Auswahl der wissenschaftlichen Literatur zum Thema „RNA-DNA FISH“

Abstract Fluorescence in situ hybridization (FISH) can be used for the intracellular detection of DNA or RNA molecules. The detection of DNA sequences by DNA FISH requires the denaturation of the DNA double helix to allow the hybridization of the fluorescent probe with DNA in a single stranded form. These hybridization conditions require high temperature and low pH that can damage RNA, and therefore RNA is not typically detectable by DNA FISH. In contrast, RNA FISH does not require a denaturation step since RNA is single stranded, and therefore DNA molecules are not detectable by RNA FISH. Hence, DNA FISH and RNA FISH are mutually exclusive. In this study, we show that plasmid DNA transiently transfected into cells is readily detectable in the cytoplasm by RNA FISH without need for denaturation, shortly after transfection and for several hours. The plasmids, however, are usually not detectable in the nucleus except when the plasmids are efficiently directed into the nucleus, which may imply a more open packaging state for DNA after transfection. This detection of plasmid DNA in the cytoplasm has implications for RNA FISH experiments and opens a window to study conditions when DNA is present in the cytoplasm.

The concentrations of RNA, DNA, and protein in white muscle from 240 uniquely tagged rainbow trout (Oncorhynchus mykiss) held at three temperatures (5, 8 (control), and 11 °C) were measured. Both RNA and RNA/DNA ratios were better predictors of recent length- and weight-specific growth rates than they were of absolute fish size. Furthermore, RNA concentrations were better predictors of growth than RNA/DNA ratios. The strength of the regression between either RNA/DNA ratio or RNA and growth rate did not differ consistently among temperatures. Fish reared at warmer temperatures had lower concentrations of RNA for both a given growth rate and a given DNA concentration compared with cold-reared trout. Warm-reared fish also had lower concentrations of DNA and higher protein/DNA ratios than cold-reared trout when fish size was standardized. The concomitant decrease in both RNA and DNA concentrations resulted in marginally lower RNA/DNA ratios in warm-reared fish.

An experiment was conducted at Corona of Agriculture (COA)”, Gunjanagar-3, Chitwan, Nepal to complete a project granted by University Grant Commission (UGC), Second Higher Education Project (SHEP) in 2013. Four (average 380 m2) ponds (W, X, Y and Z) with four different diets as D1 (20%), D2 (30%), D3 (40%) and D4 (50%) protein contents were used to conduct the experiment properly to determine the varied proportion of dietary protein on the growth performance of major carp rohu Labeo Rohita (H) in relation with RNA: DNA ratio. After 12th weeks of culture, average length, average weight and specific growth rate of fish were found significantly (P<0.05) higher in the carp with D3 and D4 diet fed fish. Similar results were observed in average total protein, albumin and globulin contents. RNA content increased rapidly with age. The average RNA content increased highest in D2 and D3 diet fed fish while, DNA content were highest in D3 diet fed fish. RNA: DNA ratio was recorded highest in D2 and D3 diet fed fish. RNA: DNA ratio, an indicator of protein synthesis and have been used to accurately estimate the growth rate and feeding condition of fish hence, as the dose of protein increased RNA and DNA contents also increased with age of carp cultured during experiment. Thus, it was clear from this study that the incorporation of protein in diet enhances the growth of fish regardless of species weight groups and the doses, as the average weight of fish was significantly lower in control diet fed fish as compared to the treated one.

Fusion transcripts are used as biomarkers in companion diagnoses. Although more than 15,000 fusion RNAs have been identified from diverse cancer types, few common features have been reported. Here, we compared 16,410 fusion transcripts detected in cancer (from a published cohort of 9,966 tumor samples of 33 cancer types) with genome-wide RNA–DNA interactions mapped in two normal, noncancerous cell types [using iMARGI, an enhanced version of the mapping of RNA–genome interactions (MARGI) assay]. Among the top 10 most significant RNA–DNA interactions in normal cells, 5 colocalized with the gene pairs that formed fusion RNAs in cancer. Furthermore, throughout the genome, the frequency of a gene pair to exhibit RNA–DNA interactions is positively correlated with the probability of this gene pair to present documented fusion transcripts in cancer. To test whether RNA–DNA interactions in normal cells are predictive of fusion RNAs, we analyzed these in a validation cohort of 96 lung cancer samples using RNA sequencing (RNA-seq). Thirty-seven of 42 fusion transcripts in the validation cohort were found to exhibit RNA–DNA interactions in normal cells. Finally, by combining RNA-seq, single-molecule RNA FISH, and DNA FISH, we detected a cancer sample with EML4-ALK fusion RNA without forming the EML4-ALK fusion gene. Collectively, these data suggest an RNA-poise model, where spatial proximity of RNA and DNA could poise for the creation of fusion transcripts.

l-DNA tagged FISH (LT-FISH), including two-step hybridization processes with a l–d chimera oligonucleotide and a fluorescence-labeled PCR product tethering a l-DNA tag, has realized sensitive RNA detection in fixed cells.

Abstract Spatially resolved visualization of RNA processing and structures is important for better studying single-cell RNA function and landscape. However, currently available RNA imaging methods are limited to sequence analysis, and not capable of identifying RNA processing events and structures. Here, we developed click-encoded rolling FISH (ClickerFISH) for visualizing RNA polyadenylation and structures in single cells. In ClickerFISH, RNA 3′ polyadenylation tails, single-stranded and duplex regions are chemically labeled with different clickable DNA barcodes. These barcodes then initiate DNA rolling amplification, generating repetitive templates for FISH to image their subcellular distributions. Combined with single-molecule FISH, the proposed strategy can also obtain quantitative information of RNA of interest. Finally, we found that RNA poly(A) tailing and higher-order structures are spatially organized in a cell type-specific style with cell-to-cell heterogeneity. We also explored their spatiotemporal patterns during cell cycle stages, and revealed the highly dynamic organization especially in S phase. This method will help clarify the spatiotemporal architecture of RNA polyadenylation and structures.

We cloned and sequenced a 2.35-kilobase EcoRI fragment of genomic DNA from a local freshwater fish (Perca fluviatilis) that strongly hybridized to probes derived from the murine influenza virus resistance gene Mx. The cloned fish DNA contained blocks of sequences related to Mx gene exons 3 to 8, which appeared to represent exons of a bona fide fish gene because they were separated by intron sequences flanked by consensus splice acceptor and donor sites. Injection of double-stranded RNA into the peritoneal cavity of trouts resulted in 5- to 10-fold elevated levels of two liver mRNAs of about 2.0 to 2.5 kilobases in length that hybridized to the cloned genomic DNA. High sequence similarity between this fish gene and the murine Mx gene, identical exon lengths, and similar inducibilities in vivo by double-stranded RNA indicate that we isolated a fragment of a fish Mx gene.

The nucleic acid content in different tissues such as brain, liver, kidney & testis of both control and copper sulphate (CuSO4) exposed freshwater fish N. notopterus has been studied. The following observation is made in both control and copper sulphate (CuSO4) exposed fish. The testis contain large amount of DNA in comparison to other tissues. The degree of DNA content in control and copper sulphate (CuSO4) exposed fish testis >liver>brain>kidney. The RNA content also exhibited similar to that of DNA, having higher amount in the testis. The degree of RNA content in control and copper sulphate (Cuso4) exposed fish testis >liver>brain>kidney. The nucleic acid content of tissues get reduced under copper sulphate (CuSO4) exposed in the male freshwater fish N. notopterus indicating copper sulphate as a pollutant effect the nucleic acid content in the tissue.

We cloned and sequenced a 2.35-kilobase EcoRI fragment of genomic DNA from a local freshwater fish (Perca fluviatilis) that strongly hybridized to probes derived from the murine influenza virus resistance gene Mx. The cloned fish DNA contained blocks of sequences related to Mx gene exons 3 to 8, which appeared to represent exons of a bona fide fish gene because they were separated by intron sequences flanked by consensus splice acceptor and donor sites. Injection of double-stranded RNA into the peritoneal cavity of trouts resulted in 5- to 10-fold elevated levels of two liver mRNAs of about 2.0 to 2.5 kilobases in length that hybridized to the cloned genomic DNA. High sequence similarity between this fish gene and the murine Mx gene, identical exon lengths, and similar inducibilities in vivo by double-stranded RNA indicate that we isolated a fragment of a fish Mx gene.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The marine fish farming and culture of snook, Centropomus undecimalis, have good prospects for commercial development in Brazil. In this regard, there is a need to expand studies on the evaluation of growth on snook, which can be realized by morphological or biochemical methods. The nutritional status and growth of fish can be influenced by several factors and their interactions, including genetic and environmental conditions, such as diet, different temperatures and salinities. Morphological analysis allows an investigation of the biometric data obtained from the body dimensions which are analyzed based on a mathematical relationship through correlation and linear regression equation. Regarding the biochemical methods, the most used are the quantification of DNA, RNA, proteins and determination of the RNA/DNA, protein/DNA ratios. Once the cell metabolism is usually associated with the nutritional status of the organism higher amounts of these components indicate greater biochemical activity of cells and protein synthesis. The results from this study will provide useful information related to the biology and cultivation of snook, promoting the application of biometric analysis and promoting a jumpstart for the application of biochemical analysis, cellular metabolism involved in the culture snook
A piscicultura marinha e o cultivo do robalo-flecha possuem boas perspectivas para o desenvolvimento comercial no Brasil. Neste sentido, existe a necessidade da ampliação de estudos relacionados com a avaliação do crescimento em robalos, que pode ser realizado por métodos morfológicos ou bioquímicos. A condição nutricional e o crescimento dos peixes podem ser influenciados por vários fatores e suas interações, incluindo a genética e as condições ambientais, como a alimentação, diferentes temperaturas e salinidades. A análise morfológica permite uma investigação das características biométricas, obtidas por meio das dimensões corporais, com base na sua relação matemática pela correlação e equação de regressão linear. Em relação aos métodos bioquímicos, os mais utilizados são a quantificação do DNA, RNA, proteínas e determinação das razões RNA/DNA, proteína/DNA. Uma vez que o metabolismo celular normalmente está relacionado com a situação nutricional do organismo, maiores quantidades desses componentes indicam maior atividade bioquímica das células e síntese proteica. Os resultados deste estudo permitem incrementar informações relacionadas à biologia e ao cultivo dos robalos aperfeiçoando a aplicação das análises biométricas e promovendo um salto inicial para a aplicação de análises bioquímicas, envolvidas com o metabolismo celular, no cultivo do robalo-flecha

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Os cromossomos sexuais se originam independentemente de um par de homólogos autossômicos e em várias linhagens apresentam características comuns, tais como acúmulo de vários tipos de DNA repetitivo, restrição da recombinação e perda ou ganho de genes devido á diferenciação morfológica e genética entre os cromossomos sexuais X e Y ou Z e W. Estas características representam um exemplo fascinante de convergência evolutiva. Em Orthoptera, o sistema cromossômico sexual comumente encontrado na maioria das espécies estudadas é do tipo X0♂/XX♀. Entretanto, sistemas cromossômicos sexuais derivados dos tipos neo-XY♂/XX♀ e neo- X1X2Y♂/X1X1X2X2♀ são também observados, surgindo repetidamente por fusões cêntricas e em tandem, inversões e dissociações envolvendo cromossomos sexuais ancestrais e autossomos. O presente trabalho teve três objetivos. Primeiro, entender o possível papel dos DNAs repetitivos na estrutura/diversificação dos cromossomos sexuais simples e derivados, a partir do isolamento e mapeamento físico de sequências, tais como, famílias multigênicas, DNA satélite (DNAsat) e microssatélites, nas espécies Gryllus assimilis, Cycloptiloides americanus e Eneoptera surinamensis. Segundo, testar e comparar transcrição diferencial de DNAsat entre diferentes tecidos, sexos e espécies a partir de transcriptomas de Gryllus assimilis, G. bimaculatus, G. firmus e G. rubens, com o objetivo de entender os possíveis papéis funcionais destas sequências na regulação gênica, modulação da cromatina e como componentes funcionais de importantes estruturas como telômeros, centrômeros e cromossomos sexuais. Terceiro, a partir de transcriptomas de espécies de grilos (Gryllus assimilis, G. bimaculatus e G. firmus) prospectar genes codificadores de proteínas relacionados com a determinação sexual, envolvidos com o fitness reprodutivo e genes enviesados do sexo, responsáveis pelas diferenças fenotípicas entre machos e fêmeas, e tentar elucidar de uma maneira comparativa os fatores evolutivos atuando nestes loci. Origem de novo de cromossomos sexuais mediante rearranjos cromossômicos, assim como acúmulo de DNA repetitivo que levaram a diferenciação entre cromossomos sexuais são relatados em C. americanus (X1X20) e E. surianmensis (neo-X1X2Y). Estas características observadas em grilos representam outro caso notável de convergência evolutiva devido os cromossomos sexuais não relacionados compartilharem muitas propriedades entre táxons distantes. Acúmulo surpreendente de loci de DNAsat foi encontrado no neo-Y altamente diferenciado de E. surinamensis, incluindo 39 DNAsat representados em excesso neste cromossomo, que é a maior diversidade de DNAsat até agora relatada para cromossomos sexuais. Foi documentado que, particularmente os DNAsat, contribuíram grandemente para o aumento de tamanho genômico entre G. assimilis e E. surinamensis. Um achado interessante foi a identificação de DNAsat conservados entre espécies de grilos (Gryllus assimilis, G. bimaculatus e G. firmus), mas transcritos diferencialmente. Os dados relativos à presença de DNAsat no genoma de G. assimilis foram discutidos em um contexto evolutivo, com dados transcricionais permitindo comparações entre os sexos e entre os tecidos quando possível. Foram discutidas hipóteses para a conservação e transcrição de DNAsat em Gryllus, que podem resultar do seu papel na diferenciação sexual no nível da cromatina, na formação da heterocromatina e na função centromérica. Outra descoberta foi a identificação de genes determinantes do sexo e outros genes relacionados ao fitness reprodutivo, como a biossíntese de hormônios de insetos e ritmo circadiano entre espécies de Gryllus. Os efetores e os alvos downstream das vias de determinação do sexo foram previamente identificados em outros insetos, mas nunca em Orthoptera. Usando G. assimilis como modelo para estudar genes enviesados do sexo foi possível identificar um conjunto de genes altamente expressos que podem explicar diferenças fenotípicas entre os sexos. Estimou-se que os genes codificadores de proteínas relacionadas com a diferenciação sexual e com o fitness reprodutivo evoluem mais rapidamente do que os genes não reprodutivos (genes housekeeping) como resultado de uma forte seleção positiva nos primeiros. Além disso, foi encontrado que as espécies estudadas apresentam níveis excepcionalmente elevados de duplicações gênicas. As descobertas sugerem que as duplicações gênicas podem desempenhar um papel na expressão de genes enviesados do sexo no grilo de campo G. assimilis, uma espécie que no futuro provavelmente irá fornecer informações sobre genômica funcional e epigenética da determinação do sexo.
Sex chromosomes have arisen independently from an ordinary autosomal pair and in several lineages they present common characteristics, such as accumulation of distinct classes of repetitive DNAs, restriction of the recombination and loss or gain of genes due to the morphological and genetic differentiation between the sexual chromosomes X and Y or Z and W. These characteristics represent a fascinating example of evolutionary convergence. In Orthoptera, the X0♂/XX♀ sex-determining system is considered modal but eventually, diverse sex chromosome systems evolved several times, such as neo-XY♂/XX♀, X1X20♂/X1X1X2X2♀ and even neo- X1X2Y♂/X1X1X2X2♀. It was found that particularly centric fusions (i.e., Robertsonian translocations) and tandem fusions with autosomes, dissociations and inversions contributed to the formation of neo-sex chromosomes in Orthoptera. The present work had three objectives. First, get insights of the role of repetitive DNAs in the structure/diversification of simple and derivative sex-chromosomes by isolation and physical mapping of repetitive DNA sequences, such as multigene families, satellite DNA (satDNA) and microsatellites using Gryllus assimilis, Cycloptiloides americanus e Eneoptera surinamensis, as models. Second, looking at differential satDNA transcription between different tissues, sexes, and species from transcriptomes of Gryllus assimilis, G. bimaculatus, G. firmus and G. rubens, I tried to understand the possible functional roles of these sequences in gene regulation, chromatin modulation and as functional components of important structures such as telomeres, centromeres and sex chromosomes. Third, using transcriptomes from cricket species (Gryllus assimilis, G. bimaculatus and G. firmus), I searched for genes encoding proteins related to sexual determination, reproductive fitness and sex-biased genes which are responsible for the phenotypic differences between males and females. I also tried to elucidate in a comparative way the evolutionary factors acting at these loci. De novo origin of sex chromosomes by chromosomal rearrangements, as well as repetitive DNA accumulation that led to the differentiation between sex chromosomes are reported for C. americanus (X1X20) e E. surianmensis (neo-X1X2Y). These features observed in crickets represent another remarkable case of evolutionary convergence because unrelated sex chromosomes share many common properties among distant taxa. 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. It has been documented that, particularly the satDNA, contributed greatly to the increase in genomic size between G. assimilis and E. surinamensis. An interesting finding was the identification of satDNA conserved among species of crickets (Gryllus assimilis, G. bimaculatus and G. firmus), but differentially transcribed. The data regarding satDNA presence in G. assimilis genome was discussed in an evolutionary context, with transcriptional data enabling comparisons between sexes and across tissues when possible. I discussed hypotheses for the conservation and transcription of satDNAs in Gryllus, which might result from their role in sexual differentiation at the chromatin level, heterochromatin formation, and centromeric function. Another finding was the identification of sex-determining genes and other genes related to reproductive fitness, such as biosynthesis of insect hormones and circadian rhythm among Gryllus species. The effectors as well as downstream targets of sex-determination pathways have been previously identified in other insects but never in Orthoptera. Using G. assimilis to study sex-biased genes I identified a set of highly expressed genes that might account for phenotypic differences between sexes. Furthermore, I estimated that proteinencoding reproductive genes evolve faster than non-reproductive genes as result of strong positive selection at those loci. It was documented that the species studied harbor exceptionally high levels of gene duplications. The findings suggest that gene duplications may play a role in sex-biased genes expression in the field cricket G. assimilis, a species likely to yield insights into the functional genomics and epigenetics of sex determination.
FAPESP: 2014/02038-8

The chromosomal translocation of t(14;18)(14q32;18q21) is a characteristic aberration of follicular lymphoma and Diffuse Large B cells lymphoma. By PCR, it was proved that the rearrangement of chromosomes 14 and 18 leads to an overexpression of BCL2, an anti-apoptotic protein, which is one of the factors responsible for the maturation of the diseases. The translocation involves the promoter region of IGH gene and the transcriptional unit of BCL2 gene. Previous studies carried out in Dr Tosi’s lab showed a looping out of the BCL2 gene from its chromosome territory in 15% of the nuclei analysed. This looping out could be possibly responsible for the transcriptional activity of the gene. A further relevant finding concerns the spatial distribution of the genes involved in the translocation in the interphase nuclei. In the Pfeiffer cell line, harbouring the t(14;18) rearrangement, the translocated BCL2 gene was positioned in the cell nuclei according to a bimodal distribution. One could speculate that the distribution in the periphery and in the centre of the nuclei could divide the Pfeiffer cell line in two different subpopulations, consequently from the transcriptional activity. These preliminary data set the ground for more experimental work to test whether genes associated with the nuclear interior were transcriptionally active as opposed to the genes positioned towards the nuclear periphery, transcriptionally inactive. The work here presented focuses on this investigation using RNA-DNA FISH (Fluorescence in situ hybridization). My work enabled the detection of IGH, BCL2 and t(14;18) genes along with their transcripts inside of the nuclei of Pfeiffer cell line. Contrary to what had been hinted by previous work, my results showed multiple nuclear positions of transcriptionally active IGH/BCL2 translocation. The result will need to be further supported by software analysis in order to define its specific nuclear position and to ensure the perfect localization of the genes inside each nucleus.

Le génome eucaryote est organisé dans l’espace et le long de sa séquence. Du chromosome aux gènes, son organisation 3D est étroitement liée à son activité transcriptionnelle. A une échelle inférieure à la mégabase, le génome est organisé physiquement en domaines d’auto-interaction (TAD, pour topologically associating domains), que l’on pense influencer l’action de séquences régulatrices des gènes, appelées ‘amplificateurs’. Les TAD sont parfois vus comme des ‘unités de régulation’ permettant de coréguler plusieurs gènes en les exposant aux mêmes amplificateurs. L’expression de gènes d’un même TAD est en effet souvent corrélée entre tissus et types cellulaires. On sait d’autre part que l’expression de gènes fonctionnellement liés est aussi souvent corrélée. Cependant, le rôle que joue l’organisation 3D d’un locus génomique dans la corégulation de gènes fonctionnellement liés reste mal compris. Par des techniques d’imagerie de molécules uniques, j’ai observé, dans des cellules individuelles, la position et la transcription de trois gènes adjacents et fonctionnellement liés, partageant –ou non– les mêmes amplificateurs. J’ai utilisé comme système modèle la réponse à l’œstrogène dans des cellules MCF7. En combinant les techniques de FISH ARN et ADN, j’ai mesuré le couplage transcriptionnel de paires de gènes en cis, mettant en évidence une augmentation de la corrélation entre gènes d’un même TAD en réponse à l’œstrogène. Une perturbation de la frontière entre TAD indique également le rôle des contacts génomiques dans la corégulation de gènes. Ces travaux posent les bases pour comprendre comment les amplificateurs et les gènes communiquent et coordonnent leur activité en 3D
The eukaryotic genome is highly organized in both space and sequence. From entire chromosomes to individual genes the 3D organization of the genome is linked to transcription and many regulatory mechanisms likely coexist at different scales. At the sub-megabase scale, the genome is physically organized into self-interacting topologically associating domains (TADs) that are thought to constrain the range of action of gene regulatory elements called ‘enhancers’. Current data suggest that TADs serve as ‘regulatory units’ to coregulate multiple genes by exposing them to the same enhancers. Genes from the same TAD indeed often display correlated expression across different tissues and cell types. Interestingly, correlated expression is seen between functionally related genes. However, how 3D organization at an individual locus plays a mechanistic role in coregulating functionally related genes is unknown. Using single-molecule imaging, I observed in single cells the spatial positions and transcription of three adjacent functionally related genes regulated by the same/different enhancers. I used estrogen stimulation in MCF7 cells as a model system to study hormone-responsive genes and enhancers. Using combined RNA-DNA FISH, I measured the coupling between genes as the correlation in cis of their transcription. I found, that stimulation with estrogen increases the correlation in cis between genes belonging to the same TAD. Perturbation of the TAD boundary revealed the contribution of contact insulation to gene coregulation. Together, this work lays the ground towards an understanding of how enhancers and genes communicate and coordinate their activity within the 3D genome

Snakehead fishes in the family Channidae are obligate freshwater fishes represented by two extant genera, the African Parachannna and the Asian Channa. These species prefer still or slow flowing water bodies, where they are top predators that exercise high levels of parental care, have the ability to breathe air, can tolerate poor water quality, and interestingly, can aestivate or traverse terrestrial habitat in response to seasonal changes in freshwater habitat availability. These attributes suggest that snakehead fishes may possess high dispersal potential, irrespective of the terrestrial barriers that would otherwise constrain the distribution of most freshwater fishes. A number of biogeographical hypotheses have been developed to account for the modern distributions of snakehead fishes across two continents, including ancient vicariance during Gondwanan break-up, or recent colonisation tracking the formation of suitable climatic conditions. Taxonomic uncertainty also surrounds some members of the Channa genus, as geographical distributions for some taxa across southern and Southeast (SE) Asia are very large, and in one case is highly disjunct. The current study adopted a molecular genetics approach to gain an understanding of the evolution of this group of fishes, and in particular how the phylogeography of two Asian species may have been influenced by contemporary versus historical levels of dispersal and vicariance. First, a molecular phylogeny was constructed based on multiple DNA loci and calibrated with fossil evidence to provide a dated chronology of divergence events among extant species, and also within species with widespread geographical distributions. The data provide strong evidence that trans-continental distribution of the Channidae arose as a result of dispersal out of Asia and into Africa in the mid–Eocene. Among Asian Channa, deep divergence among lineages indicates that the Oligocene-Miocene boundary was a time of significant species radiation, potentially associated with historical changes in climate and drainage geomorphology. Mid-Miocene divergence among lineages suggests that a taxonomic revision is warranted for two taxa. Deep intra-specific divergence (~8Mya) was also detected between C. striata lineages that occur sympatrically in the Mekong River Basin. The study then examined the phylogeography and population structure of two major taxa, Channa striata (the chevron snakehead) and the C. micropeltes (the giant snakehead), across SE Asia. Species specific microsatellite loci were developed and used in addition to a mitochondrial DNA marker (Cyt b) to screen neutral genetic variation within and among wild populations. C. striata individuals were sampled across SE Asia (n=988), with the major focus being the Mekong Basin, which is the largest drainage basin in the region. The distributions of two divergent lineages were identified and admixture analysis showed that where they co-occur they are interbreeding, indicating that after long periods of evolution in isolation, divergence has not resulted in reproductive isolation. One lineage is predominantly confined to upland areas of northern Lao PDR to the north of the Khorat Plateau, while the other, which is more closely related to individuals from southern India, has a widespread distribution across mainland SE Asian and Sumatra. The phylogeographical pattern recovered is associated with past river networks, and high diversity and divergence among all populations sampled reveal that contemporary dispersal is very low for this taxon, even where populations occur in contiguous freshwater habitats. C. micropeltes (n=280) were also sampled from across the Mekong River Basin, focusing on the lower basin where it constitutes an important wild fishery resource. In comparison with C. striata, allelic diversity and genetic divergence among populations were extremely low, suggesting very recent colonisation of the greater Mekong region. Populations were significantly structured into at least three discrete populations in the lower Mekong. Results of this study have implications for establishing effective conservation plans for managing both species, that represent economically important wild fishery resources for the region. For C. micropeltes, it is likely that a single fisheries stock in the Tonle Sap Great Lake is being exploited by multiple fisheries operations, and future management initiatives for this species in this region will need to account for this. For C. striata, conservation of natural levels of genetic variation will require management initiatives designed to promote population persistence at very localised spatial scales, as the high level of population structuring uncovered for this species indicates that significant unique diversity is present at this fine spatial scale.

From the introduction: "... This thesis reports on simplified models (`coarse grained') of proteins and nucleic acids in which many degrees of freedom (DOF) are eliminated, to speed up the computation of the interactions. The resolution of these models is minimal, with respect to the representation of the phenomena studied, in order to extend the time and length scales accessible to atomistic simulations. Furthermore the existence of a coarse- to fine-grain backmapping ensures to switch between diff erent resolutions of the same system, naturally leading to an integrative approach used for multi-scale simulations. Careful parameterization and optimization of the interactions between the interactive centers additionally allow to overcome the limitations of the reductionist first stage by including directly experimental and theoretical data.

Transcriptomics and gene expression profiling enables the elucidation of the genetic response of an organism to various environmental cues. Transcriptomics enables the deciphering of differences between two closely related organisms to the same environment and in contrast, enables the elucidation of genetic responses of the same organism to different environmental cues. Two major methods are utilized for the study of transcriptomes, high-throughput sequencing and microarray analysis. High-throughput sequencing technologies such as the Illumina platform are relatively new and protocols must be developed for the analyses of transcriptomes (RNA-sequencing). A RNA-seq protocol was developed and refined for the Illumina sequencing platform. This protocol was then utilized for the de novo sequencing of the steelhead salmon transcriptome. Hatchery steelhead exhibit a reduced fitness compared to wild steelhead that has been shown to be genetically based. Consequently, the steelhead transcriptome was assembled, annotated, and used to identify gene expression differences between hatchery and wild fish. We uncovered many differentially expressed genes involved in metabolic processes and growth and development. This work has created a better understanding of the genetic differences between hatchery and wild steelhead salmon. Brachypodium distachyon is a monocot grass important as a model for cereal crops and potential biofuels feedstocks. To better understand the genetic response of this plant to different environmental cues, a comprehensive assessment of the transcriptomic response was conducted under a variety of conditions including diurnal/circadian light/dark/temperature environments and different abiotic stress conditions. Using a whole-genome tiling DNA microarray, we identified that the majority of transcripts in Brachypodium exhibit a daily rhythm in their abundance that is conserved between rice and Brachypodium. We also identified numerous cis-regulatory elements dictating these rhythmic expression patterns. We also identified the genetic response to abiotic stresses such as salinity, drought, cold, heat, and high light. We uncovered a core set of genes which responds to all stresses, indicating a core stress response. A large number of transcription factors were uncovered as potential nodes for regulating the abiotic stress response in Brachypodium. Moreover, promoter elements that drive specific responses to discrete abiotic stresses were uncovered. Altogether, the transcriptome analyses in this work furthers our understandings of how particular organisms respond to environmental cues and better elucidates the relationship between genes and the environment.
Graduation date: 2012
Access restricted to the OSU Community at author's request from Oct. 5, 2011 - April 5, 2012.

Abstract Gene drives, particularly synthetic gene drives, may help to address some important challenges, by efficiently altering specific sections of DNA in entire populations of wild organisms. Here we review the current development of the synthetic gene drives, especially those RNA-guided synthetic gene drives based on the CRISPR nuclease Cas. Particular focuses are on their possible applications in agriculture (e.g. disease resistance, weed control management), ecosystem conservation (e.g. evasion species control), health (e.g. to combat insect-borne and fungal infections), and for basic research in model organisms (e.g. Saccharomyces, fruit fly, and zebra fish). The physical, chemical, biological, and ecological containment strategies that might help to confine these gene drive-modified organisms are then explored. The gene flow issues, those from gene drive-derived organisms to the environment, are discussed, while possible mitigation strategies for gene drive research are explored. Last but not least, the regulatory context and opinions from key stakeholders (regulators, scientists, and concerned organizations) are reviewed, aiming to provide a more comprehensive overview of the field.

Abstract This chapter focuses on the fluorescence in situ hybridization (FISH) of DNA probes to three-dimensionally preserved cells, so-called 3D FISH. This technique allows three-dimensional visualization of specific DNA and RNA targets within the nucleus at all stages of the cell cycle. It provides information about the arrangement of chromosome territories and the organization of sub chromosomal domains, about the pattern of chromatin density within a chromosome territory, about positions of individual genes and RNA transcripts read from them. Accumulation of such data is necessary for understanding relationships between the spatial organization of the genome and its functioning in the interphase nucleus.

This chapter examines molecular biology, which is generally concerned with the structures, functions, and interactions of the two major groups of macromolecules, the nucleic acids DNA and RNA, and proteins. Nucleic acids can be purified from organisms using a variety of techniques. Restriction endonucleases (REs) can digest the isolated DNA into smaller-sized fragments suitable for analysis and for use in a number of techniques of clinical interest. Molecular biology techniques are used in biomedical science and they rely on the complementary binding of nucleic acids to provide a convenient way of recognizing and isolating specific base sequences within fragments of DNA or RNA molecules include the sequencing of isolated DNA, Southern and Northern blotting, fluorescence in situ hybridization (FISH), the cloning of DNA by recombination and polymerase chain reaction (PCR) technologies, and DNA microarray analysis. The chapter then looks at CRISPR–Cas9, which is a quick and efficient method for editing genes.