and the pistachio specific satellite DNAs PIVE-40, and PIVE-180, together with DAPI-staining. PIVE-180 has a monomeric unit of 176–178 bp and high sequence homology between family members; PIVE-40 has a 43 bp consensus monomeric unit, and is most likely arranged in higher order repeats (HORs) of two units. The P. vera genome is highly heterochromatic, and prominent DAPI positive blocks are detected in most chromosomes. Despite the difficulty in classifying chromosomes according to morphology, 10 out of 15 pairs (2n = 30) could be distinguished by their unique banding patterns using a combination of FISH probes. Significantly, the largest pair, designated HC1, is strongly heteropycnotic, shows differential condensation, and has massive enrichment in PIVE-40 repeats. There are two types of HC1 chromosomes (type-I and type-II) with differing PIVE-40 hybridization signal. Only type-I/II heterozygotes and type-I homozygotes individuals were found. We speculate that the differentiation between the two HC1 chromosomes is due to suppression of homologous recombination at meiosis, reinforced by the presence of PIVE-40 HORs and differences in PIVE-40 abundance. This would be compatible with a ZW sex-determination system in the pistachio tree. PMID:26633808

Reassociation and hybridization properties of DNAs from several species of fish

USGS Publications Warehouse

Gharrett, A.J.; Simon, R.C.; McIntyre, J.D.

1977-01-01

Reassociation and hybridization properties from spectrophotometric studies of DNAs from 10 species of fish indicate:1. Great diversity in the amounts of repeated sequences in the genomes of different species - more specialized fish had less redundancy.2. Large differences in the complexities of the DNAs - more specialized fish had less information.3. Little homology between sequences of remotely related species but substantial homology between sequences of closely related species.

Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar Micro-Tom, a reference system for the Solanaceae genomics.

PubMed

Aoki, Koh; Yano, Kentaro; Suzuki, Ayako; Kawamura, Shingo; Sakurai, Nozomu; Suda, Kunihiro; Kurabayashi, Atsushi; Suzuki, Tatsuya; Tsugane, Taneaki; Watanabe, Manabu; Ooga, Kazuhide; Torii, Maiko; Narita, Takanori; Shin-I, Tadasu; Kohara, Yuji; Yamamoto, Naoki; Takahashi, Hideki; Watanabe, Yuichiro; Egusa, Mayumi; Kodama, Motoichiro; Ichinose, Yuki; Kikuchi, Mari; Fukushima, Sumire; Okabe, Akiko; Arie, Tsutomu; Sato, Yuko; Yazawa, Katsumi; Satoh, Shinobu; Omura, Toshikazu; Ezura, Hiroshi; Shibata, Daisuke

2010-03-30

The Solanaceae family includes several economically important vegetable crops. The tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. Recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. In particular, a miniature cultivar, Micro-Tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the Micro-Tom-background, such as ESTs and mutagenized lines, have been established by an international alliance. To accelerate the progress in tomato genomics, we developed a collection of fully-sequenced 13,227 Micro-Tom full-length cDNAs. By checking redundant sequences, coding sequences, and chimeric sequences, a set of 11,502 non-redundant full-length cDNAs (nrFLcDNAs) was generated. Analysis of untranslated regions demonstrated that tomato has longer 5'- and 3'-untranslated regions than most other plants but rice. Classification of functions of proteins predicted from the coding sequences demonstrated that nrFLcDNAs covered a broad range of functions. A comparison of nrFLcDNAs with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. Mapping of the nrFLcDNAs onto currently available tomato genome sequences facilitated prediction of exon-intron structure. Introns of tomato genes were longer than those of Arabidopsis and rice. According to a comparison of exon sequences between the nrFLcDNAs and the tomato genome sequences, the frequency of nucleotide mismatch in exons between Micro-Tom and the genome-sequencing cultivar (Heinz 1706) was estimated to be 0.061%. The collection of Micro-Tom nrFLcDNAs generated in this study will serve as a valuable genomic tool for plant biologists to bridge the gap between basic and applied studies. The nrFLcDNA sequences will help annotation of the tomato whole-genome sequence and aid in tomato functional genomics and molecular breeding. Full-length cDNA sequences and their annotations are provided in the database KaFTom http://www.pgb.kazusa.or.jp/kaftom/ via the website of the National Bioresource Project Tomato http://tomato.nbrp.jp.

Characterization of cDNAs and genomic DNAs for human threonyl- and cysteinyl-tRNA synthetases

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

Cruzen, M.E.

1993-01-01

Techniques of molecular biology were used to clone, sequence and map two human aminoacyl-tRNA synthetase (aaRS) cDNAs: threonyl-tRNA synthetase (ThrRS) a class II enzyme and cysteinyl-tRNA synthetase (CysRS) a class I enzyme. The predicted protein sequence of human ThrRS is highly homologous to that of lower eukaryotic and prokaryotic ThRSs, particularly in the regions containing the three structural motifs common to all class II synthetases. Signature regions 1 and 2, which characterize the class IIa subgroup (SerRS, ThrRS and HisRS) are highly conserved from bacteria to human. Structural predictions for human ThrRS based on the known structure of the closelymore » related SerRS from E.coli implicate strongly conserved residues in the signature sequences to be important in substrate binding. The amino terminal 100 residues of the deduced amino acid sequence of ThrRS shares structural similarity to SerRS consistent with forming an antiparallel helix implicated in tRNA binding. The 5' untranslated sequence of the human ThrRS gene shares short stretches of common sequence with the gene for hamster HisRS including a binding site for the promoter specific transcription factor sp-1. The deduced amino acid sequence of human CysRS has a high degree of sequence identify to E. coli CysRS. Human CysRS possesses the classic characteristics of a class I synthetase and is most closely related to the MetRS subgroup. The amino terminal half of human CysRS can be modeled as a nucleotide binding fold and shares significant sequence and structural similarity to the other enzymes in this subgroup. The CysRS structural gene (CARS) was mapped to human chromosome 11p15.5 by fluorescent in situ hybridization. CARS is the first aaRS gene to be mapped to chromosome 11. The steady state of both CysRS and ThrRs mRNA were quantitated in several human tissues. Message levels for these enzymes appear to be subjected to differential regulation in different cell types.« less

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

Culbert, A.A.; Wallis, G.A.; Kadler, K.E.

The brittleness of bone in people with lethal (type II) osteogenesis imperfecta, a heritable disorder caused by mutations in the type I collagen genes, arises from the deposition of abnormal collagen in the bone matrix. The inability of the abnormal collagen to participate in mineralization may be caused by its failure to interact with other bone proteins. Here, we have designed a strategy to isolate the genes important for mineralization of collagen during bone formation. Cells isolated from 16-day embryonic chick calvaria and seeded post-confluence in culture deposited a mineralized matrix over a period of 2 weeks. Chick skin fibroblastsmore » seeded and cultured under the same conditions did not mineralize. Using RT-PCR, we prepared short cDNAs ({approximately}300 bp) corresponding to the 3{prime} ends of mRNA from fibroblasts and separately from the mineralizing calvarial cells. Subtractive cDNA hybridization generated a pool of cDNAs that were specific to mineralizing calvarial cells but not to fibroblasts. Screening of 100,000 plaques of a chick bone ZAP Express cDNA library with this pool of mineralizing-specific cDNAs identified ten clones which comprised full-length cDNAs for the bone proteins osteopontin (eight of the ten positives), bone sialoprotein II (one of the ten positives), and cystatin (one of the ten positives). cDNAs for type I collagen, fibronectin, alkaline phosphatase, house-keeping genes, and other genes expressed in fibroblasts were not identified in this preliminary screen. The pool of short cDNAs is likely to comprise cDNAs for further bone-specific genes and will be used to screen the entire bone cDNA library of 4.2 million clones. 30 refs., 4 figs.« less

Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

PubMed

Hong, Y K; Kim, D H; Beletskii, A; Lee, C; Memili, E; Strauss, W M

2001-04-01

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells. Copyright 2001 Academic Press.

Altered Subcellular Localization of a Tobacco Membrane Raft-Associated Remorin Protein by Tobamovirus Infection and Transient Expression of Viral Replication and Movement Proteins

PubMed Central

Sasaki, Nobumitsu; Takashima, Eita; Nyunoya, Hiroshi

2018-01-01

Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexvirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by tomato mosaic virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamovirus movement. PMID:29868075

Isolation and characterization of cDNAs encoding wheat 3-hydroxy-3-methylglutaryl coenzyme A reductase.

PubMed Central

Aoyagi, K; Beyou, A; Moon, K; Fang, L; Ulrich, T

1993-01-01

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is a key enzyme in the isoprenoid biosynthetic pathway. We have isolated partial cDNAs from wheat (Triticum aestivum) using the polymerase chain reaction. Comparison of deduced amino acid sequences of these cDNAs shows that they represent a small family of genes that share a high degree of sequence homology among themselves as well as among genes from other organisms including tomato, Arabidopsis, hamster, human, Drosophila, and yeast. Southern blot analysis reveals the presence of at least four genes. Our results concerning the tissue-specific expression as well as developmental regulation of these HMGR cDNAs highlight the important role of this enzyme in the growth and development of wheat. PMID:8108513

Profiling of wheat class III peroxidase genes derived from powdery mildew-attacked epidermis reveals distinct sequence-associated expression patterns.

PubMed

Liu, Guosheng; Sheng, Xiaoyan; Greenshields, David L; Ogieglo, Adam; Kaminskyj, Susan; Selvaraj, Gopalan; Wei, Yangdou

2005-07-01

A cDNA library was constructed from leaf epidermis of diploid wheat (Triticum monococcum) infected with the powdery mildew fungus (Blumeria graminis f. sp. tritici) and was screened for genes encoding peroxidases. From 2,500 expressed sequence tags (ESTs), 36 cDNAs representing 10 peroxidase genes (designated TmPRX1 to TmPRX10) were isolated and further characterized. Alignment of the deduced amino acid sequences and phylogenetic clustering with peroxidases from other plant species demonstrated that these peroxidases fall into four distinct groups. Differential expression and tissue-specific localization among the members were observed during the B. graminis f. sp. tritici attack using Northern blots and reverse-transcriptase polymerase chain reaction analyses. Consistent with its abundance in the EST collection, TmPRX1 expression showed the highest induction during pathogen attack and fluctuated in response to the fungal parasitic stages. TmPRX1 to TmPRX6 were expressed predominantly in mesophyll cells, whereas TmPRX7 to TmPRX10, which feature a putative C-terminal propeptide, were detectable mainly in epidermal cells. Using TmPRX8 as a representative, we demonstrated that its C-terminal propeptide was sufficient to target a green fluorescent protein fusion protein to the vacuoles in onion cells. Finally, differential expression profiles of the TmPRXs after abiotic stresses and signal molecule treatments were used to dissect the potential role of these peroxidases in multiple stress and defense pathways.

Idiopathic slow transit constipation and megacolon are not associated with neurturin mutations.

PubMed

Chen, B; Knowles, C H; Scott, M; Anand, P; Williams, N S; Milbrandt, J; Tam, P K H

2002-10-01

Chronic idiopathic slow-transit constipation (ISTC) and idiopathic megacolon (IMC) are early-onset gastrointestinal motility disorders of unknown aetiology. The gene encoding the neurotrophic factor neurturin may be a candidate for these disorders, as neurturin-deficient mice have a similar enteric phenotype. In the present study, we tested this hypothesis. Genomic DNA from 26 cases of chronic idiopathic STC [with a family history of constipation in 15 (58%) and Hirschsprung's disease in two (8%)], and five cases of IMC [two familial (40%)] was screened by direct DNA sequencing using the fluorescent dideoxy terminator method. Results were compared with published sequence data and 24 control DNAs. Our results revealed several previously unreported common sequence polymorphisms, but overall frequencies were comparable between patients and controls. We conclude that mutation of neurturin is not a frequent cause of ISTC or IMC.

Gaucher disease: Pseudoreversion of a disease mutation`s effects--implications for structure/function and genotype/phenotype correlations

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

Ponce, E.; Mear, J; Grabowski, G.A.

1994-09-01

Numerous mutations ({approximately}45) of the acid {beta}-glucosidase gene have been identified in patients with Gaucher disease. Many of these have been characterized by partial sequencing of cDNAs derived by RT-PCR or PCR of genomic DNA. In addition, genotype/phenotype correlations have been based on screening for known mutations. Thus, only a part of the gene is characterized in any population of affected patients. Several Gaucher disease alleles contain multiple, authentic point mutations that raises concern about conclusions based on only partial genetic characterization. Several wild-type cDNAs for acid {beta}-glucosidase have been sequenced. One contained a cloning artifact encoding R495H. We expressedmore » this cDNA and showed that the R495H enzyme had normal kinetic and stability properties. A disease-associated allele encoding R496H has been found by several groups. The close association and similarities of these two substitutions led us to question the disease casuality of the R496H allele. To evaluate this, we created and/or expressed cDNAs encoding R495, R496 (wild-type), (R495H, R496), (R495, R496H) and (R495H, R496H). The (wild-type) and (R495H, R496) enzymes had indistinguishable properties whereas the (R495, R496H) enzyme was essentially inactive. The introduction of both mutations (R495H, R496H) produced an enzyme whose activity was 25 to 50% of the wild-type. These results indicate that a pseudoreversion to a functional enzyme can occur by introducing a functionally neutral mutation together with a severe mutation. These results have major implications to structure/function and genotype/phenotype correlations in this disease.« less

Missing genes, multiple ORFs, and C-to-U type RNA editing in Acrasis kona (Heterolobosea, Excavata) mitochondrial DNA.

PubMed

Fu, Cheng-Jie; Sheikh, Sanea; Miao, Wei; Andersson, Siv G E; Baldauf, Sandra L

2014-08-21

Discoba (Excavata) is an ancient group of eukaryotes with great morphological and ecological diversity. Unlike the other major divisions of Discoba (Jakobida and Euglenozoa), little is known about the mitochondrial DNAs (mtDNAs) of Heterolobosea. We have assembled a complete mtDNA genome from the aggregating heterolobosean amoeba, Acrasis kona, which consists of a single circular highly AT-rich (83.3%) molecule of 51.5 kb. Unexpectedly, A. kona mtDNA is missing roughly 40% of the protein-coding genes and nearly half of the transfer RNAs found in the only other sequenced heterolobosean mtDNAs, those of Naegleria spp. Instead, over a quarter of A. kona mtDNA consists of novel open reading frames. Eleven of the 16 protein-coding genes missing from A. kona mtDNA were identified in its nuclear DNA and polyA RNA, and phylogenetic analyses indicate that at least 10 of these 11 putative nuclear-encoded mitochondrial (NcMt) proteins arose by direct transfer from the mitochondrion. Acrasis kona mtDNA also employs C-to-U type RNA editing, and 12 homologs of DYW-type pentatricopeptide repeat (PPR) proteins implicated in plant organellar RNA editing are found in A. kona nuclear DNA. A mapping of mitochondrial gene content onto a consensus phylogeny reveals a sporadic pattern of relative stasis and rampant gene loss in Discoba. Rampant loss occurred independently in the unique common lineage leading to Heterolobosea + Tsukubamonadida and later in the unique lineage leading to Acrasis. Meanwhile, mtDNA gene content appears to be remarkably stable in the Acrasis sister lineage leading to Naegleria and in their distant relatives Jakobida. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Identification of Genes Potentially Responsible for extra-Oral Digestion and Overcoming Plant Defense from Salivary Glands of the Tarnished Plant Bug (Hemiptera: Miridae) Using cDNA Sequencing

PubMed Central

Zhu, Yu-Cheng; Yao, Jianxiu; Luttrell, Randall

2016-01-01

Saliva is known to play a crucial role in tarnished plant bug (TPB, Lygus lineolaris [Palisot de Beauvois]) feeding. By facilitating the piercing, the enzyme-rich saliva may be used for extra-oral digestion and for overcoming plant defense before the plant fluids are ingested by TPBs. To identify salivary gland genes, mRNA was extracted from salivary glands and cDNA library clones were sequenced. A de novo-assembling of 7,000 Sanger sequences revealed 666 high-quality unique cDNAs with an average size of 624 bp, in which the identities of 347 cDNAs were determined using Blast2GO. Kyoto Encyclopedia of Genes and Genomes analysis indicated that these genes participate in eighteen metabolic pathways. Identifications of large number of enzyme genes in TPB salivary glands evidenced functions for extra-oral digestion and feeding damage mechanism, including 45 polygalacturonase, two α- amylase, one glucosidase, one glycan enzyme, one aminopeptidase, four lipase, and many serine protease cDNAs. The presence of multiple transcripts, multigene members, and high abundance of cell wall degradation enzymes (polygalacturonases) indicated that the enzyme-rich saliva may cause damage to plants by breaking down plant cell walls to make nutrients available for feeding. We also identified genes potentially involved in insect adaptation and detoxifying xenobiotics that may allow insects to overcome plant defense responses, including four glutathione S-transferases, three esterases, one cytochrome P450, and several serine proteases. The gene profiles of TPB salivary glands revealed in this study provides a foundation for further understanding and potential development of novel enzymatic inhibitors, or other RNAi approaches that may interrupt or minimize TPB feeding damage. PMID:27324587

Extracellular vesicle-mediated transfer of donor genomic DNA to recipient cells is a novel mechanism for genetic influence between cells

PubMed Central

Cai, Jin; Han, Yu; Ren, Hongmei; Chen, Caiyu; He, Duofen; Zhou, Lin; Eisner, Gilbert M.; Asico, Laureano D.; Jose, Pedro A.; Zeng, Chunyu

2013-01-01

Extracellular vesicles (EVs) carry signals within or at their limiting membranes, providing a mechanism by which cells can exchange more complex information than what was previously thought. In addition to mRNAs and microRNAs, there are DNA fragments in EVs. Solexa sequencing indicated the presence of at least 16434 genomic DNA (gDNA) fragments in the EVs from human plasma. Immunofluorescence study showed direct evidence that acridine orange-stained EV DNAs could be transferred into the cells and localize to and inside the nuclear membrane. However, whether the transferred EV DNAs are functional or not is not clear. We found that EV gDNAs could be homologously or heterologously transferred from donor cells to recipient cells, and increase gDNA-coding mRNA, protein expression, and function (e.g. AT1 receptor). An endogenous promoter of the AT1 receptor, NF-κB, could be recruited to the transferred DNAs in the nucleus, and increase the transcription of AT1 receptor in the recipient cells. Moreover, the transferred EV gDNAs have pathophysiological significance. BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia, could be transferred from K562 EVs to HEK293 cells or neutrophils. Our present study shows that the gDNAs transferred from EVs to cells have physiological significance, not only to increase the gDNA-coding mRNA and protein levels, but also to influence function in recipient cells. PMID:23580760

The past, present and future of mitochondrial genomics: have we sequenced enough mtDNAs?

PubMed

Smith, David Roy

2016-01-01

The year 2014 saw more than a thousand new mitochondrial genome sequences deposited in GenBank-an almost 15% increase from the previous year. Hundreds of peer-reviewed articles accompanied these genomes, making mitochondrial DNAs (mtDNAs) the most sequenced and reported type of eukaryotic chromosome. These mtDNA data have advanced a wide range of scientific fields, from forensics to anthropology to medicine to molecular evolution. But for many biological lineages, mtDNAs are so well sampled that newly published genomes are arguably no longer contributing significantly to the progression of science, and in some cases they are tying up valuable resources, particularly journal editors and referees. Is it time to acknowledge that as a research community we have published enough mitochondrial genome papers? Here, I address this question, exploring the history, milestones and impacts of mitochondrial genomics, the benefits and drawbacks of continuing to publish mtDNAs at a high rate and what the future may hold for such an important and popular genetic marker. I highlight groups for which mtDNAs are still poorly sampled, thus meriting further investigation, and recommend that more energy be spent characterizing aspects of mitochondrial genomes apart from the DNA sequence, such as their chromosomal and transcriptional architectures. Ultimately, one should be mindful before writing a mitochondrial genome paper. Consider perhaps sending the sequence directly to GenBank instead, and be sure to annotate it correctly before submission. © The Author 2015. Published by Oxford University Press.

DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication.

PubMed Central

Saunders, K; Lucy, A; Stanley, J

1991-01-01

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2). Images PMID:2041773

Quantitative fluorescence-polymerase chain reaction assay for the detection of the duplication of the Charcot Marie Tooth disease type 1A critical region.

PubMed

De Toffol, Simona; Bellone, Emilia; Dulcetti, Francesca; Ruggeri, Anna Maria; Maggio, Pietro Paolo; Pulimeno, Maria Rosaria; Mandich, Paola; Maggi, Federico; Simoni, Giuseppe; Grati, Francesca Romana

2010-04-01

Charcot Marie Tooth (CMT) syndrome is the most common hereditary peripheral neuropathy, with an incidence of about 1 in 2500. The subtype 1A (CMT1A) is caused by a tandem duplication of a 1.5-Mb region encompassing the PMP22 gene. Conventional short tandem repeat (STR) analysis can reveal this imbalance if a triallelic pattern, defining with certainty the presence of duplication, is present. In case of duplication with a biallelic pattern, it can only indicate a semiquantitative dosage of the fluorescence intensity ratio of the two fragments. In this study we developed a quantitative fluorescence-PCR using seven highly informative STRs within the CMT1A critical region that successfully disclosed or excluded the presence of the pathogenic imbalance in a cohort of 60 samples including 40 DNAs from samples with the CMT1A duplication previously characterized with two different molecular approaches, and 20 diagnostic samples from 10 members of a five-generation pedigree segregating CMT1A, 8 unrelated cases and 2 prenatal samples. The application of the quantitative fluorescence-PCR using STRs located in the critical region could be a reliable method to evaluate the presence of the PMP22 duplication for the diagnosis and classification of hereditary neuropathies in asymptomatic subjects with a family history of inherited neuropathy, in prenatal samples in cases with one affected parent, and in unrelated patients with a sporadic demyelinating neuropathy with clinical features resembling CMT (i.e., pes cavus with hammer toes) or with conduction velocities in the range of CMT1A.

Hairpin DNA-Templated Silver Nanoclusters as Novel Beacons in Strand Displacement Amplification for MicroRNA Detection.

PubMed

Zhang, Jingpu; Li, Chao; Zhi, Xiao; Ramón, Gabriel Alfranca; Liu, Yanlei; Zhang, Chunlei; Pan, Fei; Cui, Daxiang

2016-01-19

MicroRNA (miRNA) biomarkers display great potential for cancer diagnosis and prognosis. The development of rapid and specific methods for miRNA detection has become a hotspot. Herein, hairpin DNA-templated silver nanoclusters (AgNCs/HpDNA) were prepared and integrated into strand-displacement amplification (SDA) as a novel beacon for miRNA detection. The light-up platform was established based on guanine (G)-rich fluorescence enhancement that essentially converted the excitation/emission pair of AgNCs/HpDNAs from a shorter wavelength to a longer wavelength, and then achieved fluorescent enhancement at longer wavelength. On the basis of the validation of the method, the single and duplex detection were conducted in two plasma biomarkers (miR-16-5p and miR-19b-3p) for the diagnosis of gastric cancer. The probe (AgNCs/RED 16(7s)C) utilized for miR-16-5p detection adopted a better conformation with high specificity to recognize single-base mismatches by producing dramatically opposite signals (increase or decrease at 580 nm ex/640 nm em) while the probe (AgNCs/GRE 19b(5s)C) for miR-19b-3p generated dual signals (increase at 490 nm ex/570 nm em and decrease at 430 nm ex/530 nm em) with bright fluorescence in one reaction during the amplification, but unexpectedly was partially digested. This is for the first time to allow the generation of enhanced fluorescent AgNCs and the target recognition integrated into a single process, which offers great opportunity for specific miRNA detection in an easy and rapid way.

Tuning Fluorescence Direction with Plasmonic Metal–Dielectric– Metal Substrates

PubMed Central

Choudhury, Sharmistha Dutta; Badugu, Ramachandram; Nowaczyk, Kazimierz; Ray, Krishanu; Lakowicz, Joseph R.

2013-01-01

Controlling the emission properties of fluorophores is essential for improving the performance of fluorescence-based techniques in modern biochemical research, medical diagnosis, and sensing. Fluorescence emission is isotropic in nature, which makes it difficult to capture more than a small fraction of the total emission. Metal– dielectric–metal (MDM) substrates, discussed in this Letter, convert isotropic fluorescence into beaming emission normal to the substrate. This improves fluorescence collection efficiency and also opens up new avenues for a wide range of fluorescence-based applications. We suggest that MDM substrates can be readily adapted for multiple uses, such as in microarray formats, for directional fluorescence studies of multiple probes or for molecule-specific sensing with a high degree of spatial control over the fluorescence emission. SECTION: Physical Processes in Nanomaterials and Nanostructures PMID:24013521

Different visible colors and green fluorescence were obtained from the mutated purple chromoprotein isolated from sea anemone.

PubMed

Chiang, Cheng-Yi; Chen, Yi-Lin; Tsai, Huai-Jen

2014-08-01

Green fluorescent protein (GFP)-like proteins have been studied with the aim of developing fluorescent proteins. Since the property of color variation is understudied, we isolated a novel GFP-like chromoprotein from the carpet anemone Stichodactyla haddoni, termed shCP. Its maximum absorption wavelength peak (λ(max)) is located at 574 nm, resulting in a purple color. The shCP protein consists of 227 amino acids (aa), sharing 96 % identity with the GFP-like chromoprotein of Heteractis crispa. We mutated aa residues to examine any alteration in color. When E63, the first aa of the chromophore, was replaced by serine (E63S), the λ(max) of the mutated protein shCP-E63S was shifted to 560 nm and exhibited a pink color. When Q39, T194, and I196, which reside in the surrounding 5 Å of the chromophore's microenvironment, were mutated, we found that (1) the λ(max) of the mutated protein shCP-Q39S was shifted to 518 nm and exhibited a red color, (2) shCP-T194I exhibited a purple-blue color, and (3) an additional mutation at I196H of the mutated protein shCP-E63L exhibited green fluorescence. In contrast, when the aa located neither at the chromophore nor within its microenvironment were mutated, the resultant proteins shCP-L122H, -E138G, -S137D, -T95I, -D129N, -T194V, -E138Q, -G75E, -I183V, and -I70V never altered their purple color, suggesting that mutations at the shCP chromophore and the surrounding 5 Å microenvironment mostly control changes in color expression or cause fluorescence to develop. Additionally, we found that the cDNAs of shCP and its mutated varieties are faithfully and stably expressed both in Escherichia coli and zebrafish embryos.

Distribution and threshold expression of the tRNA[sup Lys] mutation in skeletal muscle of patients with myoclonic epilepsy and ragged-red fibers (MERRF)

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

Boulet, L.; Karpati, G.; Shoubridge, E.A.

1992-12-01

The authors investigated the distribution and expression of mutant mtDNAs carrying the A-to-G mutation at position 8344 in the tRNA[sup Lys] gene in the skeletal muscle of four patients with myoclonus epilepsy and ragged-red fibers (MERRF). The proportion of mutant genomes was greater than 80% of total mtDNAs in muscle samples of all patients and was associated with a decrease in the activity of cytochrome c oxidase (COX). The vast majority of myoblasts, cloned from the satellite-cell population in the same muscles, were homoplasmic for the mutation. The overall proportion of mutant mtDNAs in this population was similar to thatmore » in differentiated muscle, suggesting that the ratio of mutant to wild-type mtDNAs in skeletal muscle is determined either in the ovum or during early development and changes little with age. Translation of all mtDNA-encoded genes was severely depressed in homoplasmic mutant myoblast clones but not in heteroplasmic or wild-type clones. The threshold for biochemical expression of the mutation was determined in heteroplasmic myotubes formed by fusion of different proportions of mutant and wild-type myoblasts. The magnitude of the decrease in translation in myotubes containing mutant mtDNAs was protein specific. Complex I and IV subunits were more affected than complex V subunits, and there was a rough correlation with both protein size and number of lysine residues. Approximately 15% wild-type mtDNAs restored translation and COX activity to near normal levels. These results show that the A-to-G substitution in tRNA[sup Lys] is a functionally recessive mutation that can be rescued by intraorganellar complementation with a small proportion of wild-type mtDNAs and explain the steep threshold for expression of the MERRF clinical phenotype. 40 refs., 7 figs., 2 tabs.« less

Double-hairpin molecular-beacon-based amplification detection for gene diagnosis linked to cancer.

PubMed

Xu, Huo; Zhang, Rongbo; Li, Feng; Zhou, Yingying; Peng, Ting; Wang, Xuedong; Shen, Zhifa

2016-09-01

A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related KRAS gene detection based on the one-to-two stoichiometry. During target DNA detection, DHMB can execute signal transduction even if no any exogenous element is involved. Unlike the conventional molecular beacon based on the one-to-one interaction, one target DNA not only hybridizes with one DHMB and opens its hairpin but also promotes the interaction between two DHMBs, causing the separation of two fluorophores from quenchers. This leads to an enhanced fluorescence signal. As a result, the target KRAS gene is able to be detected within a wide dynamic range from 0.05 to 200 nM with the detection limit of 50 pM, indicating a dramatic improvement compared with traditional molecular beacons. Moreover, the point mutations existing in target DNAs can be easily screened. The potential application for target species in real samples was indicated by the analysis of PCR amplicons of DNAs from the DNA extracted from SW620 cell. Besides becoming a promising candidate probe for molecular biology research and clinical diagnosis of genetic diseases, the DHMB is expected to provide a significant insight into the design of DNA probe-based homogenous sensing systems. Graphical Abstract A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related gene KRAS detection based on the one-to-two stoichiometry. Without the help of any exogenous probe, the point mutation is easily screened, and the target DNA can be quantified down to 50 pM, indicating a dramatic improvement compared with traditional molecular beacons.

Initial genome sequencing and analysis of multiple myeloma

PubMed Central

Chapman, Michael A.; Lawrence, Michael S.; Keats, Jonathan J.; Cibulskis, Kristian; Sougnez, Carrie; Schinzel, Anna C.; Harview, Christina L.; Brunet, Jean-Philippe; Ahmann, Gregory J.; Adli, Mazhar; Anderson, Kenneth C.; Ardlie, Kristin G.; Auclair, Daniel; Baker, Angela; Bergsagel, P. Leif; Bernstein, Bradley E.; Drier, Yotam; Fonseca, Rafael; Gabriel, Stacey B.; Hofmeister, Craig C.; Jagannath, Sundar; Jakubowiak, Andrzej J.; Krishnan, Amrita; Levy, Joan; Liefeld, Ted; Lonial, Sagar; Mahan, Scott; Mfuko, Bunmi; Monti, Stefano; Perkins, Louise M.; Onofrio, Robb; Pugh, Trevor J.; Vincent Rajkumar, S.; Ramos, Alex H.; Siegel, David S.; Sivachenko, Andrey; Trudel, Suzanne; Vij, Ravi; Voet, Douglas; Winckler, Wendy; Zimmerman, Todd; Carpten, John; Trent, Jeff; Hahn, William C.; Garraway, Levi A.; Meyerson, Matthew; Lander, Eric S.; Getz, Gad; Golub, Todd R.

2013-01-01

Multiple myeloma is an incurable malignancy of plasma cells, and its pathogenesis is poorly understood. Here we report the massively parallel sequencing of 38 tumor genomes and their comparison to matched normal DNAs. Several new and unexpected oncogenic mechanisms were suggested by the pattern of somatic mutation across the dataset. These include the mutation of genes involved in protein translation (seen in nearly half of the patients), genes involved in histone methylation, and genes involved in blood coagulation. In addition, a broader than anticipated role of NF-κB signaling was suggested by mutations in 11 members of the NF-κB pathway. Of potential immediate clinical relevance, activating mutations of the kinase BRAF were observed in 4% of patients, suggesting the evaluation of BRAF inhibitors in multiple myeloma clinical trials. These results indicate that cancer genome sequencing of large collections of samples will yield new insights into cancer not anticipated by existing knowledge. PMID:21430775

Ferrocenemonocarboxylic-HRP@Pt nanoparticles labeled RCA for multiple amplification of electro-immunosensing.

PubMed

Su, Huilan; Yuan, Ruo; Chai, Yaqin; Mao, Li; Zhuo, Ying

2011-07-15

A multiple amplification immunoassay was proposed to detect alpha-fetoprotein (AFP), which was based on ferrocenemonocarboxylic-HRP conjugated on Pt nanoparticles as labels for rolling circle amplification (RCA). Firstly, the capture antibody (anti-AFP) was immobilized on glass carbon electrode (GCE) deposited nano-sized gold particles. After a typical immuno-sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which acted as a precursor to initiate RCA. The products of RCA provide large amount of sites to link detection DNAs, which were labeled by signal probes (ferrocenemonocarboxylic) and horseradish peroxidase (HRP). Moreover, the enzymatic amplification signals could be produced by the catalysis of HRP and Pt nanoparticles with the addition of H₂O₂. These lead to multiple amplification signals monitoring by electrochemical instrument and further resulted in high sensitivity of the immunoassay with the detection limit of 1.7 pg/mL. Copyright © 2011 Elsevier B.V. All rights reserved.

A Modular Lentiviral and Retroviral Construction System to Rapidly Generate Vectors for Gene Expression and Gene Knockdown In Vitro and In Vivo

PubMed Central

Geiling, Benjamin; Vandal, Guillaume; Posner, Ada R.; de Bruyns, Angeline; Dutchak, Kendall L.; Garnett, Samantha; Dankort, David

2013-01-01

The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems. PMID:24146852

eTumorType, An Algorithm of Discriminating Cancer Types for Circulating Tumor Cells or Cell-free DNAs in Blood.

PubMed

Zou, Jinfeng; Wang, Edwin

2017-04-01

With the technology development on detecting circulating tumor cells (CTCs) and cell-free DNAs (cfDNAs) in blood, serum, and plasma, non-invasive diagnosis of cancer becomes promising. A few studies reported good correlations between signals from tumor tissues and CTCs or cfDNAs, making it possible to detect cancers using CTCs and cfDNAs. However, the detection cannot tell which cancer types the person has. To meet these challenges, we developed an algorithm, eTumorType, to identify cancer types based on copy number variations (CNVs) of the cancer founding clone. eTumorType integrates cancer hallmark concepts and a few computational techniques such as stochastic gradient boosting, voting, centroid, and leading patterns. eTumorType has been trained and validated on a large dataset including 18 common cancer types and 5327 tumor samples. eTumorType produced high accuracies (0.86-0.96) and high recall rates (0.79-0.92) for predicting colon, brain, prostate, and kidney cancers. In addition, relatively high accuracies (0.78-0.92) and recall rates (0.58-0.95) have also been achieved for predicting ovarian, breast luminal, lung, endometrial, stomach, head and neck, leukemia, and skin cancers. These results suggest that eTumorType could be used for non-invasive diagnosis to determine cancer types based on CNVs of CTCs and cfDNAs. Copyright © 2017 Beijing Institute of Genomics, Chinese Academy of Sciences and Genetics Society of China. Production and hosting by Elsevier B.V. All rights reserved.

Multiple components in restriction enzyme digests of mammalian (insectivore), avian and reptilian genomic DNA hybridize with murine immunoglobulin VH probes.

PubMed

Litman, G W; Berger, L; Jahn, C L

1982-06-11

High molecular weight genomic DNAs isolated from an insectivore, Tupaia, and a representative reptilian, Caiman, and avian, Gallus, were digested with restriction endonucleases transferred to nitrocellulose and hybridized with nick-translated probes of murine VH genes. The derivations of the probes designated S107V (1) and mu 107V (2,3) have been described previously. Under conditions of reduced stringency, multiple hybridizing components were observed with Tupaia and Caiman; only mu mu 107V exhibited significant hybridization with the separated fragments of Gallus DNA. The nick-translated S107V probe was digested with Fnu4H1 and subinserts corresponding to the 5' and 3' regions both detected multiple hybridizing components in Tupaia and Caiman DNA. A 5' probe lacking the leader sequence identified the same components as the intact 5' probe, suggesting that VH coding regions distant as the reptilians may possess multiple genetic components which exhibit significant homology with murine immunoglobulin in VH regions.

Multiple components in restriction enzyme digests of mammalian (insectivore), avian and reptilian genomic DNA hybridize with murine immunoglobulin VH probes.

PubMed Central

Litman, G W; Berger, L; Jahn, C L

1982-01-01

High molecular weight genomic DNAs isolated from an insectivore, Tupaia, and a representative reptilian, Caiman, and avian, Gallus, were digested with restriction endonucleases transferred to nitrocellulose and hybridized with nick-translated probes of murine VH genes. The derivations of the probes designated S107V (1) and mu 107V (2,3) have been described previously. Under conditions of reduced stringency, multiple hybridizing components were observed with Tupaia and Caiman; only mu mu 107V exhibited significant hybridization with the separated fragments of Gallus DNA. The nick-translated S107V probe was digested with Fnu4H1 and subinserts corresponding to the 5' and 3' regions both detected multiple hybridizing components in Tupaia and Caiman DNA. A 5' probe lacking the leader sequence identified the same components as the intact 5' probe, suggesting that VH coding regions distant as the reptilians may possess multiple genetic components which exhibit significant homology with murine immunoglobulin in VH regions. Images PMID:6285298

Automatic cytometric device using multiple wavelength excitations

NASA Astrophysics Data System (ADS)

Rongeat, Nelly; Ledroit, Sylvain; Chauvet, Laurence; Cremien, Didier; Urankar, Alexandra; Couderc, Vincent; Nérin, Philippe

2011-05-01

Precise identification of eosinophils, basophils, and specific subpopulations of blood cells (B lymphocytes) in an unconventional automatic hematology analyzer is demonstrated. Our specific apparatus mixes two excitation radiations by means of an acousto-optics tunable filter to properly control fluorescence emission of phycoerythrin cyanin 5 (PC5) conjugated to antibodies (anti-CD20 or anti-CRTH2) and Thiazole Orange. This way our analyzer combining techniques of hematology analysis and flow cytometry based on multiple fluorescence detection, drastically improves the signal to noise ratio and decreases the spectral overlaps impact coming from multiple fluorescence emissions.

Tree-hierarchy of DNA and distribution of Holliday junctions.

PubMed

Rozikov, U A

2017-12-01

We define a DNA as a sequence of [Formula: see text]'s and embed it on a path of Cayley tree. Using group representation of the Cayley tree, we give a hierarchy of a countable set of DNAs each of which 'lives' on the same Cayley tree. This hierarchy has property that each vertex of the Cayley tree belongs only to one of DNA. Then we give a model (energy, Hamiltonian) of this set of DNAs by an analogue of Ising model with three spin values (considered as DNA base pairs) on a set of admissible configurations. To study thermodynamic properties of the model of DNAs we describe corresponding translation invariant Gibbs measures (TIGM) of the model on the Cayley tree of order two. We show that there is a critical temperature [Formula: see text] such that (i) if temperature [Formula: see text] then there exists unique TIGM; (ii) if [Formula: see text] then there are two TIGMs; (iii) if [Formula: see text] then there are three TIGMs. Each such measure describes a phase of the set of DNAs. We use these results to study distributions of Holliday junctions and branches of DNAs. In case of very high and very low temperatures we give stationary distributions and typical configurations of the Holliday junctions.

The isolation of cDNAs from OATL1 at Xp11.2 using a 480-kb YAC

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

Geraghty, M.T.; Brody, L.C.; Martin, L.S.

1993-05-01

Using an ornithine-{delta}-aminotransferase (OAT) cDNA, the authors identified five YACs that cover two nonadjacent OAT-related loci in Xp11.2-p11.3, designated OATL1 (distal) and OATL2 (proximal). Because several retinal degenerative disorders map to this region, they used YAC2 (480 kb), which covers the most distal part of OATL1, as a probe to screen a retinal cDNA library. From 8 {times} 10{sup 4} plaques screened, they isolated 13 clones. Two were OAT cDNAs. The remaining 11 were divided into eight groups by cross-hybridization. Groups 1-4 contain cDNAs that originate from single-copy X-linked genes in YAC2. Each has an open reading frame of >500more » bp and detects one or more transcripts on a Northern blot. The gene for each was sublocalized and ordered in YAC2. The cDNAs in groups 5-8 contained two or more Alu sequences, had no open reading frames, and did not detect transcripts. The cDNAs from groups 1-4 provide expressed sequence tags and identify candidate genes for the genetic disorders that map to this region. 28 refs., 5 figs., 1 tab.« less

Factor Structure Evaluation of the French Version of the Digital Natives Assessment Scale.

PubMed

Wagner, Vincent; Acier, Didier

2017-03-01

"Digital natives" concept defines young adults particularly familiar with emerging technologies such as computers, smartphones, or Internet. This notion is still controversial and so far, the primary identifying criterion was to consider their date of birth. However, literature highlighted the need to describe specific characteristics. The purpose of this research was to evaluate the factor structure of a French version of the Digital Natives Assessment Scale (DNAS). The sample of this study includes 590 participants from a 6-week massive open online course and from Web sites, electronic forums, and social networks. The DNAS was translated in French and then back-translated to English. A principal component analysis with orthogonal rotation followed by a confirmatory factorial analysis showed that a 15-item four-correlated component model provided the best fit for the data of our sample. Factor structure of this French-translated version of the DNAS was rather similar than those found in earlier studies. This study provides evidence of the DNAS robustness through cross-cultural and cross-generational validation. The French version of the DNAS appears to be appropriate as a quick and effective questionnaire to assess digital natives. More studies are needed to better define further features of this particular group.

DNA Motion Capture Reveals the Mechanical Properties of DNA at the Mesoscale

PubMed Central

Price, Allen C.; Pilkiewicz, Kevin R.; Graham, Thomas G.W.; Song, Dan; Eaves, Joel D.; Loparo, Joseph J.

2015-01-01

Single-molecule studies probing the end-to-end extension of long DNAs have established that the mechanical properties of DNA are well described by a wormlike chain force law, a polymer model where persistence length is the only adjustable parameter. We present a DNA motion-capture technique in which DNA molecules are labeled with fluorescent quantum dots at specific sites along the DNA contour and their positions are imaged. Tracking these positions in time allows us to characterize how segments within a long DNA are extended by flow and how fluctuations within the molecule are correlated. Utilizing a linear response theory of small fluctuations, we extract elastic forces for the different, ∼2-μm-long segments along the DNA backbone. We find that the average force-extension behavior of the segments can be well described by a wormlike chain force law with an anomalously small persistence length. PMID:25992731

Single cell electroporation using proton beam fabricated biochips

NASA Astrophysics Data System (ADS)

Homhuan, S.; Zhang, B.; Sheu, F.-S.; Bettiol, A. A.; Watt, F.

2010-05-01

We report the design and fabrication of a novel single cell electroporation biochip fabricated by the Proton Beam Writing technique (PBW), a new technique capable of direct-writing high-aspect-ratio nano and microstructures. The biochip features nickel micro-electrodes with straight-side walls between which individual cells are positioned. By applying electrical impulses across the electrodes, SYTOX® Green nucleic acid stain is incorporated into mouse neuroblastoma (N2a) cells. When the stain binds with DNA inside the cell nucleus, green fluorescence is observed upon excitation from a halogen lamp. Three parameters; electric field strength, pulse duration, and the number of pulses have been considered and optimized for the single cell electroporation. The results show that our biochip gives successfully electroporated cells . This single cell electroporation system represents a promising method for investigating the introduction of a wide variety of fluorophores, nanoparticles, quantum dots, DNAs and proteins into cells.

DNA Molecules Adsorbed on Rippled Supported Cationic Lipid Membranes -- A new way to stretch DNAs

NASA Astrophysics Data System (ADS)

Golubovic, Leonardo

2005-03-01

We discuss a novel approach to control to shapes of DNA molecules. We elucidate the recent experimental work of M. Hochrein, L. Golubovic and J. Raedler, on the conformational behavior of DNA molecules adsorbed on lipid membranes that are supported on grooved micro-structured surfaces. We explain the striking ability of the edges formed on these supported membranes to adsorb and completely orient (stretch) very long DNA molecules. Here we explain the experimentally observed DNA stretching effect in terms of the surface curvature dependent electrostatic potential seen by the adsorbed DNA molecules. On the curved, rippled membrane, we show that the DNA molecules undergo localization transitions causing them to stretch by binding to the ripple edges of the supported membrane. In the future, this stretching will allow to directly image, by the common fluorescence microscopy, fundamental biological processes of the interactions between DNA and single protein molecules.

Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries

PubMed Central

Schmidt, Thorsten L.; Beliveau, Brian J.; Uca, Yavuz O.; Theilmann, Mark; Da Cruz, Felipe; Wu, Chao-Ting; Shih, William M.

2015-01-01

Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes. PMID:26567534

Polyfluorophore Excimers and Exciplexes as FRET Donors in DNA

PubMed Central

Teo, Yin Nah; Kool, Eric T.

2009-01-01

We describe studies aimed at testing whether oligomeric exciplex- and excimer fluorophores conjugated to DNA have the potential to act as donors for energy transfer by the Förster mechanism. Oligodeoxyfluorosides (ODFs) are composed of stacked, electronically interacting fluorophores replacing the bases on a DNA scaffold. The monomer chromophores in the twenty tetramer-length ODFs studied here include pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and a nonfluorescent spacer (S); these are conjugated in varied combinations at the 3’ end of a 14mer DNA probe sequence. In the absence of an acceptor chromophore, many of the ODF-DNAs show broad, unstructured long-wavelength emission peaks characteristic of excimer and exciplex excited states, similar to what has been observed for unconjugated ODFs. Although such delocalized excited states have been widely studied, we know of no prior report of their use in FRET. We tested the ability of the twenty ODFs to donate energy to Cy5 and TAMRA dyes conjugated to a complementary strand of DNA, with these acceptors oriented either at the near or far end of the ODF-conjugated probes. Results showed that a number of the ODF fluorophores exhibited relatively efficient energy transfer characteristic of the Förster mechanism, as judged by drops in donor emission quantum yield and fluorescence lifetime, accompanied by increases in intensity of acceptor emission bands. Excimer/exciplex bands in the donors were selectively quenched while shorter-wavelength monomer emission stayed relatively constant, consistent with the notion that the delocalized excited states, rather than individual fluorophores, are the donors. Interestingly, only specific sequences of ODFs were able to act as donors, while others did not, even though their emission wavelengths were similar. The new FRET donors possess large Stokes shifts, which can be beneficial for multiple applications. In addition, all ODFs can be excited at a single wavelength; thus, ODFs may be candidates as “universal FRET donors”, thus allowing multicolor FRET of multiple species to be carried out with one excitation. PMID:19916519

Sequencing and annotation of the chloroplast DNAs and identification of polymorphisms distinguishing normal male-fertile and male-sterile cytoplasms of onion.

PubMed

von Kohn, Christopher; Kiełkowska, Agnieszka; Havey, Michael J

2013-12-01

Male-sterile (S) cytoplasm of onion is an alien cytoplasm introgressed into onion in antiquity and is widely used for hybrid seed production. Owing to the biennial generation time of onion, classical crossing takes at least 4 years to classify cytoplasms as S or normal (N) male-fertile. Molecular markers in the organellar DNAs that distinguish N and S cytoplasms are useful to reduce the time required to classify onion cytoplasms. In this research, we completed next-generation sequencing of the chloroplast DNAs of N- and S-cytoplasmic onions; we assembled and annotated the genomes in addition to identifying polymorphisms that distinguish these cytoplasms. The sizes (153 538 and 153 355 base pairs) and GC contents (36.8%) were very similar for the chloroplast DNAs of N and S cytoplasms, respectively, as expected given their close phylogenetic relationship. The size difference was primarily due to small indels in intergenic regions and a deletion in the accD gene of N-cytoplasmic onion. The structures of the onion chloroplast DNAs were similar to those of most land plants with large and small single copy regions separated by inverted repeats. Twenty-eight single nucleotide polymorphisms, two polymorphic restriction-enzyme sites, and one indel distributed across 20 chloroplast genes in the large and small single copy regions were selected and validated using diverse onion populations previously classified as N or S cytoplasmic using restriction fragment length polymorphisms. Although cytoplasmic male sterility is likely associated with the mitochondrial DNA, maternal transmission of the mitochondrial and chloroplast DNAs allows for polymorphisms in either genome to be useful for classifying onion cytoplasms to aid the development of hybrid onion cultivars.

Bacteriophage of Haemophilus influenzae III. Morphology, DNA Homology, and Immunity Properties of HP1c1, S2, and the Defective Bacteriophage from Strain Rd

PubMed Central

Boling, Maxon E.; Allison, David P.; Setlow, Jane K.

1973-01-01

The phages HP1c1 and S2 and a defective phage of Haemophilus influenzae have been compared. The morphology of the phages and the mol wt of their DNAs are similar, although the defective phage appears to have a different tail plate region. Electron microscope observation indicates that the defective phage does not attach to the cell surface, and its DNA appears to lack cohesive ends. The homology of the DNAs of the phages has been measured by hydridization. DNA from the defective phage shows little or no homology with the other phage DNAs. HP1c1 and S2 DNAs show a high level of homology. Each of these phages can form plaques on lawns of the lysogen of the other phage but at reduced plating efficiencies, suggesting that the two phages have related but not identical immunity systems. Images PMID:4540713

Amerindian mitochondrial DNAs have rare Asian mutations at high frequencies, suggesting they derived from four primary maternal lineages.

PubMed Central

Schurr, T G; Ballinger, S W; Gan, Y Y; Hodge, J A; Merriwether, D A; Lawrence, D N; Knowler, W C; Weiss, K M; Wallace, D C

1990-01-01

The mitochondrial DNA (mtDNA) sequence variation of the South American Ticuna, the Central American Maya, and the North American Pima was analyzed by restriction-endonuclease digestion and oligonucleotide hybridization. The analysis revealed that Amerindian populations have high frequencies of mtDNAs containing the rare Asian RFLP HincII morph 6, a rare HaeIII site gain, and a unique AluI site gain. In addition, the Asian-specific deletion between the cytochrome c oxidase subunit II (COII) and tRNA(Lys) genes was also prevalent in both the Pima and the Maya. These data suggest that Amerindian mtDNAs derived from at least four primary maternal lineages, that new tribal-specific variants accumulated as these mtDNAs became distributed throughout the Americas, and that some genetic variation may have been lost when the progenitors of the Ticuna separated from the North and Central American populations. Images Figure 1 PMID:1968708

Existence of host-related DNA sequences in the schistosome genome.

PubMed

Iwamura, Y; Irie, Y; Kominami, R; Nara, T; Yasuraoka, K

1991-06-01

DNA sequences homologous to the mouse intracisternal A particle and endogenous type C retrovirus were detected in the DNAs of Schistosoma japonicum adults and S. mansoni eggs. Furthermore, other kinds of repetitive sequences in the host genome such as mouse type 1 Alu sequence (B1), mouse type 2 Alu sequence (B2) and mo-2 sequence, a mouse mini-satellite, were also detected in the DNAs from adults and eggs of S. japonicum and eggs of S. mansoni. Almost all of the sequences described above were absent in the DNAs of S. mansoni adults. The DNA fingerprints of schistosomes, using the mo-2 sequence, were indistinguishable from each other and resembled those of their murine hosts. Moreover, the mo-2 sequence was hypermethylated in the DNAs of schistosomes and its amount was variable in them. These facts indicate that host-related sequences are actually present in schistosomes and that the mo-2 repetitive sequence exists probably in extra-chromosome.

A portable fluorescent sensing system using multiple LEDs

NASA Astrophysics Data System (ADS)

Shin, Young-Ho; Barnett, Jonathan Z.; Gutierrez-Wing, M. Teresa; Rusch, Kelly A.; Choi, Jin-Woo

2017-02-01

This paper presents a portable fluorescent sensing system that utilizes different light emitting diode (LED) excitation lights for multiple target detection. In order to identify different analytes, three different wavelengths (385 nm, 448 nm, and 590 nm) of excitation light emitting diodes were used to selectively stimulate the target analytes. A highly sensitive silicon photomultiplier (SiPM) was used to detect corresponding fluorescent signals from each analyte. Based on the unique fluorescent response of each analyte, it is possible to simultaneously differentiate one analyte from the other in a mixture of target analytes. A portable system was designed and fabricated consisting of a display module, battery, data storage card, and sample loading tray into a compact 3D-printed jig. The portable sensor system was demonstrated for quantification and differentiation of microalgae (Chlorella vulgaris) and cyanobacteria (Spirulina) by measuring fluorescent responses of chlorophyll a in microalgae and phycocyanin in cyanobacteria. Obtained results suggest that the developed portable sensor system could be used as a generic fluorescence sensor platform for on-site detection of multiple analytes of interest.

Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus.

PubMed

Qian, Shasha; Chen, Xiaolan; Sun, Kai; Zhang, Yang; Li, Zhenghe

2017-06-13

Recovery of recombinant negative-stranded RNA viruses from cloned cDNAs is an inefficient process as multiple viral components need to be delivered into cells for reconstitution of infectious entities. Previously studies have shown that authentic viral RNA termini are essential for efficient virus rescue. However, little is known about the activity of viral RNAs processed by different strategies in supporting recovery of plant negative-stranded RNA virus. In this study, we used several versions of hammerhead ribozymes and a truncated cauliflower mosaic virus 35S promoter to generate precise 5' termini of sonchus yellow net rhabdovirus (SYNV) antigenomic RNA (agRNA) derivatives. These agRNAs were co-expressed with the SYNV core proteins in Nicotiana benthamiana leaves to evaluate their efficiency in supporting fluorescent reporter gene expression from an SYNV minireplicon (MR) and rescue of full-length virus. Optimization of hammerhead ribozyme cleavage activities led to improved SYNV MR reporter gene expression. Although the MR agRNA processed by the most active hammerhead variants is comparable to the capped, precisely transcribed agRNA in supporting MR activity, efficient recovery of recombinant SYNV was only achieved with capped agRNA. Further studies showed that the capped SYNV agRNA permitted transient expression of the nucleocapsid (N) protein, and an agRNA derivatives unable to express the N protein in cis exhibited dramatically reduced rescue efficiency. Our study reveals superior activity of precisely transcribed, capped SYNV agRNAs to uncapped, hammerhead ribozyme-processed agRNAs, and suggests a cis-acting function for the N protein expressed from the capped agRNA during recovery of SYNV from plasmids.

Gene expression profiles in rainbow trout, Onchorynchus mykiss, exposed to a simple chemical mixture.

PubMed

Hook, Sharon E; Skillman, Ann D; Gopalan, Banu; Small, Jack A; Schultz, Irvin R

2008-03-01

Among proposed uses for microarrays in environmental toxiciology is the identification of key contributors to toxicity within a mixture. However, it remains uncertain whether the transcriptomic profiles resulting from exposure to a mixture have patterns of altered gene expression that contain identifiable contributions from each toxicant component. We exposed isogenic rainbow trout Onchorynchus mykiss, to sublethal levels of ethynylestradiol, 2,2,4,4-tetrabromodiphenyl ether, and chromium VI or to a mixture of all three toxicants Fluorescently labeled complementary DNA (cDNA) were generated and hybridized against a commercially available Salmonid array spotted with 16,000 cDNAs. Data were analyzed using analysis of variance (p<0.05) with a Benjamani-Hochberg multiple test correction (Genespring [Agilent] software package) to identify up and downregulated genes. Gene clustering patterns that can be used as "expression signatures" were determined using hierarchical cluster analysis. The gene ontology terms associated with significantly altered genes were also used to identify functional groups that were associated with toxicant exposure. Cross-ontological analytics approach was used to assign functional annotations to genes with "unknown" function. Our analysis indicates that transcriptomic profiles resulting from the mixture exposure resemble those of the individual contaminant exposures, but are not a simple additive list. However, patterns of altered genes representative of each component of the mixture are clearly discernible, and the functional classes of genes altered represent the individual components of the mixture. These findings indicate that the use of microarrays to identify transcriptomic profiles may aid in the identification of key stressors within a chemical mixture, ultimately improving environmental assessment.

A fluorescent bimolecular complementation screen reveals MAF1, RNF7 and SETD3 as PCNA-associated proteins in human cells

PubMed Central

Cooper, Simon E; Hodimont, Elsie; Green, Catherine M

2015-01-01

The proliferating cell nuclear antigen (PCNA) is a conserved component of DNA replication factories, and interactions with PCNA mediate the recruitment of many essential DNA replication enzymes to these sites of DNA synthesis. A complete description of the structure and composition of these factories remains elusive, and a better knowledge of them will improve our understanding of how the maintenance of genome and epigenetic stability is achieved. To fully characterize the set of proteins that interact with PCNA we developed a bimolecular fluorescence complementation (BiFC) screen for PCNA-interactors in human cells. This 2-hybrid type screen for interactors from a human cDNA library is rapid and efficient. The fluorescent read-out for protein interaction enables facile selection of interacting clones, and we combined this with next generation sequencing to identify the cDNAs encoding the interacting proteins. This method was able to reproducibly identify previously characterized PCNA-interactors but importantly also identified RNF7, Maf1 and SetD3 as PCNA-interacting proteins. We validated these interactions by co-immunoprecipitation from human cell extracts and by interaction analyses using recombinant proteins. These results show that the BiFC screen is a valuable method for the identification of protein-protein interactions in living mammalian cells. This approach has potentially wide application as it is high throughput and readily automated. We suggest that, given this interaction with PCNA, Maf1, RNF7, and SetD3 are potentially involved in DNA replication, DNA repair, or associated processes. PMID:26030842

Simultaneous multicolor imaging of wide-field epi-fluorescence microscopy with four-bucket detection

PubMed Central

Park, Kwan Seob; Kim, Dong Uk; Lee, Jooran; Kim, Geon Hee; Chang, Ki Soo

2016-01-01

We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization. PMID:27375944

CRE Activation in Antiestrogen Resistance

DTIC Science & Technology

2005-05-01

differences in transfection efficiency, 0.1 ug of a plasmid containing the Renilla luciferase gene was also cotransfected into the cells. Both plasmid DNAs...containing the Renilla luciferase gene was also cotransfected into the cells. Both plasmid DNAs were added to a mix of Fugene 6 (Roche) and serum free

Recent African origin of modern humans revealed by complete sequences of hominoid mitochondrial DNAs.

PubMed Central

Horai, S; Hayasaka, K; Kondo, R; Tsugane, K; Takahata, N

1995-01-01

We analyzed the complete mitochondrial DNA (mtDNA) sequences of three humans (African, European, and Japanese), three African apes (common and pygmy chimpanzees, and gorilla), and one orangutan in an attempt to estimate most accurately the substitution rates and divergence times of hominoid mtDNAs. Nonsynonymous substitutions and substitutions in RNA genes have accumulated with an approximately clock-like regularity. From these substitutions and under the assumption that the orangutan and African apes diverged 13 million years ago, we obtained a divergence time for humans and chimpanzees of 4.9 million years. This divergence time permitted calibration of the synonymous substitution rate (3.89 x 10(-8)/site per year). To obtain the substitution rate in the displacement (D)-loop region, we compared the three human mtDNAs and measured the relative abundance of substitutions in the D-loop region and at synonymous sites. The estimated substitution rate in the D-loop region was 7.00 x 10(-8)/site per year. Using both synonymous and D-loop substitutions, we inferred the age of the last common ancestor of the human mtDNAs as 143,000 +/- 18,000 years. The shallow ancestry of human mtDNAs, together with the observation that the African sequence is the most diverged among humans, strongly supports the recent African origin of modern humans, Homo sapiens sapiens. PMID:7530363

Transfection of gene regulation nanoparticles complexed with pDNA and shRNA controls multilineage differentiation of hMSCs.

PubMed

Kim, Hye Jin; Yi, Se Won; Oh, Hyun Jyung; Lee, Jung Sun; Park, Ji Sun; Park, Keun-Hong

2018-05-29

Overexpression and knockdown of specific proteins can control stem cell differentiation for therapeutic purposes. In this study, we fabricated RUNX2, SOX9, and C/EBPα plasmid DNAs (pDNAs) and ATF4-targeting shRNA (shATF4) to induce osteogenesis, chondrogenesis, and adipogenesis of human mesenchymal stem cells (hMSCs). The pDNAs and shATF4 were complexed with TRITC-gene regulation nanoparticles (GRN). Osteogenesis-related gene expression was reduced at early (12 h) and late (36 h) time points after co-delivery of shATF4 and SOX9 or C/EBPα pDNA, respectively, and osteogenesis was inhibited in these hMSCs. By contrast, osteogenesis-related genes were highly expressed upon co-delivery of RUNX2 and ATF4 pDNAs. DEX in GRN enhanced chondrogenic differentiation. Expression of osteogenesis-, chondrogenesis-, and adipogenesis-related genes was higher in hMSCs transfected with NPs complexed with RUNX2 and ATF4 pDNAs, shATF4 and SOX9 pDNA, and shATF4 and C/EBPα pDNA for 72 h than in control hMSCs, respectively. Moreover, delivery of these NPs also increased expression of osteogenesis-, chondrogenesis-, and adipogenesis-related proteins. These alterations in expression led to morphological changes, indicating that hMSCs differentiated into osteoblasts, chondrocytes, and adipose cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Analysis of complex repeat sequences within the spinal muscular atrophy (SMA) candidate region in 5q13

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

Davies, K.E.; Morrison, K.E.; Daniels, R.I.

1994-09-01

We previously reported that the 400 kb interval flanked the polymorphic loci D5S435 and D5S557 contains blocks of a chromosome 5 specific repeat. This interval also defines the SMA candidate region by genetic analysis of recombinant families. A YAC contig of 2-3 Mb encompassing this area has been constructed and a 5.5 kb conserved fragment, isolated from a YAC end clone within the above interval, was used to obtain cDNAs from both fetal and adult brain libraries. We describe the identification of cDNAs with stretches of high DNA sequence homology to exons of {beta} glucuronidase on human chromosome 7. Themore » cDNAs map both to the candidate region and to an area of 5p using FISH and deletion hybrid analysis. Hybridization to bacteriophage and cosmid clones from the YACs localizes the {beta} glucuronidase related sequences within the 400 kb region of the YAC contig. The cDNAs show a polymorphic pattern on hybridization to genomic BamH1 fragments in the size range of 10-250 kb. Further analysis using YAC fragmentation vectors is being used to determine how these {beta} glucuronidase related cDNAs are distributed within 5q13. Dinucleotide repeats within the region are being investigated to determine linkage disequilibrium with the disease locus.« less

Monte Carlo based method for fluorescence tomographic imaging with lifetime multiplexing using time gates

PubMed Central

Chen, Jin; Venugopal, Vivek; Intes, Xavier

2011-01-01

Time-resolved fluorescence optical tomography allows 3-dimensional localization of multiple fluorophores based on lifetime contrast while providing a unique data set for improved resolution. However, to employ the full fluorescence time measurements, a light propagation model that accurately simulates weakly diffused and multiple scattered photons is required. In this article, we derive a computationally efficient Monte Carlo based method to compute time-gated fluorescence Jacobians for the simultaneous imaging of two fluorophores with lifetime contrast. The Monte Carlo based formulation is validated on a synthetic murine model simulating the uptake in the kidneys of two distinct fluorophores with lifetime contrast. Experimentally, the method is validated using capillaries filled with 2.5nmol of ICG and IRDye™800CW respectively embedded in a diffuse media mimicking the average optical properties of mice. Combining multiple time gates in one inverse problem allows the simultaneous reconstruction of multiple fluorophores with increased resolution and minimal crosstalk using the proposed formulation. PMID:21483610

Sequential Superresolution Imaging of Multiple Targets Using a Single Fluorophore

PubMed Central

Lidke, Diane S.; Lidke, Keith A.

2015-01-01

Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the x-y imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique. PMID:25860558

The mosaic mutants of cucumber: A system to produce mitochondrial knock-downs

USDA-ARS?s Scientific Manuscript database

The mitochondrial (mt) DNA of cucumber has several unique attributes, including paternal transmission and large size due in part to the accumulation of repetitive DNAs. Recombination among these repetitive motifs generates structural rearrangements in the mt DNAs. When the highly inbred line ‘B’ of ...

Poly(amidoamine) Dendrimers Modified with 1,2-Epoxyhexane or 1,2-Epoxydodecane for Enhanced Gene Delivery Applications.

PubMed

Xiao, Tongyu; Cao, Xueyan; Hou, Wenxiu; Peng, Chen; Qiu, Jieru; Shi, Xiangyang

2015-12-01

We report a new non-viral gene delivery system based on hydrophobically modified poly(amidoamine) (PAMAM) dendrimers. In this study, the periphery of amine-terminated generation 5 (G5) PAMAM dendrimers was partially reacted with 1,2-epoxyhexane and 1,2-epoxydodecane, respectively. The formed hydrophobically modified G5 dendrimers (denoted as G5.NH2-C6 or G5.NH2-C12) were used to complex two different plasmid DNAs (pDNAs) encoding luciferase (Luc) and enhanced green fluorescent protein (EGFP), respectively for gene transfection studies. The polyplexes formed between vectors and pDNA were characterized by gel retardation assay, dynamic light scattering, and zeta potential measurements. We show that the G5.NH2-C6 and G5.NH2-C12 vectors are able to effectively compact the pDNA, allowing for highly efficient gene transfection into a model cell line (HeLa cells) as demonstrated by both Luc assay and confocal microscopic imaging of the EGFP expression. Under the studied N/P ratios (the molar ratio of primary amines of the dendrimers to phosphates in the pDNA backbone) at 2.5 or 5, the transfection efficiency of the dendrimer-based vectors followed the order of G5.NH2-C12 > G5.NH2-C6 > G5.NH2. This enhanced gene transfection capacity is believed to be associated with the enhanced hydrophobic interaction between the vector/pDNA complexes and the relatively hydrophobic cell membranes. The developed hydrophobically modified dendrimers may be used as a promising non-viral vector for enhanced gene delivery applications.

Phylogenetic Diversity Analysis of Subterranean Hot Springs in Iceland

PubMed Central

Marteinsson, Viggó Thór; Hauksdóttir, Sigurbjörg; Hobel, Cédric F. V.; Kristmannsdóttir, Hrefna; Hreggvidsson, Gudmundur Oli; Kristjánsson, Jakob K.

2001-01-01

Geothermal energy has been harnessed and used for domestic heating in Iceland. In wells that are typically drilled to a depth of 1,500 to 2,000 m, the temperature of the source water is 50 to 130°C. The bottoms of the boreholes can therefore be regarded as subterranean hot springs and provide a unique opportunity to study the subterranean biosphere. Large volumes of geothermal fluid from five wells and a mixture of geothermal water from 50 geothermal wells (hot tap water) were sampled and concentrated through a 0.2-μm-pore-size filter. Cells were observed in wells RG-39 (91.4°C) and MG-18 (71.8°C) and in hot tap water (76°C), but no cells were detected in wells SN-4, SN-5 (95 to 117°C), and RV-5 (130°C). Archaea and Bacteria were detected by whole-cell fluorescent in situ hybridization. DNAs were extracted from the biomass, and small-subunit rRNA genes (16S rDNAs) were amplified by PCR using primers specific for the Archaea and Bacteria domains. The PCR products were cloned and sequenced. The sequence analysis showed 11 new operational taxonomic units (OTUs) out of 14, 3 of which were affiliated with known surface OTUs. Samples from RG-39 and hot tap water were inoculated into enrichment media and incubated at 65 and 85°C. Growth was observed only in media based on geothermal water. 16S rDNA analysis showed enrichments dominated with Desulfurococcales relatives. Two strains belonging to Desulfurococcus mobilis and to the Thermus/Deinococcus group were isolated from borehole RG-39. The results indicate that subsurface volcanic zones are an environment that provides a rich subsurface for novel thermophiles. PMID:11526029

Recombinant Saccharomyces cerevisiae serves as novel carrier for oral DNA vaccines in Carassius auratus.

PubMed

Yan, Nana; Xu, Kun; Li, Xinyi; Liu, Yuwan; Bai, Yichun; Zhang, Xiaohan; Han, Baoquan; Chen, Zhilong; Zhang, Zhiying

2015-12-01

Oral delivery of DNA vaccines represents a promising vaccinating method for fish. Recombinant yeast has been proved to be a safe carrier for delivering antigen proteins and DNAs to some species in vivo. However, whether recombinant yeast can be used to deliver functional DNAs for vaccination to fish is still unknown. In this study, red crucian carp (Carassius auratus) was orally administrated with recombinant Saccharomyces cerevisiae harboring CMV-EGFP expression cassette. On day 5 post the first vaccination, EGFP expression in the hindgut was detected under fluorescence microscope. To further study whether the delivered gene could induce specific immune responses, the model antigen ovalbumin (OVA) was used as immunogen, and oral administrations were conducted with recombinant S. cerevisiae harboring pCMV-OVA mammalian gene expression cassette as gene delivery or pADH1-OVA yeast gene expression cassette as protein delivery. Each administration was performed with three different doses, and the OVA-specific serum antibody was detected in all the experimental groups by western blotting and enzyme-linked immunosorbent assay (ELISA). ELISA assay also revealed that pCMV-OVA group with lower dose (pCMV-OVA-L) and pADH1-OVA group with moderate dose (pADH1-OVA-M) triggered relatively stronger antibody response than the other two doses. Moreover, the antibody level induced by pCMV-OVA-L group was significantly higher than pADH1-OVA-M group at the same serum dilutions. All the results suggested that recombinant yeast can be used as a potential carrier for oral DNA vaccines and would help to develop more practical strategies to control infectious diseases in aquaculture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phylogenetic diversity analysis of subterranean hot springs in Iceland.

PubMed

Marteinsson, V T; Hauksdóttir, S; Hobel, C F; Kristmannsdóttir, H; Hreggvidsson, G O; Kristjánsson, J K

2001-09-01

Geothermal energy has been harnessed and used for domestic heating in Iceland. In wells that are typically drilled to a depth of 1,500 to 2,000 m, the temperature of the source water is 50 to 130 degrees C. The bottoms of the boreholes can therefore be regarded as subterranean hot springs and provide a unique opportunity to study the subterranean biosphere. Large volumes of geothermal fluid from five wells and a mixture of geothermal water from 50 geothermal wells (hot tap water) were sampled and concentrated through a 0.2-microm-pore-size filter. Cells were observed in wells RG-39 (91.4 degrees C) and MG-18 (71.8 degrees C) and in hot tap water (76 degrees C), but no cells were detected in wells SN-4, SN-5 (95 to 117 degrees C), and RV-5 (130 degrees C). Archaea and Bacteria were detected by whole-cell fluorescent in situ hybridization. DNAs were extracted from the biomass, and small-subunit rRNA genes (16S rDNAs) were amplified by PCR using primers specific for the Archaea and Bacteria domains. The PCR products were cloned and sequenced. The sequence analysis showed 11 new operational taxonomic units (OTUs) out of 14, 3 of which were affiliated with known surface OTUs. Samples from RG-39 and hot tap water were inoculated into enrichment media and incubated at 65 and 85 degrees C. Growth was observed only in media based on geothermal water. 16S rDNA analysis showed enrichments dominated with Desulfurococcales relatives. Two strains belonging to Desulfurococcus mobilis and to the Thermus/Deinococcus group were isolated from borehole RG-39. The results indicate that subsurface volcanic zones are an environment that provides a rich subsurface for novel thermophiles.

Algorithms for differentiating between images of heterogeneous tissue across fluorescence microscopes.

PubMed

Chitalia, Rhea; Mueller, Jenna; Fu, Henry L; Whitley, Melodi Javid; Kirsch, David G; Brown, J Quincy; Willett, Rebecca; Ramanujam, Nimmi

2016-09-01

Fluorescence microscopy can be used to acquire real-time images of tissue morphology and with appropriate algorithms can rapidly quantify features associated with disease. The objective of this study was to assess the ability of various segmentation algorithms to isolate fluorescent positive features (FPFs) in heterogeneous images and identify an approach that can be used across multiple fluorescence microscopes with minimal tuning between systems. Specifically, we show a variety of image segmentation algorithms applied to images of stained tumor and muscle tissue acquired with 3 different fluorescence microscopes. Results indicate that a technique called maximally stable extremal regions followed by thresholding (MSER + Binary) yielded the greatest contrast in FPF density between tumor and muscle images across multiple microscopy systems.

A simple and novel method for RNA-seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase.

PubMed

Brouilette, Scott; Kuersten, Scott; Mein, Charles; Bozek, Monika; Terry, Anna; Dias, Kerith-Rae; Bhaw-Rosun, Leena; Shintani, Yasunori; Coppen, Steven; Ikebe, Chiho; Sawhney, Vinit; Campbell, Niall; Kaneko, Masahiro; Tano, Nobuko; Ishida, Hidekazu; Suzuki, Ken; Yashiro, Kenta

2012-10-01

Deep sequencing of single cell-derived cDNAs offers novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA-seq analysis requires multiple steps with consequent sample loss and stochastic variation at each step significantly affecting output. Thus, a simpler and better protocol is desirable. The recently developed hyperactive Tn5-mediated library preparation, which brings high quality libraries, is likely one of the solutions. Here, we tested the applicability of hyperactive Tn5-mediated library preparation to deep sequencing of single cell cDNA, optimized the protocol, and compared it with the conventional method based on sonication. This new technique does not require any expensive or special equipment, which secures wider availability. A library was constructed from only 100 ng of cDNA, which enables the saving of precious specimens. Only a few steps of robust enzymatic reaction resulted in saved time, enabling more specimens to be prepared at once, and with a more reproducible size distribution among the different specimens. The obtained RNA-seq results were comparable to the conventional method. Thus, this Tn5-mediated preparation is applicable for anyone who aims to carry out deep sequencing for single cell cDNAs. Copyright © 2012 Wiley Periodicals, Inc.

Molecular Cloning of Drebrin: Progress and Perspectives.

PubMed

Kojima, Nobuhiko

2017-01-01

Chicken drebrin isoforms were first identified in the optic tectum of developing brain. Although the time course of protein expression was different in each drebrin isoform, the similarity between their protein structures was suggested by biochemical analysis of purified protein. To determine their protein structures, the cloning of drebrin cDNAs was conducted. Comparison between the cDNA sequences shows that all drebrin cDNAs are identical except that the internal insertion sequences are present or absent in their sequences. Chicken drebrin are now classified into three isoforms, namely, drebrins E1, E2, and A. Genomic cloning demonstrated that the three isoforms are generated by an alternative splicing of individual exons encoding the insertion sequences from single drebrin gene. The mechanism should be precisely regulated in cell-type-specific and developmental stage-specific fashion. Drebrin protein, which is well conserved in various vertebrate species, although mammalian drebrin has only two isoforms, namely, drebrin E and drebrin A, is different from chicken drebrin that has three isoforms. Drebrin belongs to an actin-depolymerizing factor homology (ADF-H) domain protein family. Besides the ADF-H domain, drebrin has other domains, including the actin-binding domain and Homer-binding motifs. Diversity of protein isoform and multiple domains of drebrin could interact differentially with the actin cytoskeleton and other intracellular proteins and regulate diverse cellular processes.

Actin genes and their expression in pacific white shrimp, Litopenaeus vannamei.

PubMed

Zhang, Xiaoxi; Zhang, Xiaojun; Yuan, Jianbo; Du, Jiangli; Li, Fuhua; Xiang, Jianhai

2018-04-01

Actin is a multi-functional gene family that can be divided into muscle-type actins and non-muscle-type actins. In this study, 37 unigenes encoding actins were identified from RNA-Seq data of Pacific white shrimp, Litopenaeus vannamei. According to phylogenetic analysis, four and three cDNAs belong to cytoplasmic- and heart-type actins and were named LvActinCT and LvActinHT, respectively. 10 cDNAs belong to the slow-type skeletal muscle actins, and 18 belong to the fast-type skeletal muscle actins; they were designated LvActinSSK and LvActinFSK, respectively. Some muscle actin genes formed gene clusters in the genome. Multiple alternative transcription starts sites (ATSSs) were found for LvActinCT1. Based on the early developmental expression profile, almost all LvActins were highly expressed between the early limb bud and post-larval stages. Using LvActinSSK5 as probes, slow-type muscle was localized in pleopod muscle and superficial ventral muscle. We also found three actin genes that were down-regulated in the hemocytes of white spot syndrome virus (WSSV)- and Vibrio parahaemolyticus-infected L. vannamei. This study provides valuable information on the actin gene structure of shrimp, furthers our understanding of the shrimp muscle system and helps us develop strategies for disease control and sustainable shrimp farming.

Multiple Origins and Nested Cycles of Hybridization Result in High Tetraploid Diversity in the Monocot Prospero.

PubMed

Jang, Tae-Soo; Parker, John S; Emadzade, Khatere; Temsch, Eva M; Leitch, Andrew R; Weiss-Schneeweiss, Hanna

2018-01-01

Polyploidy is a major driving force in angiosperm evolution, but our understanding of establishment and early diversification processes following allo- vs. auto-polyploidy is limited. An excellent system to address such questions is the monocot plant Prospero autumnale , as it comprises several genomically and chromosomally distinct diploid cytotypes and their auto- and allotetraploid derivatives. To infer origins and evolutionary trajectories of the tetraploids, we use genome size data, in situ hybridization with parental genomic DNAs and specific probes (satDNA, rDNAs), as well as molecular-phylogenetic analyses. Thus, we demonstrate that an astounding range of allotetraploid lineages has been formed recurrently by chromosomal re-patterning, interactions of chromosomally variable parental genomes and nested cycles of extensive hybridization, whereas autotetraploids have originated at least twice and are cytologically stable. During the recurrent formation and establishment across wide geographic areas hybridization in some populations could have inhibited lineage diversification and nascent speciation of such a hybrid swarm. However, cytotypes that became fixed in populations enhanced the potential for species diversification, possibly exploiting the extended allelic base, and fixed heterozygosity that polyploidy confers. The time required for polyploid cytotype fixation may in part reflect the lag phase reported for polyploids between their formation and species diversification.

Curation of microarray oligonucleotides and corresponding ESTs/cDNAs used for gene expression analysis in zebra finches.

PubMed

Lovell, Peter V; Huizinga, Nicole A; Getachew, Abel; Mees, Brianna; Friedrich, Samantha R; Wirthlin, Morgan; Mello, Claudio V

2018-05-18

Zebra finches are a major model organism for investigating mechanisms of vocal learning, a trait that enables spoken language in humans. The development of cDNA collections with expressed sequence tags (ESTs) and microarrays has allowed for extensive molecular characterizations of circuitry underlying vocal learning and production. However, poor database curation can lead to errors in transcriptome and bioinformatics analyses, limiting the impact of these resources. Here we used genomic alignments and synteny analysis for orthology verification to curate and reannotate ~ 35% of the oligonucleotides and corresponding ESTs/cDNAs that make-up Agilent microarrays for gene expression analysis in finches. We found that: (1) 5475 out of 43,084 oligos (a) failed to align to the zebra finch genome, (b) aligned to multiple loci, or (c) aligned to Chr_un only, and thus need to be flagged until a better genome assembly is available, or (d) reflect cloning artifacts; (2) Out of 9635 valid oligos examined further, 3120 were incorrectly named, including 1533 with no known orthologs; and (3) 2635 oligos required name update. The resulting curated dataset provides a reference for correcting gene identification errors in previous finch microarrays studies, and avoiding such errors in future studies.

Supercoiled Minivector DNA resists shear forces associated with gene therapy delivery

PubMed Central

Catanese, D J; Fogg, J M; Schrock, D E; Gilbert, B E; Zechiedrich, L

2012-01-01

Supercoiled DNAs varying from 281 to 5302 bp were subjected to shear forces generated by aerosolization or sonication. DNA shearing strongly correlated with length. Typical sized plasmids (⩾3000 bp) degraded rapidly. DNAs 2000–3000 bp persisted ∼10 min. Even in the absence of condensing agents, supercoiled DNA <1200 bp survived nebulization, and increased forces of sonication were necessary to shear it. Circular vectors were considerably more resistant to shearing than linear vectors of the same length. DNA supercoiling afforded additional protection. These results show the potential of shear-resistant Minivector DNAs to overcome one of the major challenges associated with gene therapy delivery. PMID:21633394

tDNA insulators and the emerging role of TFIIIC in genome organization

PubMed Central

Van Bortle, Kevin; Corces, Victor G.

2012-01-01

Recent findings provide evidence that tDNAs function as chromatin insulators from yeast to humans. TFIIIC, a transcription factor that interacts with the B-box in tDNAs as well as thousands of ETC sites in the genome, is responsible for insulator function. Though tDNAs are capable of enhancer-blocking and barrier activities for which insulators are defined, new insights into the relationship between insulators and chromatin structure suggest that TFIIIC serves a complex role in genome organization. We review the role of tRNA genes and TFIIIC as chromatin insulators, and highlight recent findings that have broadened our understanding of insulators in genome biology. PMID:22889843

Unprecedented multiplicity of Ig transmembrane and secretory mRNA forms in the cartilaginous fish.

PubMed

Rumfelt, Lynn L; Diaz, Marilyn; Lohr, Rebecca L; Mochon, Evonne; Flajnik, Martin F

2004-07-15

In most jawed vertebrates including cartilaginous fish, membrane-bound IgM is expressed as a five Ig superfamily (Igsf)-domain H chain attached to a transmembrane (Tm) region. Heretofore, bony fish IgM was the one exception with IgM mRNA spliced to produce a four-domain Tm H chain. We now demonstrate that the Tm and secretory (Sec) mRNAs of the novel cartilaginous fish Ig isotypes, IgW and IgNAR, are present in multiple forms, most likely generated by alternative splicing. In the nurse shark, Ginglymostoma cirratum, and horn shark, Heterodontus francisci, alternative splicing of Tm exons to the second or the fourth constant (C(H)) exons produces two distinct IgW Tm cDNAs. Although the seven-domain IgW Sec cDNA form contains a canonical secretory tail shared with IgM, IgNAR, and IgA, we report a three-domain cDNA form of shark IgW (IgW(short)) having an unusual Sec tail, which is orthologous to skate IgX(short) cDNA. The IgW and IgW(short) Sec transcripts are restricted in their tissue distribution and expression levels vary among individual sharks, with all forms expressed early in ontogeny. IgNAR mRNA is alternatively spliced to produce a truncated four-domain Tm cDNA and a second Tm cDNA is expressed identical in Igsf domains as the Sec form. PBL is enriched in the Tm cDNA of these Igs. These molecular data suggest that cartilaginous fish have augmented their humoral immune repertoire by diversifying the sizes of their Ig isotypes. Furthermore, these Tm cDNAs are prototypical and the truncated variants may translate as more stable protein at the cell surface.

Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms, Actively Released via Membrane Vesicles, and Influenced by Components of the Protein Secretion Machinery

PubMed Central

Liao, Sumei; Klein, Marlise I.; Heim, Kyle P.; Fan, Yuwei; Bitoun, Jacob P.; Ahn, San-Joon; Burne, Robert A.; Koo, Hyun; Brady, L. Jeannine

2014-01-01

Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA. PMID:24748612

Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli.

PubMed

Zerbini, Francesca; Zanella, Ilaria; Fraccascia, Davide; König, Enrico; Irene, Carmela; Frattini, Luca F; Tomasi, Michele; Fantappiè, Laura; Ganfini, Luisa; Caproni, Elena; Parri, Matteo; Grandi, Alberto; Grandi, Guido

2017-04-24

The exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a "brute force" validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions. We first set up our own version of the CRISPR/Cas9 protocol and then we evaluated the mutagenesis efficiency by changing different parameters including sequence of guide RNAs, length and concentration of donor DNAs, and use of single stranded and double stranded donor DNAs. We then validated the optimized conditions targeting 78 "dispensable" genes. This work led to the definition of a protocol, featuring the use of double stranded synthetic donor DNAs, which guarantees mutagenesis efficiencies consistently higher than 10% and a robustness of 100%. The procedure can be applied also for simultaneous gene deletions. This work defines for the first time the robustness of a CRISPR/Cas9-based protocol based on a large sample size. Since the technical solutions here proposed can be applied to other similar procedures, the data could be of general interest for the scientific community working on bacterial genome editing and, in particular, for those involved in synthetic biology projects requiring high throughput procedures.

Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection

PubMed Central

Bates, Mark; Dempsey, Graham T; Chen, Kok Hao; Zhuang, Xiaowei

2012-01-01

Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution. PMID:22213647

[Combined application of multiple fluorescence in research on the degradation of fluoranthene by potassium ferrate].

PubMed

Li, Si; Yu, Dan-Ni; Ji, Fang-Ying; Zhou, Guang-Ming; He, Qiang

2012-11-01

The degradation of fluoranthene was researched by combined means of multiple fluorescence spectra, including emission, synchronous, excitation emission matrix (EEM), time-scan and photometry. The characteristics of the degradation and fluoranthene molecular changes within the degradation's process were also discussed according to the information about the degradation provided by all of the fluorescence spectra mentioned above. The equations of fluoranthene's degradation by potassium ferrate were obtained on the bases of fitting time-scan fluorescence curves at different time, and the degradation's kinetic was speculated accordingly. From the experimental results, multiple fluorescence data commonly reflected that it had same degradation rate at the same reaction time. t = 10 s, and the degradation rate is -55%, t = 25 s, -81%, t = 40 s, -91%. No new fluorescent characteristic was observed within every degradation' stage. The reaction stage during t < or = 20 s was crucial, in which the degradation process is closest to linear relationship. After this beginning stage, the linear relationship deviated gradually with the development of the degradation process. The degradation of fluoranthene by potassium ferrate was nearly in accord with the order of the first order reaction.

ESTIMATION OF BACTERIAL CELL NUMBERS IN HUMIC ACID-RICH SALT MARSH SEDIMENTS WITH PROBES DIRECTED TO 16S RIBOSOMAL DNA

EPA Science Inventory

The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membr...

High Efficiency Hydrodynamic DNA Fragmentation in a Bubbling System

PubMed Central

Li, Lanhui; Jin, Mingliang; Sun, Chenglong; Wang, Xiaoxue; Xie, Shuting; Zhou, Guofu; van den Berg, Albert; Eijkel, Jan C. T.; Shui, Lingling

2017-01-01

DNA fragmentation down to a precise fragment size is important for biomedical applications, disease determination, gene therapy and shotgun sequencing. In this work, a cheap, easy to operate and high efficiency DNA fragmentation method is demonstrated based on hydrodynamic shearing in a bubbling system. We expect that hydrodynamic forces generated during the bubbling process shear the DNA molecules, extending and breaking them at the points where shearing forces are larger than the strength of the phosphate backbone. Factors of applied pressure, bubbling time and temperature have been investigated. Genomic DNA could be fragmented down to controllable 1–10 Kbp fragment lengths with a yield of 75.30–91.60%. We demonstrate that the ends of the genomic DNAs generated from hydrodynamic shearing can be ligated by T4 ligase and the fragmented DNAs can be used as templates for polymerase chain reaction. Therefore, in the bubbling system, DNAs could be hydrodynamically sheared to achieve smaller pieces in dsDNAs available for further processes. It could potentially serve as a DNA sample pretreatment technique in the future. PMID:28098208

High Efficiency Hydrodynamic DNA Fragmentation in a Bubbling System.

PubMed

Li, Lanhui; Jin, Mingliang; Sun, Chenglong; Wang, Xiaoxue; Xie, Shuting; Zhou, Guofu; van den Berg, Albert; Eijkel, Jan C T; Shui, Lingling

2017-01-18

DNA fragmentation down to a precise fragment size is important for biomedical applications, disease determination, gene therapy and shotgun sequencing. In this work, a cheap, easy to operate and high efficiency DNA fragmentation method is demonstrated based on hydrodynamic shearing in a bubbling system. We expect that hydrodynamic forces generated during the bubbling process shear the DNA molecules, extending and breaking them at the points where shearing forces are larger than the strength of the phosphate backbone. Factors of applied pressure, bubbling time and temperature have been investigated. Genomic DNA could be fragmented down to controllable 1-10 Kbp fragment lengths with a yield of 75.30-91.60%. We demonstrate that the ends of the genomic DNAs generated from hydrodynamic shearing can be ligated by T4 ligase and the fragmented DNAs can be used as templates for polymerase chain reaction. Therefore, in the bubbling system, DNAs could be hydrodynamically sheared to achieve smaller pieces in dsDNAs available for further processes. It could potentially serve as a DNA sample pretreatment technique in the future.

An efficient method for visualization and growth of fluorescent Xanthomonas oryzae pv. oryzae in planta

PubMed Central

Han, Sang-Wook; Park, Chang-Jin; Lee, Sang-Won; Ronald, Pamela C

2008-01-01

Background Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight disease, is a serious pathogen of rice. Here we describe a fluorescent marker system to study virulence and pathogenicity of X. oryzae pv. oryzae. Results A fluorescent X. oryzae pv. oryzae Philippine race 6 strain expressing green fluorescent protein (GFP) (PXO99GFP) was generated using the gfp gene under the control of the neomycin promoter in the vector, pPneo-gfp. The PXO99GFPstrain displayed identical virulence and avirulence properties as the wild type control strain, PXO99. Using fluorescent microscopy, bacterial multiplication and colonization were directly observed in rice xylem vessels. Accurate and rapid determination of bacterial growth was assessed using fluoremetry and an Enzyme-Linked ImmunoSorbant Assay (ELISA). Conclusion Our results indicate that the fluorescent marker system is useful for assessing bacterial infection and monitoring bacterial multiplication in planta. PMID:18826644

Quantitative, spectrally-resolved intraoperative fluorescence imaging

PubMed Central

Valdés, Pablo A.; Leblond, Frederic; Jacobs, Valerie L.; Wilson, Brian C.; Paulsen, Keith D.; Roberts, David W.

2012-01-01

Intraoperative visual fluorescence imaging (vFI) has emerged as a promising aid to surgical guidance, but does not fully exploit the potential of the fluorescent agents that are currently available. Here, we introduce a quantitative fluorescence imaging (qFI) approach that converts spectrally-resolved data into images of absolute fluorophore concentration pixel-by-pixel across the surgical field of view (FOV). The resulting estimates are linear, accurate, and precise relative to true values, and spectral decomposition of multiple fluorophores is also achieved. Experiments with protoporphyrin IX in a glioma rodent model demonstrate in vivo quantitative and spectrally-resolved fluorescence imaging of infiltrating tumor margins for the first time. Moreover, we present images from human surgery which detect residual tumor not evident with state-of-the-art vFI. The wide-field qFI technique has broad implications for intraoperative surgical guidance because it provides near real-time quantitative assessment of multiple fluorescent biomarkers across the operative field. PMID:23152935

The complete chloroplast DNA sequences of the charophycean green algae Staurastrum and Zygnema reveal that the chloroplast genome underwent extensive changes during the evolution of the Zygnematales

PubMed Central

Turmel, Monique; Otis, Christian; Lemieux, Claude

2005-01-01

Background The Streptophyta comprise all land plants and six monophyletic groups of charophycean green algae. Phylogenetic analyses of four genes from three cellular compartments support the following branching order for these algal lineages: Mesostigmatales, Chlorokybales, Klebsormidiales, Zygnematales, Coleochaetales and Charales, with the last lineage being sister to land plants. Comparative analyses of the Mesostigma viride (Mesostigmatales) and land plant chloroplast genome sequences revealed that this genome experienced many gene losses, intron insertions and gene rearrangements during the evolution of charophyceans. On the other hand, the chloroplast genome of Chaetosphaeridium globosum (Coleochaetales) is highly similar to its land plant counterparts in terms of gene content, intron composition and gene order, indicating that most of the features characteristic of land plant chloroplast DNA (cpDNA) were acquired from charophycean green algae. To gain further insight into when the highly conservative pattern displayed by land plant cpDNAs originated in the Streptophyta, we have determined the cpDNA sequences of the distantly related zygnematalean algae Staurastrum punctulatum and Zygnema circumcarinatum. Results The 157,089 bp Staurastrum and 165,372 bp Zygnema cpDNAs encode 121 and 125 genes, respectively. Although both cpDNAs lack an rRNA-encoding inverted repeat (IR), they are substantially larger than Chaetosphaeridium and land plant cpDNAs. This increased size is explained by the expansion of intergenic spacers and introns. The Staurastrum and Zygnema genomes differ extensively from one another and from their streptophyte counterparts at the level of gene order, with the Staurastrum genome more closely resembling its land plant counterparts than does Zygnema cpDNA. Many intergenic regions in Zygnema cpDNA harbor tandem repeats. The introns in both Staurastrum (8 introns) and Zygnema (13 introns) cpDNAs represent subsets of those found in land plant cpDNAs. They represent 16 distinct insertion sites, only five of which are shared by the two zygnematalean genomes. Three of these insertions sites have not been identified in Chaetosphaeridium cpDNA. Conclusion The chloroplast genome experienced substantial changes in overall structure, gene order, and intron content during the evolution of the Zygnematales. Most of the features considered earlier as typical of land plant cpDNAs probably originated before the emergence of the Zygnematales and Coleochaetales. PMID:16236178

Fluorescence bio-barcode DNA assay based on gold and magnetic nanoparticles for detection of Exotoxin A gene sequence.

PubMed

Amini, Bahram; Kamali, Mehdi; Salouti, Mojtaba; Yaghmaei, Parichehreh

2017-06-15

Bio-barcode DNA based on gold nanoparticle (bDNA-GNPs) as a new generation of biosensor based detection tools, holds promise for biological science studies. They are of enormous importance in the emergence of rapid and sensitive procedures for detecting toxins of microorganisms. Exotoxin A (ETA) is the most toxic virulence factor of Pseudomonas aeruginosa. ETA has ADP-ribosylation activity and decisively affects the protein synthesis of the host cells. In the present study, we developed a fluorescence bio-barcode technology to trace P. aeruginosa ETA. The GNPs were coated with the first target-specific DNA probe 1 (1pDNA) and bio-barcode DNA, which acted as a signal reporter. The magnetic nanoparticles (MNPs) were coated with the second target-specific DNA probe 2 (2pDNA) that was able to recognize the other end of the target DNA. After binding the nanoparticles with the target DNA, the following sandwich structure was formed: MNP 2pDNA/tDNA/1pDNA-GNP-bDNA. After isolating the sandwiches by a magnetic field, the DNAs of the probes which have been hybridized to their complementary DNA, GNPs and MNPs, via the hydrogen, electrostatic and covalently bonds, were released from the sandwiches after dissolving in dithiothreitol solution (DTT 0.8M). This bio-barcode DNA with known DNA sequence was then detected by fluorescence spectrophotometry. The findings showed that the new method has the advantages of fast, high sensitivity (the detection limit was 1.2ng/ml), good selectivity, and wide linear range of 5-200ng/ml. The regression analysis also showed that there was a good linear relationship (∆F=0.57 [target DNA]+21.31, R 2 =0.9984) between the fluorescent intensity and the target DNA concentration in the samples. Copyright © 2016. Published by Elsevier B.V.

Fluorescent in situ hybridization shows DIPLOSPOROUS located on one of the NOR chromosomes in apomictic dandelions (Taraxacum) in the absence of a large hemizygous chromosomal region.

PubMed

Vašut, Radim J; Vijverberg, Kitty; van Dijk, Peter J; de Jong, Hans

2014-11-01

Apomixis in dandelions (Taraxacum: Asteraceae) is encoded by two unlinked dominant loci and a third yet undefined genetic factor: diplosporous omission of meiosis (DIPLOSPOROUS, DIP), parthenogenetic embryo development (PARTHENOGENESIS, PAR), and autonomous endosperm formation, respectively. In this study, we determined the chromosomal position of the DIP locus in Taraxacum by using fluorescent in situ hybridization (FISH) with bacterial artificial chromosomes (BACs) that genetically map within 1.2-0.2 cM of DIP. The BACs showed dispersed fluorescent signals, except for S4-BAC 83 that displayed strong unique signals as well. Under stringent blocking of repeats by C0t-DNA fragments, only a few fluorescent foci restricted to defined chromosome regions remained, including one on the nucleolus organizer region (NOR) chromosomes that contains the 45S rDNAs. FISH with S4-BAC 83 alone and optimal blocking showed discrete foci in the middle of the long arm of one of the NOR chromosomes only in triploid and tetraploid diplosporous dandelions, while signals in sexual diploids were lacking. This agrees with the genetic model of a single dose, dominant DIP allele, absent in sexuals. The length of the DIP region is estimated to cover a region of 1-10 Mb. FISH in various accessions of Taraxacum and the apomictic sister species Chondrilla juncea, confirmed the chromosomal position of DIP within Taraxacum but not outside the genus. Our results endorse that, compared to other model apomictic species, expressing either diplospory or apospory, the genome of Taraxacum shows a more similar and less diverged chromosome structure at the DIP locus. The different levels of allele sequence divergence at apomeiosis loci may reflect different terms of asexual reproduction. The association of apomeiosis loci with repetitiveness, dispersed repeats, and retrotransposons commonly observed in apomictic species may imply a functional role of these shared features in apomictic reproduction, as is discussed.

Smallpox and pan-Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms

PubMed Central

Kulesh, David A.; Baker, Robert O.; Loveless, Bonnie M.; Norwood, David; Zwiers, Susan H.; Mucker, Eric; Hartmann, Chris; Herrera, Rafael; Miller, David; Christensen, Deanna; Wasieloski, Leonard P.; Huggins, John; Jahrling, Peter B.

2004-01-01

We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3′-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 × 107, 1.24 × 105, 1.24 × 103, and 1.24 × 101 genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs. However, all five assays had nearly 100% sensitivity on both machines with samples above the LOD (>12 gene copies). These real-time PCR assays represent a battery of tests to screen for and confirm the presence of variola virus DNA. The early detection of a smallpox outbreak is crucial whether the incident is an act of bioterrorism or an accidental occurrence. PMID:14766823

Smallpox and pan-orthopox virus detection by real-time 3'-minor groove binder TaqMan assays on the roche LightCycler and the Cepheid smart Cycler platforms.

PubMed

Kulesh, David A; Baker, Robert O; Loveless, Bonnie M; Norwood, David; Zwiers, Susan H; Mucker, Eric; Hartmann, Chris; Herrera, Rafael; Miller, David; Christensen, Deanna; Wasieloski, Leonard P; Huggins, John; Jahrling, Peter B

2004-02-01

We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3'-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 x 10(7), 1.24 x 10(5), 1.24 x 10(3), and 1.24 x 10(1) genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs. However, all five assays had nearly 100% sensitivity on both machines with samples above the LOD (>12 gene copies). These real-time PCR assays represent a battery of tests to screen for and confirm the presence of variola virus DNA. The early detection of a smallpox outbreak is crucial whether the incident is an act of bioterrorism or an accidental occurrence.

A molecular-sized optical logic circuit for digital modulation of a fluorescence signal

NASA Astrophysics Data System (ADS)

Nishimura, Takahiro; Tsuchida, Karin; Ogura, Yusuke; Tanida, Jun

2018-03-01

Fluorescence measurement allows simultaneous detection of multiple molecular species by using spectrally distinct fluorescence probes. However, due to the broad spectra of fluorescence emission, the multiplicity of fluorescence measurement is generally limited. To overcome this limitation, we propose a method to digitally modulate fluorescence output signals with a molecular-sized optical logic circuit by using optical control of fluorescence resonance energy transfer (FRET). The circuit receives a set of optical inputs represented with different light wavelengths, and then it switches high and low fluorescence intensity from a reporting molecule according to the result of the logic operation. By using combinational optical inputs in readout of fluorescence signals, the number of biomolecular species that can be identified is increased. To implement the FRET-based circuits, we designed two types of basic elements, YES and NOT switches. An YES switch produces a high-level output intensity when receiving a designated light wavelength input and a low-level intensity without the light irradiation. A NOT switch operates inversely to the YES switch. In experiments, we investigated the operation of the YES and NOT switches that receive a 532-nm light input and modulate the fluorescence intensity of Alexa Fluor 488. The experimental result demonstrates that the switches can modulate fluorescence signals according to the optical input.

Complete suppression of the fluorophore fluorescence by combined effect of multiple fluorescence quenching groups: A fluorescent sensor for Cu²⁺ with zero background signals.

PubMed

Long, Lingliang; Wu, Yanjun; Wang, Lin; Gong, Aihua; Hu, Rongfeng; Zhang, Chi

2016-02-18

The reaction-based fluorescent sensors have attracted increasing attention in the past decades. However, the application of these sensors for accurate sensing was significantly retarded by the background fluorescence from the sensors themselves. In this work, we demonstrated a novel strategy that the background fluorescence of the sensor could be completely eliminated by the combined effect of multiple fluorescence quenching groups. Based on this new strategy, as proof-of-principle study, a fluorescent sensor (CuFS) for Cu(2+) was judiciously developed. In CuFS, three types of fluorescence quenching groups were directly tethered to a commonly used coumarin fluorophore. The fluorescence of coumarin fluorophore in CuFS was completely suppressed by the combined effect of these fluorescence quenching groups. Upon treatment with 22 μM Cu(2+), sensor CuFS achieved a dramatic fluorescence enhancement (fluorescence intensity enhanced up to 811-fold) centered at 469 nm. The detection limits was determined to be 12.3 nM. The fluorescence intensity enhancement also showed a good linearity with the Cu(2+) concentration in the range of 12.3 nM to 2 μM. By fabricating test strips, sensor CuFS can be utilized as a simple tool to detect Cu(2+) in water samples. Furthermore, the fluorescent sensor was successfully applied in detecting different concentration of Cu(2+) in living cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Difference in trafficking of brain-derived neurotrophic factor between axons and dendrites of cortical neurons, revealed by live-cell imaging

PubMed Central

Adachi, Naoki; Kohara, Keigo; Tsumoto, Tadaharu

2005-01-01

Background Brain-derived neurotrophic factor (BDNF), which is sorted into a regulated secretory pathway of neurons, is supposed to act retrogradely through dendrites on presynaptic neurons or anterogradely through axons on postsynaptic neurons. Depending on which is the case, the pattern and direction of trafficking of BDNF in dendrites and axons are expected to be different. To address this issue, we analyzed movements of green fluorescent protein (GFP)-tagged BDNF in axons and dendrites of living cortical neurons by time-lapse imaging. In part of the experiments, the expression of BDNF tagged with cyan fluorescent protein (CFP) was compared with that of nerve growth factor (NGF) tagged with yellow fluorescent protein (YFP), to see whether fluorescent protein-tagged BDNF is expressed in a manner specific to this neurotrophin. Results We found that BDNF tagged with GFP or CFP was expressed in a punctated manner in dendrites and axons in about two-thirds of neurons into which plasmid cDNAs had been injected, while NGF tagged with GFP or YFP was diffusely expressed even in dendrites in about 70% of the plasmid-injected neurons. In neurons in which BDNF-GFP was expressed as vesicular puncta in axons, 59 and 23% of the puncta were moving rapidly in the anterograde and retrograde directions, respectively. On the other hand, 64% of BDNF-GFP puncta in dendrites did not move at all or fluttered back and forth within a short distance. The rest of the puncta in dendrites were moving relatively smoothly in either direction, but their mean velocity of transport, 0.47 ± 0.23 (SD) μm/s, was slower than that of the moving puncta in axons (0.73 ± 0.26 μm/s). Conclusion The present results show that the pattern and velocity of the trafficking of fluorescence protein-tagged BDNF are different between axons and dendrites, and suggest that the anterograde transport in axons may be the dominant stream of BDNF to release sites. PMID:15969745

[Retrospective, descriptive, observational study of treatment of multiple actinic keratoses with topical methyl aminolevulinate and red light: results in clinical practice and correlation with fluorescence imaging].

PubMed

Fernández-Guarino, M; Harto, A; Sánchez-Ronco, M; Pérez-García, B; Marquet, A; Jaén, P

2008-12-01

Actinic keratosis (AK) is one of the most common skin diseases seen in clinical practice. In the last 5 years, several studies assessing the efficacy of photodynamic therapy in the treatment of multiple AKs have been published. We aimed to assess the clinical outcomes of photodynamic therapy in patients with multiple AKs and the correlation of those outcomes with fluorescence imaging. In this retrospective, descriptive, observational study of 57 patients treated in our hospital with photodynamic therapy for multiple AKs, we recorded age, sex, and lesion site (face, scalp, and dorsum of the hands). All patients were treated in the same way: methyl aminolevulinic acid (Metvix) was applied for 3 hours and the skin then irradiated with red light at 630 nm, 37 J/cm(2), for 7.5 minutes (Aktilite). The response, remission duration, tolerance, number of sessions, and fluorescence images were recorded by site. The chi(2) test was used to assess between-site differences and the correlation between fluorescence imaging and clinical response. The greatest improvements were obtained for facial lesions; these required fewer sessions and remission lasted longer than lesions at other sites. The treatment was best tolerated on the dorsum of the hands. The fluorescence area and the reduction in intensity on applying treatment were found to be strongly and significantly correlated with the extent of clinical response. Overall, the outcomes of treatment of multiple AKs with photodynamic therapy are better for the face than for the scalp and dorsum of the hands. Fluorescence imaging may be an effective tool for predicting response to treatment.

Live Cell Visualization of Multiple Protein-Protein Interactions with BiFC Rainbow.

PubMed

Wang, Sheng; Ding, Miao; Xue, Boxin; Hou, Yingping; Sun, Yujie

2018-05-18

As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.

DNA strand-displacement-induced fluorescence enhancement for highly sensitive and selective assay of multiple microRNA in cancer cells.

PubMed

Wu, Ping; Tu, Yunqiu; Qian, Yingdan; Zhang, Hui; Cai, Chenxin

2014-01-28

We report a new strategy for evaluating multiple miRNA expressions in cancer cells based on DNA strand-displacement-induced fluorescence enhancement. This assay has the ability to discriminate the target from even single-base mismatched sequences or other miRNAs.

Characterization and expression analysis of two cDNAs encoding Xa1 and oxysterol binding proteins in sorghum (Sorghum bicolor)

USDA-ARS?s Scientific Manuscript database

Using suppression subtractive hybridization (SSH) and subsequent microarray analysis, expression profiles of sorghum genes responsive to greenbug phloem-feeding were obtained and identified. Among the profiles, two cDNAs designated to MM73 and MM95 were identified to encode Xa1 (Xa1) and oxysterol ...

Inheritance of restriction fragment length polymorphisms, random amplified polymorphic DNAs and isozymes in coastal Douglas-fir

Treesearch

K.D. Jermstad; A.M. Reem; J.R. Henifin; N.C. Wheeler; D.B Neale

1994-01-01

A total of 225 new genetic loci [151 restriction fragment length polymorphisms (RFLP) and 74 random amplified polymorphic DNAs (RAPD)] in coastal Douglas- fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] have been identified using a three-generation outbred pedigree. The Mendelian inheritance of 16 RFLP loci and 29...

Characterization of cDNAs encoding serine proteases and their transcriptional responses to Cry1Ab protoxin in the gut of Ostrinia nubilalis larvae

USDA-ARS?s Scientific Manuscript database

Serine proteases, such as trypsin and chymotrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt) protoxin activation and protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding trypsins, chymotrypsins and th...

Methylated bases in mycoplasmal DNA.

PubMed Central

Razin, A; Razin, S

1980-01-01

The DNAs of four Mycoplasma and one Acholeplasma species were found to contain methylated bases. All of the five species contained 6-methyladenine (m6Ade), the methylated base characteristic of prokaryotic DNA. The extent of methylation of adenine residues in the mycoplasmal DNA ranged from 0.2% in Mycoplasma capricolum to about 2% in Mycoplasma arginini and Mycoplasma hyorhinis with intermediate methylation values for Mycoplasma orale and Acholeplasma laidlawii DNAs. About 5.8% of the cytosine residues in M. hyorhinis DNA were methylated also. Analysis of cell culture DNA for the presence of m6Ade as a means for detection of contamination by mycoplasmas, and the phylogenetic implications of the finding of methylated bases in mycoplasmal DNAs are discussed. PMID:7433124

Genomic in situ hybridization in interspecific hybrids of scallops (Bivalvia, Pectinidae) and localization of the satellite DNA Cf303, and the vertebrate telomeric sequences (TTAGGG)n on chromosomes of scallop Chlamys farreri (Jones & Preston, 1904)

PubMed Central

Hu, Liping; Jiang, Liming; Bi, Ke; Liao, Huan; Yang, Zujing; Huang, Xiaoting; Bao, Zhenmin

2018-01-01

Abstract Mitotic chromosome preparations of the interspecific hybrids Chlamys farreri (Jones & Preston, 1904) × Patinopecten yessoensis (Jay, 1857), C. farreri × Argopecten irradians (Lamarck, 1819) and C. farreri × Mimachlamys nobilis (Reeve, 1852) were used to compare two different scallop genomes in a single slide. Although genomic in situ hybridization (GISH) using genomic DNA from each scallop species as probe painted mitotic chromosomes of the interspecific hybrids, the painting results were not uniform; instead it showed species-specific distribution patterns of fluorescent signals among the chromosomes. The most prominent GISH-bands were mainly located at centromeric or telomeric regions of scallop chromosomes. In order to illustrate the sequence constitution of the GISH-bands, the satellite Cf303 sequences of C. farreri and the vertebrate telomeric (TTAGGG)n sequences were used to map mitotic chromosomes of C. farreri by fluorescence in situ hybridization (FISH). The results indicated that the GISH-banding pattern presented by the chromosomes of C. farreri is mainly due to the distribution of the satellite Cf303 DNA, therefore suggesting that the GISH-banding patterns found in the other three scallops could also be the result of the chromosomal distribution of other species-specific satellite DNAs. PMID:29675138

Genomic in situ hybridization in interspecific hybrids of scallops (Bivalvia, Pectinidae) and localization of the satellite DNA Cf303, and the vertebrate telomeric sequences (TTAGGG)n on chromosomes of scallop Chlamys farreri (Jones & Preston, 1904).

PubMed

Hu, Liping; Jiang, Liming; Bi, Ke; Liao, Huan; Yang, Zujing; Huang, Xiaoting; Bao, Zhenmin

2018-01-01

Mitotic chromosome preparations of the interspecific hybrids Chlamys farreri (Jones & Preston, 1904) × Patinopecten yessoensis (Jay, 1857), C. farreri × Argopecten irradians (Lamarck, 1819) and C. farreri × Mimachlamys nobilis (Reeve, 1852) were used to compare two different scallop genomes in a single slide. Although genomic in situ hybridization (GISH) using genomic DNA from each scallop species as probe painted mitotic chromosomes of the interspecific hybrids, the painting results were not uniform; instead it showed species-specific distribution patterns of fluorescent signals among the chromosomes. The most prominent GISH-bands were mainly located at centromeric or telomeric regions of scallop chromosomes. In order to illustrate the sequence constitution of the GISH-bands, the satellite Cf303 sequences of C. farreri and the vertebrate telomeric (TTAGGG) n sequences were used to map mitotic chromosomes of C. farreri by fluorescence in situ hybridization (FISH). The results indicated that the GISH-banding pattern presented by the chromosomes of C. farreri is mainly due to the distribution of the satellite Cf303 DNA, therefore suggesting that the GISH-banding patterns found in the other three scallops could also be the result of the chromosomal distribution of other species-specific satellite DNAs.

Atomistic Free Energy Model for Nucleic Acids: Simulations of Single-Stranded DNA and the Entropy Landscape of RNA Stem-Loop Structures.

PubMed

Mak, Chi H

2015-11-25

While single-stranded (ss) segments of DNAs and RNAs are ubiquitous in biology, details about their structures have only recently begun to emerge. To study ssDNA and RNAs, we have developed a new Monte Carlo (MC) simulation using a free energy model for nucleic acids that has the atomisitic accuracy to capture fine molecular details of the sugar-phosphate backbone. Formulated on the basis of a first-principle calculation of the conformational entropy of the nucleic acid chain, this free energy model correctly reproduced both the long and short length-scale structural properties of ssDNA and RNAs in a rigorous comparison against recent data from fluorescence resonance energy transfer, small-angle X-ray scattering, force spectroscopy and fluorescence correlation transport measurements on sequences up to ∼100 nucleotides long. With this new MC algorithm, we conducted a comprehensive investigation of the entropy landscape of small RNA stem-loop structures. From a simulated ensemble of ∼10(6) equilibrium conformations, the entropy for the initiation of different size RNA hairpin loops was computed and compared against thermodynamic measurements. Starting from seeded hairpin loops, constrained MC simulations were then used to estimate the entropic costs associated with propagation of the stem. The numerical results provide new direct molecular insights into thermodynaimc measurement from macroscopic calorimetry and melting experiments.

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

Nishimura, Takahiro, E-mail: t-nishimura@ist.osaka-u.ac.jp; Fujii, Ryo; Ogura, Yusuke

Molecular logic circuits represent a promising technology for observation and manipulation of biological systems at the molecular level. However, the implementation of molecular logic circuits for temporal and programmable operation remains challenging. In this paper, we demonstrate an optically controllable logic circuit that uses fluorescence resonance energy transfer (FRET) for signaling. The FRET-based signaling process is modulated by both molecular and optical inputs. Based on the distance dependence of FRET, the FRET pathways required to execute molecular logic operations are formed on a DNA nanostructure as a circuit based on its molecular inputs. In addition, the FRET pathways on themore » DNA nanostructure are controlled optically, using photoswitching fluorescent molecules to instruct the execution of the desired operation and the related timings. The behavior of the circuit can thus be controlled using external optical signals. As an example, a molecular logic circuit capable of executing two different logic operations was studied. The circuit contains functional DNAs and a DNA scaffold to construct two FRET routes for executing Input 1 AND Input 2 and Input 1 AND NOT Input 3 operations on molecular inputs. The circuit produced the correct outputs with all possible combinations of the inputs by following the light signals. Moreover, the operation execution timings were controlled based on light irradiation and the circuit responded to time-dependent inputs. The experimental results demonstrate that the circuit changes the output for the required operations following the input of temporal light signals.« less

A generalized model on the effects of nanoparticles on fluorophore fluorescence in solution

USDA-ARS?s Scientific Manuscript database

Nanoparticles (NP) can modify fluorophore fluorescence in solution through multiple pathways that include fluorescence inner filter effect (IFE), dynamic and static quenching, surface enhancement, and fluorophore quantum yield variation associated with structural and conformational modifications ind...

Some properties of human neuronal alpha 7 nicotinic acetylcholine receptors fused to the green fluorescent protein.

PubMed

Palma, Eleonora; Mileo, Anna M; Martinez-Torres, Ataulfo; Eusebi, Fabrizio; Miledi, Ricardo

2002-03-19

The functional properties and cellular localization of the human neuronal alpha7 nicotinic acetylcholine (AcCho) receptor (alpha7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mutalpha7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtalpha7 receptors decay much faster than those elicited by the mutalpha7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated alpha7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable "run-down" of the AcCho currents generated by mutalpha7-GFP receptors, whereas those of the wtalpha7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mutalpha7-GFP oocytes was accompanied by a marked decrease of alpha-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wtalpha7 and mutalpha7 receptors provides powerful tools to study the distribution and function of alpha7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins.

Tetris Is a Foldback Transposon that Provided the Building Blocks for an Emerging Satellite DNA of Drosophila virilis

PubMed Central

Dias, Guilherme B.; Svartman, Marta; Delprat, Alejandra; Ruiz, Alfredo; Kuhn, Gustavo C.S.

2014-01-01

Transposable elements (TEs) and satellite DNAs (satDNAs) are abundant components of most eukaryotic genomes studied so far and their impact on evolution has been the focus of several studies. A number of studies linked TEs with satDNAs, but the nature of their evolutionary relationships remains unclear. During in silico analyses of the Drosophila virilis assembled genome, we found a novel DNA transposon we named Tetris based on its modular structure and diversity of rearranged forms. We aimed to characterize Tetris and investigate its role in generating satDNAs. Data mining and sequence analysis showed that Tetris is apparently nonautonomous, with a structure similar to foldback elements, and present in D. virilis and D. americana. Herein, we show that Tetris shares the final portions of its terminal inverted repeats (TIRs) with DAIBAM, a previously described miniature inverted transposable element implicated in the generation of chromosome inversions. Both elements are likely to be mobilized by the same autonomous TE. Tetris TIRs contain approximately 220-bp internal tandem repeats that we have named TIR-220. We also found TIR-220 repeats making up longer (kb-size) satDNA-like arrays. Using bioinformatic, phylogenetic and cytogenomic tools, we demonstrated that Tetris has contributed to shaping the genomes of D. virilis and D. americana, providing internal tandem repeats that served as building blocks for the amplification of satDNA arrays. The β-heterochromatic genomic environment seemed to have favored such amplification. Our results imply for the first time a role for foldback elements in generating satDNAs. PMID:24858539

TFIIIC Bound DNA Elements in Nuclear Organization and Insulation

PubMed Central

Kirkland, Jacob G.; Raab, Jesse R.

2012-01-01

tRNA genes (tDNAs) have been known to have barrier insulator function in budding yeast, Saccharomyces cerevisiae, for over a decade. tDNAs also play a role in genome organization by clustering at sites in the nucleus and both of these functions are dependent on the transcription factor TFIIIC. More recently TFIIIC bound sites devoid of pol III, termed Extra-TFIIIC sites (ETC) have been identified in budding yeast and these sites also function as insulators and affect genome organization. Subsequent studies in Schizosaccharomyces pombe showed that TFIIIC bound sites were insulators and also functioned as Chromosome Organization Clamps (COC); tethering the sites to the nuclear periphery. Very recently studies have moved to mammalian systems where pol III genes and their associated factors have been investigated in both mouse and human cells. Short Interspersed Nuclear Elements (SINEs) that bind TFIIIC, function as insulator elements and tDNAs can also function as both enhancer -blocking and barrier insulators in these organisms. It was also recently shown that tDNAs cluster with other tDNAs and with ETCs but not with pol II transcribed genes. Intriguingly, TFIIIC is often found near pol II transcription start sites and it remains unclear what the consequences of TFIIIC based genomic organization are and what influence pol III factors have on pol II transcribed genes and vise versa. In this review we provide a comprehensive overview of the known data on pol III factors in insulation and genome organization and identify the many open questions that require further investigation. \\ PMID:23000638

RiceFOX: a database of Arabidopsis mutant lines overexpressing rice full-length cDNA that contains a wide range of trait information to facilitate analysis of gene function.

PubMed

Sakurai, Tetsuya; Kondou, Youichi; Akiyama, Kenji; Kurotani, Atsushi; Higuchi, Mieko; Ichikawa, Takanari; Kuroda, Hirofumi; Kusano, Miyako; Mori, Masaki; Saitou, Tsutomu; Sakakibara, Hitoshi; Sugano, Shoji; Suzuki, Makoto; Takahashi, Hideki; Takahashi, Shinya; Takatsuji, Hiroshi; Yokotani, Naoki; Yoshizumi, Takeshi; Saito, Kazuki; Shinozaki, Kazuo; Oda, Kenji; Hirochika, Hirohiko; Matsui, Minami

2011-02-01

Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named 'RiceFOX'. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/.

RiceFOX: A Database of Arabidopsis Mutant Lines Overexpressing Rice Full-Length cDNA that Contains a Wide Range of Trait Information to Facilitate Analysis of Gene Function

PubMed Central

Sakurai, Tetsuya; Kondou, Youichi; Akiyama, Kenji; Kurotani, Atsushi; Higuchi, Mieko; Ichikawa, Takanari; Kuroda, Hirofumi; Kusano, Miyako; Mori, Masaki; Saitou, Tsutomu; Sakakibara, Hitoshi; Sugano, Shoji; Suzuki, Makoto; Takahashi, Hideki; Takahashi, Shinya; Takatsuji, Hiroshi; Yokotani, Naoki; Yoshizumi, Takeshi; Saito, Kazuki; Shinozaki, Kazuo; Oda, Kenji; Hirochika, Hirohiko; Matsui, Minami

2011-01-01

Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named ‘RiceFOX’. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/. PMID:21186176

Sequencing and annotation of the chloroplast DNAs and identification of polymorphisms distinguishing normal male-fertile and male-sterile cytoplasms of onion

USDA-ARS?s Scientific Manuscript database

Due the biennial generation time of onion, classical crossing takes at least four years to classify cytoplasms as normal (N) male-fertile or male-sterile (S). Molecular markers in the organellar DNAs that distinguish N and S cytoplasms are useful to reduce the time required to classify onion cytopla...

Do Digital Natives Differ by Computer Self-Efficacy and Experience? An Empirical Study

ERIC Educational Resources Information Center

Teo, Timothy

2016-01-01

This study serves to validate a Chinese translation of the Digital Native Assessment Scale (C-DNAS) and assess if significant differences exist between a sample of students and teachers from a culture different than the one used in the development of the DNAS. Participants were 402 university students from one province in Mainland China. Results…

Structurally Complex Organization of Repetitive DNAs in the Genome of Cobia (Rachycentron canadum).

PubMed

Costa, Gideão W W F; Cioffi, Marcelo de B; Bertollo, Luiz A C; Molina, Wagner F

2015-06-01

Repetitive DNAs comprise the largest fraction of the eukaryotic genome. They include microsatellites or simple sequence repeats (SSRs), which play an important role in the chromosome differentiation among fishes. Rachycentron canadum is the only representative of the family Rachycentridae. This species has been focused on several multidisciplinary studies in view of its important potential for marine fish farming. In the present study, distinct classes of repetitive DNAs, with emphasis on SSRs, were mapped in the chromosomes of this species to improve the knowledge of its genome organization. Microsatellites exhibited a diversified distribution, both dispersed in euchromatin and clustered in the heterochromatin. The multilocus location of SSRs strengthened the heterochromatin heterogeneity in this species, as suggested by some previous studies. The colocalization of SSRs with retrotransposons and transposons pointed to a close evolutionary relationship between these repetitive sequences. A number of heterochromatic regions highlighted a greater complex organization than previously supposed, harboring a diversity of repetitive elements. In this sense, there was also evidence of colocalization of active genetic regions and different classes of repetitive DNAs in a common heterochromatic region, which offers a potential opportunity for further researches regarding the interaction of these distinct fractions in fish genomes.

Bacteriophage T4 capsid packaging and unpackaging of DNA and proteins.

PubMed

Mullaney, Julienne M; Black, Lindsay W

2014-01-01

Bacteriophage T4 has proven itself readily amenable to phage-based DNA and protein packaging, expression, and display systems due to its physical resiliency and genomic flexibility. As a large dsDNA phage with dispensable internal proteins and dispensable outer capsid proteins it can be adapted to package both DNA and proteins of interest within the capsid and to display peptides and proteins externally on the capsid. A single 170 kb linear DNA, or single or multiple copies of shorter linear DNAs, of any sequence can be packaged by the large terminase subunit in vitro into protein-containing proheads and give full or partially full capsids. The prohead receptacles for DNA packaging can also display peptides or full-length proteins from capsid display proteins HOC and SOC. Our laboratory has also developed a protein expression, packaging, and processing (PEPP) system which we have found to have advantages over mammalian and bacterial cell systems, including high yield, increased stability, and simplified downstream processing. Proteins that we have produced by the phage PEPP platform include human HIV-1 protease, micrococcal endonuclease from Staphylococcus aureus, restriction endonuclease EcoRI, luciferase, human granulocyte colony stimulating factor (GCSF), green fluorescent protein (GFP), and the 99 amino acid C-terminus of amyloid precursor protein (APP). Difficult to produce proteins that are toxic in mammalian protein expression systems are easily produced, packaged, and processed with the PEPP platform. APP is one example of such a highly refractory protein that has been produced successfully. The methods below describe the procedures for in vitro packaging of proheads with DNA and for producing recombinant T4 phage that carry a gene of interest in the phage genome and produce and internally package the corresponding protein of interest.

Pseudomonas syringae effector avrB confers soybean cultivar-specific avirulence on Soybean mosaic virus adapted for transgene expression but effector avrPto does not.

PubMed

Wang, Li; Eggenberger, Alan; Hill, John; Bogdanove, Adam J

2006-03-01

Soybean mosaic virus (SMV) was adapted for transgene expression in soybean and used to examine the function of avirulence genes avrB and avrPto of Pseudomonas syringae pvs. glycinea and tomato, respectively. A cloning site was introduced between the P1 and HC-Pro genes in 35S-driven infectious cDNAs of strains SMV-N and SMV-G7. Insertion of the uidA gene or the green fluorescent protein gene into either modified cDNA and bombardment into primary leaves resulted in systemic expression that reflected the pattern of viral movement into uninoculated leaves. Insertion of avrB blocked symptom development and detectable viral movement in cv. Harosoy, which carries the Rpg1-b resistance gene corresponding to avrB, but not in cvs. Keburi or Hurrelbrink, which lack Rpg1-b. In Keburi and Hurrelbrink, symptoms caused by SMV carrying avrB appeared more quickly and were more severe than those caused by the virus without avrB. Insertion of avrPto enhanced symptoms in Harosoy, Hurrelbrink, and Keburi. This result was unexpected because avrPto was reported to confer avirulence on P. syringae pv. glycinea inoculated to Harosoy. We inoculated Harosoy with P syringae pv. glycinea expressing avrPto, but observed no hypersensitive reaction, avrPto-dependent induction of pathogenesis-related protein la, or limitation of bacterial population growth. In Hurrelbrink, avrPto enhanced bacterial multiplication and exacerbated symptoms. Our results establish SMV as an expression vector for soybean. They demonstrate that resistance triggered by avrB is effective against SMV, and that avrB and avrPto have general virulence effects in soybean. The results also led to a reevaluation of the reported avirulence activity of avrPto in this plant.

DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

PubMed

Yamagishi, J; Isobe, R; Takebuchi, T; Bando, H

2003-03-01

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

DNA motion capture reveals the mechanical properties of DNA at the mesoscale.

PubMed

Price, Allen C; Pilkiewicz, Kevin R; Graham, Thomas G W; Song, Dan; Eaves, Joel D; Loparo, Joseph J

2015-05-19

Single-molecule studies probing the end-to-end extension of long DNAs have established that the mechanical properties of DNA are well described by a wormlike chain force law, a polymer model where persistence length is the only adjustable parameter. We present a DNA motion-capture technique in which DNA molecules are labeled with fluorescent quantum dots at specific sites along the DNA contour and their positions are imaged. Tracking these positions in time allows us to characterize how segments within a long DNA are extended by flow and how fluctuations within the molecule are correlated. Utilizing a linear response theory of small fluctuations, we extract elastic forces for the different, ∼2-μm-long segments along the DNA backbone. We find that the average force-extension behavior of the segments can be well described by a wormlike chain force law with an anomalously small persistence length. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Controlled enzymatic cutting of DNA molecules adsorbed on surfaces using soft lithography

NASA Astrophysics Data System (ADS)

Auerbach, Alyssa; Budassi, Julia; Shea, Emily; Zhu, Ke; Sokolov, Jonathan

2013-03-01

The enzyme DNase I was applied to adsorbed and aligned DNA molecules (Lamda, 48.5 kilobase pairs (kbp), and T4, 165.6 kbp), stretched linearly on a surface, by stamping with a polydimethylsiloxane (PDMS) grating. The DNAs were cut by the enzyme into separated, micron-sized segments along the length of the molecules at positions determined by the grating dimensions (3-20 microns). Ozone-treated PDMS stamps were coated with DNase I solutions and placed in contact with surface-adsorbed DNA molecules deposited on a 750 polymethylmethacrylate (PMMA) film spun-cast onto a silicon substrate. The stamps were applied under pressure for times up to 15 minutes at 37 C. The cutting was observed by fluorescence microscopy imaging of DNA labeled with YOYO dye. Cutting was found to be efficient despite the steric hindrance due to surface attachment of the molecules. Methods for detaching and separating the cut segments for sequencing applications will be discussed. Supported by NSF-DMR program.

A multifaceted FISH approach to study endogenous RNAs and DNAs in native nuclear and cell structures.

PubMed

Byron, Meg; Hall, Lisa L; Lawrence, Jeanne B

2013-01-01

Fluorescence in situ hybridization (FISH) is not a singular technique, but a battery of powerful and versatile tools for examining the distribution of endogenous genes and RNAs in precise context with each other and in relation to specific proteins or cell structures. This unit offers the details of highly sensitive and successful protocols that were initially developed largely in our lab and honed over a number of years. Our emphasis is on analysis of nuclear RNAs and DNA to address specific biological questions about nuclear structure, pre-mRNA metabolism, or the role of noncoding RNAs; however, cytoplasmic RNA detection is also discussed. Multifaceted molecular cytological approaches bring precise resolution and sensitive multicolor detection to illuminate the organization and functional roles of endogenous genes and their RNAs within the native structure of fixed cells. Solutions to several common technical pitfalls are discussed, as are cautions regarding the judicious use of digital imaging and the rigors of analyzing and interpreting complex molecular cytological results.

cDNAs for the synthesis of cyclic carotenoids in petals of Gentiana lutea and their regulation during flower development.

PubMed

Zhu, Changfu; Yamamura, Saburo; Nishihara, Masashiro; Koiwa, Hiroyuki; Sandmann, Gerhard

2003-02-20

cDNAs encoding lycopene epsilon -cyclase, lycopene beta-cyclase, beta-carotene hydroxylase and zeaxanthin epoxidase were isolated from a Gentiana lutea petal cDNA library. The function of all cDNAs was analyzed by complementation in Escherichia coli. Transcript levels during different stages of flower development of G. lutea were determined and compared to the carotenoid composition. Expression of all genes increased by a factor of up to 2, with the exception of the lycopene epsilon -cyclase gene. The transcript amount of the latter was strongly decreased. These results indicate that during flower development, carotenoid formation is enhanced. Moreover, metabolites are shifted away from the biosynthetic branch to lutein and are channeled into beta-carotene and derivatives.

Excitation of surface plasmon polariton modes with multiple nitrogen vacancy centers in single nanodiamonds

NASA Astrophysics Data System (ADS)

Kumar, Shailesh; Lausen, Jens L.; Garcia-Ortiz, Cesar E.; Andersen, Sebastian K. H.; Roberts, Alexander S.; Radko, Ilya P.; Smith, Cameron L. C.; Kristensen, Anders; Bozhevolnyi, Sergey I.

2016-02-01

Nitrogen-vacancy (NV) centers in diamonds are interesting due to their remarkable characteristics that are well suited to applications in quantum-information processing and magnetic field sensing, as well as representing stable fluorescent sources. Multiple NV centers in nanodiamonds (NDs) are especially useful as biological fluorophores due to their chemical neutrality, brightness and room-temperature photostability. Furthermore, NDs containing multiple NV centers also have potential in high-precision magnetic field and temperature sensing. Coupling NV centers to propagating surface plasmon polariton (SPP) modes gives a base for lab-on-a-chip sensing devices, allows enhanced fluorescence emission and collection which can further enhance the precision of NV-based sensors. Here, we investigate coupling of multiple NV centers in individual NDs to the SPP modes supported by silver surfaces protected by thin dielectric layers and by gold V-grooves (VGs) produced via the self-terminated silicon etching. In the first case, we concentrate on monitoring differences in fluorescence spectra obtained from a source ND, which is illuminated by a pump laser, and from a scattering ND illuminated only by the fluorescence-excited SPP radiation. In the second case, we observe changes in the average NV lifetime when the same ND is characterized outside and inside a VG. Fluorescence emission from the VG terminations is also observed, which confirms the NV coupling to the VG-supported SPP modes.

Fluorescent image tracking velocimeter

DOEpatents

Shaffer, Franklin D.

1994-01-01

A multiple-exposure fluorescent image tracking velocimeter (FITV) detects and measures the motion (trajectory, direction and velocity) of small particles close to light scattering surfaces. The small particles may follow the motion of a carrier medium such as a liquid, gas or multi-phase mixture, allowing the motion of the carrier medium to be observed, measured and recorded. The main components of the FITV include: (1) fluorescent particles; (2) a pulsed fluorescent excitation laser source; (3) an imaging camera; and (4) an image analyzer. FITV uses fluorescing particles excited by visible laser light to enhance particle image detectability near light scattering surfaces. The excitation laser light is filtered out before reaching the imaging camera allowing the fluoresced wavelengths emitted by the particles to be detected and recorded by the camera. FITV employs multiple exposures of a single camera image by pulsing the excitation laser light for producing a series of images of each particle along its trajectory. The time-lapsed image may be used to determine trajectory and velocity and the exposures may be coded to derive directional information.

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

PubMed

Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

Cs2SO4 Gradients Containing Both Hg2+ and Ag+ Effect the Complete Separation of Satellite Deoxyribonucleic Acids Having Identical Densities in Neutral CsCl Gradients

PubMed Central

Skinner, Dorothy M.; Beattie, Wanda G.

1973-01-01

A combination of both Ag+ and Hg2+ in Cs2SO4 effects the complete separation of two DNAs having identical densities in CsCl. Satellite DNAs of hermit crab, Pagurus pollicaris, and lobster, Homarus americanus, have been isolated by this means. PMID:4522292

Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species.

PubMed

Zheng, X; Seiliez, I; Hastings, N; Tocher, D R; Panserat, S; Dickson, C A; Bergot, P; Teale, A J

2004-10-01

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.

Two RNAs or DNAs May Artificially Fuse Together at a Short Homologous Sequence (SHS) during Reverse Transcription or Polymerase Chain Reactions, and Thus Reporting an SHS-Containing Chimeric RNA Requires Extra Caution

PubMed Central

Xie, Bingkun; Yang, Wei; Ouyang, Yongchang; Chen, Lichan; Jiang, Hesheng; Liao, Yuying; Liao, D. Joshua

2016-01-01

Tens of thousands of chimeric RNAs have been reported. Most of them contain a short homologous sequence (SHS) at the joining site of the two partner genes but are not associated with a fusion gene. We hypothesize that many of these chimeras may be technical artifacts derived from SHS-caused mis-priming in reverse transcription (RT) or polymerase chain reactions (PCR). We cloned six chimeric complementary DNAs (cDNAs) formed by human mitochondrial (mt) 16S rRNA sequences at an SHS, which were similar to several expression sequence tags (ESTs).These chimeras, which could not be detected with cDNA protection assay, were likely formed because some regions of the 16S rRNA are reversely complementary to another region to form an SHS, which allows the downstream sequence to loop back and anneal at the SHS to prime the synthesis of its complementary strand, yielding a palindromic sequence that can form a hairpin-like structure.We identified a 16S rRNA that ended at the 4th nucleotide(nt) of the mt-tRNA-leu was dominant and thus should be the wild type. We also cloned a mouse Bcl2-Nek9 chimeric cDNA that contained a 5-nt unmatchable sequence between the two partners, contained two copies of the reverse primer in the same direction but did not contain the forward primer, making it unclear how this Bcl2-Nek9 was formed and amplified. Moreover, a cDNA was amplified because one primer has 4 nts matched to the template, suggesting that there may be many more artificial cDNAs than we have realized, because the nuclear and mt genomes have many more 4-nt than 5-nt or longer homologues. Altogether, the chimeric cDNAs we cloned are good examples suggesting that many cDNAs may be artifacts due to SHS-caused mis-priming and thus greater caution should be taken when new sequence is obtained from a technique involving DNA polymerization. PMID:27148738

Isolation and characterisation of mRNA encoding the salmon- and chicken-II type gonadotrophin-releasing hormones in the teleost fish Rutilus rutilus (Cyprinidae).

PubMed

Penlington, M C; Williams, M A; Sumpter, J P; Rand-Weaver, M; Hoole, D; Arme, C

1997-12-01

The complementary DNAs (cDNA) encoding the [Trp7,Leu8]-gonadotrophin-releasing hormone (salmon-type GnRH; sGnRH:GeneBank accession no. u60667) and the [His5,Trp7,Tyr8]-GnRH (chicken-II-type GnRH; cGnRH-II: GeneBank accession no. u60668) precursor in the roach (Rutilus rutilus) were isolated and sequenced following reverse transcription and rapid amplification of cDNA ends (RACE). The sGnRH and cGnRH-II precursor cDNAs consisted of 439 and 628 bp, and included open reading frames of 282 and 255 bp respectively. The structures of the encoded peptides were the same as GnRHs previously identified in other vertebrates. The sGnRH and cGnRH-II precursor cDNAs, including the non-coding regions, had 88.6 and 79.9% identity respectively, to those identified in goldfish (Carassius auratus). However, significant similarity was not observed between the non-coding regions of the GnRH cDNAs of Cyprinidae and other fish. The presumed third exon, encoding partial sGnRH associated peptide (GAP) of roach, demonstrated significant nucleotide and amino acid similarity with the appropriate regions in the goldfish, but not with other species, and this may indicate functional differences of GAP between different families of fish. cGnRH-II precursor cDNAs from roach had relatively high nucleotide similarity across this GnRH variant. Cladistic analysis classified the sGnRH and cGnRH-II precursor cDNAs into three and two groups respectively. However, the divergence between nucleotide sequences within the sGnRH variant was greater than those encoding the cGnRH-II precursors. Consistent with the consensus developed from previous studies, Northern blot analysis demonstrated that expression of sGnRH and cGnRH-II was restricted to the olfactory bulbs and midbrain of roach respectively. This work forms the basis for further study on the mechanisms by which the tapeworm, Ligula intestinalis, interacts with the pituitary-gonadal axis of its fish host.

Apparatus for eliminating background interference in fluorescence measurements

DOEpatents

Martin, J.C.; Jett, J.H.

1984-01-06

The disclosure is directed to an apparatus for eliminating background interference during fluorescence measurements in a multiple laser flow cytometer. A biological particle stained with fluorescent dyes is excited by a laser. A fluorescence detector detects the fluorescence. The particle scatters light and a gate signal is generated and delayed until the biological particle reaches the next laser. The delayed signal turns on this next laser which excites a different stained component of the same biological particle.

Apparatus for eliminating background interference in fluorescence measurements

DOEpatents

Martin, John C.; Jett, James H.

1986-01-01

The disclosure is directed to an apparatus for eliminating background interference during fluorescence measurements in a multiple laser flow cytometer. A biological particle stained with fluorescent dyes is excited by a laser. A fluorescence detector detects the fluorescence. The particle scatters light and a gate signal is generated and delayed until the biological particle reaches the next laser. The delayed signal turns on this next laser, which excites a different stained component of the same biological particle.

Apparatus for eliminating background interference in fluorescence measurements

DOEpatents

Martin, J.C.; Jett, J.H.

1986-03-04

The disclosure is directed to an apparatus for eliminating background interference during fluorescence measurements in a multiple laser flow cytometer. A biological particle stained with fluorescent dyes is excited by a laser. A fluorescence detector detects the fluorescence. The particle scatters light and a gate signal is generated and delayed until the biological particle reaches the next laser. The delayed signal turns on this next laser, which excites a different stained component of the same biological particle. 8 figs.

Tetris is a foldback transposon that provided the building blocks for an emerging satellite DNA of Drosophila virilis.

PubMed

Dias, Guilherme B; Svartman, Marta; Delprat, Alejandra; Ruiz, Alfredo; Kuhn, Gustavo C S

2014-05-24

Transposable elements (TEs) and satellite DNAs (satDNAs) are abundant components of most eukaryotic genomes studied so far and their impact on evolution has been the focus of several studies. A number of studies linked TEs with satDNAs, but the nature of their evolutionary relationships remains unclear. During in silico analyses of the Drosophila virilis assembled genome, we found a novel DNA transposon we named Tetris based on its modular structure and diversity of rearranged forms. We aimed to characterize Tetris and investigate its role in generating satDNAs. Data mining and sequence analysis showed that Tetris is apparently nonautonomous, with a structure similar to foldback elements, and present in D. virilis and D. americana. Herein, we show that Tetris shares the final portions of its terminal inverted repeats (TIRs) with DAIBAM, a previously described miniature inverted transposable element implicated in the generation of chromosome inversions. Both elements are likely to be mobilized by the same autonomous TE. Tetris TIRs contain approximately 220-bp internal tandem repeats that we have named TIR-220. We also found TIR-220 repeats making up longer (kb-size) satDNA-like arrays. Using bioinformatic, phylogenetic and cytogenomic tools, we demonstrated that Tetris has contributed to shaping the genomes of D. virilis and D. americana, providing internal tandem repeats that served as building blocks for the amplification of satDNA arrays. The β-heterochromatic genomic environment seemed to have favored such amplification. Our results imply for the first time a role for foldback elements in generating satDNAs. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Direct Determination of Molecular Weight Distribution of Calf-Thymus DNAs and Study of Their Fragmentation under Ultrasonic and Low-Energy IR Irradiations. A Charge Detection Mass Spectrometry Investigation.

PubMed

Halim, Mohammad A; Bertorelle, Franck; Doussineau, Tristan; Antoine, Rodolphe

2018-06-09

Calf-thymus (CT-DNA) is widely used as binding agent. The commercial samples are known to be "highly polymerized DNA" samples. CT-DNA is known to be fragile in particular upon ultrasonic wave irradiation. Degradation products might have dramatic consequence on its bio-sensing activity, and an accurate determination of the molecular weight distribution and stability of commercial samples is highly demanded. We investigated the sensitivity of charge detection mass spectrometry (CDMS), a single-molecule MS method, both with single-pass and ion trap CDMS ("Benner" trap) modes to the determination of the composition and stability (under multiphoton IR irradiation) of calf-thymus DNAs. We also investigated the changes of molecular weight distributions in the course of sonication by irradiating ultrasonic wave to CT-DNA. We report for the first time, the direct molecular weight (MW) distribution of DNA sodium salt from calf-thymus revealing two populations at high (~10 MDa) and low (~3 MDa) molecular weights. We evidence a transition between the high-MW to the low-MW distribution, confirming that the low-MW distribution results from degradation of CT-DNA. Finally, we report also IRMPD experiments carried out on trapped single-stranded linear DNAs from calf-thymus allowing to extract their activation energy for unimolecular dissociation. We show that single-pass CDMS is a direct, efficient and accurate MS-based approach to determine the composition of calf-thymus DNAs. Furthermore, ion trap CDMS allows us to evaluate the stability (both under multiphoton IR irradiation and in the course of sonication by irradiating ultrasonic wave) of calf-thymus DNAs. This article is protected by copyright. All rights reserved.

The complete chloroplast DNA sequence of the green alga Nephroselmis olivacea: Insights into the architecture of ancestral chloroplast genomes

PubMed Central

Turmel, Monique; Otis, Christian; Lemieux, Claude

1999-01-01

Green plants seem to form two sister lineages: Chlorophyta, comprising the green algal classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae, and Streptophyta, comprising the Charophyceae and land plants. We have determined the complete chloroplast DNA (cpDNA) sequence (200,799 bp) of Nephroselmis olivacea, a member of the class (Prasinophyceae) thought to include descendants of the earliest-diverging green algae. The 127 genes identified in this genome represent the largest gene repertoire among the green algal and land plant cpDNAs completely sequenced to date. Of the Nephroselmis genes, 2 (ycf81 and ftsI, a gene involved in peptidoglycan synthesis) have not been identified in any previously investigated cpDNA; 5 genes [ftsW, rnE, ycf62, rnpB, and trnS(cga)] have been found only in cpDNAs of nongreen algae; and 10 others (ndh genes) have been described only in land plant cpDNAs. Nephroselmis and land plant cpDNAs share the same quadripartite structure—which is characterized by the presence of a large rRNA-encoding inverted repeat and two unequal single-copy regions—and very similar sets of genes in corresponding genomic regions. Given that our phylogenetic analyses place Nephroselmis within the Chlorophyta, these structural characteristics were most likely present in the cpDNA of the common ancestor of chlorophytes and streptophytes. Comparative analyses of chloroplast genomes indicate that the typical quadripartite architecture and gene-partitioning pattern of land plant cpDNAs are ancient features that may have been derived from the genome of the cyanobacterial progenitor of chloroplasts. Our phylogenetic data also offer insight into the chlorophyte ancestor of euglenophyte chloroplasts. PMID:10468594

TFIIIC bound DNA elements in nuclear organization and insulation.

PubMed

Kirkland, Jacob G; Raab, Jesse R; Kamakaka, Rohinton T

2013-01-01

tRNA genes (tDNAs) have been known to have barrier insulator function in budding yeast, Saccharomyces cerevisiae, for over a decade. tDNAs also play a role in genome organization by clustering at sites in the nucleus and both of these functions are dependent on the transcription factor TFIIIC. More recently TFIIIC bound sites devoid of pol III, termed Extra-TFIIIC sites (ETC) have been identified in budding yeast and these sites also function as insulators and affect genome organization. Subsequent studies in Schizosaccharomyces pombe showed that TFIIIC bound sites were insulators and also functioned as Chromosome Organization Clamps (COC); tethering the sites to the nuclear periphery. Very recently studies have moved to mammalian systems where pol III genes and their associated factors have been investigated in both mouse and human cells. Short interspersed nuclear elements (SINEs) that bind TFIIIC, function as insulator elements and tDNAs can also function as both enhancer - blocking and barrier insulators in these organisms. It was also recently shown that tDNAs cluster with other tDNAs and with ETCs but not with pol II transcribed genes. Intriguingly, TFIIIC is often found near pol II transcription start sites and it remains unclear what the consequences of TFIIIC based genomic organization are and what influence pol III factors have on pol II transcribed genes and vice versa. In this review we provide a comprehensive overview of the known data on pol III factors in insulation and genome organization and identify the many open questions that require further investigation. This article is part of a Special Issue entitled: Transcription by Odd Pols. Copyright © 2012 Elsevier B.V. All rights reserved.

Electrochemical detection of methylated DNA on a microfluidic chip with nanoelectrokinetic pre-concentration.

PubMed

Hong, Sung A; Kim, Yong-June; Kim, Sung Jae; Yang, Sung

2018-06-01

DNA methylation is considered to be a promising marker for the early diagnosis and prognosis of cancer. However, direct detection of the methylated DNAs in clinically relevant samples is still challenging because of its extremely low concentration (~fM). Here, an integrated microfluidic chip is reported, which is capable of pre-concentrating the methylated DNAs using ion concentration polarization (ICP) and electrochemically detecting the pre-concentrated DNAs on a single chip. The proposed chip is the first demonstration of an electrochemical detection of both level and concentration of the methylated DNAs by integrating a DNA pre-concentration unit without gene amplification. Using the proposed chip, 500 fM to 500 nM of methylated DNAs is pre-concentrated by almost 100-fold in 10 min, resulting in a drastic improvement of the electrochemical detection threshold down to the fM level. The proposed chip is able to measure not only the DNA concentration, but also the level of methylation using human urine sample by performing a consecutive electrochemical sensing on a chip. For clinical application, the level as well as the concentration of methylation of glutathione-S transferase-P1 (GSTP1) and EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1), which are known to be closely associated with prostate cancer diagnosis, are electrochemically detected in human urine spiked with these genes. The developed chip shows a limit of detection (LoD) of 7.9 pM for GSTP1 and 11.8 pM for EFEMP1 and is able to detect the level of methylation in a wide range from 10% to 100% with the concentration variation from 50 pM to 500 nM. Copyright © 2018 Elsevier B.V. All rights reserved.

A nano grating tunable MEMS optical filter for high-speed on-chip multispectral fluorescent detection.

PubMed

Truxal, Steven C; Huang, Nien-Tsu; Kurabayashi, Katsuo

2009-01-01

We report a microelectromechanical (MEMS) tunable optical filter and its integration in a fluorescence microscope for high speed on-chip spectral measurements. This integration allows for measurements of any fluorescence sample placed onto the microscope stage. We demonstrate the system capabilities by taking spectral measurements of multicolor fluorescent beads and fluorescently labeled cells passing through a microfluidic cytometer. The system has applications in biological studies where the measurement of multiple fluorescent peaks is restricted by the detection method's speed and sensitivity.

Four rice seed cDNA clones belonging to the alpha-amylase/trypsin inhibitor gene family encode potential rice allergens.

PubMed

Alvarez, A M; Fukuhara, E; Nakase, M; Adachi, T; Aoki, N; Nakamura, R; Matsuda, T

1995-07-01

Four rice seed proteins encoded by cDNAs belonging to the alpha-amylase/trypsin inhibitor gene family were overexpressed as TrpE-fusion proteins in E. coli. The expressed rice proteins were detected by SDS-PAGE as major proteins in bacterial cell lysates. Western blot analyses showed that all the recombinant proteins were immunologically reactive to rabbit polyclonal antibodies and to a mouse monoclonal antibody (25B9) specific for a previously isolated rice allergen of 16 kDa. Some truncated proteins from deletion mutants of the cDNAs retained their reactivity to the specific antibodies. These results suggest that the cDNAs encode potential rice allergens and that some epitopes of the recombinant proteins are still immunoreactive when they are expressed as their fragments.

Reconstructing the complex evolutionary history of mobile plasmids in red algal genomes

PubMed Central

Lee, JunMo; Kim, Kyeong Mi; Yang, Eun Chan; Miller, Kathy Ann; Boo, Sung Min; Bhattacharya, Debashish; Yoon, Hwan Su

2016-01-01

The integration of foreign DNA into algal and plant plastid genomes is a rare event, with only a few known examples of horizontal gene transfer (HGT). Plasmids, which are well-studied drivers of HGT in prokaryotes, have been reported previously in red algae (Rhodophyta). However, the distribution of these mobile DNA elements and their sites of integration into the plastid (ptDNA), mitochondrial (mtDNA), and nuclear genomes of Rhodophyta remain unknown. Here we reconstructed the complex evolutionary history of plasmid-derived DNAs in red algae. Comparative analysis of 21 rhodophyte ptDNAs, including new genome data for 5 species, turned up 22 plasmid-derived open reading frames (ORFs) that showed syntenic and copy number variation among species, but were conserved within different individuals in three lineages. Several plasmid-derived homologs were found not only in ptDNA but also in mtDNA and in the nuclear genome of green plants, stramenopiles, and rhizarians. Phylogenetic and plasmid-derived ORF analyses showed that the majority of plasmid DNAs originated within red algae, whereas others were derived from cyanobacteria, other bacteria, and viruses. Our results elucidate the evolution of plasmid DNAs in red algae and suggest that they spread as parasitic genetic elements. This hypothesis is consistent with their sporadic distribution within Rhodophyta. PMID:27030297

Continuous in vitro evolution of bacteriophage RNA polymerase promoters

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Banerji, A.; Joyce, G. F.

1994-01-01

Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.

Solution Hybrid Selection Capture for the Recovery of Functional Full-Length Eukaryotic cDNAs From Complex Environmental Samples

PubMed Central

Bragalini, Claudia; Ribière, Céline; Parisot, Nicolas; Vallon, Laurent; Prudent, Elsa; Peyretaillade, Eric; Girlanda, Mariangela; Peyret, Pierre; Marmeisse, Roland; Luis, Patricia

2014-01-01

Eukaryotic microbial communities play key functional roles in soil biology and potentially represent a rich source of natural products including biocatalysts. Culture-independent molecular methods are powerful tools to isolate functional genes from uncultured microorganisms. However, none of the methods used in environmental genomics allow for a rapid isolation of numerous functional genes from eukaryotic microbial communities. We developed an original adaptation of the solution hybrid selection (SHS) for an efficient recovery of functional complementary DNAs (cDNAs) synthesized from soil-extracted polyadenylated mRNAs. This protocol was tested on the Glycoside Hydrolase 11 gene family encoding endo-xylanases for which we designed 35 explorative 31-mers capture probes. SHS was implemented on four soil eukaryotic cDNA pools. After two successive rounds of capture, >90% of the resulting cDNAs were GH11 sequences, of which 70% (38 among 53 sequenced genes) were full length. Between 1.5 and 25% of the cloned captured sequences were expressed in Saccharomyces cerevisiae. Sequencing of polymerase chain reaction-amplified GH11 gene fragments from the captured sequences highlighted hundreds of phylogenetically diverse sequences that were not yet described, in public databases. This protocol offers the possibility of performing exhaustive exploration of eukaryotic gene families within microbial communities thriving in any type of environment. PMID:25281543

SINGLE STRAND-CONTAINING REPLICATING MOLECULES OF CIRCULAR MITOCHONDRIAL DNA

PubMed Central

Wolstenholme, David R.; Koike, Katsuro; Cochran-Fouts, Patricia

1973-01-01

Mitochondrial DNAs (mtDNAs) from Chang rat solid hepatomas and Novikoff rat ascites hepatomas were examined in the electron microscope after preparation by the aqueous and by the formamide protein monolayer techniques. MtDNAs from both tumors were found to include double-forked circular molecules with a form and size suggesting they were replicative intermediates. These molecules were of two classes. In molecules of one class, all three segments were apparently totally double stranded. Molecules of the second class were distinguished by the fact that one of the segments spanning the region between the forks in which replication had occurred (the daughter segments) was either totally single stranded, or contained a single-stranded region associated with one of the forks. Daughter segments of both totally double-stranded and single strand-containing replicating molecules varied in length from about 3 to about 80% of the circular contour length of the molecule. Similar classes of replicating molecules were found in mtDNA from regenerating rat liver and chick embryos, indicating them to be normal intermediates in the replication of mtDNA All of the mtDNAs examined included partially single-stranded simple (nonforked) circular molecules. A possible scheme for the replication of mtDNA is presented, based on the different molecular forms observed PMID:4345165

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

Combined RT-qPCR of mRNA and microRNA Targets within One Fluidigm Integrated Fluidic Circuit.

PubMed

Baldwin, Don A; Horan, Annamarie D; Hesketh, Patrick J; Mehta, Samir

2016-07-01

The ability to profile expression levels of a large number of mRNAs and microRNAs (miRNAs) within the same sample, using a single assay method, would facilitate investigations of miRNA effects on mRNA abundance and streamline biomarker screening across multiple RNA classes. A protocol is described for reverse transcription of long RNA and miRNA targets, followed by preassay amplification of the pooled cDNAs and quantitative PCR (qPCR) detection for a mixed panel of candidate RNA biomarkers. The method provides flexibility for designing custom target panels, is robust over a range of input RNA amounts, and demonstrated a high assay success rate.

Two potential calmodulin-binding sequences in the ryanodine receptor contribute to a mobile, intra-subunit calmodulin-binding domain

PubMed Central

Huang, Xiaojun; Liu, Ying; Wang, Ruiwu; Zhong, Xiaowei; Liu, Yingjie; Koop, Andrea; Chen, S. R. Wayne; Wagenknecht, Terence; Liu, Zheng

2013-01-01

Summary Calmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the calcium release channel/ryanodine receptor (RyR), and modulate RyR function. CaM binding studies using RyR fragments or synthetic peptides have revealed the presence of multiple, potential CaM-binding regions in the primary sequence of RyR. In the present study, we inserted GFP into two of these proposed CaM-binding sequences and mapped them onto the three-dimensional structure of intact cardiac RyR2 by cryo-electron microscopy. Interestingly, we found that the two potential CaM-binding regions encompassing, Arg3595 and Lys4269, respectively, are in close proximity and are adjacent to the previously mapped CaM-binding sites. To monitor the conformational dynamics of these CaM-binding regions, we generated a fluorescence resonance energy transfer (FRET) pair, a dual CFP- and YFP-labeled RyR2 (RyR2R3595-CFP/K4269-YFP) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with the RyR2R3595-CFP/K4269-YFP cDNA, and examined their FRET signal in live cells. We detected significant FRET signals in transfected cells that are sensitive to the channel activator caffeine, suggesting that caffeine is able to induce conformational changes in these CaM-binding regions. Importantly, no significant FRET signals were detected in cells co-transfected with cDNAs encoding the single CFP (RyR2R3595-CFP) and single YFP (RyR2K4269-YFP) insertions, indicating that the FRET signal stemmed from the interaction between R3595–CFP and K4269–YFP that are in the same RyR subunit. These observations suggest that multiple regions in the RyR2 sequence may contribute to an intra-subunit CaM-binding pocket that undergoes conformational changes during channel gating. PMID:23868982

Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish.

PubMed

Callard, G V; Tchoudakova, A V; Kishida, M; Wood, E

2001-12-01

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish (Carassius auratus) and zebrafish (Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (P450aromB>A) and ovary (P450aromA>B) and have a different developmental program (B>A) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24-48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (alpha, beta, and gamma). The 5'-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (a>b) are opposite to fish pituitary (b>a). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30-48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

PubMed

Ma, Qian; Khademhosseinieh, Bahar; Huang, Eric; Qian, Haoliang; Bakowski, Malina A; Troemel, Emily R; Liu, Zhaowei

2016-08-16

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

PubMed Central

Migita, M; Medin, J A; Pawliuk, R; Jacobson, S; Nagle, J W; Anderson, S; Amiri, M; Humphries, R K; Karlsson, S

1995-01-01

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected. Images Fig. 2 Fig. 3 PMID:8618847

Chair-side detection of Prevotella Intermedia in mature dental plaque by its fluorescence.

PubMed

Nomura, Yoshiaki; Takeuchi, Hiroaki; Okamoto, Masaaki; Sogabe, Kaoru; Okada, Ayako; Hanada, Nobuhiro

2017-06-01

Prevotella intermedia/nigrescens is one of the well-known pathogens causing periodontal diseases, and the red florescence excited by the visible blue light caused by the protoporphyrin IX in the bacterial cells could be useful for the chair-side detection. The aim of this study was to evaluated levels of periodontal pathogen, especially P. intermedia in clinical samples of red fluorescent dental plaque. Thirty two supra gingival plaque samples from six individuals were measured its fluorescence at 640nm wavelength excited by 409nm. Periodontopathic bacteria were counted by the Invader PLUS PCR assay. Co-relations the fluorescence intensity and bacterial counts were analyzed by Person's correlation coefficient and simple and multiple regression analysis. Positive and negative predictive values of the fluorescence intensities for with or without P. intermedia in supragingival plaque was calculated. When relative fluorescence unit (RFU) were logarithmic transformed, statistically significant linear relations between RFU and bacterial counts were obtained for P. intermedia, Porphyromonas gingivalis and Tannerella forsythia. By the multiple regression analysis, only P. intermedia had statistically significant co-relation with fluorescence intensities. All of the fluorescent dental plaque contained P. intermedia m. In contrast, 28% of non-fluorescent plaques contained P. intermedia. To check the fluorescence dental plaque in the oral cavity could be the simple chair-side screening of the mature dental plaque before examining the periodontal pathogens especially P. intermedia by the PCR method. Copyright © 2017 Elsevier B.V. All rights reserved.

Estimating the Biodegradability of Treated Sewage Samples Using Synchronous Fluorescence Spectra

PubMed Central

Lai, Tien M.; Shin, Jae-Ki; Hur, Jin

2011-01-01

Synchronous fluorescence spectra (SFS) and the first derivative spectra of the influent versus the effluent wastewater samples were compared and the use of fluorescence indices is suggested as a means to estimate the biodegradability of the effluent wastewater. Three distinct peaks were identified from the SFS of the effluent wastewater samples. Protein-like fluorescence (PLF) was reduced, whereas fulvic and/or humic-like fluorescence (HLF) were enhanced, suggesting that the two fluorescence characteristics may represent biodegradable and refractory components, respectively. Five fluorescence indices were selected for the biodegradability estimation based on the spectral features changing from the influent to the effluent. Among the selected indices, the relative distribution of PLF to the total fluorescence area of SFS (Index II) exhibited the highest correlation coefficient with total organic carbon (TOC)-based biodegradability, which was even higher than those obtained with the traditional oxygen demand-based parameters. A multiple regression analysis using Index II and the area ratio of PLF to HLF (Index III) demonstrated the enhancement of the correlations from 0.558 to 0.711 for TOC-based biodegradability. The multiple regression equation finally obtained was 0.148 × Index II − 4.964 × Index III − 0.001 and 0.046 × Index II − 1.128 × Index III + 0.026. The fluorescence indices proposed here are expected to be utilized for successful development of real-time monitoring using a simple fluorescence sensing device for the biodegradability of treated sewage. PMID:22164023

Estimating the biodegradability of treated sewage samples using synchronous fluorescence spectra.

PubMed

Lai, Tien M; Shin, Jae-Ki; Hur, Jin

2011-01-01

Synchronous fluorescence spectra (SFS) and the first derivative spectra of the influent versus the effluent wastewater samples were compared and the use of fluorescence indices is suggested as a means to estimate the biodegradability of the effluent wastewater. Three distinct peaks were identified from the SFS of the effluent wastewater samples. Protein-like fluorescence (PLF) was reduced, whereas fulvic and/or humic-like fluorescence (HLF) were enhanced, suggesting that the two fluorescence characteristics may represent biodegradable and refractory components, respectively. Five fluorescence indices were selected for the biodegradability estimation based on the spectral features changing from the influent to the effluent. Among the selected indices, the relative distribution of PLF to the total fluorescence area of SFS (Index II) exhibited the highest correlation coefficient with total organic carbon (TOC)-based biodegradability, which was even higher than those obtained with the traditional oxygen demand-based parameters. A multiple regression analysis using Index II and the area ratio of PLF to HLF (Index III) demonstrated the enhancement of the correlations from 0.558 to 0.711 for TOC-based biodegradability. The multiple regression equation finally obtained was 0.148 × Index II - 4.964 × Index III - 0.001 and 0.046 × Index II - 1.128 × Index III + 0.026. The fluorescence indices proposed here are expected to be utilized for successful development of real-time monitoring using a simple fluorescence sensing device for the biodegradability of treated sewage.

Generation of HIV-1 based bi-cistronic lentiviral vectors for stable gene expression and live cell imaging.

PubMed

Sehgal, Lalit; Budnar, Srikanth; Bhatt, Khyati; Sansare, Sneha; Mukhopadhaya, Amitabha; Kalraiya, Rajiv D; Dalal, Sorab N

2012-10-01

The study of protein-protein interactions, protein localization, protein organization into higher order structures and organelle dynamics in live cells, has greatly enhanced the understanding of various cellular processes. Live cell imaging experiments employ plasmid or viral vectors to express the protein/proteins of interest fused to a fluorescent protein. Unlike plasmid vectors, lentiviral vectors can be introduced into both dividing and non dividing cells, can be pseudotyped to infect a broad or narrow range of cells, and can be used to generate transgenic animals. However, the currently available lentiviral vectors are limited by the choice of fluorescent protein tag, choice of restriction enzyme sites in the Multiple Cloning Sites (MCS) and promoter choice for gene expression. In this report, HIV-1 based bi-cistronic lentiviral vectors have been generated that drive the expression of multiple fluorescent tags (EGFP, mCherry, ECFP, EYFP and dsRed), using two different promoters. The presence of a unique MCS with multiple restriction sites allows the generation of fusion proteins with the fluorescent tag of choice, allowing analysis of multiple fusion proteins in live cell imaging experiments. These novel lentiviral vectors are improved delivery vehicles for gene transfer applications and are important tools for live cell imaging in vivo.

Method for determining surface coverage by materials exhibiting different fluorescent properties

NASA Technical Reports Server (NTRS)

Chappelle, Emmett W. (Inventor); Daughtry, Craig S. T. (Inventor); Mcmurtrey, James E., III (Inventor)

1995-01-01

An improved method for detecting, measuring, and distinguishing crop residue, live vegetation, and mineral soil is presented. By measuring fluorescence in multiple bands, live and dead vegetation are distinguished. The surface of the ground is illuminated with ultraviolet radiation, inducing fluorescence in certain molecules. The emitted fluorescent emission induced by the ultraviolet radiation is measured by means of a fluorescence detector, consisting of a photodetector or video camera and filters. The spectral content of the emitted fluorescent emission is characterized at each point sampled, and the proportion of the sampled area covered by residue or vegetation is calculated.

Cloning of the heat shock protein 90 and 70 genes from the beet armyworm, Spodoptera exigua, and expression characteristics in relation to thermal stress and development

USDA-ARS?s Scientific Manuscript database

Two full-length complementary DNAs (cDNAs) of heat shock protein (HSP) genes (Se-hsp90 and Se-hsp70) were cloned from the beet armyworm, Spodoptera exigua, and their expression was investigated in relation to cold shock, heat shock, and development. The open reading frames of Se-hsp90 and Sehsp70 ar...

Inversions and Gene Order Shuffling in Anopheles gambiae and A. funestus

NASA Astrophysics Data System (ADS)

Sharakhov, Igor V.; Serazin, Andrew C.; Grushko, Olga G.; Dana, Ali; Lobo, Neil; Hillenmeyer, Maureen E.; Westerman, Richard; Romero-Severson, Jeanne; Costantini, Carlo; Sagnon, N'Fale; Collins, Frank H.; Besansky, Nora J.

2002-10-01

In tropical Africa, Anopheles funestus is one of the three most important malaria vectors. We physically mapped 157 A. funestus complementary DNAs (cDNAs) to the polytene chromosomes of this species. Sequences of the cDNAs were mapped in silico to the A. gambiae genome as part of a comparative genomic study of synteny, gene order, and sequence conservation between A. funestus and A. gambiae. These species are in the same subgenus and diverged about as recently as humans and chimpanzees. Despite nearly perfect preservation of synteny, we found substantial shuffling of gene order along corresponding chromosome arms. Since the divergence of these species, at least 70 chromosomal inversions have been fixed, the highest rate of rearrangement of any eukaryote studied to date. The high incidence of paracentric inversions and limited colinearity suggests that locating genes in one anopheline species based on gene order in another may be limited to closely related taxa.

Equilibrious Strand Exchange Promoted by DNA Conformational Switching

NASA Astrophysics Data System (ADS)

Wu, Zhiguo; Xie, Xiao; Li, Puzhen; Zhao, Jiayi; Huang, Lili; Zhou, Xiang

2013-01-01

Most of DNA strand exchange reactions in vitro are based on toehold strategy which is generally nonequilibrium, and intracellular strand exchange mediated by proteins shows little sequence specificity. Herein, a new strand exchange promoted by equilibrious DNA conformational switching is verified. Duplexes containing c-myc sequence which is potentially converted into G-quadruplex are designed in this strategy. The dynamic equilibrium between duplex and G4-DNA is response to the specific exchange of homologous single-stranded DNA (ssDNA). The SER is enzyme free and sequence specific. No ATP is needed and the displaced ssDNAs are identical to the homologous ssDNAs. The SER products and exchange kenetics are analyzed by PAGE and the RecA mediated SER is performed as the contrast. This SER is a new feature of G4-DNAs and a novel strategy to utilize the dynamic equilibrium of DNA conformations.

Characterization of two new plasmid DNAs found in mitochondria of wild-type Neurospora intermedia strains.

PubMed Central

Stohl, L L; Collins, R A; Cole, M D; Lambowitz, A M

1982-01-01

Mitochondria from two Neurospora intermedia strains (P4O5-Labelle and Fiji N6-6) were found to contain plasmid DNAs in addition to the standard mitochondrial DNA species. The plasmid DNAs consist of monomeric circles (4.1-4.3 kbp and 5.2-5.3 kbp for Labelle and Fiji, respectively) and oligomers in which monomers are organized as head-to-tail repeats. DNA-DNA hybridization experiments showed that the plasmids have no substantial sequence homology to mtDNA, to each other, or to a previously characterized mitochondrial plasmid from N. crassa strain Mauriceville-lc (Collins et al. Cell 24, 443-452, 1981). The intramitochondrial location of the plasmids was established by cell fractionation and nuclease protection experiments. In sexual crosses, the plasmids showed strict maternal inheritance, the same as Neurospora mitochondrial DNA. The plasmids may represent a novel class of mitochondrial genetic elements. Images PMID:6280144

Segregation and recombination of a multipartite mitochondrial DNA in populations of the potato cyst nematode Globodera pallida.

PubMed

Armstrong, Miles R; Husmeier, Dirk; Phillips, Mark S; Blok, Vivian C

2007-06-01

The discovery that the potato cyst nematode Globodera pallida has a multipartite mitochondrial DNA (mtDNA) composed, at least in part, of six small circular mtDNAs (scmtDNAs) raised a number of questions concerning the population-level processes that might act on such a complex genome. Here we report our observations on the distribution of some scmtDNAs among a sample of European and South American G. pallida populations. The occurrence of sequence variants of scmtDNA IV in population P4A from South America, and that particular sequence variants are common to the individuals within a single cyst, is described. Evidence for recombination of sequence variants of scmtDNA IV in P4A is also reported. The mosaic structure of P4A scmtDNA IV sequences was revealed using several detection methods and recombination breakpoints were independently detected by maximum likelihood and Bayesian MCMC methods.

Anchoring plant metallothioneins to the inner face of the plasma membrane of Saccharomyces cerevisiae cells leads to heavy metal accumulation.

PubMed

Ruta, Lavinia Liliana; Lin, Ya-Fen; Kissen, Ralph; Nicolau, Ioana; Neagoe, Aurora Daniela; Ghenea, Simona; Bones, Atle M; Farcasanu, Ileana Cornelia

2017-01-01

In this study we engineered yeast cells armed for heavy metal accumulation by targeting plant metallothioneins to the inner face of the yeast plasma membrane. Metallothioneins (MTs) are cysteine-rich proteins involved in the buffering of excess metal ions, especially Cu(I), Zn(II) or Cd(II). The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) were each translationally fused to the C-terminus of a myristoylation green fluorescent protein variant (myrGFP) and expressed in Saccharomyces cerevisiae cells. The myrGFP cassette introduced a yeast myristoylation sequence which allowed directional targeting to the cytosolic face of the plasma membrane along with direct monitoring of the intracellular localization of the recombinant protein by fluorescence microscopy. The yeast strains expressing plant MTs were investigated against an array of heavy metals in order to identify strains which exhibit the (hyper)accumulation phenotype without developing toxicity symptoms. Among the transgenic strains which could accumulate Cu(II), Zn(II) or Cd(II), but also non-canonical metal ions, such as Co(II), Mn(II) or Ni(II), myrGFP-NcMT3 qualified as the best candidate for bioremediation applications, thanks to the robust growth accompanied by significant accumulative capacity.

In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage

PubMed Central

Edwards, Sarah K.; Ono, Toshikazu; Wang, Shenliang; Jiang, Wei; Franzini, Raphael M.; Jung, Jong Wha; Chan, Ke Min; Kool, Eric T.

2015-01-01

The repair of oxidative damage to DNA is essential to avoidance of mutations that lead to cancer. Oxidized DNA bases, such as 8-oxoguanine, are a chief source of these mutations, and the enzyme 8-oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8-oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel-based measurements of radiolabeled DNAs. Here we report on the design and properties of fluorogenic probes that directly report on OGG1 (and bacterial homologue Fpg) activity in real time as the oxidized base is excised. The probes are short modified DNA oligomers containing fluorescent DNA bases and are designed to utilize the damaged DNA base itself as a fluorescence quencher. Screening of combinations of fluorophores and 8-oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probe designs containing these fluorophores, and we found an optimized probe OGR1 that yields a 60-fold light-up signal in vitro with OGG1 and Fpg, and can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes may be useful in quantifying enzyme activity and performing competitive inhibition assays. PMID:26073452

Polyethylenimine-coated iron oxide magnetic nanoparticles for high efficient gene delivery

NASA Astrophysics Data System (ADS)

Nguyen, Anh H.; Abdelrasoul, Gaser N.; Lin, Donghai; Maadi, Hamid; Tong, Junfeng; Chen, Grace; Wang, Richard; Anwar, Afreen; Shoute, Lian; Fang, Qiang; Wang, Zhixiang; Chen, Jie

2018-04-01

Properties of magnetic nanoparticles (MNPs) are of notable interest in many fields of biomedical engineering, especially for gene therapy. In this paper, we report a method for synthesis and delivery of MNPs loaded with DNAs, which overcomes the drawbacks of high cost and cytotoxicity associated with current delivery techniques (chemical- and liposome-based designs). 24-nm MNPs (Fe3O4) were synthesized, functionalized and characterized by analytical techniques to understand the surface properties for DNA binding and cellular uptake. The simple surface functionalization with polyethylenimine (PEI) through glutaraldehyde linker activation gave the complex of PEI-coated MNPs, resulting in high stability with a positive surface charge of about + 31 mV. Under the guidance of an external magnetic field, the functionalized MNPs with a loaded isothiocyanate (FITC) or green fluorescent protein (GFP) will enter the cells, which can be visualized by the fluorescence of FITC or GFP. We also examined the cytotoxicity of our synthesized MNPs by MTT assay. We showed that the IC50s of these MNPs for COS-7 and CHO cells were low and at 0.2 and 0.26 mg/mL, respectively. Moreover, our synthesized MNPs that were loaded with plasmids encoding GFP showed high transfection rate, 38.3% for COS-7cells and 27.6% for CHO cells. In conclusion, we established a promising method with low cost, low toxicity, and high transfection efficiency for siRNA and gene delivery.

Suppression of Red Blood Cell Autofluorescence for Immunocytochemistry on Fixed Embryonic Mouse Tissue.

PubMed

Whittington, Niteace C; Wray, Susan

2017-10-23

Autofluorescence is a problem that interferes with immunofluorescent staining and complicates data analysis. Throughout the mouse embryo, red blood cells naturally fluoresce across multiple wavelengths, spanning the emission and excitation spectra of many commonly used fluorescent reporters, including antibodies, dyes, stains, probes, and transgenic proteins, making it difficult to distinguish assay fluorescence from endogenous fluorescence. Several tissue treatment methods have been developed to bypass this issue with varying degrees of success. Sudan Black B dye has been commonly used to quench autofluorescence, but can also introduce background fluorescence. Here we present a protocol for an alternative called TrueBlack Lipofuscin Autofluorescence Quencher. The protocol described in this unit demonstrates how TrueBlack efficiently quenches red blood cell autofluorescence across red and green wavelengths in fixed embryonic tissue without interfering with immunofluorescent signal intensity or introducing background staining. We also identify optimal incubation, concentration, and multiple usage conditions for routine immunofluorescence microscopy. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

A novel two T-DNA binary vector allows efficient generation of marker-free transgenic plants in three elite cultivars of rice (Oryza sativa L.).

PubMed

Breitler, Jean-Christophe; Meynard, Donaldo; Van Boxtel, Jos; Royer, Monique; Bonnot, François; Cambillau, Laurence; Guiderdoni, Emmanuel

2004-06-01

A pilot binary vector was constructed to assess the potential of the 2 T-DNA system for generating selectable marker-free progeny plants in three elite rice cultivars (ZhongZuo321, Ariete and Khao Dawk Mali 105) known to exhibit contrasting amenabilities to transformation. The first T-DNA of the vector, delimited by Agrobacterium tumefaciens borders, contains the hygromycin phosphotransferase (hpt) selectable gene and the green fluorescent protein (gfp) reporter gene while the second T-DNA, delimited by Agrobacterium rhizogenes borders, bears the phosphinothricin acetyl transferase (bar) gene, featuring the gene of interest. 82-90% of the hygromycin-resistant primary transformants exhibited tolerance to ammonium glufosinate mediated by the bar gene suggesting very high co-transformation frequency in the three cultivars. All of the regenerated plants were analyzed by Southern blot which confirmed co-integration of the T-DNAs at frequencies consistent with those of co-expression and allowed determination of copy number for each gene as well as detection of two different vector backbone fragments extending between the two T-DNAs. Hygromycin susceptible, ammonium glufosinate tolerant phenotypes represented 14.4, 17.4 and 14.3% of the plants in T1 progenies of ZZ321, Ariete and KDML105 primary transformants, respectively. We developed a statistical model for deducing from the observed copy number of each T-DNA in T0 plants and phenotypic segregations in T1 progenies the most likely constitution and linkage of the T-DNA integration locus. Statistical analysis identified in 40 out of 42 lines a most likely linkage configuration theoretically allowing genetic separation of the two T-DNA types and out segregation of the T-DNA bearing the bar gene. Overall, though improvements of the technology would be beneficial, the 2 T-DNA system appeared to be a useful approach to generate selectable marker-free rice plants with a consistent frequency among cultivars.

M-DNA is stabilised in G•C tracts or by incorporation of 5-fluorouracil

PubMed Central

Wood, David O.; Dinsmore, Michael J.; Bare, Grant A.; Lee, Jeremy S.

2002-01-01

M-DNA is a complex between the divalent metal ions Zn2+, Ni2+ and Co2+ and duplex DNA which forms at a pH of ∼8.5. The stability and formation of M-DNA was monitored with an ethidium fluorescence assay in order to assess the relationship between pH, metal ion concentration, DNA concentration and the base composition. The dismutation of calf thymus DNA exhibits hysteresis with the formation of M-DNA occurring at a higher pH than the reconversion of M-DNA back to B-DNA. Hysteresis is most prominent with the Ni form of M-DNA where complete reconversion to B-DNA takes several hours even in the presence of EDTA. Increasing the DNA concentration leads to an increase in the metal ion concentration required for M-DNA formation. Both poly(dG)•poly(dC) and poly(dA)•poly(dT) formed M-DNA more readily than the corresponding mixed sequence DNAs. For poly(dG)•(poly(dC) M-DNA formation was observed at pH 7.4 with 0.5 mM ZnCl2. Modified bases were incorporated into a 500 bp fragment of phage λ DNA by polymerase chain reaction. DNAs in which guanine was replaced with hypoxanthine or thymine with 5-fluorouracil formed M-DNA at pHs below 8 whereas substitutions such as 2-aminoadenine and 5-methylcytosine had little effect. Poly[d(A5FU)] also formed a very stable M-DNA duplex as judged from Tm measurements. It is evident that the lower the pKa of the imino proton of the base, the lower the pH at which M-DNA will form; a finding that is consistent with the replacement of the imino proton with the metal ion. PMID:12000844

Photonic reagents for concentration measurement of flu-orescent proteins with overlapping spectra

NASA Astrophysics Data System (ADS)

Goun, Alexei; Bondar, Denys I.; Er, Ali O.; Quine, Zachary; Rabitz, Herschel A.

2016-05-01

By exploiting photonic reagents (i.e., coherent control by shaped laser pulses), we employ Optimal Dynamic Discrimination (ODD) as a novel means for quantitatively characterizing mixtures of fluorescent proteins with a large spectral overlap. To illustrate ODD, we simultaneously measured concentrations of in vitro mixtures of Enhanced Blue Fluorescent Protein (EBFP) and Enhanced Cyan Fluorescent Protein (ECFP). Building on this foundational study, the ultimate goal is to exploit the capabilities of ODD for parallel monitoring of genetic and protein circuits by suppressing the spectral cross-talk among multiple fluorescent reporters.

High resolution multiple excitation spot optical microscopy

NASA Astrophysics Data System (ADS)

Dilipkumar, Shilpa; Mondal, Partha Pratim

2011-06-01

We propose fundamental improvements in three-dimensional (3D) resolution of multiple excitation spot optical microscopy. The excitation point spread function (PSF) is generated by two interfering counter-propagating depth-of-focus beams along the optical axis. Detection PSF is obtained by coherently interfering the emitted fluorescent light (collected by both the objectives) at the detector. System PSF shows upto 14-fold reduction in focal volume as compared to confocal, and almost 2-fold improvement in lateral resolution. Proposed PSF has the ability to simultaneously excite multiple 3D-spots of sub-femtoliter volume. Potential applications are in fluorescence microscopy and nanobioimaging.

Enzymes that cleave non-glycosidic ether bonds between lignins or derivatives thereof and saccharides

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

Kravit, Nancy G.; Schmidt, Katherine A.

The patent application relates to isolated polypeptides that specifically cleave non-glycosidic ether bonds between lignins or derivatives thereof and saccharides, and to cDNAs encoding the polypeptides. The patent application also relates to nucleic acid constructs, expression vectors and host cells comprising the cDNAs, as well as methods of producing and using the isolated polypeptides for treating pulp and biomass to increase soluble saccharide yield and enrich lignin fractions.

Mitochondrial DNA analysis reveals substantial Native American ancestry in Puerto Rico.

PubMed

Martínez-Cruzado, J C; Toro-Labrador, G; Ho-Fung, V; Estévez-Montero, M A; Lobaina-Manzanet, A; Padovani-Claudio, D A; Sánchez-Cruz, H; Ortiz-Bermúdez, P; Sánchez-Crespo, A

2001-08-01

To estimate the maternal contribution of Native Americans to the human gene pool of Puerto Ricans--a population of mixed African, European, and Amerindian ancestry--the mtDNAs of two sample sets were screened for restriction fragment length polymorphisms (RFLPs) defining the four major Native American haplogroups. The sample set collected from people who claimed to have a maternal ancestor with Native American physiognomic traits had a statistically significant higher frequency of Native American mtDNAs (69.6%) than did the unbiased sample set (52.6%). This higher frequency suggests that, despite the fact that the native Taíno culture has been extinct for centuries, the Taíno contribution to the current population is considerable and some of the Taíno physiognomic traits are still present. Native American haplogroup frequency analysis shows a highly structured distribution, suggesting that the contribution of Native Americans foreign to Puerto Rico is minimal. Haplogroups A and C cover 56.0% and 35.6% of the Native American mtDNAs, respectively. No haplogroup D mtDNAs were found. Most of the linguistic, biological, and cultural evidence suggests that the Ceramic culture of the Taínos originated in or close to the Yanomama territory in the Amazon. However, the absence of haplogroup A in the Yanomami suggests that the Yanomami are not the only Taíno ancestors.

Cloning and molecular characterization of the cDNAs encoding the variable regions of an anti-CD20 monoclonal antibody.

PubMed

Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili

2017-01-01

CD20-based targeting of B-cells in hematologic malignancies and autoimmune disorders is associated with outstanding clinical outcomes. Isolation and characterization of VH and VL cDNAs encoding the variable regions of the heavy and light chains of monoclonal antibodies (MAb) is necessary to produce next generation MAbs and their derivatives such as bispecific antibodies (bsAb) and single-chain variable fragments (scFv). This study was aimed at cloning and characterization of the VH and VL cDNAs from a hybridoma cell line producing an anti-CD20 MAb. VH and VL fragments were amplified, cloned and characterized. Furthermore, amino acid sequences of VH, VL and corresponding complementarity-determining regions (CDR) were determined and compared with those of four approved MAbs including Rituximab (RTX), Ibritumomab tiuxetan, Ofatumumab and GA101. The cloned VH and VL cDNAs were found to be functional and follow a consensus pattern. Amino acid sequences corresponding to the VH and VL fragments also indicated noticeable homologies to those of RTX and Ibritumomab. Furthermore, amino acid sequences of the relating CDRs had remarkable similarities to their counterparts in RTX and Ibritumomab. Successful recovery of VH and VL fragments encourages the development of novel CD20 targeting bsAbs, scFvs, antibody conjugates and T-cells armed with chimeric antigen receptors.

Large-Scale Collection and Analysis of Full-Length cDNAs from Brachypodium distachyon and Integration with Pooideae Sequence Resources

PubMed Central

Mochida, Keiichi; Uehara-Yamaguchi, Yukiko; Takahashi, Fuminori; Yoshida, Takuhiro; Sakurai, Tetsuya; Shinozaki, Kazuo

2013-01-01

A comprehensive collection of full-length cDNAs is essential for correct structural gene annotation and functional analyses of genes. We constructed a mixed full-length cDNA library from 21 different tissues of Brachypodium distachyon Bd21, and obtained 78,163 high quality expressed sequence tags (ESTs) from both ends of ca. 40,000 clones (including 16,079 contigs). We updated gene structure annotations of Brachypodium genes based on full-length cDNA sequences in comparison with the latest publicly available annotations. About 10,000 non-redundant gene models were supported by full-length cDNAs; ca. 6,000 showed some transcription unit modifications. We also found ca. 580 novel gene models, including 362 newly identified in Bd21. Using the updated transcription start sites, we searched a total of 580 plant cis-motifs in the −3 kb promoter regions and determined a genome-wide Brachypodium promoter architecture. Furthermore, we integrated the Brachypodium full-length cDNAs and updated gene structures with available sequence resources in wheat and barley in a web-accessible database, the RIKEN Brachypodium FL cDNA database. The database represents a “one-stop” information resource for all genomic information in the Pooideae, facilitating functional analysis of genes in this model grass plant and seamless knowledge transfer to the Triticeae crops. PMID:24130698

Recombination of mitochondrial DNA without selection pressure among compatible strains of the Aspergillus niger species aggregate.

PubMed

Tóth, B; Hamari, Z; Ferenczy, L; Varga, J; Kevei, F

1998-03-01

Previous mitochondrial transmission experiments between oligomycin-resistant and oligomycin-sensitive incompatible strains of the A. niger aggregate bearing various mtDNA RFLP profiles resulted in a great variety of mitochondrial recombinants under selection pressure. Apart from the recombinant mtDNAs, resistant clones harbouring unchanged RFLP profiles of resistant donor mtDNAs with the recipient nuclear backgrounds were rarely isolated. These strains were anastomosed with nuclearly isogenic oligomycin-sensitive recipient partners and the mitochondria of the resulting progeny were examined under non-selective conditions. These experiments provide insights into events which are possibly similar to those occurring in nature. The heterokaryons obtained formed both oligomycin-resistant and -sensitive sectors, most of which were found to be homoplasmons. Progenies harbouring oligomycin-resistant and -sensitive mtDNAs may originate either from individual recombination events or be due to parental segregation. MtDNA recombination might take place in the heterokaryons without selection by oligomycin. The most frequent recombinant types of mtDNA RFLP profiles were indistinguishable from those recombinant mtDNAs which were frequently obtained under selection pressure from directed transfer experiments between incompatible strains. We present evidence that mixed mitochondrial populations may influence the compatibility reactions in the presence of an isogenic nuclear background, that recombination may take place without selection pressure, and that the process does not require specific nuclear sequences of both parental strains.

Extrachromosomal circular DNA-based amplification and transmission of herbicide resistance in crop weed Amaranthus palmeri

PubMed Central

Koo, Dal-Hoe; Molin, William T.; Saski, Christopher A.; Jiang, Jiming; Putta, Karthik; Friebe, Bernd; Gill, Bikram S.

2018-01-01

Gene amplification has been observed in many bacteria and eukaryotes as a response to various selective pressures, such as antibiotics, cytotoxic drugs, pesticides, herbicides, and other stressful environmental conditions. An increase in gene copy number is often found as extrachromosomal elements that usually contain autonomously replicating extrachromosomal circular DNA molecules (eccDNAs). Amaranthus palmeri, a crop weed, can develop herbicide resistance to glyphosate [N-(phosphonomethyl) glycine] by amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, the molecular target of glyphosate. However, biological questions regarding the source of the amplified EPSPS, the nature of the amplified DNA structures, and mechanisms responsible for maintaining this gene amplification in cells and their inheritance remain unknown. Here, we report that amplified EPSPS copies in glyphosate-resistant (GR) A. palmeri are present in the form of eccDNAs with various conformations. The eccDNAs are transmitted during cell division in mitosis and meiosis to the soma and germ cells and the progeny by an as yet unknown mechanism of tethering to mitotic and meiotic chromosomes. We propose that eccDNAs are one of the components of McClintock’s postulated innate systems [McClintock B (1978) Stadler Genetics Symposium] that can rapidly produce soma variation, amplify EPSPS genes in the sporophyte that are transmitted to germ cells, and modulate rapid glyphosate resistance through genome plasticity and adaptive evolution. PMID:29531028

Derivatization of DNAs with Selenium at 6-Position of Guanine for Function and Crystal Structure Studies

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

Salon, J.; Jiang, J; Sheng, J

2008-01-01

To investigate nucleic acid base pairing and stacking via atom-specific mutagenesis and crystallography, we have synthesized for the first time the 6-Se-deoxyguanosine phosphoramidite and incorporated it into DNAs via solid-phase synthesis with a coupling yield over 97%. We found that the UV absorption of the Se-DNAs red-shifts over 100 nm to 360 nm ({Epsilon} = 2.3 x 10{sup 4} M{sup -1} cm{sup -1}), the Se-DNAs are yellow colored, and this Se modification is relatively stable in water and at elevated temperature. Moreover, we successfully crystallized a ternary complex of the Se-G-DNA, RNA and RNase H. The crystal structure determination andmore » analysis reveal that the overall structures of the native and Se-modified nucleic acid duplexes are very similar, the selenium atom participates in a Se-mediated hydrogen bond (Se H-N), and the {sup Se}G and C form a base pair similar to the natural G-C pair though the Se-modification causes the base-pair to shift (approximately 0.3 {angstrom}). Our biophysical and structural studies provide new insights into the nucleic acid flexibility, duplex recognition and stability. Furthermore, this novel selenium modification of nucleic acids can be used to investigate chemogenetics and structure of nucleic acids and their protein complexes.« less

Beta Human Papillomavirus Infection Is Prevalent in Elephantiasis and Exhibits a Productive Phenotype: A Case-Control Study.

PubMed

Carlson, John Andrew; Rady, Peter; Kadam, Pooja; He, Qin; Simonette, Rebecca; Tyring, Stephen

2017-06-01

Elephantiasis is considered a cutaneous region of immune deficiency with cobblestone-like surface caused by a wart-like eruption. Verrucosis is a diffuse human papillomavirus (HPV) infection linked to immunodeficiency disorders. The objective of this study was to examine the prevalence of HPV infection in lymphedema and its pathogenic role in elephantiasis. A retrospective case-control study was performed examining lymphedematous skin and controls of peritumoral normal skin. HPV infection was evaluated at the DNA, protein, and histopathologic levels by polymerase chain reaction, immunohistochemistry, and light microscopy, respectively. Overall, 540 HPV DNAs were detected in 120 of 122 cutaneous samples (median 4 HPV DNAs per sample, range 0-9). Compared with controls, no differences existed in type or number of HPVs identified. Instead, a diverse spectrum of HPV-related histopathologies were evident, likely reflecting the multiplicity of HPV genotypes detected. Most notably, increasing histopathologic lymphedema stage significantly correlated with markers of productive HPV infection such as altered keratohyaline granules and HPV L1 capsid expression. Limitations of this study are the absence of normal skin controls not associated with neoplasia or subclinical lymphedema, and lack of assessment of HPV copy number per keratinocyte infected. In conclusion, productive HPV infection, not HPV type or numbers detected, distinguished lymphedematous skin from controls. These findings support the theory that lymphedema creates a region of depressed immunity that permits productive HPV infection, manifested clinically by diffuse papillomatosis, characteristic of elephantiasis.

Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging.

PubMed

Zhao, Ming; Li, Yu; Peng, Leilei

2014-05-05

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community.

Failure to detect genomic material of HTLV-I or HTLV-II in mononuclear cells of Italian patients with multiple sclerosis and chronic progressive myelopathy.

PubMed

Merelli, E; Sola, P; Marasca, R; Salati, R; Torelli, G

1993-01-01

To contribute to the undecided question if a retrovirus of the human T-cell lymphotropic virus (HTLV) family may be involved in the development of multiple sclerosis (MS), we investigated by the polymerase chain reaction (PCR) the presence of HTLV-I and HTLV-II sequences in the peripheral blood mononuclear cell DNAs from 30 patients affected by MS and 15 by chronic progressive myelopathy. Moreover a control group of 14 blood donors was examined. All these patients were devoid of anti-HTLV-I antibody in the serum and cerebrospinal fluid at ELISA. For the PCR, primers and probes specific for the tax region common to HTLV-I and HTLV-II, for the pol region of HTLV-I, and for the pol region of HTLV-II were used. In spite of the high sensitivity of the technique used, the three groups of subjects were negative for HTLV-I and HTLV-II genomic sequences.

De novo transcriptome sequencing in Bixa orellana to identify genes involved in methylerythritol phosphate, carotenoid and bixin biosynthesis

DOE PAGES

Cárdenas-Conejo, Yair; Carballo-Uicab, Víctor; Lieberman, Meric; ...

2015-10-28

Bixin or annatto is a commercially important natural orange-red pigment derived from lycopene that is produced and stored in seeds of Bixa orellana L. An enzymatic pathway for bixin biosynthesis was inferred from homology of putative proteins encoded by differentially expressed seed cDNAs. Some activities were later validated in a heterologous system. Nevertheless, much of the pathway remains to be clarified. For example, it is essential to identify the methylerythritol phosphate (MEP) and carotenoid pathways genes. In order to investigate the MEP, carotenoid, and bixin pathways genes, total RNA from young leaves and two different developmental stages of seeds frommore » B. orellana were used for the construction of indexed mRNA libraries, sequenced on the Illumina HiSeq 2500 platform and assembled de novo using Velvet, CLC Genomics Workbench and CAP3 software. A total of 52,549 contigs were obtained with average length of 1,924 bp. Two phylogenetic analyses of inferred proteins, in one case encoded by thirteen general, single-copy cDNAs, in the other from carotenoid and MEP cDNAs, indicated that B. orellana is closely related to sister Malvales species cacao and cotton. Using homology, we identified 7 and 14 core gene products from the MEP and carotenoid pathways, respectively. Surprisingly, previously defined bixin pathway cDNAs were not present in our transcriptome. Here we propose a new set of gene products involved in bixin pathway. In conclusion, the identification and qRT-PCR quantification of cDNAs involved in annatto production suggest a hypothetical model for bixin biosynthesis that involve coordinated activation of some MEP, carotenoid and bixin pathway genes. These findings provide a better understanding of the mechanisms regulating these pathways and will facilitate the genetic improvement of B. orellana.« less

De novo transcriptome sequencing in Bixa orellana to identify genes involved in methylerythritol phosphate, carotenoid and bixin biosynthesis

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

Cárdenas-Conejo, Yair; Carballo-Uicab, Víctor; Lieberman, Meric

Bixin or annatto is a commercially important natural orange-red pigment derived from lycopene that is produced and stored in seeds of Bixa orellana L. An enzymatic pathway for bixin biosynthesis was inferred from homology of putative proteins encoded by differentially expressed seed cDNAs. Some activities were later validated in a heterologous system. Nevertheless, much of the pathway remains to be clarified. For example, it is essential to identify the methylerythritol phosphate (MEP) and carotenoid pathways genes. In order to investigate the MEP, carotenoid, and bixin pathways genes, total RNA from young leaves and two different developmental stages of seeds frommore » B. orellana were used for the construction of indexed mRNA libraries, sequenced on the Illumina HiSeq 2500 platform and assembled de novo using Velvet, CLC Genomics Workbench and CAP3 software. A total of 52,549 contigs were obtained with average length of 1,924 bp. Two phylogenetic analyses of inferred proteins, in one case encoded by thirteen general, single-copy cDNAs, in the other from carotenoid and MEP cDNAs, indicated that B. orellana is closely related to sister Malvales species cacao and cotton. Using homology, we identified 7 and 14 core gene products from the MEP and carotenoid pathways, respectively. Surprisingly, previously defined bixin pathway cDNAs were not present in our transcriptome. Here we propose a new set of gene products involved in bixin pathway. In conclusion, the identification and qRT-PCR quantification of cDNAs involved in annatto production suggest a hypothetical model for bixin biosynthesis that involve coordinated activation of some MEP, carotenoid and bixin pathway genes. These findings provide a better understanding of the mechanisms regulating these pathways and will facilitate the genetic improvement of B. orellana.« less

Multi-Phenotypic subtyping of circulating tumor cells using sequential fluorescent quenching and restaining

NASA Astrophysics Data System (ADS)

Adams, Daniel L.; Alpaugh, R. Katherine; Tsai, Susan; Tang, Cha-Mei; Stefansson, Steingrimur

2016-09-01

In tissue biopsies formalin fixed paraffin embedded cancer blocks are micro-sectioned producing multiple semi-identical specimens which are analyzed and subtyped proteomically, and genomically, with numerous biomarkers. In blood based biopsies (BBBs), blood is purified for circulating tumor cells (CTCs) and clinical utility is typically limited to cell enumeration, as only 2-3 positive fluorescent markers and 1 negative marker can be used. As such, increasing the number of subtyping biomarkers on each individual CTC could dramatically enhance the clinical utility of BBBs, allowing in depth interrogation of clinically relevant CTCs. We describe a simple and inexpensive method for quenching the specific fluors of fluorescently stained CTCs followed by sequential restaining with additional biomarkers. As proof of principle a CTC panel, immunosuppression panel and stem cell panel were used to sequentially subtype individual fluorescently stained patient CTCs, suggesting a simple and universal technique to analyze multiple clinically applicable immunomarkers from BBBs.

Nucleic acid encoding a self-assembling split-fluorescent protein system

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2014-04-01

The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

Nucleic acid encoding a self-assembling split-fluorescent protein system

DOEpatents

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2011-06-07

The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

Nucleic acid encoding a self-assembling split-fluorescent protein system

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2015-07-14

The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

Highly Efficient Multiple-Anchored Fluorescent Probe for the Detection of Aniline Vapor Based on Synergistic Effect: Chemical Reaction and PET.

PubMed

Jiao, Zinuo; Zhang, Yu; Xu, Wei; Zhang, Xiangtao; Jiang, Haibo; Wu, Pengcheng; Fu, Yanyan; He, Qingguo; Cao, Huimin; Cheng, Jiangong

2017-05-26

A multiple-anchored fluorescent probe ((((hexane-1,6-diylbis(2,7-bis(4-formyl)-phenyl)-9H-fluorine-9,9-diyl))-bis(hexane-6,1-diyl))-bis(9H-carbazole-9,3,6-triyl))-tetrakis(benzene-4,1-diyl))-tetraformyl-(8FP-2F) with eight aldehyde groups was designed and synthesized. The molecule has four branches and highly twisted structure. Furthermore, it tends to self-assemble into nanospheres, which is beneficial for gaseous analyte penetration and high fluorescence quantum efficiency. Among gaseous analytes, detection of aniline vapor is extraordinarily important in the control of environmental issues and human diseases. Herein, 8FP-2F was introduced to detect aniline vapor with distinguished sensitivity and selectivity via simple Schiff base reaction at room temperature. After exposure to saturate aniline vapor, the 89% fluorescence of 8FP-2F was quenched in 50 s and the detection limit was as low as 3 ppb. Further study showed the suitable HOMO/LUMO energy levels and matched orbital symmetry between probe and aniline molecules ensured chemical reaction and PET process work together. The synergistic effect resulted in a significant sensing performance and fluorescence quenching toward aniline vapor. Moreover, the multiple active sites structure of 8FP-2F means it could be applied for constructing many interesting structures and highly efficient organic optoelectronic functional materials.

Appalachian spring: variations on ancient gastro-entero-pancreatic themes in New World mammals.

PubMed

Seino, S; Blackstone, C D; Chan, S J; Whittaker, J; Bell, G I; Steiner, D F

1988-07-01

Studies of guinea pig genomic and/or cDNA clones encoding the gastro-entero-pancreatic (GEP) hormones--insulin, glucagon and pancreatic polypeptide--as well as portions of the insulin receptor, are described. Multiple clustered substitutions (localized rapid mutation acceptance) altering the biological properties of both insulin and glucagon have been revealed, but this does not appear to be the case with either pancreatic polypeptide or those regions of guinea pig insulin receptor cDNAs that have been examined thus far. These findings suggest that novel selective pressures operative in the New World environment, in which these animals evolved in isolation from Old World mammalian species, have led to altered solutions to problems related to the regulation of growth and carbohydrate metabolism.

Exploring the Presence of microDNAs in Prostate Cancer Cell Lines, Tissue, and Sera of Prostate Cancer Patients and its Possible Application as Biomarker

DTIC Science & Technology

2015-08-01

Sequence tags were mapped on the human reference genome using the Novoalign software. Only those tags... the linear islands to create a novel junctional sequence that does not exist in the genome . Thus the PE- sequence of a fragment that breaks at or...identified in cancer cell lines. (b) Median percent GC content of microDNAs and the genomic sequences up- or downstream of the source loci are

Graphical classification of DNA sequences of HLA alleles by deep learning.

PubMed

Miyake, Jun; Kaneshita, Yuhei; Asatani, Satoshi; Tagawa, Seiichi; Niioka, Hirohiko; Hirano, Takashi

2018-04-01

Alleles of human leukocyte antigen (HLA)-A DNAs are classified and expressed graphically by using artificial intelligence "Deep Learning (Stacked autoencoder)". Nucleotide sequence data corresponding to the length of 822 bp, collected from the Immuno Polymorphism Database, were compressed to 2-dimensional representation and were plotted. Profiles of the two-dimensional plots indicate that the alleles can be classified as clusters are formed. The two-dimensional plot of HLA-A DNAs gives a clear outlook for characterizing the various alleles.

The fate of UV-induced pyrimidine dimers in the nuclear and mitochondrial DNAs of Saccharomyces cerevisiae on various postirradiation treatments and its influence on survival and cytoplasmic "petite" induction.

PubMed

Waters, R; Moustacchi, E

1975-01-01

The photoreactivability of UV-induced pyrimidine dimers in the nuclear and mitochondrial DNAs of Saccharomyces cerevisiae has been investigated in conjunction with the fate of these photoproducts following postirradiation dark incubation in saline and nutrient media. In all instances, survival and "petite" induction were measured. An attempt has been made to relate these results to present ideas on the repair of UV damages in DNA.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy.

PubMed

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy

NASA Astrophysics Data System (ADS)

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

Photonic reagents for concentration measurement of flu-orescent proteins with overlapping spectra

PubMed Central

Goun, Alexei; Bondar, Denys I.; Er, Ali O.; Quine, Zachary; Rabitz, Herschel A.

2016-01-01

By exploiting photonic reagents (i.e., coherent control by shaped laser pulses), we employ Optimal Dynamic Discrimination (ODD) as a novel means for quantitatively characterizing mixtures of fluorescent proteins with a large spectral overlap. To illustrate ODD, we simultaneously measured concentrations of in vitro mixtures of Enhanced Blue Fluorescent Protein (EBFP) and Enhanced Cyan Fluorescent Protein (ECFP). Building on this foundational study, the ultimate goal is to exploit the capabilities of ODD for parallel monitoring of genetic and protein circuits by suppressing the spectral cross-talk among multiple fluorescent reporters. PMID:27181496

Isolation and expression analysis of cDNAs that are associated with alternate bearing in Olea europaea L. cv. Ayvalık

PubMed Central

2013-01-01

Background Olive cDNA libraries to isolate candidate genes that can help enlightening the molecular mechanism of periodicity and / or fruit production were constructed and analyzed. For this purpose, cDNA libraries from the leaves of trees in “on year” and in “off year” in July (when fruits start to appear) and in November (harvest time) were constructed. Randomly selected 100 positive clones from each library were analyzed with respect to sequence and size. A fruit-flesh cDNA library was also constructed and characterized to confirm the reliability of each library’s temporal and spatial properties. Results Quantitative real-time RT-PCR (qRT-PCR) analyses of the cDNA libraries confirmed cDNA molecules that are associated with different developmental stages (e. g. “on year” leaves in July, “off year” leaves in July, leaves in November) and fruits. Hence, a number of candidate cDNAs associated with “on year” and “off year” were isolated. Comparison of the detected cDNAs to the current EST database of GenBank along with other non - redundant databases of NCBI revealed homologs of previously described genes along with several unknown cDNAs. Of around 500 screened cDNAs, 48 cDNA elements were obtained after eliminating ribosomal RNA sequences. These independent transcripts were analyzed using BLAST searches (cutoff E-value of 1.0E-5) against the KEGG and GenBank nucleotide databases and 37 putative transcripts corresponding to known gene functions were annotated with gene names and Gene Ontology (GO) terms. Transcripts in the biological process were found to be related with metabolic process (27%), cellular process (23%), response to stimulus (17%), localization process (8.5%), multicellular organismal process (6.25%), developmental process (6.25%) and reproduction (4.2%). Conclusions A putative P450 monooxigenase expressed fivefold more in the “on year” than that of “off year” leaves in July. Two putative dehydrins expressed significantly more in “on year” leaves than that of “off year” leaves in November. Homologs of UDP – glucose epimerase, acyl - CoA binding protein, triose phosphate isomerase and a putative nuclear core anchor protein were significant in fruits only, while a homolog of an embryo binding protein / small GTPase regulator was detected in “on year” leaves only. One of the two unknown cDNAs was specific to leaves in July while the other was detected in all of the libraries except fruits. KEGG pathway analyses for the obtained sequences correlated with essential metabolisms such as galactose metabolism, amino sugar and nucleotide sugar metabolisms and photosynthesis. Detailed analysis of the results presents candidate cDNAs that can be used to dissect further the genetic basis of fruit production and / or alternate bearing which causes significant economical loss for olive growers. PMID:23552171

How a short double-stranded DNA bends

NASA Astrophysics Data System (ADS)

Shin, Jaeoh; Lee, O.-Chul; Sung, Wokyung

2015-04-01

A recent experiment using fluorescence microscopy showed that double-stranded DNA fragments shorter than 100 base pairs loop with the probabilities higher by the factor of 102-106 than predicted by the worm-like chain (WLC) model [R. Vafabakhsh and T. Ha, Science 337, 1101(2012)]. Furthermore, the looping probabilities were found to be nearly independent of the loop size. The results signify a breakdown of the WLC model for DNA mechanics which works well on long length scales and calls for fundamental understanding for stressed DNA on shorter length scales. We develop an analytical, statistical mechanical model to investigate what emerges to the short DNA under a tight bending. A bending above a critical level initiates nucleation of a thermally induced bubble, which could be trapped for a long time, in contrast to the bubbles in both free and uniformly bent DNAs, which are either transient or unstable. The trapped bubble is none other than the previously hypothesized kink, which releases the bending energy more easily as the contour length decreases. It leads to tremendous enhancement of the cyclization probabilities, in a reasonable agreement with experiment.

Epstein-Barr virus nuclear antigen-1 is highly colocalized with interphase chromatin and its newly replicated regions in particular.

PubMed

Ito, Sayuri; Gotoh, Eisuke; Ozawa, Shigeru; Yanagi, Kazuo

2002-10-01

Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1), which binds to both the EBV origin of replication (oriP) and metaphase chromosomes, is essential for the replication/retention and segregation/partition of oriP-containing plasmids. Here the chromosomal localization of EBNA-1 fused to green fluorescent protein (GFP-EBNA-1) is examined by confocal microscopy combined with a 'premature chromosome condensation' (PCC) procedure. Analyses show that GFP-EBNA-1 expressed in living cells that lack oriP plasmids is associated with cellular chromatin that has been condensed rapidly by the PCC procedure into identifiable forms that are unique to each phase of interphase as well as metaphase chromosomes. Studies of cellular chromosomal DNAs labelled with BrdU or Cy3-dUTP indicate that GFP-EBNA-1 colocalizes highly with the labelled, newly replicated regions of interphase chromatin in cells. These results suggest that EBNA-1 is associated not only with cellular metaphase chromosomes but also with condensing chromatin/chromosomes and probably with interphase chromatin, especially with its newly replicated regions.

An Elegant Biosensor Molecular Beacon Probe: Challenges and Recent Solutions

PubMed Central

Kolpashchikov, Dmitry M.

2012-01-01

Molecular beacon (MB) probes are fluorophore- and quencher-labeled short synthetic DNAs folded in a stem-loop shape. Since the first report by Tyagi and Kramer, it has become a widely accepted tool for nucleic acid analysis and triggered a cascade of related developments in the field of molecular sensing. The unprecedented success of MB probes stems from their ability to detect specific DNA or RNA sequences immediately after hybridization with no need to wash out the unbound probe (instantaneous format). Importantly, the hairpin structure of the probe is responsible for both the low fluorescent background and improved selectivity. Furthermore, the signal is generated in a reversible manner; thus, if the analyte is removed, the signal is reduced to the background. This paper highlights the advantages of MB probes and discusses the approaches that address the challenges in MB probe design. Variations of MB-based assays tackle the problem of stem invasion, improve SNP genotyping and signal-to-noise ratio, as well as address the challenges of detecting folded RNA and DNA. PMID:24278758

Dose-response relationships in gene expression profiles in rainbow trout, Oncorhyncus mykiss, exposed to ethynylestradiol.

PubMed

Hook, Sharon E; Skillman, Ann D; Small, Jack A; Schultz, Irvin R

2006-07-01

Determining how gene expression profiles change with toxicant dose will improve the utility of arrays in identifying biomarkers and modes of toxic action. Isogenic rainbow trout, Oncorhyncus mykiss,were exposed to 10, 50 or 100 ng/L ethynylestradiol (a xeno-estrogen) for 7 days. Following exposure hepatic RNA was extracted. Fluorescently labeled cDNA were generated and hybridized against a commercially available Atlantic Salmon/Trout array (GRASP project, University of Victoria) spotted with 16,000 cDNAs. Transcript expression in treated vs control fish was analyzed via Genespring (Silicon Genetics) to identify genes with altered expression, as well as to determine gene clustering patterns that can be used as "expression signatures". Array results were confirmed via qRT PCR. Our analysis indicates that gene expression profiles varied somewhat with dose. Established biomarkers of exposure to estrogenic chemicals, such as vitellogenin, vitelline envelope proteins, and the estrogen receptor alpha, were induced at every dose. Other genes were dose specific, suggesting that different doses induce distinct physiological responses. These findings demonstrate that cDNA microarrays could be used to identify both toxicant class and relative dose.

Studying DNA looping by single-molecule FRET.

PubMed

Le, Tung T; Kim, Harold D

2014-06-28

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

FISH and AgNor mapping of the 45S and 5S rRNA genes in wild and cultivated species of Capsicum (Solananceae).

PubMed

Scaldaferro, Marisel A; da Cruz, M Victoria Romero; Cecchini, Nicolás M; Moscone, Eduardo A

2016-02-01

Chromosome number and position of rDNA were studied in 12 wild and cultivated species of the genus Capsicum with chromosome numbers x = 12 and x = 13 (22 samples). For the first time in these species, the 5S and 45S rRNA loci were localized and physically mapped using two-color fluorescence in situ hybridization and AgNOR banding. We focused on the comparison of the results obtained with both methods with the aim of accurately revealing the real functional rRNA genes. The analyzes were based on a previous work that reported that the 18S-5.8S-25S loci mostly coincide with GC-rich heterochromatic regions and likely have given rise to satellite DNAs, which are not active genes. These data show the variability of rDNA within karyotypes of the genus Capsicum, providing anchor points for (comparative) genetic maps. In addition, the obtained information might be useful for studies on evolution of repetitive DNA.

Studying DNA Looping by Single-Molecule FRET

PubMed Central

Le, Tung T.; Kim, Harold D.

2014-01-01

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA. PMID:24998459

Localization of the human mitochondrial citrate transporter protein gene to chromosome 22q11 in the DiGeorge syndrome critical region

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

Heisterkamp, N.; Hoeve, J.T.; Groffen, J.

A high percentage of patients with DiGeorge syndrome and velo-cardio-facial syndrome have interstitial deletions on chromosome 22q11. The shortest region of overlap is currently estimated to be around 500 kb. Two segments of DNA from chromosome 22q11, located 160 kb apart, were cloned because they contained NotI restriction enzyme sites. In the current study we demonstrate that these segments are absent from chromosomes 22 carrying microdeletions of two different DiGeorge patients. Fluorescence in situ and Southern blot hybridization was further used to show that this locus is within the DiGeorge critical region. Phylogenetically conserved sequences adjacent to one of themore » two NotI sites hybridized to mRNAs in different human cell lines. cDNAs isolated with a probe from this segment showed it to contain the gene for the human mitochondrial citrate transporter protein. Deletion of this gene in DiGeorge may contribute to the mental deficiency seen in the patients. 35 refs., 5 figs.« less

Array biosensor for detection of toxins

NASA Technical Reports Server (NTRS)

Ligler, Frances S.; Taitt, Chris Rowe; Shriver-Lake, Lisa C.; Sapsford, Kim E.; Shubin, Yura; Golden, Joel P.

2003-01-01

The array biosensor is capable of detecting multiple targets rapidly and simultaneously on the surface of a single waveguide. Sandwich and competitive fluoroimmunoassays have been developed to detect high and low molecular weight toxins, respectively, in complex samples. Recognition molecules (usually antibodies) were first immobilized in specific locations on the waveguide and the resultant patterned array was used to interrogate up to 12 different samples for the presence of multiple different analytes. Upon binding of a fluorescent analyte or fluorescent immunocomplex, the pattern of fluorescent spots was detected using a CCD camera. Automated image analysis was used to determine a mean fluorescence value for each assay spot and to subtract the local background signal. The location of the spot and its mean fluorescence value were used to determine the toxin identity and concentration. Toxins were measured in clinical fluids, environmental samples and foods, with minimal sample preparation. Results are shown for rapid analyses of staphylococcal enterotoxin B, ricin, cholera toxin, botulinum toxoids, trinitrotoluene, and the mycotoxin fumonisin. Toxins were detected at levels as low as 0.5 ng mL(-1).

Functional analysis of two genes coding for distinct cation diffusion facilitators of the ectomycorrhizal Zn-accumulating fungus Russula atropurpurea.

PubMed

Sácký, Jan; Leonhardt, Tereza; Kotrba, Pavel

2016-04-01

Russula atropurpurea can accumulate remarkably high concentrations of Zn in its sporocarps. We have previously demonstrated that 40 % of the intracellular Zn in this species is sequestered by MT-like RaZBP peptides. To see what other mechanisms for the handling of the accumulated Zn are available to R. atropurpurea, we searched its transcriptome for cDNAs coding for transporters of the cation diffusion facilitator (CDF) family. The transcriptome search enabled us to identify RaCDF1 and RaCDF2, which were further subjected to functional studies in metal sensitive Saccharomyces cerevisiae. The expression of RaCDF1 and its translational fusion with green fluorescent protein (GFP) protected the yeasts against Zn and Co, but not Cd or Mn, toxicity and led to increased Zn accumulation in the cells. The GFP fluorescence, observed in the RaCDF1::GFP-expressing yeasts on tonoplasts, indicated that the RaCDF1-mediated Zn and Co tolerance was a result of vacuolar sequestration of the metals. The expression of RaCDF2 supported Zn, but not Mn, tolerance in the yeasts and reduced the cellular uptake of Zn, which is congruent with the proposed idea of the Zn-efflux function of RaCDF2, supported by the localization of GFP-derived fluorescence on the plasma membrane of the yeasts expressing functional RaCDF2::GFP. Contrarily, RaCDF2 increased the sensitivity to Co and Cd in the yeasts and significantly promoted Cd uptake, which suggested that it can act as a bidirectional metal transporter. The notion that RaCDF1 and RaCDF2 are genuine CDF transporters in R. atropurputrea was further reinforced by the fact that the RaCDF-associated metal tolerance and uptake phenotypes were lost upon the replacement of histidyl (in RaCDF1) and aspartyl (in RaCDF2), which are highly conserved in the second transmembrane domain and known to be essential for the function of CDF proteins.

Some properties of human neuronal α7 nicotinic acetylcholine receptors fused to the green fluorescent protein

PubMed Central

Palma, Eleonora; Mileo, Anna M.; Martínez-Torres, Ataúlfo; Eusebi, Fabrizio; Miledi, Ricardo

2002-01-01

The functional properties and cellular localization of the human neuronal α7 nicotinic acetylcholine (AcCho) receptor (α7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mutα7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtα7 receptors decay much faster than those elicited by the mutα7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated α7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable “run-down” of the AcCho currents generated by mutα7-GFP receptors, whereas those of the wtα7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mutα7-GFP oocytes was accompanied by a marked decrease of α-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wtα7 and mutα7 receptors provides powerful tools to study the distribution and function of α7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins. PMID:11891308

The GC-rich mitochondrial and plastid genomes of the green alga Coccomyxa give insight into the evolution of organelle DNA nucleotide landscape

DOE PAGES

Smith, David Roy; Burki, Fabien; Yamada, Takashi; ...

2011-08-26

Here, most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features ofmore » this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.« less

Thermodynamics for the Formation of Double-Stranded DNA-Single-Walled Carbon Nanotube Hybrids.

PubMed

Shiraki, Tomohiro; Tsuzuki, Akiko; Toshimitsu, Fumiyuki; Nakashima, Naotoshi

2016-03-24

For the first time, the thermodynamics are described for the formation of double-stranded DNA (ds-DNA)-single-walled carbon nanotube (SWNT) hybrids. This treatment is applied to the exchange reaction of sodium cholate (SC) molecules on SWNTs and the ds-DNAs d(A)20 -d(T)20 and nuclear factor (NF)-κB decoy. UV/Vis/near-IR spectroscopy with temperature variations was used for analyzing the exchange reaction on the SWNTs with four different chiralities: (n,m)=(8,3), (6,5), (7,5), and (8,6). Single-stranded DNAs (ss-DNAs), including d(A)20 and d(T)20, are also used for comparison. The d(A)20-d(T)20 shows a drastic change in its thermodynamic parameters around the melting temperature (Tm ) of the DNA oligomer. No such Tm dependency was measured, owing to high Tm in the NF-κB decoy DNA and no Tm in the ss-DNA. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A general strategy for cloning viroids and other small circular RNAs that uses minimal amounts of template and does not require prior knowledge of its sequence.

PubMed

Navarro, B; Daròs, J A; Flores, R

1996-01-01

Two PCR-based methods are described for obtaining clones of small circular RNAs of unknown sequence and for which only minute amounts are available. To avoid introducing any assumption about the RNA sequence, synthesis of the cDNAs is initiated with random primers. The cDNA population is then PCR-amplified using a primer whose sequence is present at both sides of the cDNAs, since they have been obtained with random hexamers and then a linker with the sequence of the PCR primer has been ligated to their termini, or because the cDNAs have been synthesized with an oligonucleotide that contains the sequence of the PCR primer at its 5' end and six randomized positions at its 3' end. The procedures need only approximately 50 ng of purified RNA template. The reasons for the emergence of cloning artifacts and precautions to avoid them are discussed.

Dermatoxin and phylloxin from the waxy monkey frog, Phyllomedusa sauvagei: cloning of precursor cDNAs and structural characterization from lyophilized skin secretion.

PubMed

Chen, Tianbao; Walker, Brian; Zhou, Mei; Shaw, Chris

2005-07-15

Amphibian skin is a morphologically, biochemically and physiologically complex organ that performs the wide range of functions necessary for amphibian survival. Here we describe the primary structures of representatives of two novel classes of amphibian skin antimicrobials, dermatoxin and phylloxin, from the skin secretion of Phyllomedusa sauvagei, deduced from their respective precursor encoding cDNAs cloned from a lyophilized skin secretion library. A degenerate primer, designed to a highly conserved domain in the 5'-untranslated region of analogous peptide precursor cDNAs from Phyllomedusa bicolor, was employed in a 3'-RACE reaction. Peptides with molecular masses coincident with precursor-deduced mature toxin peptides were identified in LC/MS fractions of skin secretion and primary structures were confirmed by MS/MS fragmentation. This integrated experimental approach can thus rapidly expedite the primary structural characterization of amphibian skin peptides in a manner that circumvents specimen sacrifice whilst preserving robustness of scientific data.

Functional domains of the poliovirus receptor

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

Koike, Satoshi; Ise, Iku; Nomoto, Akio

1991-05-15

A number of mutant cDNAs of the human poliovirus receptor were constructed to identify essential regions of the molecule as the receptor. All mutant cDNAs carrying the sequence coding for the entire N-terminal immunoglobulin-like domain (domain I) confer permissiveness for poliovirus to mouse L cells, but a mutant cDNA lacking the sequence for domain I does not. The transformants permissive for poliovirus were able to bind the virus and were also recognized by monoclonal antibody D171, which competes with poliovirus for the cellular receptor. These results strongly suggest that the poliovirus binding site resides in domain I of the receptor.more » Mutant cDNAs for the sequence encoding the intracellular peptide were also constructed and expressed in mouse L cells. Susceptibility of these cells to poliovirus revealed that the entire putative cytoplasmic domain is not essential for virus infection. Thus, the cytoplasmic domain of the molecule appears not to play a role in the penetration of poliovirus.« less

The GC-Rich Mitochondrial and Plastid Genomes of the Green Alga Coccomyxa Give Insight into the Evolution of Organelle DNA Nucleotide Landscape

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

Smith, David Roy; Burki, Fabien; Yamada, Takashi

2011-05-13

Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of thismore » species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.« less

The organization of repeating units in mitochondrial DNA from yeast petite mutants.

PubMed

Bos, J L; Heyting, C; Van der Horst, G; Borst, P

1980-04-01

We have reinvestigated the linkage orientation of repeating units in mtDNAs of yeast ρ(-) petite mutants containing an inverted duplication. All five petite mtDNAs studied contain a continuous segment of wild-type mtDNA, part of which is duplicated and present in inverted form in the repeat. We show by restriction enzyme analysis that the non-duplicated segments between the inverted duplications are present in random orientation in all five petite mtDNAs. There is no segregation of sub-types with unique orientation. We attribute this to the high rate of intramolecular recombination between the inverted duplications. The results provide additional evidence for the high rate of recombination of yeast mtDNA even in haploid ρ(-) petite cells.We conclude that only two types of stable sequence organization exist in petite mtDNA: petites without an inverted duplication have repeats linked in straight head-to-tail arrangement (abcabc); petites with an inverted duplication have repeats in which the non-duplicated segments are present in random orientation.

Development of DNA probes for Candida albicans

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

Cheung, L.L.; Hudson, J.B.

1988-07-01

An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves.more » It became evident that, although both /sup 32/P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis.« less

Assignment of two mitochondrially synthesized polypeptides to human mitochondrial DNA and their use in the study of intracellular mitochondrial interaction.

PubMed Central

Oliver, N A; Wallace, D C

1982-01-01

Two mitochondrially synthesized marker polypeptides, MV-1 and MV-2, were found in human HeLa and HT1080 cells. These were assigned to the mitochondrial DNA in HeLa-HT1080 cybrids and hybrids by demonstrating their linkage to cytoplasmic genetic markers. These markers include mitochondrial DNA restriction site polymorphisms and resistance to chloramphenicol, an inhibitor of mitochondrial protein synthesis. In the absence of chloramphenicol, the expression of MV-1 and MV-2 in cybrids and hybrids was found to be directly proportional to the ratio of the parental mitochondrial DNAs. In the presence of chloramphenicol, the marker polypeptide linked to the chloramphenicol-sensitive mitochondrial DNA continued to be expressed. This demonstrated that resistant and sensitive mitochondrial DNAs can cooperate within a cell for gene expression and that the CAP-resistant allele was dominant or codominant to sensitive. Such cooperation suggests that mitochondrial DNAs can be exchanged between mitochondria. Images PMID:6955589

SV40 host-substituted variants: a new look at the monkey DNA inserts and recombinant junctions.

PubMed

Singer, Maxine; Winocour, Ernest

2011-04-10

The available monkey genomic data banks were examined in order to determine the chromosomal locations of the host DNA inserts in 8 host-substituted SV40 variant DNAs. Five of the 8 variants contained more than one linked monkey DNA insert per tandem repeat unit and in all cases but one, the 19 monkey DNA inserts in the 8 variants mapped to different locations in the monkey genome. The 50 parental DNAs (32 monkey and 18 SV40 DNA segments) which spanned the crossover and flanking regions that participated in monkey/monkey and monkey/SV40 recombinations were characterized by substantial levels of microhomology of up to 8 nucleotides in length; the parental DNAs also exhibited direct and inverted repeats at or adjacent to the crossover sequences. We discuss how the host-substituted SV40 variants arose and the nature of the recombination mechanisms involved. Copyright © 2011 Elsevier Inc. All rights reserved.

LaRbp38: A Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs

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

Lira, C.B.B.; Instituto de Biologia, UNICAMP, Campinas, SP; Siqueira Neto, J.L.

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38 kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA andmore » to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.« less

An investigation of nonsimultaneous laser-induced fluorescence

NASA Technical Reports Server (NTRS)

Fletcher, D. G.

1993-01-01

An alternative to simultaneous, two-line laser-induced fluorescence for thermodynamic property measurement is presented. This spectroscopic approach is similar to multiple-overheat hot-wire anemometry and is based on laser excitation of different fluorescence transitions for separate, sequential wind tunnel runs. Both fluctuating and mean thermodynamic property measurements seem to be achievable with this method without exciting the transitions during the same laser pulse.

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, E.S.; Taylor, J.A.

1996-03-12

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis. 14 figs.

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, E.S.; Taylor, J.A.

1994-06-28

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis. 14 figures.

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, Edward S.; Taylor, John A.

1996-03-12

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis.

Multiplexed fluorescence detector system for capillary electrophoresis

DOEpatents

Yeung, Edward S.; Taylor, John A.

1994-06-28

A fluorescence detection system for capillary electrophoresis is provided wherein the detection system can simultaneously excite fluorescence and substantially simultaneously monitor separations in multiple capillaries. This multiplexing approach involves laser irradiation of a sample in a plurality of capillaries through optical fibers that are coupled individually with the capillaries. The array is imaged orthogonally through a microscope onto a charge-coupled device camera for signal analysis.

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

Li, Gaoming; Key Laboratory of OptoElectronic Science and Technology for Medicine, Ministry of Education, Fujian Normal University, Fuzhou 350007; Gao, Fei

Multiple stimulated emission fluorescence photoacoustic (MSEF-PA) phenomenon is demonstrated in this letter. Under simultaneous illumination of pumping light and stimulated emission light, the fluorescence emission process is speeded up by the stimulated emission effect. This leads to nonlinear enhancement of photoacoustic signal while the quantity of absorbed photons is more than that of fluorescent molecules illuminated by pumping light. The electronic states' specificity of fluorescent molecular can also be labelled by the MSEF-PA signals, which can potentially be used to obtain fluorescence excitation spectrum in deep scattering tissue with nonlinearly enhanced photoacoustic detection. In this preliminary study, the fluorescence excitationmore » spectrum is reconstructed by MSEF-PA signals through sweeping the wavelength of exciting light, which confirms the theoretical derivation well.« less

Localization of pulmonary nodules using navigation bronchoscope and a near-infrared fluorescence thoracoscope.

PubMed

Anayama, Takashi; Qiu, Jimmy; Chan, Harley; Nakajima, Takahiro; Weersink, Robert; Daly, Michael; McConnell, Judy; Waddell, Thomas; Keshavjee, Shaf; Jaffray, David; Irish, Jonathan C; Hirohashi, Kentaro; Wada, Hironobu; Orihashi, Kazumasa; Yasufuku, Kazuhiro

2015-01-01

Video-assisted thoracoscopic wedge resection of multiple small, non-visible, and nonpalpable pulmonary nodules is a clinical challenge. We propose an ultra-minimally invasive technique for localization of pulmonary nodules using the electromagnetic navigation bronchoscope (ENB)-guided transbronchial indocyanine green (ICG) injection and intraoperative fluorescence detection with a near-infrared (NIR) fluorescence thoracoscope. Fluorescence properties of ICG topically injected into the lung parenchyma were determined using a resected porcine lung. The combination of ENB-guided ICG injection and NIR fluorescence detection was tested using a live porcine model. An electromagnetic sensor integrated flexible bronchoscope was geometrically registered to the three-dimensional chest computed tomographic image data by way of a real-time electromagnetic tracking system. The ICG mixed with iopamidol was injected into the pulmonary nodules by ENB guidance; ICG fluorescence was visualized by a near-infrared (NIR) thoracoscope. The ICG existing under 24-mm depth of inflated lung was detectable by the NIR fluorescence thoracoscope. The size of the fluorescence spot made by 0.1 mL of ICG was 10.4 ± 2.2 mm. An ICG or iopamidol spot remained at the injected point of the lung for more than 6 hours in vivo. The ICG fluorescence spot injected into the pulmonary nodule with ENB guidance was identified at the pulmonary nodule with the NIR thoracoscope. The ENB-guided transbronchial ICG injection and intraoperative NIR thoracoscopic detection is a feasible method to localize multiple pulmonary nodules. Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging

PubMed Central

Zhao, Ming; Li, Yu; Peng, Leilei

2014-01-01

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community. PMID:24921725

Detection of Myelination Using a Novel Histological Probe

PubMed Central

Xiang, Zhongmin; Nesterov, Evgueni E.; Skoch, Jesse; Lin, Tong; Hyman, Bradley T.; Swager, Timothy M.; Bacskai, Brian J.; Reeves, Steven A.

2005-01-01

Current methods for myelin staining in tissue sections include both histological and immunohistochemical techniques. Fluorescence immunohistochemistry, which uses antibodies against myelin components such as myelin basic protein, is often used because of the convenience for multiple labeling. To facilitate studies on myelin, this paper describes a quick and easy method for direct myelin staining in rodent and human tissues using novel near-infrared myelin (NIM) dyes that are comparable to other well-characterized histochemical reagents. The near-infrared fluorescence spectra of these probes allow fluorescent staining of tissue sections in multiple channels using visible light fluorophores commonly used in immunocytochemistry. These dyes have been used successfully to detect normal myelin structure and myelin loss in a mouse model of demyelination disease. PMID:16046669

Characterization of human carotid atherosclerotic tissues imaged by combining multiple multiphoton microscopy techniques

NASA Astrophysics Data System (ADS)

Baria, E.; Cicchi, R.; Nesi, G.; Massi, D.; Pavone, F. S.

2017-07-01

We combined Second Harmonic Generation, Two-Photon Fluorescence and Fluorescence Lifetime Imaging Microscopy for studying human carotid ex vivo tissue sections affected by atherosclerosis, resulting in the discrimination of different arterial regions within the plaques.

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

PubMed Central

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

1990-01-01

Protein 4.2 (P4.2) comprises approximately 5% of the protein mass of human erythrocyte (RBC) membranes. Anemia occurs in patients with RBCs deficient in P4.2, suggesting a role for this protein in maintaining RBC stability and integrity. We now report the molecular cloning and characterization of human RBC P4.2 cDNAs. By immunoscreening a human reticulocyte cDNA library and by using the polymerase chain reaction, two cDNA sequences of 2.4 and 2.5 kilobases (kb) were obtained. These cDNAs differ only by a 90-base-pair insert in the longer isoform located three codons downstream from the putative initiation site. The 2.4- and 2.5-kb cDNAs predict proteins of approximately 77 and approximately 80 kDa, respectively, and the authenticity was confirmed by sequence identity with 46 amino acids of three cyanogen bromide-cleaved peptides of P4.2. Northern blot analysis detected a major 2.4-kb RNA species in reticulocytes. Isolation of two P4.2 cDNAs implies existence of specific regulation of P4.2 expression in human RBCs. Human RBC P4.2 has significant homology with human factor XIII subunit a and guinea pig liver transglutaminase. Sequence alignment of P4.2 with these two transglutaminases, however, revealed that P4.2 lacks the critical cysteine residue required for the enzymatic crosslinking of substrates. Images PMID:1689063

Surface-enhanced Raman spectroscopy of genomic DNA from in vitro grown tomato (Lycopersicon esculentum Mill.) cultivars before and after plant cryopreservation.

PubMed

Muntean, Cristina M; Leopold, Nicolae; Tripon, Carmen; Coste, Ana; Halmagyi, Adela

2015-06-05

In this work the surface-enhanced Raman scattering (SERS) spectra of five genomic DNAs from non-cryopreserved control tomato plants (Lycopersicon esculentum Mill. cultivars Siriana, Darsirius, Kristin, Pontica and Capriciu) respectively, have been analyzed in the wavenumber range 400-1800 cm(-1). Structural changes induced in genomic DNAs upon cryopreservation were discussed in detail for four of the above mentioned tomato cultivars. The surface-enhanced Raman vibrational modes for each of these cases, spectroscopic band assignments and structural interpretations of genomic DNAs are reported. We have found, that DNA isolated from Siriana cultivar leaf tissues suffers the weakest structural changes upon cryogenic storage of tomato shoot apices. On the contrary, genomic DNA extracted from Pontica cultivar is the most responsive system to cryopreservation process. Particularly, both C2'-endo-anti and C3'-endo-anti conformations have been detected. As a general observation, the wavenumber range 1511-1652 cm(-1), being due to dA, dG and dT residues seems to be influenced by cryopreservation process. These changes could reflect unstacking of DNA bases. However, not significant structural changes of genomic DNAs from Siriana, Darsirius and Kristin have been found upon cryopreservation process of tomato cultivars. Based on this work, specific plant DNA-ligand interactions or accurate local structure of DNA in the proximity of a metallic surface, might be further investigated using surface-enhanced Raman spectroscopy. Copyright © 2015 Elsevier B.V. All rights reserved.

Surface-enhanced Raman spectroscopy of genomic DNA from in vitro grown tomato (Lycopersicon esculentum Mill.) cultivars before and after plant cryopreservation

NASA Astrophysics Data System (ADS)

Muntean, Cristina M.; Leopold, Nicolae; Tripon, Carmen; Coste, Ana; Halmagyi, Adela

2015-06-01

In this work the surface-enhanced Raman scattering (SERS) spectra of five genomic DNAs from non-cryopreserved control tomato plants (Lycopersicon esculentum Mill. cultivars Siriana, Darsirius, Kristin, Pontica and Capriciu) respectively, have been analyzed in the wavenumber range 400-1800 cm-1. Structural changes induced in genomic DNAs upon cryopreservation were discussed in detail for four of the above mentioned tomato cultivars. The surface-enhanced Raman vibrational modes for each of these cases, spectroscopic band assignments and structural interpretations of genomic DNAs are reported. We have found, that DNA isolated from Siriana cultivar leaf tissues suffers the weakest structural changes upon cryogenic storage of tomato shoot apices. On the contrary, genomic DNA extracted from Pontica cultivar is the most responsive system to cryopreservation process. Particularly, both C2‧-endo-anti and C3'-endo-anti conformations have been detected. As a general observation, the wavenumber range 1511-1652 cm-1, being due to dA, dG and dT residues seems to be influenced by cryopreservation process. These changes could reflect unstacking of DNA bases. However, not significant structural changes of genomic DNAs from Siriana, Darsirius and Kristin have been found upon cryopreservation process of tomato cultivars. Based on this work, specific plant DNA-ligand interactions or accurate local structure of DNA in the proximity of a metallic surface, might be further investigated using surface-enhanced Raman spectroscopy.

Target innervation is necessary for neuronal polyploidization in the terrestrial slug Limax.

PubMed

Matsuo, Ryota; Yamagishi, Miki; Wakiya, Kyoko; Tanaka, Yoko; Ito, Etsuro

2013-08-01

The brain of gastropod mollusks contains many giant neurons with polyploid genomic DNAs. Such DNAs are generated through repeated DNA endoreplication during body growth. However, it is not known what triggers DNA endoreplication in neurons. There are two possibilities: (1) DNAs are replicated in response to some unknown molecules in the hemolymph that reflect the nutritive status of the animal; or (2) DNAs are replicated in response to some unknown factors that are retrogradely transported through axons from the innervated target organs. We first tested whether hemolymph with rich nutrition could induce DNA endoreplication. We tested whether the transplanted brain exhibits enhanced DNA endoreplication like an endogenous brain does when transplanted into the homocoel of the body of a slug whose body growth is promoted by an increased food supply. However, no enhancement was observed in the frequency of DNA endoreplication when we compared the transplanted brains in the growth-promoted and growth-suppressed host slugs, suggesting that the humoral environment is irrelevant to triggering the body growth-dependent DNA endoreplication. Next, we tested the requirement of target innervation by surgically dissecting a unilateral posterior pedal nerve of an endogenous brain. Substantially lower number of neurons exhibited DNA endoreplication in the pedal ganglion ipsilateral to the dissected nerve. These results support the view that enhanced DNA endoreplication is mediated by target innervation and is not brought about through the direct effect of humoral factors in the hemolymph during body growth. Copyright © 2013 Wiley Periodicals, Inc.

Disulfide-induced self-assembled targets: A novel strategy for the label free colorimetric detection of DNAs/RNAs via unmodified gold nanoparticles

NASA Astrophysics Data System (ADS)

Shokri, Ehsan; Hosseini, Morteza; Davari, Mehdi D.; Ganjali, Mohammad R.; Peppelenbosch, Maikel P.; Rezaee, Farhad

2017-04-01

A modified non-cross-linking gold-nanoparticles (Au-NPs) aggregation strategy has been developed for the label free colorimetric detection of DNAs/RNAs based on self-assembling target species in the presence of thiolated probes. Two complementary thiol- modified probes, each of which specifically binds at one half of the target introduced SH groups at both ends of dsDNA. Continuous disulfide bond formation at 3‧ and 5‧ terminals of targets leads to the self-assembly of dsDNAs into the sulfur- rich and flexible products with different lengths. These products have a high affinity for the surface of Au-NPs and efficiently protect the surface from salt induced aggregation. To evaluate the assay efficacy, a small part of the citrus tristeza virus (CTV) genome was targeted, leading to a detection limit of about 5 × 10-9 mol.L-1 over a linear ranged from 20 × 10-9 to 10 × 10-7 mol.L-1. This approach also exhibits good reproducibility and recovery levels in the presence of plant total RNA or human plasma total circulating RNA extracts. Self-assembled targets can be then sensitively distinguished from non-assembled or mismatched targets after gel electrophoresis. The disulfide reaction method and integrating self-assembled DNAs/RNAs targets with bare AuNPs as a sensitive indicator provide us a powerful and simple visual detection tool for a wide range of applications.

Isolation of expressed sequences from the region commonly deleted in Velo-cardio-facial syndrome

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

Sirotkin, H.; Morrow, B.; DasGupta, R.

Velo-cardio-facial syndrome (VCFS) is a relatively common autosomal dominant genetic disorder characterized by cleft palate, cardiac abnormalities, learning disabilities and a characteristic facial dysmorphology. Most VCFS patients have interstitial deletions of 22q11 of 1-2 mb. In an effort to isolate the gene(s) responsible for VCFS we have utilized a hybrid selection protocol to recover expressed sequences from three non-overlapping YACs comprising almost 1 mb of the commonly deleted region. Total yeast genomic DNA or isolated YAC DNA was immobilized on Hybond-N filters, blocked with yeast and human ribosomal and human repetitive sequences and hybridized with a mixture of random primedmore » short fragment cDNA libraries. Six human short fragment libraries derived from total fetus, fetal brain, adult brain, testes, thymus and spleen have been used for the selections. Short fragment cDNAs retained on the filter were passed through a second round of selection and cloned into lambda gt10. cDNAs shown to originate from the YACs and from chromosome 22 are being used to isolate full length cDNAs. Three genes known to be present on these YACs, catechol-O-methyltransferase, tuple 1 and clathrin heavy chain have been recovered. Additionally, a gene related to the murine p120 gene and a number of novel short cDNAs have been isolated. The role of these genes in VCFS is being investigated.« less

Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

PubMed

Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

1995-08-01

Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

Tracking multiple particles in fluorescence time-lapse microscopy images via probabilistic data association.

PubMed

Godinez, William J; Rohr, Karl

2015-02-01

Tracking subcellular structures as well as viral structures displayed as 'particles' in fluorescence microscopy images yields quantitative information on the underlying dynamical processes. We have developed an approach for tracking multiple fluorescent particles based on probabilistic data association. The approach combines a localization scheme that uses a bottom-up strategy based on the spot-enhancing filter as well as a top-down strategy based on an ellipsoidal sampling scheme that uses the Gaussian probability distributions computed by a Kalman filter. The localization scheme yields multiple measurements that are incorporated into the Kalman filter via a combined innovation, where the association probabilities are interpreted as weights calculated using an image likelihood. To track objects in close proximity, we compute the support of each image position relative to the neighboring objects of a tracked object and use this support to recalculate the weights. To cope with multiple motion models, we integrated the interacting multiple model algorithm. The approach has been successfully applied to synthetic 2-D and 3-D images as well as to real 2-D and 3-D microscopy images, and the performance has been quantified. In addition, the approach was successfully applied to the 2-D and 3-D image data of the recent Particle Tracking Challenge at the IEEE International Symposium on Biomedical Imaging (ISBI) 2012.

Intraoperative Identification of a Normal Pituitary Gland and an Adenoma Using Near-Infrared Fluorescence Imaging and Low-Dose Indocyanine Green.

PubMed

Verstegen, Marco J T; Tummers, Quirijn R J G; Schutte, Pieter J; Pereira, Alberto M; van Furth, Wouter R; van de Velde, Cornelis J H; Malessy, Martijn J A; Vahrmeijer, Alexander L

2016-09-01

The intraoperative distinction between normal and abnormal pituitary tissue is crucial during pituitary adenoma surgery to obtain a complete tumor resection while preserving endocrine function. Near-infrared (NIR) fluorescence imaging is a technique to intraoperatively visualize tumors by using indocyanine green (ICG), a contrast agent allowing visualization of differences in tissue vascularization. Although NIR fluorescence imaging has been described in pituitary surgery, it has, in contrast to other surgical areas, never become widely used. To evaluate NIR fluorescence imaging in pituitary surgery, both qualitatively and quantitatively, and to assess the additional value of resecting adenoma tissue under NIR fluorescence guidance. We included 10 patients planned to undergo transnasal transsphenoidal selective adenomectomy. Patients received multiple intravenous administrations of 5 mg ICG, up to a maximum of 15 mg per patient. Endoscopic NIR fluorescence imaging was performed at multiple points in time. The NIR fluorescent signal in both the adenoma and pituitary gland was obtained, and the fluorescence contrast ratio was assessed. Four patients had Cushing disease, 1 had acromegaly, and 1 had a prolactinoma. Four patients had a nonfunctioning macroadenoma. In 9 of 10 patients with a histologically proven pituitary adenoma, the normal pituitary gland showed a stronger fluorescent signal than the adenoma. A fluorescence contrast ratio of normal pituitary gland to adenoma of 1.5 ± 0.2 was obtained. In 2 patients; adenoma resection was actually performed under NIR fluorescence guidance instead of under white light. NIR fluorescence imaging can easily and safely be implemented in pituitary surgery. The timing of ICG administration is important for optimal results and warrants further study. It appears that injection of ICG can best be postponed until some part of the normal pituitary gland is identified. Subsequent repeated low-dose ICG administrations improved the distinction between adenoma and gland.

Laser-induced fluorescence imaging of subsurface tissue structures with a volume holographic spatial-spectral imaging system.

PubMed

Luo, Yuan; Gelsinger-Austin, Paul J; Watson, Jonathan M; Barbastathis, George; Barton, Jennifer K; Kostuk, Raymond K

2008-09-15

A three-dimensional imaging system incorporating multiplexed holographic gratings to visualize fluorescence tissue structures is presented. Holographic gratings formed in volume recording materials such as a phenanthrenquinone poly(methyl methacrylate) photopolymer have narrowband angular and spectral transmittance filtering properties that enable obtaining spatial-spectral information within an object. We demonstrate this imaging system's ability to obtain multiple depth-resolved fluorescence images simultaneously.

Morphological spot counting from stacked images for automated analysis of gene copy numbers by fluorescence in situ hybridization.

PubMed

Grigoryan, Artyom M; Dougherty, Edward R; Kononen, Juha; Bubendorf, Lukas; Hostetter, Galen; Kallioniemi, Olli

2002-01-01

Fluorescence in situ hybridization (FISH) is a molecular diagnostic technique in which a fluorescent labeled probe hybridizes to a target nucleotide sequence of deoxyribose nucleic acid. Upon excitation, each chromosome containing the target sequence produces a fluorescent signal (spot). Because fluorescent spot counting is tedious and often subjective, automated digital algorithms to count spots are desirable. New technology provides a stack of images on multiple focal planes throughout a tissue sample. Multiple-focal-plane imaging helps overcome the biases and imprecision inherent in single-focal-plane methods. This paper proposes an algorithm for global spot counting in stacked three-dimensional slice FISH images without the necessity of nuclei segmentation. It is designed to work in complex backgrounds, when there are agglomerated nuclei, and in the presence of illumination gradients. It is based on the morphological top-hat transform, which locates intensity spikes on irregular backgrounds. After finding signals in the slice images, the algorithm groups these together to form three-dimensional spots. Filters are employed to separate legitimate spots from fluorescent noise. The algorithm is set in a comprehensive toolbox that provides visualization and analytic facilities. It includes simulation software that allows examination of algorithm performance for various image and algorithm parameter settings, including signal size, signal density, and the number of slices.

Multiple scattering and the density distribution of a Cs MOT.

PubMed

Overstreet, K; Zabawa, P; Tallant, J; Schwettmann, A; Shaffer, J

2005-11-28

Multiple scattering is studied in a Cs magneto-optical trap (MOT). We use two Abel inversion algorithms to recover density distributions of the MOT from fluorescence images. Deviations of the density distribution from a Gaussian are attributed to multiple scattering.

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae.

PubMed

Higuchi-Sanabria, Ryo; Garcia, Enrique J; Tomoiaga, Delia; Munteanu, Emilia L; Feinstein, Paul; Pon, Liza A

2016-01-01

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.

Quantitative measurement of binary liquid distributions using multiple-tracer x-ray fluorescence and radiography

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

Halls, Benjamin R.; Meyer, Terrence R.; Kastengren, Alan L.

2015-01-01

The complex geometry and large index-of-refraction gradients that occur near the point of impingement of binary liquid jets present a challenging environment for optical interrogation. A simultaneous quadruple-tracer x-ray fluorescence and line-of-sight radiography technique is proposed as a means of distinguishing and quantifying individual liquid component distributions prior to, during, and after jet impact. Two different pairs of fluorescence tracers are seeded into each liquid stream to maximize their attenuation ratio for reabsorption correction and differentiation of the two fluids during mixing. This approach for instantaneous correction of x-ray fluorescence reabsorption is compared with a more time-intensive approach of usingmore » stereographic reconstruction of x-ray attenuation along multiple lines of sight. The proposed methodology addresses the need for a quantitative measurement technique capable of interrogating optically complex, near-field liquid distributions in many mixing systems of practical interest involving two or more liquid streams.« less

Multiresolution multiscale active mask segmentation of fluorescence microscope images

NASA Astrophysics Data System (ADS)

Srinivasa, Gowri; Fickus, Matthew; Kovačević, Jelena

2009-08-01

We propose an active mask segmentation framework that combines the advantages of statistical modeling, smoothing, speed and flexibility offered by the traditional methods of region-growing, multiscale, multiresolution and active contours respectively. At the crux of this framework is a paradigm shift from evolving contours in the continuous domain to evolving multiple masks in the discrete domain. Thus, the active mask framework is particularly suited to segment digital images. We demonstrate the use of the framework in practice through the segmentation of punctate patterns in fluorescence microscope images. Experiments reveal that statistical modeling helps the multiple masks converge from a random initial configuration to a meaningful one. This obviates the need for an involved initialization procedure germane to most of the traditional methods used to segment fluorescence microscope images. While we provide the mathematical details of the functions used to segment fluorescence microscope images, this is only an instantiation of the active mask framework. We suggest some other instantiations of the framework to segment different types of images.

Quantitative measurement of binary liquid distributions using multiple-tracer x-ray fluorescence and radiography

DOE PAGES

Halls, Benjamin R.; Meyer, Terrence R.; Kastengren, Alan L.

2015-01-23

The complex geometry and large index-of-refraction gradients that occur near the point of impingement of binary liquid jets present a challenging environment for optical interrogation. A simultaneous quadruple-tracer x-ray fluorescence and line-of-sight radiography technique is proposed as a means of distinguishing and quantifying individual liquid component distributions prior to, during, and after jet impact. Two different pairs of fluorescence tracers are seeded into each liquid stream to maximize their attenuation ratio for reabsorption correction and differentiation of the two fluids during mixing. This approach for instantaneous correction of x-ray fluorescence reabsorption is compared with a more time-intensive approach of usingmore » stereographic reconstruction of x-ray attenuation along multiple lines of sight. The proposed methodology addresses the need for a quantitative measurement technique capable of interrogating optically complex, near-field liquid distributions in many mixing systems of practical interest involving two or more liquid streams.« less

High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

PubMed Central

Blackburn, Jessica S; Liu, Sali; Raimondi, Aubrey R; Ignatius, Myron S; Salthouse, Christopher D; Langenau, David M

2011-01-01

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis. PMID:21293462

Multiple pathogen biomarker detection using an encoded bead array in droplet PCR.

PubMed

Periyannan Rajeswari, Prem Kumar; Soderberg, Lovisa M; Yacoub, Alia; Leijon, Mikael; Andersson Svahn, Helene; Joensson, Haakan N

2017-08-01

We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color-coded Luminex® beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads. Copyright © 2017. Published by Elsevier B.V.

Differentiation of ocular fundus fluorophores by fluorescence lifetime imaging using multiple excitation and emission wavelengths

NASA Astrophysics Data System (ADS)

Hammer, M.; Schweitzer, D.; Schenke, S.; Becker, W.; Bergmann, A.

2006-10-01

Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently. It is in use for the observation of the age pigment lipofuscin, a precursor of age - related macular degeneration (AMD). But other fluorophores may be of interest too: The redox pair FAD - FADH II provides information on the retinal energy metabolism, advanced glycation end products (AGE) indicate protein glycation associated with pathologic processes in diabetes as well as AMD, and alterations in the fluorescence of collagen and elastin in connective tissue give us the opportunity to observe fibrosis by fluorescence imaging. This, however, needs techniques able to differentiate particular fluorophores despite limited permissible ocular exposure as well as excitation wavelength (limited by the transmission of the human ocular lens to >400 nm). We present an ophthalmic laser scanning system (SLO), equipped with picosecond laser diodes (FWHM 100 ps, 446 nm or 468 nm respectively) and time correlated single photon counting (TCSPC) in two emission bands (500 - 560 nm and 560 - 700 nm). The decays were fitted by a bi-exponential model. Fluorescence spectra were measured by a fluorescence spectrometer fluorolog. Upon excitation at 446 nm, the fluorescence of AGE, FAD, and lipofuscin were found to peak at 503 nm, 525 nm, and 600 nm respectively. Accordingly, the statistical distribution of the fluorescence decay times was found to depend on the different excitation wavelengths and emission bands used. The use of multiple excitation and emission wavelengths in conjunction with fluorescence lifetime imaging allows us to discriminate between intrinsic fluorophores of the ocular fundus. Taken together with our knowledge on the anatomical structure of the fundus, these findings suggest an association of the short, middle and long fluorescence decay time to the retinal pigment epithelium, the retina, and connective tissue respectively.

Novel transcripts of the estrogen receptor α gene in channel catfish

USGS Publications Warehouse

Patino, Reynaldo; Xia, Zhenfang; Gale, William L.; Wu, Chunfa; Maule, Alec G.; Chang, Xiaotian

2000-01-01

Complementary DNA libraries from liver and ovary of an immature female channel catfish were screened with a homologous ERα cDNA probe. The hepatic library yielded two new channel catfish ER cDNAs that encode N-terminal ERα variants of different sizes. Relative to the catfish ERα (medium size; 581 residues) previously reported, these new cDNAs encode Long-ERα (36 residues longer) and Short-ERα (389 residues shorter). The 5′-end of Long-ERα cDNA is identical to that of Medium-ERα but has an additional 503-bp segment with an upstream, in-frame translation-start codon. Recombinant Long-ERα binds estrogen with high affinity (Kd = 3.4 nM), similar to that previously reported for Medium-ERα but lower than reported for catfish ERβ. Short-ERα cDNA encodes a protein that lacks most of the receptor protein and does not bind estrogen. Northern hybridization confirmed the existence of multiple hepatic ERα RNAs that include the size range of the ERα cDNAs obtained from the libraries as well as additional sizes. Using primers for RT-PCR that target locations internal to the protein-coding sequence, we also established the presence of several ERα cDNA variants with in-frame insertions in the ligand-binding and DNA-binding domains and in-frame or out-of-frame deletions in the ligand-binding domain. These internal variants showed patterns of expression that differed between the ovary and liver. Further, the ovarian library yielded a full-length, ERα antisense cDNA containing a poly(A) signal and tail. A limited survey of histological preparations from juvenile catfish by in situ hybridization using directionally synthesized cRNA probes also suggested the expression of ERα antisense RNA in a tissue-specific manner. In conclusion, channel catfish seemingly have three broad classes of ERα mRNA variants: those encoding N-terminal truncated variants, those encoding internal variants (including C-terminal truncated variants), and antisense mRNA. The sense variants may encode functional ERα or related proteins that modulate ERα or ERβ activity. The existence of ER antisense mRNA is reported in this study for the first time. Its role may be to participate in the regulation of ER gene expression.

Chlamydomonas chloroplasts can use short dispersed repeats and multiple pathways to repair a double-strand break in the genome.

PubMed

Odom, Obed W; Baek, Kwang-Hyun; Dani, Radhika N; Herrin, David L

2008-03-01

Certain group I introns insert into intronless DNA via an endonuclease that creates a double-strand break (DSB). There are two models for intron homing in phage: synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR). The Cr.psbA4 intron homes efficiently from a plasmid into the chloroplast psbA gene in Chlamydomonas, but little is known about the mechanism. Analysis of co-transformants selected using a spectinomycin-resistant 16S gene (16S(spec)) provided evidence for both pathways. We also examined the consequences of the donor DNA having only one-sided or no homology with the psbA gene. When there was no homology with the donor DNA, deletions of up to 5 kb involving direct repeats that flank the psbA gene were obtained. Remarkably, repeats as short as 15 bp were used for this repair, which is consistent with the single-strand annealing (SSA) pathway. When the donor had one-sided homology, the DSB in most co-transformants was repaired using two DNAs, the donor and the 16S(spec) plasmid, which, coincidentally, contained a region that is repeated upstream of psbA. DSB repair using two separate DNAs provides further evidence for the SDSA pathway. These data show that the chloroplast can repair a DSB using short dispersed repeats located proximally, distally, or even on separate molecules relative to the DSB. They also provide a rationale for the extensive repertoire of repeated sequences in this genome.

Unit-length line-1 transcripts in human teratocarcinoma cells.

PubMed Central

Skowronski, J; Fanning, T G; Singer, M F

1988-01-01

We have characterized the approximately 6.5-kilobase cytoplasmic poly(A)+ Line-1 (L1) RNA present in a human teratocarcinoma cell line, NTera2D1, by primer extension and by analysis of cloned cDNAs. The bulk of the RNA begins (5' end) at the residue previously identified as the 5' terminus of the longest known primate genomic L1 elements, presumed to represent "unit" length. Several of the cDNA clones are close to 6 kilobase pairs, that is, close to full length. The partial sequences of 18 cDNA clones and full sequence of one (5,975 base pairs) indicate that many different genomic L1 elements contribute transcripts to the 6.5-kilobase cytoplasmic poly(A)+ RNA in NTera2D1 cells because no 2 of the 19 cDNAs analyzed had identical sequences. The transcribed elements appear to represent a subset of the total genomic L1s, a subset that has a characteristic consensus sequence in the 3' noncoding region and a high degree of sequence conservation throughout. Two open reading frames (ORFs) of 1,122 (ORF1) and 3,852 (ORF2) bases, flanked by about 800 and 200 bases of sequence at the 5' and 3' ends, respectively, can be identified in the cDNAs. Both ORFs are in the same frame, and they are separated by 33 bases bracketed by two conserved in-frame stop codons. ORF 2 is interrupted by at least one randomly positioned stop codon in the majority of the cDNAs. The data support proposals suggesting that the human L1 family includes one or more functional genes as well as an extraordinarily large number of pseudogenes whose ORFs are broken by stop codons. The cDNA structures suggest that both genes and pseudogenes are transcribed. At least one of the cDNAs (cD11), which was sequenced in its entirety, could, in principle, represent an mRNA for production of the ORF1 polypeptide. The similarity of mammalian L1s to several recently described invertebrate movable elements defines a new widely distributed class of elements which we term class II retrotransposons. Images PMID:2454389

Using peripheral blood circulating DNAs to detect CpG global methylation status and genetic mutations in patients with myelodysplastic syndrome

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

Iriyama, Chisako; Tomita, Akihiro, E-mail: atomita@med.nagoya-u.ac.jp; Hoshino, Hideaki

2012-03-23

Highlights: Black-Right-Pointing-Pointer Circulating DNAs (CDs) can be used to detect genetic/epigenetic abnormalities in MDS. Black-Right-Pointing-Pointer Epigenetic changes can be detected more sensitively when using plasma DNA than PBMNC. Black-Right-Pointing-Pointer Mutation ratio in CDs may reflect the ratio in stem cell population in bone marrow. Black-Right-Pointing-Pointer Using CDs can be a safer alternate strategy compared to bone marrow aspiration. -- Abstract: Myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder. Several genetic/epigenetic abnormalities are deeply associated with the pathogenesis of MDS. Although bone marrow (BM) aspiration is a common strategy to obtain MDS cells for evaluating their genetic/epigenetic abnormalities, BM aspirationmore » is difficult to perform repeatedly to obtain serial samples because of pain and safety concerns. Here, we report that circulating cell-free DNAs from plasma and serum of patients with MDS can be used to detect genetic/epigenetic abnormalities. The plasma DNA concentration was found to be relatively high in patients with higher blast cell counts in BM, and accumulation of DNA fragments from mono-/di-nucleosomes was confirmed. Using serial peripheral blood (PB) samples from patients treated with hypomethylating agents, global methylation analysis using bisulfite pyrosequencing was performed at the specific CpG sites of the LINE-1 promoter. The results confirmed a decrease of the methylation percentage after treatment with azacitidine (days 3-9) using DNAs from plasma, serum, and PB mono-nuclear cells (PBMNC). Plasma DNA tends to show more rapid change at days 3 and 6 compared with serum DNA and PBMNC. Furthermore, the TET2 gene mutation in DNAs from plasma, serum, and BM cells was quantitated by pyrosequencing analysis. The existence ratio of mutated genes in plasma and serum DNA showed almost equivalent level with that in the CD34+/38- stem cell population in BM. These data suggest that genetic/epigenetic analyses using PB circulating DNA can be a safer and painless alternative to using BM cells.« less

Multiwavelength time-resolved detection of fluorescence during the inflow of indocyanine green into the adult's brain

NASA Astrophysics Data System (ADS)

Gerega, Anna; Milej, Daniel; Weigl, Wojciech; Botwicz, Marcin; Zolek, Norbert; Kacprzak, Michal; Wierzejski, Wojciech; Toczylowska, Beata; Mayzner-Zawadzka, Ewa; Maniewski, Roman; Liebert, Adam

2012-08-01

Optical technique based on diffuse reflectance measurement combined with indocyanine green (ICG) bolus tracking is extensively tested as a method for clinical assessment of brain perfusion in adults at the bedside. Methodology of multiwavelength and time-resolved detection of fluorescence light excited in the ICG is presented and advantages of measurements at multiple wavelengths are discussed. Measurements were carried out: 1. on a physical homogeneous phantom to study the concentration dependence of the fluorescence signal, 2. on the phantom to simulate the dynamic inflow of ICG at different depths, and 3. in vivo on surface of the human head. Pattern of inflow and washout of ICG in the head of healthy volunteers after intravenous injection of the dye was observed for the first time with time-resolved instrumentation at multiple emission wavelengths. The multiwavelength detection of fluorescence signal confirms that at longer emission wavelengths, probability of reabsorption of the fluorescence light by the dye itself is reduced. Considering different light penetration depths at different wavelengths, and the pronounced reabsorption at longer wavelengths, the time-resolved multiwavelength technique may be useful in signal decomposition, leading to evaluation of extra- and intracerebral components of the measured signals.

Sun-induced chlorophyll fluorescence, photosynthesis, and light use efficiency of a soybean field from seasonally continuous measurements

USDA-ARS?s Scientific Manuscript database

Recent development of sun-induced chlorophyll fluorescence (SIF) technology is stimulating studies to remotely approximate canopy photosynthesis (measured as gross primary production, GPP). While multiple applications have advanced the empirical relationship between GPP and SIF, mechanistic understa...

repDNA: a Python package to generate various modes of feature vectors for DNA sequences by incorporating user-defined physicochemical properties and sequence-order effects.

PubMed

Liu, Bin; Liu, Fule; Fang, Longyun; Wang, Xiaolong; Chou, Kuo-Chen

2015-04-15

In order to develop powerful computational predictors for identifying the biological features or attributes of DNAs, one of the most challenging problems is to find a suitable approach to effectively represent the DNA sequences. To facilitate the studies of DNAs and nucleotides, we developed a Python package called representations of DNAs (repDNA) for generating the widely used features reflecting the physicochemical properties and sequence-order effects of DNAs and nucleotides. There are three feature groups composed of 15 features. The first group calculates three nucleic acid composition features describing the local sequence information by means of kmers; the second group calculates six autocorrelation features describing the level of correlation between two oligonucleotides along a DNA sequence in terms of their specific physicochemical properties; the third group calculates six pseudo nucleotide composition features, which can be used to represent a DNA sequence with a discrete model or vector yet still keep considerable sequence-order information via the physicochemical properties of its constituent oligonucleotides. In addition, these features can be easily calculated based on both the built-in and user-defined properties via using repDNA. The repDNA Python package is freely accessible to the public at http://bioinformatics.hitsz.edu.cn/repDNA/. bliu@insun.hit.edu.cn or kcchou@gordonlifescience.org Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Multi-organ characterization of mitochondrial genomic rearrangements in ad libitum and caloric restricted mice show striking somatic mitochondrial DNA rearrangements with age.

PubMed Central

Melov, S; Hinerfeld, D; Esposito, L; Wallace, D C

1997-01-01

Mitochondrial DNA (mtDNA) rearrangements have been shown to accumulate with age in the post-mitotic tissues of a variety of animals and have been hypothesized to result in the age-related decline of mitochondrial bioenergetics leading to tissue and organ failure. Caloric restriction in rodents has been shown to extend life span supporting an association between bioenergetics and senescence. In the present study, we use full length mtDNA amplification by long-extension polymerase chain reaction (LX-PCR) to demonstrate that mice accumulate a wide variety of mtDNA rearrangements with age in post mitotic tissues. Similarly, using an alternative PCR strategy, we have found that 2-4 kb minicircles containing the origin of heavy-strand replication accumulate with age in heart but not brain. Analysis of mtDNA structure and conformation by Southern blots of unrestricted DNA resolved by field inversion gel electrophoresis have revealed that the brain mtDNAs of young animals contain the traditional linear, nicked, and supercoiled mtDNAs while old animals accumulate substantial levels of a slower migrating species we designate age-specific mtDNAs. In old caloric restricted animals, a wide variety of rearranged mtDNAs can be detected by LX-PCR in post mitotic tissues, but Southern blots of unrestricted DNA reveals a marked reduction in the levels of the age- specific mtDNA species. These observations confirm that mtDNA mutations accumulate with age in mice and suggest that caloric restriction impedes this progress. PMID:9023106

Target innervation is necessary for neuronal polyploidization in the terrestrial slug Limax.

PubMed

Matsuo, Ryota; Yamagishi, Miki; Wakiya, Kyoko; Tanaka, Yoko; Ito, Etsuro

2013-05-30

The brain of gastropod mollusks contains many giant neurons with polyploid genomic DNAs. Such DNAs are generated through repeated DNA endoreplication during body growth. However, it is not known what triggers DNA endoreplication in neurons. There are two possibilities: (1) DNAs are replicated in response to some unknown molecules in the hemolymph that reflect the nutritive status of the animal; or (2) DNAs are replicated in response to some unknown factors that are retrogradely transported through axons from the innervated target organs. We first tested whether hemolymph with rich nutrition could induce DNA endoreplication. We tested whether the transplanted brain exhibits enhanced DNA endoreplication like an endogenous brain does when transplanted into the homocoel of the body of a slug whose body growth is promoted by an increased food supply. However, no enhancement was observed in the frequency of DNA endoreplication when we compared the transplanted brains in the growth-promoted and growth-suppressed host slugs, suggesting that the humoral environment is irrelevant to triggering the body growth-dependent DNA endoreplication. Next, we tested the requirement of target innervation by surgically dissecting a unilateral posterior pedal nerve of an endogenous brain. Substantially lower number of neurons exhibited DNA endoreplication in the pedal ganglion ipsilateral to the dissected nerve. These results support the view that enhanced DNA endoreplication is mediated by target innervation and is not brought about through the direct effect of humoral factors in the hemolymph during body growth. © 2013 Wiley Periodicals, Inc. Develop Neurobiol, 2013. Copyright © 2013 Wiley Periodicals, Inc.

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs

PubMed Central

Kakuda, Tsuneo; Shojo, Hideki; Tanaka, Mayumi; Nambiar, Phrabhakaran; Minaguchi, Kiyoshi; Umetsu, Kazuo; Adachi, Noboru

2016-01-01

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10−13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs. PMID:27355212

A Signal, from Human mtDNA, of Postglacial Recolonization in Europe

PubMed Central

Torroni, Antonio; Bandelt, Hans-Jürgen; Macaulay, Vincent; Richards, Martin; Cruciani, Fulvio; Rengo, Chiara; Martinez-Cabrera, Vicente; Villems, Richard; Kivisild, Toomas; Metspalu, Ene; Parik, Jüri; Tolk, Helle-Viivi; Tambets, Kristiina; Forster, Peter; Karger, Bernd; Francalacci, Paolo; Rudan, Pavao; Janicijevic, Branka; Rickards, Olga; Savontaus, Marja-Liisa; Huoponen, Kirsi; Laitinen, Virpi; Koivumäki, Satu; Sykes, Bryan; Hickey, Eileen; Novelletto, Andrea; Moral, Pedro; Sellitto, Daniele; Coppa, Alfredo; Al-Zaheri, Nadia; Santachiara-Benerecetti, A. Silvana; Semino, Ornella; Scozzari, Rosaria

2001-01-01

Mitochondrial HVS-I sequences from 10,365 subjects belonging to 56 populations/geographical regions of western Eurasia and northern Africa were first surveyed for the presence of the T→C transition at nucleotide position 16298, a mutation which has previously been shown to characterize haplogroup V mtDNAs. All mtDNAs with this mutation were then screened for a number of diagnostic RFLP sites, revealing two major subsets of mtDNAs. One is haplogroup V proper, and the other has been termed “pre*V,” since it predates V phylogenetically. The rather uncommon pre*V tends to be scattered throughout Europe (and northwestern Africa), whereas V attains two peaks of frequency: one situated in southwestern Europe and one in the Saami of northern Scandinavia. Geographical distributions and ages support the scenario that pre*V originated in Europe before the Last Glacial Maximum (LGM), whereas the more recently derived haplogroup V arose in a southwestern European refugium soon after the LGM. The arrival of V in eastern/central Europe, however, occurred much later, possibly with (post-)Neolithic contacts. The distribution of haplogroup V mtDNAs in modern European populations would thus, at least in part, reflect the pattern of postglacial human recolonization from that refugium, affecting even the Saami. Overall, the present study shows that the dissection of mtDNA variation into small and well-defined evolutionary units is an essential step in the identification of spatial frequency patterns. Mass screening of a few markers identified using complete mtDNA sequences promises to be an efficient strategy for inferring features of human prehistory. PMID:11517423

mtDNA mutation C1494T, haplogroup A, and hearing loss in Chinese

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

Wang Chengye; Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091; Graduate University of the Chinese Academy of Sciences, Beijing 100039

2006-09-22

Mutation C1494T in mitochondrial 12S rRNA gene was recently reported in two large Chinese families with aminoglycoside-induced and nonsyndromic hearing loss (AINHL) and was claimed to be pathogenic. This mutation, however, was first reported in a sample from central China in our previous study that was aimed to reconstruct East Asian mtDNA phylogeny. All these three mtDNAs formed a subclade defined by mutation C1494T in mtDNA haplogroup A. It thus seems that mutation C1494T is a haplogroup A-associated mutation and this matrilineal background may contribute a high risk for the penetrance of mutation C1494T in Chinese with AINHL. To testmore » this hypothesis, we first genotyped mutation C1494T in 553 unrelated individuals from three regional Chinese populations and performed an extensive search for published complete or near-complete mtDNA data sets (>3000 mtDNAs), we then screened the C1494T mutation in 111 mtDNAs with haplogroup A status that were identified from 1823 subjects across China. The search for published mtDNA data sets revealed no other mtDNA besides the above-mentioned three carrying mutation C1494T. None of the 553 randomly selected individuals and the 111 haplogroup A mtDNAs was found to bear this mutation. Therefore, our results suggest that C1494T is a very rare event. The mtDNA haplogroup A background in general is unlikely to play an active role in the penetrance of mutation C1494T in AINHL.« less

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

PubMed

Kakuda, Tsuneo; Shojo, Hideki; Tanaka, Mayumi; Nambiar, Phrabhakaran; Minaguchi, Kiyoshi; Umetsu, Kazuo; Adachi, Noboru

2016-01-01

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.

Integration of Myeloblastosis Associated Virus proviral sequences occurs in the vicinity of genes encoding signaling proteins and regulators of cell proliferation

PubMed Central

Li, Chang Long; Coullin, Philippe; Bernheim, Alain; Joliot, Véronique; Auffray, Charles; Zoroob, Rima; Perbal, Bernard

2006-01-01

Aims Myeloblastosis Associated Virus type 1 (N) [MAV 1(N)] induces specifically nephroblastomas in 8–10 weeks when injected to newborn chicken. The MAV-induced nephroblastomas constitute a unique animal model of the pediatric Wilms' tumor. We have made use of three independent nephroblastomas that represent increasing tumor grades, to identify the host DNA regions in which MAV proviral sequences were integrated. METHODS Cellular sequences localized next to MAV-integration sites in the tumor DNAs were used to screen a Bacterial Artificial Chromosomes (BACs) library and isolate BACs containing about 150 kilobases of normal DNA corresponding to MAV integration regions (MIRs). These BACs were mapped on the chicken chromosomes by Fluorescent In Situ Hybridization (FISH) and used for molecular studies. Results The different MAV integration sites that were conserved after tumor cell selection identify genes involved in the control of cell signaling and proliferation. Syntenic fragments in human DNA contain genes whose products have been involved in normal and pathological kidney development, and several oncogenes responsible for tumorigenesis in human. Conclusion The identification of putative target genes for MAV provides important clues for the understanding of the MAV pathogenic potential. These studies identified ADAMTS1 as a gene upregulated in MAV-induced nephroblastoma and established that ccn3/nov is not a preferential site of integration for MAV as previously thought. The present results support our hypothesis that the highly efficient and specific MAV-induced tumorigenesis results from the alteration of multiple target genes in differentiating blastemal cells, some of which are required for the progression to highly aggressive stages. This study reinforces our previous conclusions that the MAV-induced nephroblastoma constitutes an excellent model in which to characterize new potential oncogenes and tumor suppressors involved in the establishment and maintenance of tumors. PMID:16403231

Mapping of the 3q27 region involved in Dup(3q) syndrome

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

Rizzu, P.; Baldini, A.; Overhauser, J.

1994-09-01

The duplication 3q syndrome is characterized by partial trisomy of a segment of the long arm of chromosome 3. We have previously found that 3q26.3-3q27 is the minimal region of trisomy overlap. This critical region (CR) is delimited by two patient chromosome breakpoints, approximately 10 cM apart. In order to identify the gene(s) responsible for the Dup(3q) phenotype, we are generating a physical map of the region and identifying expressed sequences. First, we have generated a cytological map using two- and three-color fluorescence in situ hybridization on metaphase and interphase chromosomes. Results allowed us to determine the centromere-telomere orientation, ordermore » and relative distances of six cosmid clones mapped to the CR. Because some of the markers used are part of the consensus chromosome 3 map, our data were easily integrated with existing mapping information. Subsequently, we have included in the map YAC clones positive for polymorphic PCR markers identified by CEPH-Genethon, as well as newly isolated YACs. We have assigned them to the critical region 7 of the Genethon polymorphic markers and linked them to three YAC contigs. Currently our map includes two of the five genes known to map in this region. Interestingly, we found that these two functionally related genes (kininogen and histidin-rich glycoprotein) map to the same 1 Mb genomic fragment. As the physical map is being constructed we are searching for expressed sequences. Positive cDNAs have been found and their characterization is in progress. In conclusion, we will present an integrated map of 3q27 that includes genetic, physical and cytological information as well as gene annotation. As Dup(3q) syndrome is likely to be a contiguous gene syndrome, such a map will be necessary for our understanding of this multiple congenital anomaly.« less

Human tRNA genes function as chromatin insulators

PubMed Central

Raab, Jesse R; Chiu, Jonathan; Zhu, Jingchun; Katzman, Sol; Kurukuti, Sreenivasulu; Wade, Paul A; Haussler, David; Kamakaka, Rohinton T

2012-01-01

Insulators help separate active chromatin domains from silenced ones. In yeast, gene promoters act as insulators to block the spread of Sir and HP1 mediated silencing while in metazoans most insulators are multipartite autonomous entities. tDNAs are repetitive sequences dispersed throughout the human genome and we now show that some of these tDNAs can function as insulators in human cells. Using computational methods, we identified putative human tDNA insulators. Using silencer blocking, transgene protection and repressor blocking assays we show that some of these tDNA-containing fragments can function as barrier insulators in human cells. We find that these elements also have the ability to block enhancers from activating RNA pol II transcribed promoters. Characterization of a putative tDNA insulator in human cells reveals that the site possesses chromatin signatures similar to those observed at other better-characterized eukaryotic insulators. Enhanced 4C analysis demonstrates that the tDNA insulator makes long-range chromatin contacts with other tDNAs and ETC sites but not with intervening or flanking RNA pol II transcribed genes. PMID:22085927

Islander: A database of precisely mapped genomic islands in tRNA and tmRNA genes

DOE PAGES

Hudson, Corey M.; Lau, Britney Y.; Williams, Kelly P.

2014-11-05

Genomic islands are mobile DNAs that are major agents of bacterial and archaeal evolution. Integration into prokaryotic chromosomes usually occurs site-specifically at tRNA or tmRNA gene (together, tDNA) targets, catalyzed by tyrosine integrases. This splits the target gene, yet sequences within the island restore the disrupted gene; the regenerated target and its displaced fragment precisely mark the endpoints of the island. We applied this principle to search for islands in genomic DNA sequences. Our algorithm identifies tDNAs, finds fragments of those tDNAs in the same replicon and removes unlikely candidate islands through a series of filters. A search for islandsmore » in 2168 whole prokaryotic genomes produced 3919 candidates. The website Islander (recently moved to http://bioinformatics.sandia.gov/islander/) presents these precisely mapped candidate islands, the gene content and the island sequence. The algorithm further insists that each island encode an integrase, and attachment site sequence identity is carefully noted; therefore, the database also serves in the study of integrase site-specificity and its evolution.« less

Markedly lowering the viscosity of aqueous solutions of DNA by additives.

PubMed

Elkin, Igor; Weight, Alisha K; Klibanov, Alexander M

2015-10-15

Aqueous solutions of DNAs, while relevant in drug delivery and as a target of therapies, are often very viscous making them difficult to use. Since less viscous solutions could enable targeted drug delivery and/or therapies, the purpose of the present work was to explore compounds capable of "thinning" such DNA solutions under pharmaceutically relevant conditions. To this end, viscosities of aqueous solutions of DNAs and model polyanions were examined at 25 °C in the absence and presence of a number of bulky organic salts (and related compounds) previously found to substantially lower the viscosities of concentrated protein solutions. Out of two dozen compounds tested, only three were found to be effective; the FDA-approved local anesthetics lidocaine, mepivacaine, and prilocaine at near-isotonic concentrations and pH 6.4 lowered solution viscosity of three different DNAs up to about 20 fold. The observed multi-fold viscosity reductions appear to be due to these bulky organic salts' structure-specific non-covalent binding to nucleotide bases resulting in denaturation (unwinding) to, and stabilization of, single-stranded DNA. Copyright © 2015 Elsevier B.V. All rights reserved.

Plant Cell Imaging Based on Nanodiamonds with Excitation-Dependent Fluorescence

NASA Astrophysics Data System (ADS)

Su, Li-Xia; Lou, Qing; Jiao, Zhen; Shan, Chong-Xin

2016-09-01

Despite extensive work on fluorescence behavior stemming from color centers of diamond, reports on the excitation-dependent fluorescence of nanodiamonds (NDs) with a large-scale redshift from 400 to 620 nm under different excitation wavelengths are so far much fewer, especially in biological applications. The fluorescence can be attributed to the combined effects of the fraction of sp2-hybridized carbon atoms among the surface of the fine diamond nanoparticles and the defect energy trapping states on the surface of the diamond. The excitation-dependent fluorescent NDs have been applied in plant cell imaging for the first time. The results reported in this paper may provide a promising route to multiple-color bioimaging using NDs.

Plant Cell Imaging Based on Nanodiamonds with Excitation-Dependent Fluorescence.

PubMed

Su, Li-Xia; Lou, Qing; Jiao, Zhen; Shan, Chong-Xin

2016-12-01

Despite extensive work on fluorescence behavior stemming from color centers of diamond, reports on the excitation-dependent fluorescence of nanodiamonds (NDs) with a large-scale redshift from 400 to 620 nm under different excitation wavelengths are so far much fewer, especially in biological applications. The fluorescence can be attributed to the combined effects of the fraction of sp(2)-hybridized carbon atoms among the surface of the fine diamond nanoparticles and the defect energy trapping states on the surface of the diamond. The excitation-dependent fluorescent NDs have been applied in plant cell imaging for the first time. The results reported in this paper may provide a promising route to multiple-color bioimaging using NDs.

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae

PubMed Central

Higuchi-Sanabria, Ryo; Garcia, Enrique J.; Tomoiaga, Delia; Munteanu, Emilia L.; Feinstein, Paul; Pon, Liza A.

2016-01-01

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging. PMID:26727004

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

PubMed Central

Pathak, Rupak; Koturbash, Igor; Hauer-Jensen, Martin

2017-01-01

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics. PMID:28117817

Human Transposon Tectonics

PubMed Central

Burns, Kathleen H.; Boeke, Jef D.

2012-01-01

Mobile DNAs have had a central role in shaping our genome. More than half of our DNA is comprised of interspersed repeats resulting from replicative copy and paste events of retrotransposons. Although most are fixed, incapable of templating new copies, there are important exceptions to retrotransposon quiescence. De novo insertions cause genetic diseases and cancers, though reliably detecting these occurrences has been difficult. New technologies aimed at uncovering polymorphic insertions reveal that mobile DNAs provide a substantial and dynamic source of structural variation. Key questions going forward include the how and how much new transposition events affect human health and disease. PMID:22579280

Biological and Nano-Technological Applications of Artificial DNAs Made Exclusively of Nonnatural C-Nucleosides with Four Types of Nonnatural Bases

DTIC Science & Technology

2011-08-19

A) CD, (B) UV, (C) Tm, and (D) titration experiments of d(iG*)8/d(C)8. d(T/A*/T)n WC WC
 d(T/A/T)n Watson – Crick (WC)
 Hoogsteen
 Symmetrical A...base Figure 7. Triplex formation of the natural T/A/T which has one Watson - Crick (WC)-type and one Hoogsteen-type hydrogen-bondings, and the...Final Report for AOARD Grant FA2386-10-1-4033 “Biological and Nano-technological Applications of Artificial DNAs Made Exclusively of Nonnatutal C

Mesoscale Modeling of Chromatin Folding

NASA Astrophysics Data System (ADS)

Schlick, Tamar

2009-03-01

Eukaryotic chromatin is the fundamental protein/nucleic acid unit that stores the genetic material. Understanding how chromatin fibers fold and unfold in physiological conditions is important for interpreting fundamental biological processes like DNA replication and transcription regulation. Using a mesoscopic model of oligonucleosome chains and tailored sampling protocols, we elucidate the energetics of oligonucleosome folding/unfolding and the role of each histone tail, linker histones, and divalent ions in regulating chromatin structure. The resulting compact topologies reconcile features of the zigzag model with straight linker DNAs with the solenoid model with bent linker DNAs for optimal fiber organization and reveal dynamic and energetic aspects involved.

Odorant-binding proteins from a primitive termite.

PubMed

Ishida, Yuko; Chiang, Vicky P; Haverty, Michael I; Leal, Walter S

2002-09-01

Hitherto, odorant-binding proteins (OBPs) have been identified from insects belonging to more highly evolved insect orders (Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Hemiptera), whereas only chemosensory proteins have been identified from more primitive species, such as orthopteran and phasmid species. Here, we report for the first time the isolation and cloning of odorant-binding proteins from a primitive termite species, the dampwood termite. Zootermopsis nevadensis nevadensis (Isoptera: Termopsidae). A major antennae-specific protein was detected by native PAGE along with four other minor proteins, which were also absent in the extract from control tissues (hindlegs). Multiple cDNA cloning led to the full characterization of the major antennae-specific protein (ZnevOBP1) and to the identification of two other antennae-specific cDNAs, encoding putative odorant-binding proteins (ZnevOBP2 and ZnevOBP3). N-terminal amino acid sequencing of the minor antennal bands and cDNA cloning showed that olfaction in Z. n. nevadensis may involve multiple odorant-binding proteins. Database searches suggest that the OBPs from this primitive termite are homologues of the pheromone-binding proteins from scarab beetles and antennal-binding proteins from moths.

Femtosecond laser pulse optimization for multiphoton cytometry and control of fluorescence

NASA Astrophysics Data System (ADS)

Tkaczyk, Eric Robert

This body of work encompasses optimization of near infrared femtosecond laser pulses both for enhancement of flow cytometry as well as adaptive pulse shaping to control fluorescence. A two-photon system for in vivo flow cytometry is demonstrated, which allows noninvasive quantification of circulating cell populations in a single live mouse. We monitor fluorescently-labeled red blood cells for more than two weeks, and are also able to noninvasively measure circulation times of two distinct populations of breast cancer cells simultaneously in a single mouse. We build a custom laser excitation source in the form of an extended cavity mode-locked oscillator, which enables superior detection in whole blood or saline of cell lines expressing fluorescent proteins including the green fluorescent protein (GFP), tdTomato and mPlum. A mathematical model explains unique features of the signals. The ability to distinguish different fluorescent species is central to simultaneous measurement of multiple molecular targets in high throughput applications including the multiphoton flow cytometer. We demonstrate that two dyes which are not distinguishable to one-photon measurements can be differentiated and in fact quantified in mixture via phase-shaped two-photon excitation pulses found by a genetic algorithm. We also selectively enhance or suppress two-photon fluorescence of numerous common dyes with tailored pulse shapes. Using a multiplicative (rather than ratiometric) fitness parameter, we are able to control the fluorescence while maintaining a strong signal. With this method, we control the two-photon fluorescence of the blue fluorescent protein (BFP), which is of particular interest in investigations of protein-protein interactions, and has frustrated previous attempts of control. Implementing an acousto-optic interferometer, we use the same experimental setup to measure two-photon excitation cross-sections of dyes and prove that photon-photon interferences are the predominant mechanism of control. This research establishes the basis for molecularly tailored pulse shaping in multiphoton flow cytometry, which will advance our ability to probe the biology of circulating cells during disease progression and response to therapy.

Triazatruxene: A Rigid Central Donor Unit for a D-A3 Thermally Activated Delayed Fluorescence Material Exhibiting Sub-Microsecond Reverse Intersystem Crossing and Unity Quantum Yield via Multiple Singlet-Triplet State Pairs.

PubMed

Dos Santos, Paloma L; Ward, Jonathan S; Congrave, Daniel G; Batsanov, Andrei S; Eng, Julien; Stacey, Jessica E; Penfold, Thomas J; Monkman, Andrew P; Bryce, Martin R

2018-06-01

By inverting the common structural motif of thermally activated delayed fluorescence materials to a rigid donor core and multiple peripheral acceptors, reverse intersystem crossing (rISC) rates are demonstrated in an organic material that enables utilization of triplet excited states at faster rates than Ir-based phosphorescent materials. A combination of the inverted structure and multiple donor-acceptor interactions yields up to 30 vibronically coupled singlet and triplet states within 0.2 eV that are involved in rISC. This gives a significant enhancement to the rISC rate, leading to delayed fluorescence decay times as low as 103.9 ns. This new material also has an emission quantum yield ≈1 and a very small singlet-triplet gap. This work shows that it is possible to achieve both high photoluminescence quantum yield and fast rISC in the same molecule. Green organic light-emitting diode devices with external quantum efficiency >30% are demonstrated at 76 cd m -2 .

A study of MRI-guided diffuse fluorescence molecular tomography for monitoring PDT effects in pancreas cancer

NASA Astrophysics Data System (ADS)

Samkoe, Kimberley S.; Davis, Scott C.; Srinivasan, Subhadra; O'Hara, Julia A.; Hasan, Tayyaba; Pogue, Brian W.

2009-06-01

Over the last several decades little progress has been made in the therapy and treatment monitoring of pancreas adenocarcinoma, a devastating and aggressive form of cancer that has a 5-year patient survival rate of 3%. Currently, investigations for the use of interstitial Verteporfin photodynamic therapy (PDT) are being undertaken in both orthotopic xenograft mouse models and in human clinical trials. In the mouse models, magnetic resonance (MR) imaging has been used as a measure of surrogate response to Verteporfin PDT; however, MR imaging alone lacks the molecular information required to assess the metabolic function and growth rates of the tumor immediately after treatment. We propose the implementation of MR-guided fluorescence tomography in conjunction with a fluorescently labeled (IR-Dye 800 CW, LI-COR) epidermal growth factor (EGF) as a molecular measure of surrogate response. To demonstrate the effectiveness of MR-guided diffuse fluorescence tomography for molecular imaging, we have used the AsPC-1 (+EGFR) human pancreatic adenocarcinoma in an orthotopic mouse model. EGF IRDye 800CW was injected 48 hours prior to imaging. MR image sequences were collected simultaneously with the fluorescence data using a MR-coupled diffuse optical tomography system. Image reconstruction was performed multiple times with varying abdominal organ segmentation in order to obtain a optimal tomographic image. It is shown that diffuse fluorescence tomography of the orthotopic pancreas model is feasible, with consideration of confounding fluorescence signals from the multiple organs and tissues surrounding the pancreas. MR-guided diffuse fluorescence tomography will be used to monitor EGF response after photodynamic therapy. Additionally, it provide the opportunity to individualize subsequent therapies based on response to PDT as well as to evaluate the success of combination therapies, such as PDT with chemotherapy, antibody therapy or even radiation.

Synthesis-identification integration: One-pot hydrothermal preparation of fluorescent nitrogen-doped carbon nanodots for differentiating nucleobases with the aid of multivariate chemometrics analysis.

PubMed

Zhuang, Qianfen; Cao, Wei; Ni, Yongnian; Wang, Yong

2018-08-01

Most of the conventional multidimensional differential sensors currently need at least two-step fabrication, namely synthesis of probe(s) and identification of multiple analytes by mixing of analytes with probe(s), and were conducted using multiple sensing elements or several devices. In the study, we chose five different nucleobases (adenine, cytosine, guanine, thymine, and uracil) as model analytes, and found that under hydrothermal conditions, sodium citrate could react directly with various nucleobases to yield different nitrogen-doped carbon nanodots (CDs). The CDs synthesized from different nucleobases exhibited different fluorescent properties, leading to their respective characteristic fluorescence spectra. Hence, we combined the fluorescence spectra of the CDs with advanced chemometrics like principle component analysis (PCA), hierarchical cluster analysis (HCA), K-nearest neighbor (KNN) and soft independent modeling of class analogy (SIMCA), to present a conceptually novel "synthesis-identification integration" strategy to construct a multidimensional differential sensor for nucleobase discrimination. Single-wavelength excitation fluorescence spectral data, single-wavelength emission fluorescence spectral data, and fluorescence Excitation-Emission Matrices (EEMs) of the CDs were respectively used as input data of the differential sensor. The results showed that the discrimination ability of the multidimensional differential sensor with EEM data set as input data was superior to those with single-wavelength excitation/emission fluorescence data set, suggesting that increasing the number of the data input could improve the discrimination power. Two supervised pattern recognition methods, namely KNN and SIMCA, correctly identified the five nucleobases with a classification accuracy of 100%. The proposed "synthesis-identification integration" strategy together with a multidimensional array of experimental data holds great promise in the construction of differential sensors. Copyright © 2018 Elsevier B.V. All rights reserved.

Combined fluorescence and reflectance spectroscopy for in vivo quantification of cancer biomarkers in low- and high-grade glioma surgery

PubMed Central

Valdés, Pablo A.; Kim, Anthony; Leblond, Frederic; Conde, Olga M.; Harris, Brent T.; Paulsen, Keith D.; Wilson, Brian C.; Roberts, David W.

2011-01-01

Biomarkers are indicators of biological processes and hold promise for the diagnosis and treatment of disease. Gliomas represent a heterogeneous group of brain tumors with marked intra- and inter-tumor variability. The extent of surgical resection is a significant factor influencing post-surgical recurrence and prognosis. Here, we used fluorescence and reflectance spectral signatures for in vivo quantification of multiple biomarkers during glioma surgery, with fluorescence contrast provided by exogenously-induced protoporphyrin IX (PpIX) following administration of 5-aminolevulinic acid. We performed light-transport modeling to quantify multiple biomarkers indicative of tumor biological processes, including the local concentration of PpIX and associated photoproducts, total hemoglobin concentration, oxygen saturation, and optical scattering parameters. We developed a diagnostic algorithm for intra-operative tissue delineation that accounts for the combined tumor-specific predictive capabilities of these quantitative biomarkers. Tumor tissue delineation achieved accuracies of up to 94% (specificity = 94%, sensitivity = 94%) across a range of glioma histologies beyond current state-of-the-art optical approaches, including state-of-the-art fluorescence image guidance. This multiple biomarker strategy opens the door to optical methods for surgical guidance that use quantification of well-established neoplastic processes. Future work would seek to validate the predictive power of this proof-of-concept study in a separate larger cohort of patients. PMID:22112112

Combined fluorescence and reflectance spectroscopy for in vivo quantification of cancer biomarkers in low- and high-grade glioma surgery

NASA Astrophysics Data System (ADS)

Valdés, Pablo A.; Kim, Anthony; Leblond, Frederic; Conde, Olga M.; Harris, Brent T.; Paulsen, Keith D.; Wilson, Brian C.; Roberts, David W.

2011-11-01

Biomarkers are indicators of biological processes and hold promise for the diagnosis and treatment of disease. Gliomas represent a heterogeneous group of brain tumors with marked intra- and inter-tumor variability. The extent of surgical resection is a significant factor influencing post-surgical recurrence and prognosis. Here, we used fluorescence and reflectance spectral signatures for in vivo quantification of multiple biomarkers during glioma surgery, with fluorescence contrast provided by exogenously-induced protoporphyrin IX (PpIX) following administration of 5-aminolevulinic acid. We performed light-transport modeling to quantify multiple biomarkers indicative of tumor biological processes, including the local concentration of PpIX and associated photoproducts, total hemoglobin concentration, oxygen saturation, and optical scattering parameters. We developed a diagnostic algorithm for intra-operative tissue delineation that accounts for the combined tumor-specific predictive capabilities of these quantitative biomarkers. Tumor tissue delineation achieved accuracies of up to 94% (specificity = 94%, sensitivity = 94%) across a range of glioma histologies beyond current state-of-the-art optical approaches, including state-of-the-art fluorescence image guidance. This multiple biomarker strategy opens the door to optical methods for surgical guidance that use quantification of well-established neoplastic processes. Future work would seek to validate the predictive power of this proof-of-concept study in a separate larger cohort of patients.

Multiple-reflection optical gas cell

DOEpatents

Matthews, Thomas G.

1983-01-01

A multiple-reflection optical cell for Raman or fluorescence gas analysis consists of two spherical mirrors positioned transverse to a multiple-pass laser cell in a confronting plane-parallel alignment. The two mirrors are of equal diameter but possess different radii of curvature. The spacing between the mirrors is uniform and less than half of the radius of curvature of either mirror. The mirror of greater curvature possesses a small circular portal in its center which is the effective point source for conventional F1 double lens collection optics of a monochromator-detection system. Gas to be analyzed is flowed into the cell and irradiated by a multiply-reflected composite laser beam centered between the mirrors of the cell. Raman or fluorescence radiation originating from a large volume within the cell is (1) collected via multiple reflections with the cell mirrors, (2) partially collimated and (3) directed through the cell portal in a geometric array compatible with F1 collection optics.

Synthesis of SiO2-Coated Fe3O4 Nanoparticles Using Ultrasound and Its Application in DNA Extraction from Formalin-Fixed, Paraffin-Embedded Human Cancer Tissues

NASA Astrophysics Data System (ADS)

Hieu, Nguyen Minh; Nam, Nguyen Hoang; Huyen, Nguyen Thi; Van Anh, Nguyen Thi; Nghia, Phan Tuan; Khoa, Nguyen Ba; Toan, Nguyen Linh; Luong, Nguyen Hoang

2017-06-01

SiO2-coated Fe3O4 nanoparticles (Fe3O4@SiO2 NPs) were successfully synthesized using ultrasound in order to extract DNA from cancer tissues for application in diagnostics. The core 10.7-nm-diameter Fe3O4 nanoparticles were synthesized by co-precipitation of Fe3+ and Fe2+ as reaction substrates and NH4OH as precipitant, then coated with a thin layer of amorphous silica by a modified Stober method. Further SiO2 coating using alkaline hydrolysis of tetraethyl orthosilicate in ethanol and water mixture was accelerated in the presence of a 37-kHz ultrasound, resulting in the NPs having different sizes of 14.5 nm (version M1), 24.4 nm (version M2), and 34.9 nm (version M3) with saturation magnetization values of 50.2 emu/g, 18.6 emu/g, 10.3 emu/g, respectively. Among the three Fe3O4@SiO2 NPs versions, the M1 NPs allowed extraction of DNAs from 10 mg formalin-fixed and paraffin-embedded (FFPE) tissues of nasopharyngeal carcinoma patients with the highest recovery of about 100-500 ng/ μl and good purity (A260/A280: 1.8-1.9). The extracted DNAs could be used as templates for downstream amplification of 252-bp sequencing specifically for the Braf cancer biomarker gene using polymerase chain reaction (PCR), as well as detection of the pathogenic Epstein-Barr virus (EBV) and the human papilloma-virus (HPV) using real-time PCR. DNA extraction recoveries of both EBV and HPV using Fe3O4@SiO2 NPs M1 were significantly better that those using commercialized Fe3O4@SiO2 microbeads, as indicated by lower threshold cycles of all fluorescent signals including fluorescein amidite (FAM) dye representative for EBV infection, hexachlorofluorescein (HEX) dye representative for β-globin (internal control), and SYBR Green dye representative for HPV infection in tested clinical samples from patients with nasopharyngeal carcinoma (NPC).

Near-field microscopy and fluorescence spectroscopy: application to chromosomes labelled with different fluorophores.

PubMed

Mahieu-Williame, L; Falgayrettes, P; Nativel, L; Gall-Borrut, P; Costa, L; Salehzada, T; Bisbal, C

2010-04-01

We have coupled a spectrophotometer with a scanning near-field optical microscope to obtain, with a single scan, simultaneously scanning near-field optical microscope fluorescence images at different wavelengths as well as topography and transmission images. Extraction of the fluorescence spectra enabled us to decompose the different wavelengths of the fluorescence signals which normally overlap. We thus obtained images of the different fluorescence emissions of acridine orange bound to single or double stranded nucleic acids in human metaphase chromosomes before and after DNAse I or RNAse A treatment. The analysis of these images allowed us to visualize some specific chromatin areas where RNA is associated with DNA showing that such a technique could be used to identify multiple components within a cell.

Carbon Dots: A Modular Activity to Teach Fluorescence and Nanotechnology at Multiple Levels

ERIC Educational Resources Information Center

Pham, Susan N.; Kuether, Joshua E.; Gallagher, Miranda J.; Hernandez, Rodrigo Tapia; Williams, Denise N.; Zhi, Bo; Mensch, Arielle C.; Hamers, Robert J.; Rosenweig, Zeev; Fairbrother, Howard; Krause, Miriam O. P.; Feng, Z. Vivian; Haynes, Christy L.

2017-01-01

In recent years, nanomaterials have entered our daily lives via consumer products; thus, it has become increasingly important to implement activities to introduce these novel materials into chemistry curricula. Here we introduce a newly developed fluorescent nanomaterial, carbon dots, as a more environmentally friendly alternative to heavy-metal…

Multicolor fluorescence enhancement from a photonics crystal surface

NASA Astrophysics Data System (ADS)

Pokhriyal, A.; Lu, M.; Huang, C. S.; Schulz, S.; Cunningham, B. T.

2010-09-01

A photonic crystal substrate exhibiting resonant enhancement of multiple fluorophores has been demonstrated. The device, fabricated uniformly from plastic materials over a ˜3×5 in.2 surface area by nanoreplica molding, utilizes two distinct resonant modes to enhance electric field stimulation of a dye excited by a λ =632.8 nm laser (cyanine-5) and a dye excited by a λ =532 nm laser (cyanine-3). Resonant coupling of the laser excitation to the photonic crystal surface is obtained for each wavelength at a distinct incident angle. Compared to detection of a dye-labeled protein on an ordinary glass surface, the photonic crystal surface exhibited a 32× increase in fluorescent signal intensity for cyanine-5 conjugated streptavidin labeling, while a 25× increase was obtained for cyanine-3 conjugated streptavidin labeling. The photonic crystal is capable of amplifying the output of any fluorescent dye with an excitation wavelength in the 532 nm<λ<633 nm range by selection of an appropriate incident angle. The device is designed for biological assays that utilize multiple fluorescent dyes within a single imaged area, such as gene expression microarrays.

Simple and effective label-free capillary electrophoretic analysis of sugars by complexation using quinoline boronic acids.

PubMed

Kubo, Takuya; Kanemori, Koichi; Kusumoto, Risa; Kawai, Takayuki; Sueyoshi, Kenji; Naito, Toyohiro; Otsuka, Koji

2015-01-01

An effective separation and detection procedure for sugars by capillary electrophoresis (CE) using a complexation between quinolineboronic acid (QBA) and multiple hydroxyl structure of sugar alcohol is reported. We investigated the variation of fluorescence spectra of a variety of QBAs with sorbitol at a wide range of pH conditions and then found that 5-isoQBA strongly enhanced the fluorescence intensity by the complexation at basic pH conditions. The other sugar alcohols having multiple hydroxyls also revealed the enhancement of the fluorescence intensity with 5-isoQBA, whereas the alternation of the intensity was not found in the sugars such as glucose. After optimization of the 5-isoQBA concentration and pH of the buffered solution in CE analysis, 6 sugar alcohols were successfully separated in the order based on the formation constants with 5-isoQBA, which were calculated from the variation of the fluorescence intensity with each sugar alcohol and 5-isoQBA. Furthermore, the limits of detection for sorbitol and xylitol by the CE method were estimated at 15 and 27 μM, respectively.

Graphene-based aptamer logic gates and their application to multiplex detection.

PubMed

Wang, Li; Zhu, Jinbo; Han, Lei; Jin, Lihua; Zhu, Chengzhou; Wang, Erkang; Dong, Shaojun

2012-08-28

In this work, a GO/aptamer system was constructed to create multiplex logic operations and enable sensing of multiplex targets. 6-Carboxyfluorescein (FAM)-labeled adenosine triphosphate binding aptamer (ABA) and FAM-labeled thrombin binding aptamer (TBA) were first adsorbed onto graphene oxide (GO) to form a GO/aptamer complex, leading to the quenching of the fluorescence of FAM. We demonstrated that the unique GO/aptamer interaction and the specific aptamer-target recognition in the target/GO/aptamer system were programmable and could be utilized to regulate the fluorescence of FAM via OR and INHIBIT logic gates. The fluorescence changed according to different input combinations, and the integration of OR and INHIBIT logic gates provided an interesting approach for logic sensing applications where multiple target molecules were present. High-throughput fluorescence imagings that enabled the simultaneous processing of many samples by using the combinatorial logic gates were realized. The developed logic gates may find applications in further development of DNA circuits and advanced sensors for the identification of multiple targets in complex chemical environments.

Multiple capillary biochemical analyzer with barrier member

DOEpatents

Dovichi, N.J.; Zhang, J.Z.

1996-10-22

A multiple capillary biochemical analyzer is disclosed for sequencing DNA and performing other analyses, in which a set of capillaries extends from wells in a microtiter plate into a cuvette. In the cuvette the capillaries are held on fixed closely spaced centers by passing through a sandwich construction having a pair of metal shims which squeeze between them a rubber gasket, forming a leak proof seal for an interior chamber in which the capillary ends are positioned. Sheath fluid enters the chamber and entrains filament sample streams from the capillaries. The filament sample streams, and sheath fluid, flow through aligned holes in a barrier member spaced close to the capillary ends, into a collection chamber having a lower glass window. The filament streams are illuminated above the barrier member by a laser, causing them to fluoresce. The fluorescence is viewed end-on by a CCD camera chip located below the glass window. The arrangement ensures an equal optical path length from all fluorescing spots to the CCD chip and also blocks scattered fluorescence illumination, providing more uniform results and an improved signal-to-noise ratio. 12 figs.

Multicolor fluorescence enhancement from a photonics crystal surface

PubMed Central

Pokhriyal, A.; Lu, M.; Huang, C. S.; Schulz, S.; Cunningham, B. T.

2010-01-01

A photonic crystal substrate exhibiting resonant enhancement of multiple fluorophores has been demonstrated. The device, fabricated uniformly from plastic materials over a ∼3×5 in.2 surface area by nanoreplica molding, utilizes two distinct resonant modes to enhance electric field stimulation of a dye excited by a λ=632.8 nm laser (cyanine-5) and a dye excited by a λ=532 nm laser (cyanine-3). Resonant coupling of the laser excitation to the photonic crystal surface is obtained for each wavelength at a distinct incident angle. Compared to detection of a dye-labeled protein on an ordinary glass surface, the photonic crystal surface exhibited a 32× increase in fluorescent signal intensity for cyanine-5 conjugated streptavidin labeling, while a 25× increase was obtained for cyanine-3 conjugated streptavidin labeling. The photonic crystal is capable of amplifying the output of any fluorescent dye with an excitation wavelength in the 532 nm<λ<633 nm range by selection of an appropriate incident angle. The device is designed for biological assays that utilize multiple fluorescent dyes within a single imaged area, such as gene expression microarrays. PMID:20957067

Multiple capillary biochemical analyzer with barrier member

DOEpatents

Dovichi, Norman J.; Zhang, Jian Z.

1996-01-01

A multiple capillary biochemical analyzer for sequencing DNA and performing other analyses, in which a set of capillaries extends from wells in a microtiter plate into a cuvette. In the cuvette the capillaries are held on fixed closely spaced centers by passing through a sandwich construction having a pair of metal shims which squeeze between them a rubber gasket, forming a leak proof seal for an interior chamber in which the capillary ends are positioned. Sheath fluid enters the chamber and entrains filament sample streams from the capillaries. The filament sample streams, and sheath fluid, flow through aligned holes in a barrier member spaced close to the capillary ends, into a collection chamber having a lower glass window. The filament streams are illuminated above the barrier member by a laser, causing them to fluoresce. The fluorescence is viewed end-on by a CCD camera chip located below the glass window. The arrangement ensures an equal optical path length from all fluorescing spots to the CCD chip and also blocks scattered fluorescence illumination, providing more uniform results and an improved signal to noise ratio.

A multiplexed fluorescent assay for independent second-messenger systems: decoding GPCR activation in living cells.

PubMed

Tewson, Paul H; Quinn, Anne Marie; Hughes, Thomas E

2013-08-01

There is a growing need in drug discovery and basic research to measure multiple second-messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein-coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca(2+)). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence and can be combined with one another, or with existing Ca(2+) sensors, in a live-cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live-cell imaging systems.

Effects of sub-culturing on genetic and physiological parameters in different Beauveria bassiana isolates.

PubMed

Eivazian Kary, Naser; Alizadeh, Zhila

2017-05-01

Beauveria bassiana is a fungus which is widely used as a biological insecticide to control a number of economically important insect pests. Knowledge of the genetic diversity of the isolates, understanding the underlying nature of these evolutionary phenomena and finding appropriate and simple screening tools play an important role in developing effective biocontrol agents. Here, we monitored changes of electrophoretic karyotype of small molecules of extrachromosomal DNAs, presumably mitochondrial DNA or plasmids in several individual isolates of B. bassiana during the forced in vitro evolution by continual subculture on artificial media and then we evaluated these changes on the virulence of the isolates. Through agarose gel electrophoresis of the small extrachromosomal DNAs molecules, we found that mutations accumulate quickly and obvious changes take place in extrachromosomal DNAs of some isolates, although this did not always occur. This plasticity in response to culturing pressure suggests that buffering capacity of fungal genome against mutations is isolate dependent. Following the forced evolution by sub-culturing, five discriminable electrophoretic karyotype of extrachromosomal DNAs was observed among isolates. The results showed that some isolates are prone to deep mutations, but during enforced sub-culturing some others have efficiently conserved genome. These differences are influensive in screening appropriate isolates for mass production as a keystone in biocontrol program. To determine the effects of these changes on isolate traits, virulence, germination rate and spore-bound Pr1 activity were assessed parallel to sub-culturing. The results clearly revealed that parallel to sub-culturing and in correlation with karyotypic changes, isolates significantly suffered from virulence, germination rate and spore-bound Pr1 activity deficiencies. Copyright © 2017 Elsevier Inc. All rights reserved.

Mitochondrion-to-Chloroplast DNA Transfers and Intragenomic Proliferation of Chloroplast Group II Introns in Gloeotilopsis Green Algae (Ulotrichales, Ulvophyceae).

PubMed

Turmel, Monique; Otis, Christian; Lemieux, Claude

2016-09-19

To probe organelle genome evolution in the Ulvales/Ulotrichales clade, the newly sequenced chloroplast and mitochondrial genomes of Gloeotilopsis planctonica and Gloeotilopsis sarcinoidea (Ulotrichales) were compared with those of Pseudendoclonium akinetum (Ulotrichales) and of the few other green algae previously sampled in the Ulvophyceae. At 105,236 bp, the G planctonica mitochondrial DNA (mtDNA) is the largest mitochondrial genome reported so far among chlorophytes, whereas the 221,431-bp G planctonica and 262,888-bp G sarcinoidea chloroplast DNAs (cpDNAs) are the largest chloroplast genomes analyzed among the Ulvophyceae. Gains of non-coding sequences largely account for the expansion of these genomes. Both Gloeotilopsis cpDNAs lack the inverted repeat (IR) typically found in green plants, indicating that two independent IR losses occurred in the Ulvales/Ulotrichales. Our comparison of the Pseudendoclonium and Gloeotilopsis cpDNAs offered clues regarding the mechanism of IR loss in the Ulotrichales, suggesting that internal sequences from the rDNA operon were differentially lost from the two original IR copies during this process. Our analyses also unveiled a number of genetic novelties. Short mtDNA fragments were discovered in two distinct regions of the G sarcinoidea cpDNA, providing the first evidence for intracellular inter-organelle gene migration in green algae. We identified for the first time in green algal organelles, group II introns with LAGLIDADG ORFs as well as group II introns inserted into untranslated gene regions. We discovered many group II introns occupying sites not previously documented for the chloroplast genome and demonstrated that a number of them arose by intragenomic proliferation, most likely through retrohoming. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Mitochondrion-to-Chloroplast DNA Transfers and Intragenomic Proliferation of Chloroplast Group II Introns in Gloeotilopsis Green Algae (Ulotrichales, Ulvophyceae)

PubMed Central

Turmel, Monique; Otis, Christian; Lemieux, Claude

2016-01-01

Abstract To probe organelle genome evolution in the Ulvales/Ulotrichales clade, the newly sequenced chloroplast and mitochondrial genomes of Gloeotilopsis planctonica and Gloeotilopsis sarcinoidea (Ulotrichales) were compared with those of Pseudendoclonium akinetum (Ulotrichales) and of the few other green algae previously sampled in the Ulvophyceae. At 105,236 bp, the G. planctonica mitochondrial DNA (mtDNA) is the largest mitochondrial genome reported so far among chlorophytes, whereas the 221,431-bp G. planctonica and 262,888-bp G. sarcinoidea chloroplast DNAs (cpDNAs) are the largest chloroplast genomes analyzed among the Ulvophyceae. Gains of non-coding sequences largely account for the expansion of these genomes. Both Gloeotilopsis cpDNAs lack the inverted repeat (IR) typically found in green plants, indicating that two independent IR losses occurred in the Ulvales/Ulotrichales. Our comparison of the Pseudendoclonium and Gloeotilopsis cpDNAs offered clues regarding the mechanism of IR loss in the Ulotrichales, suggesting that internal sequences from the rDNA operon were differentially lost from the two original IR copies during this process. Our analyses also unveiled a number of genetic novelties. Short mtDNA fragments were discovered in two distinct regions of the G. sarcinoidea cpDNA, providing the first evidence for intracellular inter-organelle gene migration in green algae. We identified for the first time in green algal organelles, group II introns with LAGLIDADG ORFs as well as group II introns inserted into untranslated gene regions. We discovered many group II introns occupying sites not previously documented for the chloroplast genome and demonstrated that a number of them arose by intragenomic proliferation, most likely through retrohoming. PMID:27503298

Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major

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

Petrillo-Peixoto, M.L.; Beverley, S.M.

1988-12-01

We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major. Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-headmore » configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.« less

BIGEL analysis of gene expression in HL60 cells exposed to X rays or 60 Hz magnetic fields

NASA Technical Reports Server (NTRS)

Balcer-Kubiczek, E. K.; Zhang, X. F.; Han, L. H.; Harrison, G. H.; Davis, C. C.; Zhou, X. J.; Ioffe, V.; McCready, W. A.; Abraham, J. M.; Meltzer, S. J.

1998-01-01

We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.

Unique Thermal Stability of Unnatural Hydrophobic Ds Bases in Double-Stranded DNAs.

PubMed

Kimoto, Michiko; Hirao, Ichiro

2017-10-20

Genetic alphabet expansion technology, the introduction of unnatural bases or base pairs into replicable DNA, has rapidly advanced as a new synthetic biology area. A hydrophobic unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) exhibited high fidelity as a third base pair in PCR. SELEX methods using the Ds-Px pair enabled high-affinity DNA aptamer generation, and introducing a few Ds bases into DNA aptamers extremely augmented their affinities and selectivities to target proteins. Here, to further scrutinize the functions of this highly hydrophobic Ds base, the thermal stabilities of double-stranded DNAs (dsDNA) containing a noncognate Ds-Ds or G-Ds pair were examined. The thermal stability of the Ds-Ds self-pair was as high as that of the natural G-C pair, and apart from the generally higher stability of the G-C pair than that of the A-T pair, most of the 5'-pyrimidine-Ds-purine-3' sequences, such as CDsA and TDsA, exhibited higher stability than the 5'-purine-Ds-pyrimidine-3' sequences, such as GDsC and ADsC, in dsDNAs. This trait enabled the GC-content-independent control of the thermal stability of the designed dsDNA fragments. The melting temperatures of dsDNA fragments containing the Ds-Ds pair can be predicted from the nearest-neighbor parameters including the Ds base. In addition, the noncognate G-Ds pair can efficiently distinguish its neighboring cognate natural base pairs from noncognate pairs. We demonstrated that real-time PCR using primers containing Ds accurately detected a single-nucleotide mismatch in target DNAs. These unique properties of the Ds base that affect the stabilities of the neighboring base pairs could impart new functions to DNA molecules and technologies.

Plasma epidermal growth factor receptor mutation testing with a chip-based digital PCR system in patients with advanced non-small cell lung cancer.

PubMed

Kasahara, Norimitsu; Kenmotsu, Hirotsugu; Serizawa, Masakuni; Umehara, Rina; Ono, Akira; Hisamatsu, Yasushi; Wakuda, Kazushige; Omori, Shota; Nakashima, Kazuhisa; Taira, Tetsuhiko; Naito, Tateaki; Murakami, Haruyasu; Koh, Yasuhiro; Mori, Keita; Endo, Masahiro; Nakajima, Takashi; Yamada, Masanobu; Kusuhara, Masatoshi; Takahashi, Toshiaki

2017-04-01

Epidermal growth factor receptor (EGFR) mutation testing is a companion diagnostic to determine eligibility for treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). Recently, plasma-based EGFR testing by digital polymerase chain reaction (dPCR), which enables accurate quantification of target DNA, has shown promise as a minimally invasive diagnostic. Here, we aimed to evaluate the accuracy of a plasma-based EGFR mutation test developed using chip-based dPCR-based detection of 3 EGFR mutations (exon 19 deletions, L858R in exon 21, and T790M in exon 20). Forty-nine patients with NSCLC harboring EGFR-activating mutations were enrolled, and circulating free DNAs (cfDNAs) were extracted from the plasma of 21 and 28 patients before treatment and after progression following EGFR-TKI treatment, respectively. Using reference genomic DNA containing each mutation, the detection limit of each assay was determined to be 0.1%. The sensitivity and specificity of detecting exon 19 deletions and L858R mutations, calculated by comparing the mutation status in the corresponding tumors, were 70.6% and 93.3%, and 66.7% and 100%, respectively, showing similar results compared with previous studies. T790M was detected in 43% of 28 cfDNAs after progression with EGFR-TKI treatment, but in no cfDNAs before the start of the treatment. This chip-based dPCR assay can facilitate detection of EGFR mutations in cfDNA as a minimally invasive method in clinical settings. Copyright © 2017 Elsevier B.V. All rights reserved.

Highly rearranged and size-variable chloroplast genomes in conifers II clade (cupressophytes): evolution towards shorter intergenic spacers.

PubMed

Wu, Chung-Shien; Chaw, Shu-Miaw

2014-04-01

Although conifers are of immense ecological and economic value, bioengineering of their chloroplasts remains undeveloped. Understanding the chloroplast genomic organization of conifers can facilitate their bioengineering. Members of the conifer II clade (or cupressophytes) are highly diverse in both morphologic features and chloroplast genomic organization. We compared six cupressophyte chloroplast genomes (cpDNAs) that represent four of the five cupressophyte families, including three genomes that are first reported here (Agathis dammara, Calocedrus formosana and Nageia nagi). The six cupressophyte cpDNAs have lost a pair of large inverted repeats (IRs) and vary greatly in size, organization and tRNA copies. We demonstrate that cupressophyte cpDNAs have evolved towards reduced size, largely due to shrunken intergenic spacers. In cupressophytes, cpDNA rearrangements are capable of extending intergenic spacers, and synonymous mutations are negatively associated with the size and frequency of rearrangements. The variable cpDNA sizes of cupressophytes may have been shaped by mutational burden and genomic rearrangements. On the basis of cpDNA organization, our analyses revealed that in gymnosperms, cpDNA rearrangements are phylogenetically informative, which supports the 'gnepines' clade. In addition, removal of a specific IR influences the minimal rearrangements required for the gnepines and cupressophyte clades, whereby Pinaceae favours the removal of IRB but cupressophytes exclusion of IRA. This result strongly suggests that different IR copies have been lost from conifers I and II. Our data help understand the complexity and evolution of cupressophyte cpDNAs. © 2013 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology, The Association of Applied Biologists and John Wiley & Sons Ltd.

The chloroplast and mitochondrial genome sequences of the charophyte Chaetosphaeridium globosum: Insights into the timing of the events that restructured organelle DNAs within the green algal lineage that led to land plants

PubMed Central

Turmel, Monique; Otis, Christian; Lemieux, Claude

2002-01-01

The land plants and their immediate green algal ancestors, the charophytes, form the Streptophyta. There is evidence that both the chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) underwent substantial changes in their architecture (intron insertions, gene losses, scrambling in gene order, and genome expansion in the case of mtDNA) during the evolution of streptophytes; however, because no charophyte organelle DNAs have been sequenced completely thus far, the suite of events that shaped streptophyte organelle genomes remains largely unknown. Here, we have determined the complete cpDNA (131,183 bp) and mtDNA (56,574 bp) sequences of the charophyte Chaetosphaeridium globosum (Coleochaetales). At the levels of gene content (124 genes), intron composition (18 introns), and gene order, Chaetosphaeridium cpDNA is remarkably similar to land-plant cpDNAs, implying that most of the features characteristic of land-plant lineages were gained during the evolution of charophytes. Although the gene content of Chaetosphaeridium mtDNA (67 genes) closely resembles that of the bryophyte Marchantia polymorpha (69 genes), this charophyte mtDNA differs substantially from its land-plant relatives at the levels of size, intron composition (11 introns), and gene order. Our finding that it shares only one intron with its land-plant counterparts supports the idea that the vast majority of mitochondrial introns in land plants appeared after the emergence of these organisms. Our results also suggest that the events accounting for the spacious intergenic spacers found in land-plant mtDNAs took place late during the evolution of charophytes or coincided with the transition from charophytes to land plants. PMID:12161560

The Encapsidated Genome of Microplitis demolitor Bracovirus Integrates into the Host Pseudoplusia includens ▿ ‡

PubMed Central

Beck, Markus H.; Zhang, Shu; Bitra, Kavita; Burke, Gaelen R.; Strand, Michael R.

2011-01-01

Polydnaviruses (PDVs) are symbionts of parasitoid wasps that function as gene delivery vehicles in the insects (hosts) that the wasps parasitize. PDVs persist in wasps as integrated proviruses but are packaged as circularized and segmented double-stranded DNAs into the virions that wasps inject into hosts. In contrast, little is known about how PDV genomic DNAs persist in host cells. Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the host Pseudoplusia includens. MdBV infects primarily host hemocytes and also infects a hemocyte-derived cell line from P. includens called CiE1 cells. Here we report that all 15 genomic segments of the MdBV encapsidated genome exhibited long-term persistence in CiE1 cells. Most MdBV genes expressed in hemocytes were persistently expressed in CiE1 cells, including members of the glc gene family whose products transformed CiE1 cells into a suspension culture. PCR-based integration assays combined with cloning and sequencing of host-virus junctions confirmed that genomic segments J and C persisted in CiE1 cells by integration. These genomic DNAs also rapidly integrated into parasitized P. includens. Sequence analysis of wasp-viral junction clones showed that the integration of proviral segments in M. demolitor was associated with a wasp excision/integration motif (WIM) known from other bracoviruses. However, integration into host cells occurred in association with a previously unknown domain that we named the host integration motif (HIM). The presence of HIMs in most MdBV genomic DNAs suggests that the integration of each genomic segment into host cells occurs through a shared mechanism. PMID:21880747

Evolutionary force of AT-rich repeats to trap genomic and episomal DNAs into the rice genome: lessons from endogenous pararetrovirus.

PubMed

Liu, Ruifang; Koyanagi, Kanako O; Chen, Sunlu; Kishima, Yuji

2012-12-01

In plant genomes, the incorporation of DNA segments is not a common method of artificial gene transfer. Nevertheless, various segments of pararetroviruses have been found in plant genomes in recent decades. The rice genome contains a number of segments of endogenous rice tungro bacilliform virus-like sequences (ERTBVs), many of which are present between AT dinucleotide repeats (ATrs). Comparison of genomic sequences between two closely related rice subspecies, japonica and indica, allowed us to verify the preferential insertion of ERTBVs into ATrs. In addition to ERTBVs, the comparative analyses showed that ATrs occasionally incorporate repeat sequences including transposable elements, and a wide range of other sequences. Besides the known genomic sequences, the insertion sequences also represented DNAs of unclear origins together with ERTBVs, suggesting that ATrs have integrated episomal DNAs that would have been suspended in the nucleus. Such insertion DNAs might be trapped by ATrs in the genome in a host-dependent manner. Conversely, other simple mono- and dinucleotide sequence repeats (SSR) were less frequently involved in insertion events relative to ATrs. Therefore, ATrs could be regarded as hot spots of double-strand breaks that induce non-homologous end joining. The insertions within ATrs occasionally generated new gene-related sequences or involved structural modifications of existing genes. Likewise, in a comparison between Arabidopsis thaliana and Arabidopsis lyrata, the insertions preferred ATrs to other SSRs. Therefore ATrs in plant genomes could be considered as genomic dumping sites that have trapped various DNA molecules and may have exerted a powerful evolutionary force. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Efficient HOMO-LUMO separation by multiple resonance effect toward ultrapure blue thermally activated delayed fluorescence

NASA Astrophysics Data System (ADS)

Hatakeyama, Takuji; Ikuta, Toshiaki; Shiren, Kazushi; Nakajima, Kiichi; Nomura, Shintaro; Ni, Jingping

2016-09-01

Organic light-emitting diodes (OLEDs) play an important role in the new generation of flat-panel displays. Conventional OLEDs employing fluorescent materials together with triplet-triplet annihilation suffer from a relatively low internal quantum efficiency (IQE) of 62.5%. On the other hand, the IQE of OLEDs employing phosphorescent or thermally activated delayed fluorescence (TADF) materials can reach 100%. However, these materials exhibit very broad peaks with a full-width at half-maximum (FWHM) of 70-100 nm and cannot satisfy the color-purity requirements for displays. Therefore, the latest commercial OLED displays employ blue fluorescent materials with a relatively low IQE, and efficient blue emitters with a small FWHM are highly needed. In our manuscript, we present organic molecules that exhibit ultrapure blue fluorescence based on TADF. These molecules consist of three benzene rings connected by one boron and two nitrogen atoms, which establish a rigid polycyclic framework and significant localization of the highest occupied and lowest unoccupied molecular orbitals by a multiple resonance effect. An OLED device based on the new emitter exhibits ultrapure blue emission at 467 nm with an FWHM of 28 nm, Commission Internationale de l'Eclairage (CIE) coordinates of (0.12, 0.13), and an IQE of 100%, which represent record-setting performance for blue OLED devices.

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging

PubMed Central

Zhang, Ming; Chakraborty, Subhasish K.; Sampath, Padma; Rojas, Juan J.; Hou, Weizhou; Saurabh, Saumya; Thorne, Steve H.; Bruchez, Marcel P.; Waggoner, Alan S.

2015-01-01

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule–based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter–tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene. PMID:26348895

Segmentation-based retrospective shading correction in fluorescence microscopy E. coli images for quantitative analysis

NASA Astrophysics Data System (ADS)

Mai, Fei; Chang, Chunqi; Liu, Wenqing; Xu, Weichao; Hung, Yeung S.

2009-10-01

Due to the inherent imperfections in the imaging process, fluorescence microscopy images often suffer from spurious intensity variations, which is usually referred to as intensity inhomogeneity, intensity non uniformity, shading or bias field. In this paper, a retrospective shading correction method for fluorescence microscopy Escherichia coli (E. Coli) images is proposed based on segmentation result. Segmentation and shading correction are coupled together, so we iteratively correct the shading effects based on segmentation result and refine the segmentation by segmenting the image after shading correction. A fluorescence microscopy E. Coli image can be segmented (based on its intensity value) into two classes: the background and the cells, where the intensity variation within each class is close to zero if there is no shading. Therefore, we make use of this characteristics to correct the shading in each iteration. Shading is mathematically modeled as a multiplicative component and an additive noise component. The additive component is removed by a denoising process, and the multiplicative component is estimated using a fast algorithm to minimize the intra-class intensity variation. We tested our method on synthetic images and real fluorescence E.coli images. It works well not only for visual inspection, but also for numerical evaluation. Our proposed method should be useful for further quantitative analysis especially for protein expression value comparison.

Dose–response relationships in gene expression profiles in rainbow trout, Oncorhyncus mykiss, exposed to ethynylestradiol

PubMed Central

Hook, Sharon E.; Skillman, Ann D.; Small, Jack A.; Schultz, Irvin R.

2008-01-01

Determining how gene expression profiles change with toxicant dose will improve the utility of arrays in identifying biomarkers and modes of toxic action. Isogenic rainbow trout, Oncorhyncus mykiss, were exposed to 10, 50 or 100 ng/L ethynylestradiol (a xeno-estrogen) for 7 days. Following exposure hepatic RNA was extracted. Fluorescently labeled cDNA were generated and hybridized against a commercially available Atlantic Salmon/Trout array (GRASP project, University of Victoria) spotted with 16,000 cDNAs. Transcript expression in treated vs control fish was analyzed via Genespring (Silicon Genetics) to identify genes with altered expression, as well as to determine gene clustering patterns that can be used as “expression signatures”. Array results were confirmed via qRT PCR. Our analysis indicates that gene expression profiles varied somewhat with dose. Established biomarkers of exposure to estrogenic chemicals, such as vitellogenin, vitelline envelope proteins, and the estrogen receptor alpha, were induced at every dose. Other genes were dose specific, suggesting that diffierent doses induce distinct physiological responses. These findings demonstrate that cDNA microarrays could be used to identify both toxicant class and relative dose. PMID:16725192

cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/myeloid leukemia factor 2 (MLF2).

PubMed

Kuefer, M U; Look, A T; Williams, D C; Valentine, V; Naeve, C W; Behm, F G; Mullersman, J E; Yoneda-Kato, N; Montgomery, K; Kucherlapati, R; Morris, S W

1996-07-15

A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia. Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity). In contrast to the tissue-restricted expression pattern of MLF1, the MLF2 messenger RNA is expressed ubiquitously. The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal region frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage. In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9. Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

PubMed

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

2008-10-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

PubMed Central

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Žel, Jana; Gruden, Kristina

2008-01-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification. PMID:18710880

Integrated ultrasonic particle positioning and low excitation light fluorescence imaging

NASA Astrophysics Data System (ADS)

Bernassau, A. L.; Al-Rawhani, M.; Beeley, J.; Cumming, D. R. S.

2013-12-01

A compact hybrid system has been developed to position and detect fluorescent micro-particles by combining a Single Photon Avalanche Diode (SPAD) imager with an acoustic manipulator. The detector comprises a SPAD array, light-emitting diode (LED), lenses, and optical filters. The acoustic device is formed of multiple transducers surrounding an octagonal cavity. By stimulating pairs of transducers simultaneously, an acoustic landscape is created causing fluorescent micro-particles to agglomerate into lines. The fluorescent pattern is excited by a low power LED and detected by the SPAD imager. Our technique combines particle manipulation and visualization in a compact, low power, portable setup.

Differential expression and activation of a family of murine peroxisome proliferator-activated receptors.

PubMed Central

Kliewer, S A; Forman, B M; Blumberg, B; Ong, E S; Borgmeyer, U; Mangelsdorf, D J; Umesono, K; Evans, R M

1994-01-01

To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in mammals, we have cloned and characterized two PPAR alpha-related cDNAs (designated PPAR gamma and -delta, respectively) from mouse. The three PPAR isoforms display widely divergent patterns of expression during embryogenesis and in the adult. Surprisingly, PPAR gamma and -delta are not activated by pirinixic acid (Wy 14,643), a potent peroxisome proliferator and activator of PPAR alpha. However, PPAR gamma and -delta are activated by the structurally distinct peroxisome proliferator LY-171883 and linoleic acid, respectively, indicating that each of the isoforms can act as a regulated activator of transcription. These data suggest that tissue-specific responsiveness to peroxisome proliferators, including certain fatty acids, is in part a consequence of differential expression of multiple, pharmacologically distinct PPAR isoforms. Images PMID:8041794

CRISPR/Cas-mediated knock-in via non-homologous end-joining in the crustacean Daphnia magna.

PubMed

Kumagai, Hitoshi; Nakanishi, Takashi; Matsuura, Tomoaki; Kato, Yasuhiko; Watanabe, Hajime

2017-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is widely used for mediating the knock-in of foreign DNA into the genomes of various organisms. Here, we report a process of CRISPR/Cas-mediated knock-in via non-homologous end joining by the direct injection of Cas9/gRNA ribonucleoproteins (RNPs) in the crustacean Daphnia magna, which is a model organism for studies on toxicology, ecology, and evolution. First, we confirmed the cleavage activity of Cas9 RNPs comprising purified Cas9 proteins and gRNAs in D. magna. We used a gRNA that targets exon 10 of the eyeless gene. Cas9 proteins were incubated with the gRNAs and the resulting Cas9 RNPs were injected into D. magna eggs, which led to a typical phenotype of the eyeless mutant, i.e., eye deformity. The somatic and heritable mutagenesis efficiencies were up to 96% and 40%, respectively. Second, we tested the CRISPR/Cas-mediated knock-in of a plasmid by the injection of Cas9 RNPs. The donor DNA plasmid harboring the fluorescent reporter gene was designed to contain the gRNA recognition site. The co-injection of Cas9 RNPs together with the donor DNAs resulted in generation of one founder animal that produced fluorescent progenies. This transgenic Daphnia had donor DNA at the targeted genomic site, which suggested the concurrent cleavage of the injected plasmid DNA and genomic DNA. Owing to its simplicity and ease of experimental design, we suggest that the CRISPR/Cas-mediated knock-in method represents a promising tool for studying functional genomics in D. magna.

CRISPR/Cas-mediated knock-in via non-homologous end-joining in the crustacean Daphnia magna

PubMed Central

Kumagai, Hitoshi; Nakanishi, Takashi; Matsuura, Tomoaki; Kato, Yasuhiko

2017-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is widely used for mediating the knock-in of foreign DNA into the genomes of various organisms. Here, we report a process of CRISPR/Cas-mediated knock-in via non-homologous end joining by the direct injection of Cas9/gRNA ribonucleoproteins (RNPs) in the crustacean Daphnia magna, which is a model organism for studies on toxicology, ecology, and evolution. First, we confirmed the cleavage activity of Cas9 RNPs comprising purified Cas9 proteins and gRNAs in D. magna. We used a gRNA that targets exon 10 of the eyeless gene. Cas9 proteins were incubated with the gRNAs and the resulting Cas9 RNPs were injected into D. magna eggs, which led to a typical phenotype of the eyeless mutant, i.e., eye deformity. The somatic and heritable mutagenesis efficiencies were up to 96% and 40%, respectively. Second, we tested the CRISPR/Cas-mediated knock-in of a plasmid by the injection of Cas9 RNPs. The donor DNA plasmid harboring the fluorescent reporter gene was designed to contain the gRNA recognition site. The co-injection of Cas9 RNPs together with the donor DNAs resulted in generation of one founder animal that produced fluorescent progenies. This transgenic Daphnia had donor DNA at the targeted genomic site, which suggested the concurrent cleavage of the injected plasmid DNA and genomic DNA. Owing to its simplicity and ease of experimental design, we suggest that the CRISPR/Cas-mediated knock-in method represents a promising tool for studying functional genomics in D. magna. PMID:29045453

Quantum caustics in resonance-fluorescence trajectories

NASA Astrophysics Data System (ADS)

Naghiloo, M.; Tan, D.; Harrington, P. M.; Lewalle, P.; Jordan, A. N.; Murch, K. W.

2017-11-01

We employ phase-sensitive amplification to perform homodyne detection of the resonance fluorescence from a driven superconducting artificial atom. Entanglement between the emitter and its fluorescence allows us to track the individual quantum state trajectories of the emitter conditioned on the outcomes of the field measurements. We analyze the ensemble properties of these trajectories by considering trajectories that connect specific initial and final states. By applying the stochastic path-integral formalism, we calculate equations of motion for the most-likely path between two quantum states and compare these predicted paths to experimental data. Drawing on the mathematical similarity between the action formalism of the most-likely quantum paths and ray optics, we study the emergence of caustics in quantum trajectories: places where multiple extrema in the stochastic action occur. We observe such multiple most-likely paths in experimental data and find these paths to be in reasonable quantitative agreement with theoretical calculations.

Resonance fluorescence trajectories in superconducting qubit

NASA Astrophysics Data System (ADS)

Naghiloo, Mahdi; Tan, Dian; Harrington, Patrick; Lewalle, Philippe; Jordan, Andrew; Murch, Kater

We employ phase-sensitive amplification to perform homodyne detection of the resonance fluorescence from a driven superconducting artificial atom. Entanglement between the emitter and its fluorescence allows us to track the individual quantum state trajectories of the emitter. We analyze the ensemble properties of these trajectories by considering paths that connect specific initial and final states. By applying a stochastic path integral formalism, we calculate equations of motion for the most likely path between two quantum states and compare these predicted paths to experimental data. Drawing on the mathematical similarity between the action formalism of the most likely quantum paths and ray optics, we study the emergence of caustics in quantum trajectories-situations where multiple extrema in the stochastic action occur. We observe such multiple most likely paths in experimental data and find these paths to be in reasonable quantitative agreement with theoretical calculations. Supported by the John Templeton Foundation.

Dynamic labelling of neural connections in multiple colours by trans-synaptic fluorescence complementation

PubMed Central

Macpherson, Lindsey J.; Zaharieva, Emanuela E.; Kearney, Patrick J.; Alpert, Michael H.; Lin, Tzu-Yang; Turan, Zeynep; Lee, Chi-Hon; Gallio, Marco

2015-01-01

Determining the pattern of activity of individual connections within a neural circuit could provide insights into the computational processes that underlie brain function. Here, we develop new strategies to label active synapses by trans-synaptic fluorescence complementation in Drosophila. First, we demonstrate that a synaptobrevin-GRASP chimera functions as a powerful activity-dependent marker for synapses in vivo. Next, we create cyan and yellow variants, achieving activity-dependent, multi-colour fluorescence reconstitution across synapses (X-RASP). Our system allows for the first time retrospective labelling of synapses (rather than whole neurons) based on their activity, in multiple colours, in the same animal. As individual synapses often act as computational units in the brain, our method will promote the design of experiments that are not possible using existing techniques. Moreover, our strategies are easily adaptable to circuit mapping in any genetic system. PMID:26635273

Integrated photoacoustic, ultrasound and fluorescence platform for diagnostic medical imaging-proof of concept study with a tissue mimicking phantom.

PubMed

James, Joseph; Murukeshan, Vadakke Matham; Woh, Lye Sun

2014-07-01

The structural and molecular heterogeneities of biological tissues demand the interrogation of the samples with multiple energy sources and provide visualization capabilities at varying spatial resolution and depth scales for obtaining complementary diagnostic information. A novel multi-modal imaging approach that uses optical and acoustic energies to perform photoacoustic, ultrasound and fluorescence imaging at multiple resolution scales from the tissue surface and depth is proposed in this paper. The system comprises of two distinct forms of hardware level integration so as to have an integrated imaging system under a single instrumentation set-up. The experimental studies show that the system is capable of mapping high resolution fluorescence signatures from the surface, optical absorption and acoustic heterogeneities along the depth (>2cm) of the tissue at multi-scale resolution (<1µm to <0.5mm).

In-vivo multi-nonlinear optical imaging of a living cell using a supercontinuum light source generated from a photonic crystal fiber

NASA Astrophysics Data System (ADS)

Kano, Hideaki; Hamaguchi, Hiro-O.

2006-04-01

A supercontinuum light source generated with a femtosecond Ti:Sapphire oscillator has been used to obtain both vibrational and two-photon excitation fluorescence (TPEF) images of a living cell simultaneously at different wavelengths. Owing to an ultrabroadband spectral profile of the supercontinuum, multiple vibrational resonances have been detected through coherent anti-Stokes Raman scattering (CARS) process. In addition to the multiplex CARS process, multiple electronic states can be excited due to the broadband electronic two-photon excitation using the supercontinuum, giving rise to a two-photon excitation fluorescence (TPEF) signal. Using a living yeast cell whose nucleus is labeled by green fluorescent protein (GFP), we have succeeded in visualizing organelles such as mitochondria, septum, and nucleus through the CARS and the TPEF processes. The supercontinuum enables us to perform unique multi-nonlinear optical imaging through two different nonlinear optical processes.

Snapshot Hyperspectral Volumetric Microscopy

NASA Astrophysics Data System (ADS)

Wu, Jiamin; Xiong, Bo; Lin, Xing; He, Jijun; Suo, Jinli; Dai, Qionghai

2016-04-01

The comprehensive analysis of biological specimens brings about the demand for capturing the spatial, temporal and spectral dimensions of visual information together. However, such high-dimensional video acquisition faces major challenges in developing large data throughput and effective multiplexing techniques. Here, we report the snapshot hyperspectral volumetric microscopy that computationally reconstructs hyperspectral profiles for high-resolution volumes of ~1000 μm × 1000 μm × 500 μm at video rate by a novel four-dimensional (4D) deconvolution algorithm. We validated the proposed approach with both numerical simulations for quantitative evaluation and various real experimental results on the prototype system. Different applications such as biological component analysis in bright field and spectral unmixing of multiple fluorescence are demonstrated. The experiments on moving fluorescent beads and GFP labelled drosophila larvae indicate the great potential of our method for observing multiple fluorescent markers in dynamic specimens.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

PubMed Central

Gulati, Srishti; Cao, Vania Y.; Otte, Stephani

2017-01-01

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms. PMID:28654056

Dual-color multiple-particle tracking at 50-nm localization and over 100-µm range in 3D with temporal focusing two-photon microscopy

PubMed Central

Ding, Yu; Li, Chunqiang

2016-01-01

Nanoscale particle tracking in three dimensions is crucial to directly observe dynamics of molecules and nanoparticles in living cells. Here we present a three-dimensional particle tracking method based on temporally focused two-photon excitation. Multiple particles are imaged at 30 frames/s in volume up to 180 × 180 × 100 µm3. The spatial localization precision can reach 50 nm. We demonstrate its capability of tracking fast swimming microbes at speed of ~200 µm/s. Two-photon dual-color tracking is achieved by simultaneously exciting two kinds of fluorescent beads at 800 nm to demonstrate its potential in molecular interaction studies. Our method provides a simple wide-field fluorescence imaging approach for deep multiple-particle tracking. PMID:27867724

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

PubMed

Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

2008-08-22

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

The Matrilineal Ancestry of Ashkenazi Jewry: Portrait of a Recent Founder Event

PubMed Central

Behar, Doron M.; Metspalu, Ene; Kivisild, Toomas; Achilli, Alessandro; Hadid, Yarin; Tzur, Shay; Pereira, Luisa; Amorim, Antonio; Quintana-Murci, Lluís; Majamaa, Kari; Herrnstadt, Corinna; Howell, Neil; Balanovsky, Oleg; Kutuev, Ildus; Pshenichnov, Andrey; Gurwitz, David; Bonne-Tamir, Batsheva; Torroni, Antonio; Villems, Richard; Skorecki, Karl

2006-01-01

Both the extent and location of the maternal ancestral deme from which the Ashkenazi Jewry arose remain obscure. Here, using complete sequences of the maternally inherited mitochondrial DNA (mtDNA), we show that close to one-half of Ashkenazi Jews, estimated at 8,000,000 people, can be traced back to only 4 women carrying distinct mtDNAs that are virtually absent in other populations, with the important exception of low frequencies among non-Ashkenazi Jews. We conclude that four founding mtDNAs, likely of Near Eastern ancestry, underwent major expansion(s) in Europe within the past millennium. PMID:16404693

Genes from the medicinal leech (Hirudo medicinalis) coding for unusual enzymes that specifically cleave endo-epsilon (gamma-Glu)-Lys isopeptide bonds and help to dissolve blood clots.

PubMed

Zavalova, L; Lukyanov, S; Baskova, I; Snezhkov, E; Akopov, S; Berezhnoy, S; Bogdanova, E; Barsova, E; Sverdlov, E D

1996-11-27

We previously detected in salivary gland secretions of the medicinal leech (Hirudo medicinalis) a novel enzymatic activity, endo-epsilon(gamma-Glu)-Lys isopeptidase, which cleaves isopeptide bonds formed by transglutaminase (Factor XIIIa) between glutamine gamma-carboxamide and the epsilon-amino group of lysine. Such isopeptide bonds, either within or between protein polypeptide chains are formed in many biological processes. However, before we started our work no enzymes were known to be capable of specifically splitting isopeptide bonds in proteins. The isopeptidase activity we detected was specific for isopeptide bonds. The enzyme was termed destabilase. Here we report the first purification of destabilase, part of its amino acid sequence isolation and sequencing of two related cDNAs derived from the gene family that encodes destabilase proteins, and the detection of isopeptidase activity encoded by one of these cDNAs cloned in a baculovirus expression vector. The deduced mature protein products of these cDNAs contain 115 and 116 amino acid residues, including 14 highly conserved Cys residues, and are formed from precursors containing specific leader peptides. No homologous sequences were found in public databases.

First complete mitochondrial genome sequence from a box jellyfish reveals a highly fragmented linear architecture and insights into telomere evolution.

PubMed

Smith, David Roy; Kayal, Ehsan; Yanagihara, Angel A; Collins, Allen G; Pirro, Stacy; Keeling, Patrick J

2012-01-01

Animal mitochondrial DNAs (mtDNAs) are typically single circular chromosomes, with the exception of those from medusozoan cnidarians (jellyfish and hydroids), which are linear and sometimes fragmented. Most medusozoans have linear monomeric or linear bipartite mitochondrial genomes, but preliminary data have suggested that box jellyfish (cubozoans) have mtDNAs that consist of many linear chromosomes. Here, we present the complete mtDNA sequence from the winged box jellyfish Alatina moseri (the first from a cubozoan). This genome contains unprecedented levels of fragmentation: 18 unique genes distributed over eight 2.9- to 4.6-kb linear chromosomes. The telomeres are identical within and between chromosomes, and recombination between subtelomeric sequences has led to many genes initiating or terminating with sequences from other genes (the most extreme case being 150 nt of a ribosomal RNA containing the 5' end of nad2), providing evidence for a gene conversion-based model of telomere evolution. The silent-site nucleotide variation within the A. moseri mtDNA is among the highest observed from a eukaryotic genome and may be associated with elevated rates of recombination.

W-enriched satellite sequence in the Indian meal moth, Plodia interpunctella (Lepidoptera, Pyralidae).

PubMed

Dalíková, Martina; Zrzavá, Magda; Kubíčková, Svatava; Marec, František

2017-10-01

The W chromosome of most lepidopteran species represents the largest heterochromatin entity in the female genome. Although satellite DNA is a typical component of constitutive heterochromatin, there are only a few known satellite DNAs (satDNAs) located on the W chromosome in moths and butterflies. In this study, we isolated and characterized new satDNA (PiSAT1) from microdissected W chromosomes of the Indian meal moth, Plodia interpunctella. Even though the PiSAT1 is mainly localized near the female-specific segment of the W chromosome, short arrays of this satDNA also occur on autosomes and/or the Z chromosome. Probably due to the predominant location in the non-recombining part of the genome, PiSAT1 exhibits a relatively large nucleotide variability in its monomers. However, at least a part of all predicted functional motifs is located in conserved regions. Moreover, we detected polyadenylated transcripts of PiSAT1 in all developmental stages and in both sexes (female and male larvae, pupae and adults). Our results suggest a potential structural and functional role of PiSAT1 in the P. interpunctella genome, which is consistent with accumulating evidence for the important role of satDNAs in eukaryotic genomes.

Cre-lox Univector acceptor vectors for functional screening in protoplasts: analysis of Arabidopsis donor cDNAs encoding ABSCISIC ACID INSENSITIVE1-Like protein phosphatases

PubMed Central

Jia, Fan; Gampala, Srinivas S.L.; Mittal, Amandeep; Luo, Qingjun; Rock, Christopher D.

2009-01-01

The 14,200 available full length Arabidopsis thaliana cDNAs in the Universal Plasmid System (UPS) donor vector pUNI51 should be applied broadly and efficiently to leverage a “functional map-space” of homologous plant genes. We have engineered Cre-lox UPS host acceptor vectors (pCR701- 705) with N-terminal epitope tags in frame with the loxH site and downstream from the maize Ubiquitin promoter for use in transient protoplast expression assays and particle bombardment transformation of monocots. As an example of the utility of these vectors, we recombined them with several Arabidopsis cDNAs encoding Ser/Thr protein phosphatase type 2C (PP2Cs) known from genetic studies or predicted by hierarchical clustering meta-analysis to be involved in ABA and stress responses. Our functional results in Zea mays mesophyll protoplasts on ABA-inducible expression effects on the Late Embryogenesis Abundant promoter ProEm:GUS reporter were consistent with predictions and resulted in identification of novel activities of some PP2Cs. Deployment of these vectors can facilitate functional genomics and proteomics and identification of novel gene activities. PMID:19499346

Comparative Chloroplast Genome Analyses of Streptophyte Green Algae Uncover Major Structural Alterations in the Klebsormidiophyceae, Coleochaetophyceae and Zygnematophyceae.

PubMed

Lemieux, Claude; Otis, Christian; Turmel, Monique

2016-01-01

The Streptophyta comprises all land plants and six main lineages of freshwater green algae: Mesostigmatophyceae, Chlorokybophyceae, Klebsormidiophyceae, Charophyceae, Coleochaetophyceae and Zygnematophyceae. Previous comparisons of the chloroplast genome from nine streptophyte algae (including four zygnematophyceans) revealed that, although land plant chloroplast DNAs (cpDNAs) inherited most of their highly conserved structural features from green algal ancestors, considerable cpDNA changes took place during the evolution of the Zygnematophyceae, the sister group of land plants. To gain deeper insights into the evolutionary dynamics of the chloroplast genome in streptophyte algae, we sequenced the cpDNAs of nine additional taxa: two klebsormidiophyceans (Entransia fimbriata and Klebsormidium sp. SAG 51.86), one coleocheatophycean (Coleochaete scutata) and six zygnematophyceans (Cylindrocystis brebissonii, Netrium digitus, Roya obtusa, Spirogyra maxima, Cosmarium botrytis and Closterium baillyanum). Our comparative analyses of these genomes with their streptophyte algal counterparts indicate that the large inverted repeat (IR) encoding the rDNA operon experienced loss or expansion/contraction in all three sampled classes and that genes were extensively shuffled in both the Klebsormidiophyceae and Zygnematophyceae. The klebsormidiophycean genomes boast greatly expanded IRs, with the Entransia 60,590-bp IR being the largest known among green algae. The 206,025-bp Entransia cpDNA, which is one of the largest genome among streptophytes, encodes 118 standard genes, i.e., four additional genes compared to its Klebsormidium flaccidum homolog. We inferred that seven of the 21 group II introns usually found in land plants were already present in the common ancestor of the Klebsormidiophyceae and its sister lineages. At 107,236 bp and with 117 standard genes, the Coleochaete IR-less genome is both the smallest and most compact among the streptophyte algal cpDNAs analyzed thus far; it lacks eight genes relative to its Chaetosphaeridium globosum homolog, four of which represent unique events in the evolutionary scenario of gene losses we reconstructed for streptophyte algae. The 10 compared zygnematophycean cpDNAs display tremendous variations at all levels, except gene content. During zygnematophycean evolution, the IR disappeared a minimum of five times, the rDNA operon was broken at four distinct sites, group II introns were lost on at least 43 occasions, and putative foreign genes, mainly of phage/viral origin, were gained.

Comparative Chloroplast Genome Analyses of Streptophyte Green Algae Uncover Major Structural Alterations in the Klebsormidiophyceae, Coleochaetophyceae and Zygnematophyceae

PubMed Central

Lemieux, Claude; Otis, Christian; Turmel, Monique

2016-01-01

The Streptophyta comprises all land plants and six main lineages of freshwater green algae: Mesostigmatophyceae, Chlorokybophyceae, Klebsormidiophyceae, Charophyceae, Coleochaetophyceae and Zygnematophyceae. Previous comparisons of the chloroplast genome from nine streptophyte algae (including four zygnematophyceans) revealed that, although land plant chloroplast DNAs (cpDNAs) inherited most of their highly conserved structural features from green algal ancestors, considerable cpDNA changes took place during the evolution of the Zygnematophyceae, the sister group of land plants. To gain deeper insights into the evolutionary dynamics of the chloroplast genome in streptophyte algae, we sequenced the cpDNAs of nine additional taxa: two klebsormidiophyceans (Entransia fimbriata and Klebsormidium sp. SAG 51.86), one coleocheatophycean (Coleochaete scutata) and six zygnematophyceans (Cylindrocystis brebissonii, Netrium digitus, Roya obtusa, Spirogyra maxima, Cosmarium botrytis and Closterium baillyanum). Our comparative analyses of these genomes with their streptophyte algal counterparts indicate that the large inverted repeat (IR) encoding the rDNA operon experienced loss or expansion/contraction in all three sampled classes and that genes were extensively shuffled in both the Klebsormidiophyceae and Zygnematophyceae. The klebsormidiophycean genomes boast greatly expanded IRs, with the Entransia 60,590-bp IR being the largest known among green algae. The 206,025-bp Entransia cpDNA, which is one of the largest genome among streptophytes, encodes 118 standard genes, i.e., four additional genes compared to its Klebsormidium flaccidum homolog. We inferred that seven of the 21 group II introns usually found in land plants were already present in the common ancestor of the Klebsormidiophyceae and its sister lineages. At 107,236 bp and with 117 standard genes, the Coleochaete IR-less genome is both the smallest and most compact among the streptophyte algal cpDNAs analyzed thus far; it lacks eight genes relative to its Chaetosphaeridium globosum homolog, four of which represent unique events in the evolutionary scenario of gene losses we reconstructed for streptophyte algae. The 10 compared zygnematophycean cpDNAs display tremendous variations at all levels, except gene content. During zygnematophycean evolution, the IR disappeared a minimum of five times, the rDNA operon was broken at four distinct sites, group II introns were lost on at least 43 occasions, and putative foreign genes, mainly of phage/viral origin, were gained. PMID:27252715

Complete mitochondrial genomes of Taenia multiceps, T. hydatigena and T. pisiformis: additional molecular markers for a tapeworm genus of human and animal health significance.

PubMed

Jia, Wan-Zhong; Yan, Hong-Bin; Guo, Ai-Jiang; Zhu, Xing-Quan; Wang, Yu-Chao; Shi, Wan-Gui; Chen, Hao-Tai; Zhan, Fang; Zhang, Shao-Hua; Fu, Bao-Quan; Littlewood, D Timothy J; Cai, Xue-Peng

2010-07-22

Mitochondrial genomes provide a rich source of molecular variation of proven and widespread utility in molecular ecology, population genetics and evolutionary biology. The tapeworm genus Taenia includes a diversity of tapeworm parasites of significant human and veterinary importance. Here we add complete sequences of the mt genomes of T. multiceps, T. hydatigena and T. pisiformis, to a data set of 4 published mtDNAs in the same genus. Seven complete mt genomes of Taenia species are used to compare and contrast variation within and between genomes in the genus, to estimate a phylogeny for the genus, and to develop novel molecular markers as part of an extended mitochondrial toolkit. The complete circular mtDNAs of T. multiceps, T. hydatigena and T. pisiformis were 13,693, 13,492 and 13,387 bp in size respectively, comprising the usual complement of flatworm genes. Start and stop codons of protein coding genes included those found commonly amongst other platyhelminth mt genomes, but the much rarer initiation codon GTT was inferred for the gene atp6 in T. pisiformis. Phylogenetic analysis of mtDNAs offered novel estimates of the interrelationships of Taenia. Sliding window analyses showed nad6, nad5, atp6, nad3 and nad2 are amongst the most variable of genes per unit length, with the highest peaks in nucleotide diversity found in nad5. New primer pairs capable of amplifying fragments of variable DNA in nad1, rrnS and nad5 genes were designed in silico and tested as possible alternatives to existing mitochondrial markers for Taenia. With the availability of complete mtDNAs of 7 Taenia species, we have shown that analysis of amino acids provides a robust estimate of phylogeny for the genus that differs markedly from morphological estimates or those using partial genes; with implications for understanding the evolutionary radiation of important Taenia. Full alignment of the nucleotides of Taenia mtDNAs and sliding window analysis suggests numerous alternative gene regions are likely to capture greater nucleotide variation than those currently pursued as molecular markers. New PCR primers developed from a comparative mitogenomic analysis of Taenia species, extend the use of mitochondrial markers for molecular ecology, population genetics and diagnostics.

Complete mitochondrial genomes of Taenia multiceps, T. hydatigena and T. pisiformis: additional molecular markers for a tapeworm genus of human and animal health significance

PubMed Central

2010-01-01

Background Mitochondrial genomes provide a rich source of molecular variation of proven and widespread utility in molecular ecology, population genetics and evolutionary biology. The tapeworm genus Taenia includes a diversity of tapeworm parasites of significant human and veterinary importance. Here we add complete sequences of the mt genomes of T. multiceps, T. hydatigena and T. pisiformis, to a data set of 4 published mtDNAs in the same genus. Seven complete mt genomes of Taenia species are used to compare and contrast variation within and between genomes in the genus, to estimate a phylogeny for the genus, and to develop novel molecular markers as part of an extended mitochondrial toolkit. Results The complete circular mtDNAs of T. multiceps, T. hydatigena and T. pisiformis were 13,693, 13,492 and 13,387 bp in size respectively, comprising the usual complement of flatworm genes. Start and stop codons of protein coding genes included those found commonly amongst other platyhelminth mt genomes, but the much rarer initiation codon GTT was inferred for the gene atp6 in T. pisiformis. Phylogenetic analysis of mtDNAs offered novel estimates of the interrelationships of Taenia. Sliding window analyses showed nad6, nad5, atp6, nad3 and nad2 are amongst the most variable of genes per unit length, with the highest peaks in nucleotide diversity found in nad5. New primer pairs capable of amplifying fragments of variable DNA in nad1, rrnS and nad5 genes were designed in silico and tested as possible alternatives to existing mitochondrial markers for Taenia. Conclusions With the availability of complete mtDNAs of 7 Taenia species, we have shown that analysis of amino acids provides a robust estimate of phylogeny for the genus that differs markedly from morphological estimates or those using partial genes; with implications for understanding the evolutionary radiation of important Taenia. Full alignment of the nucleotides of Taenia mtDNAs and sliding window analysis suggests numerous alternative gene regions are likely to capture greater nucleotide variation than those currently pursued as molecular markers. New PCR primers developed from a comparative mitogenomic analysis of Taenia species, extend the use of mitochondrial markers for molecular ecology, population genetics and diagnostics. PMID:20649981

Anchoring a Defined Sequence to the 55' Ends of mRNAs : The Bolt to Clone Rare Full Length mRNAs and Generate cDNA Libraries porn a Few Cells.

PubMed

Baptiste, J; Milne Edwards, D; Delort, J; Mallet, J

1993-01-01

Among numerous applications, the polymerase chain reaction (PCR) (1,2) provides a convenient means to clone 5' ends of rare mRNAs and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods. Basically, the amplification of cDNAs by the PCR requires the availability of the sequences of two stretches of the molecule to be amplified. A sequence can easily be imposed at the 5' end of the first-strand cDNAs (corresponding to the 3' end of the mRNAs) by priming the reverse transcription with a specific primer (for cloning the 5' end of rare messenger) or with an oligonucleotide tailored with a poly (dT) stretch (for cDNA library construction), taking advantage of the poly (A) sequence that is located at the 3' end of mRNAs. Several strategies have been devised to tag the 3' end of the ss-cDNAs (corresponding to the 55' end of the mRNAs). We (3) and others have described strategies based on the addition of a homopolymeric dG (4,5) or dA (6,7) tail using terminal deoxyribonucleotide transferase (TdT) ("anchor-PCR" [4]). However, this strategy has important limitations. The TdT reaction is difficult to control and has a low efficiency (unpublished observations). But most importantly, the return primers containing a homopolymeric (dC or dT) tail generate nonspecific amplifications, a phenomenon that prevents the isolation of low abundance mRNA species and/or interferes with the relative abundance of primary clones in the library. To circumvent these drawbacks, we have used two approaches. First, we devised a strategy based on a cRNA enrichment procedure, which has been useful to eliminate nonspecific-PCR products and to allow detection and cloning of cDNAs of low abundance (3). More recently, to avoid the nonspecific amplification resulting from the annealing of the homopolymeric tail oligonucleotide, we have developed a novel anchoring strategy that is based on the ligation of an oligonucleotide to the 35' end of ss-cDNAs. This strategy is referred to as SLIC for single-strand ligation to ss-cDNA (8).

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

PubMed Central

Vanderperre, Benoît; Lucier, Jean-François; Bissonnette, Cyntia; Motard, Julie; Tremblay, Guillaume; Vanderperre, Solène; Wisztorski, Maxence; Salzet, Michel; Boisvert, François-Michel; Roucou, Xavier

2013-01-01

A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome. PMID:23950983

CCR6+ Th cell distribution differentiates systemic lupus erythematosus patients based on anti-dsDNA antibody status.

PubMed

Zhong, Wei; Jiang, Zhenyu; Wu, Jiang; Jiang, Yanfang; Zhao, Ling

2018-01-01

Systemic lupus erythematosus (SLE) disease has been shown to be associated with the generation of multiple auto-antibodies. Among these, anti-dsDNA antibodies (anti-DNAs) are specific and play a pathogenic role in SLE. Indeed, anti-DNA + SLE patients display a worse disease course. The generation of these pathogenic anti-DNAs has been attributed to the interaction between aberrant T helper (Th) cells and autoimmune B cells. Thus, in this study we have investigated whether CCR6 + Th cells have the ability to differentiate SLE patients based on anti-DNA status, and if their distribution has any correlation with disease activity. We recruited 25 anti-DNA + and 25 anti-DNA - treatment-naive onset SLE patients, matched for various clinical characteristics in our nested matched case-control study. CCR6 + Th cells and their additional subsets were analyzed in each patient by flow cytometry. Anti-DNA + SLE patients specifically had a higher percentage of Th cells expressing CCR6 and CXCR3. Further analysis of CCR6 + Th cell subsets showed that anti-DNA + SLE patients had elevated proportions of Th9, Th17, Th17.1 and CCR4/CXCR3 double-negative (DN) cells. However, the proportions of CCR6 - Th subsets, including Th1 and Th2 cells, did not show any association with anti-DNA status. Finally, we identified a correlation between CCR6 + Th subsets and clinical indicators, specifically in anti-DNA + SLE patients. Our data indicated that CCR6 + Th cells and their subsets were elevated and correlated with disease activity in anti-DNA + SLE patients. We speculated that CCR6 + Th cells may contribute to distinct disease severity in anti-DNA + SLE patients.

Suppression of HIV-1 Infection by APOBEC3 Proteins in Primary Human CD4+ T Cells Is Associated with Inhibition of Processive Reverse Transcription as Well as Excessive Cytidine Deamination

PubMed Central

Gillick, Kieran; Pollpeter, Darja; Phalora, Prabhjeet; Kim, Eun-Young; Wolinsky, Steven M.

2013-01-01

The Vif protein of human immunodeficiency virus type 1 (HIV-1) promotes viral replication by downregulation of the cell-encoded, antiviral APOBEC3 proteins. These proteins exert their suppressive effects through the inhibition of viral reverse transcription as well as the induction of cytidine deamination within nascent viral cDNA. Importantly, these two effects have not been characterized in detail in human CD4+ T cells, leading to controversies over their possible contributions to viral inhibition in the natural cell targets of HIV-1 replication. Here we use wild-type and Vif-deficient viruses derived from the CD4+ T cells of multiple donors to examine the consequences of APOBEC3 protein function at natural levels of expression. We demonstrate that APOBEC3 proteins impart a profound deficiency to reverse transcription from the initial stages of cDNA synthesis, as well as excessive cytidine deamination (hypermutation) of the DNAs that are synthesized. Experiments using viruses from transfected cells and a novel method for mapping the 3′ termini of cDNAs indicate that the inhibition of reverse transcription is not limited to a few specific sites, arguing that APOBEC3 proteins impede enzymatic processivity. Detailed analyses of mutation spectra in viral cDNA strongly imply that one particular APOBEC3 protein, APOBEC3G, provides the bulk of the antiviral phenotype in CD4+ T cells, with the effects of APOBEC3F and APOBEC3D being less significant. Taken together, we conclude that the dual mechanisms of action of APOBEC3 proteins combine to deliver more effective restriction of HIV-1 than either function would by itself. PMID:23152537

Daily light use efficiency in a cornfield can be related to the canopy red/far-red fluorescence ratio and leaf light use efficiency across a growing season

USDA-ARS?s Scientific Manuscript database

In multiple years (2008-2013), we collected canopy and leaf fluorescence, photosynthesis, hyperspectral reflectance spectra, and biophysical measurements along transects within a USDA/Beltsville experimental cornfield treated with optimal nitrogen application (100%N) and which has an eddy covariance...

A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis

PubMed Central

Imamura, Ryota; Murata, Naoki; Shimanouchi, Toshinori; Yamashita, Kaoru; Fukuzawa, Masayuki; Noda, Minoru

2017-01-01

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots. PMID:28714873

A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis.

PubMed

Imamura, Ryota; Murata, Naoki; Shimanouchi, Toshinori; Yamashita, Kaoru; Fukuzawa, Masayuki; Noda, Minoru

2017-07-15

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots.

Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding.

PubMed

Ohashi, Nami; Nomura, Wataru; Narumi, Tetsuo; Lewin, Nancy E; Itotani, Kyoko; Blumberg, Peter M; Tamamura, Hirokazu

2011-01-19

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.

Chamber catalogues of optical and fluorescent signatures distinguish bioaerosol classes

NASA Astrophysics Data System (ADS)

Hernandez, Mark; Perring, Anne E.; McCabe, Kevin; Kok, Greg; Granger, Gary; Baumgardner, Darrel

2016-07-01

Rapid bioaerosol characterization has immediate applications in the military, environmental and public health sectors. Recent technological advances have facilitated single-particle detection of fluorescent aerosol in near real time; this leverages controlled ultraviolet exposures with single or multiple wavelengths, followed by the characterization of associated fluorescence. This type of ultraviolet induced fluorescence has been used to detect airborne microorganisms and their fragments in laboratory studies, and it has been extended to field studies that implicate bioaerosol to compose a substantial fraction of supermicron atmospheric particles. To enhance the information yield that new-generation fluorescence instruments can provide, we report the compilation of a referential aerobiological catalogue including more than 50 pure cultures of common airborne bacteria, fungi and pollens, recovered at water activity equilibrium in a mesoscale chamber (1 m3). This catalogue juxtaposes intrinsic optical properties and select bandwidths of fluorescence emissions, which manifest to clearly distinguish between major classes of airborne microbes and pollens.

Array biosensor: recent developments

NASA Astrophysics Data System (ADS)

Golden, Joel P.; Rowe-Taitt, Chris A.; Feldstein, Mark J.; Ligler, Frances S.

1999-05-01

A fluorescence-based immunosensor has been developed for simultaneous analyses of multiple samples for 1 to 6 different antigens. A patterned array of recognition antibodies immobilized on the surface of a planar waveguide is used to 'capture' analyte present in samples. Bound analyte is then quantified by means of fluorescent detector molecules. Upon excitation of the fluorescent label by a small diode laser, a CCD camera detects the pattern of fluorescent antigen:antibody complexes on the sensor surface. Image analysis software correlates the position of fluorescent signals with the identity of the analyte. A new design for a fluidics distribution system is shown, as well as results from assays for physiologically relevant concentrations of staphylococcal enterotoxin B (SEB), F1 antigen from Yersinia pestis, and D- dimer, a marker of sepsis and thrombotic disorders.

Portable real-time fluorescence cytometry of microscale cell culture analog devices

NASA Astrophysics Data System (ADS)

Kim, Donghyun; Tatosian, Daniel A.; Shuler, Michael L.

2006-02-01

A portable fluorescence cytometric system that provides a modular platform for quantitative real-time image measurements has been used to explore the applicability to investigating cellular events on multiple time scales. For a short time scale, we investigated the real-time dynamics of uptake of daunorubicin, a chemotherapeutic agent, in cultured mouse L-cells in a micro cell culture analog compartment using the fluorescent cytometric system. The green fluorescent protein (GFP) expression to monitor induction of pre-specified genes, which occurs on a much longer time scale, has also been measured. Here GFP fluorescence from a doxycycline inducible promoter in a mouse L-cell line was determined. Additionally, a system based on inexpensive LEDs showed performance comparable to a broadband light source based system and reduced photobleaching compared to microscopic examination.

Fluorescence lifetime measurements in heterogeneous scattering medium

NASA Astrophysics Data System (ADS)

Nishimura, Goro; Awasthi, Kamlesh; Furukawa, Daisuke

2016-07-01

Fluorescence lifetime in heterogeneous multiple light scattering systems is analyzed by an algorithm without solving the diffusion or radiative transfer equations. The algorithm assumes that the optical properties of medium are constant in the excitation and emission wavelength regions. If the assumption is correct and the fluorophore is a single species, the fluorescence lifetime can be determined by a set of measurements of temporal point-spread function of the excitation light and fluorescence at two different concentrations of the fluorophore. This method is not dependent on the heterogeneity of the optical properties of the medium as well as the geometry of the excitation-detection on an arbitrary shape of the sample. The algorithm was validated by an indocyanine green fluorescence in phantom measurements and demonstrated by an in vivo measurement.

Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome.

PubMed

Piatkevich, Kiryl D; Subach, Fedor V; Verkhusha, Vladislav V

2013-01-01

The ability to modulate the fluorescence of optical probes can be used to enhance signal-to-noise ratios for imaging within highly autofluorescent environments, such as intact tissues and living organisms. Here, we report two bacteriophytochrome-based photoactivatable near-infrared fluorescent proteins, named PAiRFP1 and PAiRFP2. PAiRFPs utilize haem-derived biliverdin, ubiquitous in mammalian tissues, as the chromophore. Initially weakly fluorescent PAiRFPs undergo photoconversion into a highly fluorescent state with excitation/emission at 690/717 nm following a brief irradiation with far-red light. After photoactivation, PAiRFPs slowly revert back to initial state, enabling multiple photoactivation-relaxation cycles. Low-temperature optical spectroscopy reveals several intermediates involved in PAiRFP photocycles, which all differ from that of the bacteriophytochrome precursor. PAiRFPs can be photoactivated in a spatially selective manner in mouse tissues, and optical modulation of their fluorescence allows for substantial contrast enhancement, making PAiRFPs advantageous over permanently fluorescent probes for in vivo imaging conditions of high autofluorescence and low signal levels.

Fluorescence lifetime plate reader: Resolution and precision meet high-throughput

PubMed Central

Petersen, Karl J.; Peterson, Kurt C.; Muretta, Joseph M.; Higgins, Sutton E.; Gillispie, Gregory D.; Thomas, David D.

2014-01-01

We describe a nanosecond time-resolved fluorescence spectrometer that acquires fluorescence decay waveforms from each well of a 384-well microplate in 3 min with signal-to-noise exceeding 400 using direct waveform recording. The instrument combines high-energy pulsed laser sources (5–10 kHz repetition rate) with a photomultiplier and high-speed digitizer (1 GHz) to record a fluorescence decay waveform after each pulse. Waveforms acquired from rhodamine or 5-((2-aminoethyl)amino) naphthalene-1-sulfonic acid dyes in a 384-well plate gave lifetime measurements 5- to 25-fold more precise than the simultaneous intensity measurements. Lifetimes as short as 0.04 ns were acquired by interleaving with an effective sample rate of 5 GHz. Lifetime measurements resolved mixtures of single-exponential dyes with better than 1% accuracy. The fluorescence lifetime plate reader enables multiple-well fluorescence lifetime measurements with an acquisition time of 0.5 s per well, suitable for high-throughput fluorescence lifetime screening applications. PMID:25430092

Fluorescent Trimethoprim Conjugate Probes To Assess Drug Accumulation in Wild Type and Mutant Escherichia coli

PubMed Central

2016-01-01

Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment. PMID:27737551

Programmable oligonucleotide probes design and applications for in situ and in vivo RNA imaging in cells

NASA Astrophysics Data System (ADS)

Cheglakov, Zoya

Unequal spreading of mRNA is a frequent experience observed in varied cell lines. The study of cellular processes dynamics and precise localization of mRNAs offers a vital toolbox to target specific proteins in precise cytoplasmic areas and provides a convenient instrument to uncover their mechanisms and functions. Latest methodological innovations have allowed imaging of a single mRNA molecule in situ and in vivo. Today, Fluorescent In Situ Hybridization (FISH) methods allow the studying of mRNA expression and offer a vital toolbox for accurate biological models. Studies enable analysis of the dynamics of an individual mRNA, have uncovered the multiplex RNA transport systems. With all current approaches, a single mRNA tracking in the mammalian cells is still challenging. This thesis describes mRNA detection methods based on programmable fluorophore-labeled DNA structures complimentary to native targets providing an accurate mRNA imaging in mammalian cells. First method represents beta-actin (ACTB) transcripts in situ detection in human cells, the technique strategy is based on programmable DNA probes, amplified by rolling circle amplification (RCA). The method reports precise localization of molecule of interest with an accuracy of a single-cell. Visualization and localization of specific endogenous mRNA molecules in real-time in vivo has the promising to innovate cellular biology studies, medical analysis and to provide a vital toolbox in drugs invention area. Second method described in this thesis represents miR-21 miRNA detection within a single live-cell resolution. The method using fluorophore-labeled short synthetic DNAs probes forming a stem-loop shape and generating Fluorescent Resonance Energy Transfer (FRET) as a result of target-probes hybridization. Catalytic nucleic acid (DNAzymes) probes are cooperative tool for precise detection of different mRNA targets. With assistance of a complementary fluorophore-quencher labeled substrate, the DNAzymes provide a beneficial strategy for simultaneous tracking readily accomplished by multicolor imaging with diverse fluorescent tags. The third method in this thesis will demonstrate the advantage of DNAzymes probes amplification, and offers potential strategy to monitor miRNAs in mammalian live cells.

Utilizing a Tower Based System for Optical Sensing of Ecosystem Carbon Fluxes

NASA Astrophysics Data System (ADS)

Huemmrich, K. F.; Corp, L. A.; Middleton, E.; Campbell, P. K. E.; Landis, D.; Kustas, W. P.

2015-12-01

Optical sampling of spectral reflectance and solar induced fluorescence provide information on the physiological status of vegetation that can be used to infer stress responses and estimates of production. Multiple repeated observations are required to observe the effects of changing environmental conditions on vegetation. This study examines the use of optical signals to determine inputs to a light use efficiency (LUE) model describing productivity of a cornfield where repeated observations of carbon flux, spectral reflectance and fluorescence were collected. Data were collected at the Optimizing Production Inputs for Economic and Environmental Enhancement (OPE3) fields (39.03°N, 76.85°W) at USDA Beltsville Agricultural Research Center. Agricultural Research Service researchers measured CO2 fluxes using eddy covariance methods throughout the growing season. Optical measurements were made from the nearby tower supporting the NASA FUSION sensors. The sensor system consists of two dual channel, upward and downward looking, spectrometers used to simultaneously collect high spectral resolution measurements of reflected and fluoresced light from vegetation canopies at multiple view angles. Estimates of chlorophyll fluorescence, combined with measures of vegetation pigment content and the Photosynthetic Reflectance Index (PRI) derived from the spectral reflectance are compared with CO2 fluxes over diurnal periods for multiple days. The relationships among the different optical measurements indicate that they are providing different types of information on the vegetation and that combinations of these measurements provide improved retrievals of CO2 fluxes than any index alone

Metal-enhanced fluorescence/visual bimodal platform for multiplexed ultrasensitive detection of microRNA with reusable paper analytical devices.

PubMed

Liang, Linlin; Lan, Feifei; Yin, Xuemei; Ge, Shenguang; Yu, Jinghua; Yan, Mei

2017-09-15

Convenient biosensor for simultaneous multi-analyte detection was increasingly required in biological analysis. A novel flower-like silver (FLS)-enhanced fluorescence/visual bimodal platform for the ultrasensitive detection of multiple miRNAs was successfully constructed for the first time based on the principle of multi-channel microfluidic paper-based analytical devices (µPADs). Fluorophore-functionalized DNA 1 (DNA 1 -N-CDs) was combined with FLS, which was hybridized with quencher-carrying strand (DNA 2 -CeO 2 ) to form FLS-enhanced fluorescence biosensor. Upon the addition of the target miRNA, the fluorescent intensity of DNA 1 -N-CDs within the proximity of the FLS was strengthened. The disengaged DNA/CeO 2 complex could result in color change after joining H 2 O 2 , leading to real-time visual detection of miRNA firstly. If necessary, then the fluorescence method was applied for a accurate determination. In this strategy, the growth of FLS in µPADs not only reduced the background fluorescence but also provided an enrichment of "hot spots" for surface enhanced fluorescence detection of miRNAs. Results also showed versatility of the FLS in the enhancement of sensitivity and selectivity of the miRNA biosensor. Remarkably, this biosensor could detect as low as 0.03fM miRNA210 and 0.06fM miRNA21. Interestingly, the proposed biosensor also possessed good capability of recycling in three cycles upon change of the supplementation of DNA 2 -CeO 2 and visual substitutive device. This method opened new opportunities for further studies of miRNA related bioprocesses and will provide a new instrument for simultaneous detection of multiple low-level biomarkers. Copyright © 2017 Elsevier B.V. All rights reserved.

Multifocal Fluorescence Microscope for Fast Optical Recordings of Neuronal Action Potentials

PubMed Central

Shtrahman, Matthew; Aharoni, Daniel B.; Hardy, Nicholas F.; Buonomano, Dean V.; Arisaka, Katsushi; Otis, Thomas S.

2015-01-01

In recent years, optical sensors for tracking neural activity have been developed and offer great utility. However, developing microscopy techniques that have several kHz bandwidth necessary to reliably capture optically reported action potentials (APs) at multiple locations in parallel remains a significant challenge. To our knowledge, we describe a novel microscope optimized to measure spatially distributed optical signals with submillisecond and near diffraction-limit resolution. Our design uses a spatial light modulator to generate patterned illumination to simultaneously excite multiple user-defined targets. A galvanometer driven mirror in the emission path streaks the fluorescence emanating from each excitation point during the camera exposure, using unused camera pixels to capture time varying fluorescence at rates that are ∼1000 times faster than the camera’s native frame rate. We demonstrate that this approach is capable of recording Ca2+ transients resulting from APs in neurons labeled with the Ca2+ sensor Oregon Green Bapta-1 (OGB-1), and can localize the timing of these events with millisecond resolution. Furthermore, optically reported APs can be detected with the voltage sensitive dye DiO-DPA in multiple locations within a neuron with a signal/noise ratio up to ∼40, resolving delays in arrival time along dendrites. Thus, the microscope provides a powerful tool for photometric measurements of dynamics requiring submillisecond sampling at multiple locations. PMID:25650920

A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems

NASA Astrophysics Data System (ADS)

Narayanaswamy, Nagarjun; Kumar, Manoj; Das, Sadhan; Sharma, Rahul; Samanta, Pralok K.; Pati, Swapan K.; Dhar, Suman K.; Kundu, Tapas K.; Govindaraju, T.

2014-09-01

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology.

Fluorescence multispectral imaging-based diagnostic system for atherosclerosis.

PubMed

Ho, Cassandra Su Lyn; Horiuchi, Toshikatsu; Taniguchi, Hiroaki; Umetsu, Araya; Hagisawa, Kohsuke; Iwaya, Keiichi; Nakai, Kanji; Azmi, Amalina; Zulaziz, Natasha; Azhim, Azran; Shinomiya, Nariyoshi; Morimoto, Yuji

2016-08-20

Composition of atherosclerotic arterial walls is rich in lipids such as cholesterol, unlike normal arterial walls. In this study, we aimed to utilize this difference to diagnose atherosclerosis via multispectral fluorescence imaging, which allows for identification of fluorescence originating from the substance in the arterial wall. The inner surface of extracted arteries (rabbit abdominal aorta, human coronary artery) was illuminated by 405 nm excitation light and multispectral fluorescence images were obtained. Pathological examination of human coronary artery samples were carried out and thickness of arteries were calculated by measuring combined media and intima thickness. The fluorescence spectra in atherosclerotic sites were different from those in normal sites. Multiple regions of interest (ROI) were selected within each sample and a ratio between two fluorescence intensity differences (where each intensity difference is calculated between an identifier wavelength and a base wavelength) from each ROI was determined, allowing for discrimination of atherosclerotic sites. Fluorescence intensity and thickness of artery were found to be significantly correlated. These results indicate that multispectral fluorescence imaging provides qualitative and quantitative evaluations of atherosclerosis and is therefore a viable method of diagnosing the disease.

A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems

PubMed Central

Narayanaswamy, Nagarjun; Kumar, Manoj; Das, Sadhan; Sharma, Rahul; Samanta, Pralok K.; Pati, Swapan K.; Dhar, Suman K.; Kundu, Tapas K.; Govindaraju, T.

2014-01-01

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology. PMID:25252596

Multispectral fluorescence imaging techniques for nondestructive food safety inspection

NASA Astrophysics Data System (ADS)

Kim, Moon S.; Lefcourt, Alan M.; Chen, Yud-Ren

2004-03-01

The use of spectral sensing has gained acceptance as a rapid means for nondestructive inspection of postharvest food produce. Current technologies generally use color or a single wavelength camera technology. The applicability and sensitivity of these techniques can be expanded through the use of multiple wavelengths. Reflectance in the Vis/NIR is the prevalent spectral technique. Fluorescence, compared to reflectance, is regarded as a more sensitive technique due to its dynamic responses to subtle changes in biological entities. Our laboratory has been exploring fluorescence as a potential means for detection of quality and wholesomeness of food products. Applications of fluorescence sensing require an understanding of the spectral characteristics emanating from constituents and potential contaminants. A number of factors affecting fluorescence emission characteristics are discussed. Because of relatively low fluorescence quantum yield from biological samples, a system with a powerful pulse light source such as a laser coupled with a gated detection device is used to harvest fluorescence, in the presence of ambient light. Several fluorescence sensor platforms developed in our laboratory, including hyperspectral imaging, and laser-induced fluorescence (LIF) and steady-state fluorescence imaging systems with multispectral capabilities are presented. We demonstrate the potential uses of recently developed fluorescence imaging platforms in food safety inspection of apples contaminated with animal feces.

Clinical application of indocyanine green (ICG) fluorescent imaging of hepatoblastoma.

PubMed

Yamamichi, Taku; Oue, Takaharu; Yonekura, Takeo; Owari, Mitsugu; Nakahata, Kengo; Umeda, Satoshi; Nara, Keigo; Ueno, Takehisa; Uehara, Shuichiro; Usui, Noriaki

2015-05-01

Although the usefulness of intraoperative indocyanine green (ICG) fluorescent imaging for the resection of hepatocellular carcinoma has been reported, its usefulness for the resection of hepatoblastoma remains unclear. This study clarifies the feasibility of intraoperative ICG fluorescent imaging for the resection of hepatoblastoma. In three hepatoblastoma patients, a primary tumor, recurrent tumor, and lung metastatic lesions were intraoperatively examined using a near-infrared fluorescence imaging system after the preoperative administration of ICG. ICG fluorescent imaging was useful for the surgical navigation in hepatoblastoma patients. In the first case, the primary hepatoblastoma exhibited intense fluorescence during right hepatectomy, but no fluorescence was detected in the residual liver. In the second case, a recurrent tumor exhibited fluorescence between the residual liver and diaphragm. A complete resection of the residual liver, with a partial resection of the diaphragm, followed by liver transplantation was performed. In the third case with multiple lung metastases, each metastatic lesion showed positive fluorescence, and all were completely resected. These fluorescence-positive lesions were pathologically proven to be viable hepatoblastoma cells. Intraoperative ICG fluorescence imaging for patients with hepatoblastoma was feasible and useful for identifying small viable lesions and confirming that no remnant tumor remained after resection. Copyright © 2015 Elsevier Inc. All rights reserved.

Multiple genes for functional 6 fatty acyl desaturases (Fad) in Atlantic salmon (Salmo salar L.): gene and cDNA characterization, functional expression, tissue distribution and nutritional regulation.

PubMed

Monroig, Oscar; Zheng, Xiaozhong; Morais, Sofia; Leaver, Michael J; Taggart, John B; Tocher, Douglas R

2010-09-01

Fish are the primary source in the human food basket of the n-3 long-chain polyunsaturated fatty acids, eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), that are crucial to the health of higher vertebrates. Atlantic salmon are able to synthesize EPA and DHA from 18:3n-3 through reactions catalyzed by fatty acyl desaturases (Fad) and elongases of very long chain fatty acids. Previously, two cDNAs encoding functionally distinct Delta5 and Delta6 Fads were isolated, but screening of a genomic DNA library revealed the existence of more putative fad genes in the Atlantic salmon genome. In the present study, we show that there are at least four genes encoding putative Fad proteins in Atlantic salmon. Two genes, Delta6fad_a and Delta5fad, corresponded to the previously cloned Delta6 and Delta5 Fad cDNAs. Functional characterization by heterologous expression in yeast showed that the cDNAs for both the two further putative fad genes, Delta6fad_b and Delta6fad_c, had only Delta6 activity, converting 47 % and 12 % of 18:3n-3 to 18:4n-3, and 25 and 7 % of 18:2n-6 to 18:3n-6, for 6Fad_b and Delta6fad_c, respectively. Both 6fad_a and 6fad_b genes were highly expressed in intestine (pyloric caeca), liver and brain, with 6fad_b also highly expressed in gill, whereas 6fad_c transcript was found predominantly in brain, with lower expression levels in all other tissues. The expression levels of the 6fad_a gene in liver and the 6fad_b gene in intestine were significantly higher in fish fed diets containing vegetable oil compared to fish fed fish oil suggesting up-regulation in response to reduced dietary EPA and DHA. In contrast, no significant differences were found between transcript levels for 6fad_a in intestine, 6fad_b in liver, or 6fad_c in liver or intestine of fish fed vegetable oil compared to fish fed fish oil. The observed differences in tissue expression and nutritional regulation of the fad genes are discussed in relation to gene structures and fish physiology. 2010 Elsevier B.V. All rights reserved.

Skeletal accumulation of fluorescently tagged zoledronate is higher in animals with early stage chronic kidney disease.

PubMed

Swallow, E A; Aref, M W; Chen, N; Byiringiro, I; Hammond, M A; McCarthy, B P; Territo, P R; Kamocka, M M; Winfree, S; Dunn, K W; Moe, S M; Allen, M R

2018-06-11

This work examines the skeletal accumulation of fluorescently tagged zoledronate in an animal model of chronic kidney disease. The results show higher accumulation in 24-h post-dose animals with lower kidney function due to greater amounts of binding at individual surfaces. Chronic kidney disease (CKD) patients suffer from increased rates of skeletal-related mortality from changes driven by biochemical abnormalities. Bisphosphonates are commonly used in reducing fracture risk in a variety of diseases, yet their use is not recommended in advanced stages of CKD. This study aimed to characterize the accumulation of a single dose of fluorescently tagged zoledronate (FAM-ZOL) in the setting of reduced kidney function. At 25 weeks of age, FAM-ZOL was administered to normal and CKD rats. Twenty-four hours later, multiple bones were collected and assessed using bulk fluorescence imaging, two-photon imaging, and dynamic histomorphometry. CKD animals had significantly higher levels of FAM-ZOL accumulation in the proximal tibia, radius, and ulna, but not in lumbar vertebral body or mandible, based on multiple measurement modalities. Although a majority of trabecular bone surfaces were covered with FAM-ZOL in both normal and CKD animals, the latter had significantly higher levels of fluorescence per unit bone surface in the proximal tibia. These results provide new data regarding how reduced kidney function affects drug accumulation in rat bone.

Targeted detection of murine colonic dysplasia in vivo with flexible multispectral scanning fiber endoscopy

NASA Astrophysics Data System (ADS)

Joshi, Bishnu P.; Miller, Sharon J.; Lee, Cameron; Gustad, Adam; Seibel, Eric J.; Wang, Thomas D.

2012-02-01

We demonstrate a multi-spectral scanning fiber endoscope (SFE) that collects fluorescence images in vivo from three target peptides that bind specifically to murine colonic adenomas. This ultrathin endoscope was demonstrated in a genetically engineered mouse model of spontaneous colorectal adenomas based on somatic Apc (adenomatous polyposis coli) gene inactivation. The SFE delivers excitation at 440, 532, 635 nm with <2 mW per channel. The target 7-mer peptides were conjugated to visible organic dyes, including 7-Diethylaminocoumarin-3-carboxylic acid (DEAC) (λex=432 nm, λem=472 nm), 5-Carboxytetramethylrhodamine (5-TAMRA) (λex=535 nm, λem=568 nm), and CF-633 (λex=633 nm, λem=650 nm). Target peptides were first validated using techniques of pfu counting, flow cytometry and previously established methods of fluorescence endoscopy. Peptides were applied individually or in combination and detected with fluorescence imaging. The ability to image multiple channels of fluorescence concurrently was successful for all three channels in vitro, while two channels were resolved simultaneously in vivo. Selective binding of the peptide was evident to adenomas and not to adjacent normal-appearing mucosa. Multispectral wide-field fluorescence detection using the SFE is achievable, and this technology has potential to advance early cancer detection and image-guided therapy in human patients by simultaneously visualizing multiple over expressed molecular targets unique to dysplasia.

Caging and Photoactivation in Single-Molecule Förster Resonance Energy Transfer Experiments

PubMed Central

2017-01-01

Caged organic fluorophores are established tools for localization-based super-resolution imaging. Their use relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available caged dyes with ON switching of the fluorescent signal by ultraviolet (UV) light. Here, we establish caging of cyanine fluorophores and caged rhodamine dyes, i.e., chemical deactivation of fluorescence, for single-molecule Förster resonance energy transfer (smFRET) experiments with freely diffusing molecules. They allow temporal separation and sorting of multiple intramolecular donor–acceptor pairs during solution-based smFRET. We use this “caged FRET” methodology for the study of complex biochemical species such as multisubunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented. These reveal that chemical caging and UV reactivation allow temporal uncoupling of convoluted fluorescence signals from, e.g., multiple spectrally similar donor or acceptor molecules on nucleic acids. We also use caging without UV reactivation to remove unwanted overlabeled species in experiments with the homotrimeric membrane transporter BetP. We finally outline further possible applications of the caged FRET methodology, such as the study of weak biochemical interactions, which are otherwise impossible with diffusion-based smFRET techniques because of the required low concentrations of fluorescently labeled biomolecules. PMID:28362086

A FRET-facilitated photoswitching using an orange fluorescent protein with the fast photoconversion kinetics.

PubMed

Subach, Oksana M; Entenberg, David; Condeelis, John S; Verkhusha, Vladislav V

2012-09-12

Fluorescent proteins photoswitchable with noncytotoxic light irradiation and spectrally distinct from multiple available photoconvertible green-to-red probes are in high demand. We have developed a monomeric fluorescent protein, called PSmOrange2, which is photoswitchable with blue light from an orange (ex./em. at 546 nm/561 nm) to a far-red (ex./em. at 619 nm/651 nm) form. Compared to another orange-to-far-red photoconvertable variant, PSmOrange2 has blue-shifted photoswitching action spectrum, 9-fold higher photoconversion contrast, and up to 10-fold faster photoswitching kinetics. This results in the 4-fold more PSmOrange2 molecules being photoconverted in mammalian cells. Compared to common orange fluorescent proteins, such as mOrange, the orange form of PSmOrange has substantially higher photostability allowing its use in multicolor imaging applications to track dynamics of multiple populations of intracellular objects. The PSmOrange2 photochemical properties allow its efficient photoswitching with common two-photon lasers and, moreover, via Förster resonance energy transfer (FRET) from green fluorescent donors. We have termed the latter effect a FRET-facilitated photoswitching and demonstrated it using several sets of interacting proteins. The enhanced photoswitching properties of PSmOrange2 make it a superior photoconvertable protein tag for flow cytometry, conventional microscopy, and two-photon imaging of live cells.

A simple but highly efficient multi-formyl phenol-amine system for fluorescence detection of peroxide explosive vapour.

PubMed

Xu, Wei; Fu, Yanyan; Gao, Yixun; Yao, Junjun; Fan, Tianchi; Zhu, Defeng; He, Qingguo; Cao, Huimin; Cheng, Jiangong

2015-07-11

A simple, highly stable, sensitive and selective fluorescent system for peroxide explosives was developed via an aromatic aldehyde oxidation reaction. The high efficiency arises from its higher HOMO level and multiple H-bonding. The sensitivity is obtained to be 0.1 ppt for H2O2 and 0.2 ppb for TATP.

Endoscopic fluorescent diagnostics and PDT of early malignancies of lung and esophagus

NASA Astrophysics Data System (ADS)

Sokolov, Victor V.; Chissov, Valery I.; Trakhtenberg, A. K.; Mamontov, A. S.; Frank, George A.; Filonenko, E. V.; Telegina, L. V.; Gladunov, V. K.; Belous, T. A.; Aristarkhova, E. I.; Zharkova, Natalia N.; Smirnov, V. V.; Kozlov, Dmitrij N.

1996-01-01

In this paper the results of fluorescence diagnostics and photodynamic therapy of early stage malignancies of lung (17 patients) and esophagus (8 patients) are presented. 13 patients had multiple primary tumors. As photosensitizers the new drugs Photoheme and Photosense were used. Complete remission was obtained in 92%. The patients are followed up without relapses to 2,5 years.

Supernumerary ring chromosome 17 identified by fluorescent in situ hybridization

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

Fagan, K.; Edwards, M.

We present a patient with multiple anomalies and severe developmental delay. A small supernumerary ring chromosome was found in 40% of her lymphocyte cells at birth. The origin of the marker chromosome could not be determined by GTG banding, but fluorescent in situ hybridization (FISH) later identified the marker as deriving from chromosome 17. 20 refs., 2 figs., 1 tab.

Multiple tag labeling method for DNA sequencing

DOEpatents

Mathies, Richard A.; Huang, Xiaohua C.; Quesada, Mark A.

1995-01-01

A DNA sequencing method described which uses single lane or channel electrophoresis. Sequencing fragments are separated in said lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radio-isotope labels.

Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae.

PubMed

Ishii, Jun; Kondo, Takashi; Makino, Harumi; Ogura, Akira; Matsuda, Fumio; Kondo, Akihiko

2014-05-01

Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Method of determining the optimal dilution ratio for fluorescence fingerprint of food constituents.

PubMed

Trivittayasil, Vipavee; Tsuta, Mizuki; Kokawa, Mito; Yoshimura, Masatoshi; Sugiyama, Junichi; Fujita, Kaori; Shibata, Mario

2015-01-01

Quantitative determination by fluorescence spectroscopy is possible because of the linear relationship between the intensity of emitted fluorescence and the fluorophore concentration. However, concentration quenching may cause the relationship to become nonlinear, and thus, the optimal dilution ratio has to be determined. In the case of fluorescence fingerprint (FF) measurement, fluorescence is measured under multiple wavelength conditions and a method of determining the optimal dilution ratio for multivariate data such as FFs has not been reported. In this study, the FFs of mixed solutions of tryptophan and epicatechin of different concentrations and composition ratios were measured. Principal component analysis was applied, and the resulting loading plots were found to contain useful information about each constituent. The optimal concentration ranges could be determined by identifying the linear region of the PC score plotted against total concentration.