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The success of reef-building corals for >200 million years has been dependent on the mutualistic interaction between the coral host and its photosynthetic endosymbiont dinoflagellates (family Symbiodiniaceae) that supply the coral host with nutrients and energy for growth and calcification. While multiple light scattering in coral tissue and skeleton significantly enhance the light microenvironment for Symbiodiniaceae, the mechanisms of light propagation in tissue and skeleton remain largely unknown due to a lack of technologies to measure the intrinsic optical properties of both compartments in live corals. Here we introduce ISOCT (inverse spectroscopic optical coherence tomography), a non-invasive approach to measure optical properties and three-dimensional morphology of living corals at micron- and nano-length scales, respectively, which are involved in the control of light propagation. ISOCT enables measurements of optical properties in the visible range and thus allows for characterization of the density of light harvesting pigments in coral. We used ISOCT to characterize the optical scattering coefficient (µs) of the coral skeleton and chlorophyll a concentration of live coral tissue. ISOCT further characterized the overall micro- and nano-morphology of live tissue by measuring differences in the sub-micron spatial mass density distribution (D) that vary throughout the tissue and skeleton and give rise to light scattering, and this enabled estimates of the spatial directionality of light scattering, i.e., the anisotropy coefficient, g. Thus, ISOCT enables imaging of coral nanoscale structures and allows for quantifying light scattering and pigment absorption in live corals. ISOCT could thus be developed into an important tool for rapid, non-invasive monitoring of coral health, growth and photophysiology with unprecedented spatial resolution.
Assuntos
Antozoários/fisiologia , Monitoramento Ambiental/métodos , Tomografia de Coerência Óptica/métodos , Absorção de Radiação , Animais , Antozoários/química , Recifes de Corais , Difusão Dinâmica da Luz/métodosRESUMO
We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.
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Análise Mutacional de DNA/métodos , Testes de Mutagenicidade/métodos , Mutação , Sequência de Bases , Linhagem Celular , Corantes Fluorescentes , Genes p53 , Antígenos HLA-A/genética , Humanos , Hipoxantina Fosforribosiltransferase/genética , Perda de Heterozigosidade , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , Repetições Minissatélites , Dados de Sequência Molecular , Mutação Puntual , Proteínas Proto-Oncogênicas c-bcl-2/genética , Mapeamento por Restrição , Sensibilidade e Especificidade , Translocação GenéticaRESUMO
We have determined both the spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutational spectra in the HPRT gene of human cells (MT1) defective in the mismatch repair gene hMSH6 (GTBP). Eight of nine exons and nine of sixteen intronic flanking sequences were scanned, encompassing >900 bp of the HPRT gene. Mutant hotspots were detected and separated by differences in their melting temperatures using constant denaturant capillary electrophoresis (CDCE) or denaturing gradient gel electrophoresis (DGGE).A key finding of this work is that a high proportion of all HPRT inactivating mutations is represented by a small number of hotspots distributed over the exons and mRNA splice sites. Thirteen spontaneous hotspots and sixteen MNNG-induced hotspots accounted for 55% and 48% of all 6TG(R) point mutations, respectively. MNNG-induced hotspots were predominantly G:C-->A:T transitions. The spontaneous spectrum of cells deficient in hMSH6 contained transversions (A:T-->T:A, G:C-->T:A, A:T-->C:G), transitions (A:T-->G:C), a plus-one insertion, and a minus-one deletion. Curiously, G:C-->A:T transitions, which dominate human germinal and somatic point mutations were absent from the spontaneous hMSH6 spectra.
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Pareamento Incorreto de Bases , Reparo do DNA/genética , Hipoxantina Fosforribosiltransferase/genética , Metilnitronitrosoguanidina/toxicidade , Mutação , Alquilantes/toxicidade , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Reação em Cadeia da PolimeraseRESUMO
Human tumor samples were screened for point mutations by adapting a mobility-shift assay to automated DNA sizing. This screen identifies the type of point mutation and relative amount of mutated DNA sequences present in a sample. Test samples having known hypoxanthine-guanine phosphoribosyl transferase (hprt)/exon-3 sequence mutations were characterized by: (i) PCR amplification, (ii) fluorescent dye-primer extension with 36-atom linker derived deoxycytosine or deoxyuridine triphosphate and the remaining three natural nucleotides and (iii) sizing of the resulting fluorescently labeled modified strands, using an automated DNA sequencer. Routinely, a range of sizes is observed among the sequence variants of a single DNA target sequence. This is because nucleotide analogs are incorporated into DNA strands in a sequence-dependent manner, resulting in composition-dependent electrophoretic mobility. Thus, point mutations are identified as shifts in mobility between the fluorescently labeled modified strands of the control and test samples. The twenty different hprt/exon-3 single-base substitution mutations tested were easily identified, even at fourfold dilution with control DNA.
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Análise Mutacional de DNA/métodos , DNA de Neoplasias/genética , Testes Genéticos/métodos , Mutação Puntual , Animais , Sequência de Bases , Biópsia , Células CHO , Cor , Cricetinae , Primers do DNA , DNA de Neoplasias/análise , Éxons , Corantes Fluorescentes , Genes ras/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Dados de Sequência Molecular , Polimorfismo Conformacional de Fita Simples , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
Constant denaturant capillary electrophoresis (CDCE) permits high-resolution separation of single-base variations occurring in an approximately 100 bp isomelting DNA sequence based on their differential melting temperatures. By coupling CDCE for highly efficient enrichment of mutants with high-fidelity polymerase chain reaction (hifi PCR), we have developed an analytical approach to detecting point mutations at frequencies equal to or greater than 10(-6) in human genomic DNA. In this article, we present several applications of this approach in human genetic studies. We have measured the point mutational spectra of a 100 bp mitochondrial DNA sequence in human tissues and cultured cells. The observations have led to the conclusion that the primary causes of mutation in human mitochondrial DNA are spontaneous in origin. In the course of studying the mitochondrial somatic mutations, we have also identified several nuclear pseudogenes homologous to the analyzed mitochondrial DNA fragment. Recently, through developments of the means to isolate the desired target sequences from bulk genomic DNA and to increase the loading capacity of CDCE, we have extended the CDCE/hifi PCR approach to study a chemically induced mutational spectrum in a single-copy nuclear sequence. Future applications of the CDCE/hifi PCR approach to human genetic analysis include studies of somatic mitochondrial mutations with respect to aging, measurement of mutational spectra of nuclear genes in healthy human tissues and population screening for disease-associated single nucleotide polymorphisms (SNPs) in large pooled samples.
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DNA/análise , Eletroforese Capilar/métodos , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Humanos , Dados de Sequência MolecularRESUMO
We have observed a reproducible mitochondrial mutational spectrum in the MT1 human lymphoblastoid line treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The MNNG spectrum was distinct from the spontaneous mutational spectrum. However, our ability to observe MNNG-induced mitochondrial mutations above the high level of accumulated spontaneous mutations was dependent on the MT1 phenotype. MT1 cells are markedly resistant to the cytotoxicity but not the mutagenicity of MNNG, presumably as a result of inactivation of both copies of the hMSH6 (GTBP) mismatch repair gene. Thus, we were able to use conditions of treatment that yielded induced mitochondrial mutant fractions beyond the practical limits for human cell experiments in mismatch-proficient human cell lines. In contradistinction, when MT1 cells were treated repeatedly with maximum tolerated concentrations of (+/-) anti-benzo(a)pyrene diol-epoxide, no induced mitochondrial mutations above the spontaneous background were observed. A single dose of 4 microM MNNG (survival, 0.85) induced a mutant fraction of 8 x 10(-3) in the nuclear hypoxanthine-guanine phosphoribosyltrans. ferase gene, and a clear and reproducible pattern of seven MNNG-induced hotspot mutations was observed within the mitochondrial DNA target sequence studied (mitochondrial bp 10,030-10,130). All of the MNNG-induced hotspot mutations were G:C to A:T transitions present at frequencies between 6 x 10(-5) and 30 x 10(-5). Additional experiments supported the conclusion that MNNG-induced hotspot mutations observed were generated in living cells as a result of MNNG treatment and not from mismatch intermediates or DNA adducts converted into mutations during the PCR process.