Fluorescence energy transfer dye-labeled primers for DNA sequencing and analysis.

Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.

Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases.

Most helicases studied to date have been characterized as oligomeric, but the relation between their structure and function has not been understood. The bacteriophage T7 gene 4 helicase/primase proteins act in T7 DNA replication. We have used electron microscopy, three-dimensional reconstruction, and protein crosslinking to demonstrate that both proteins form hexameric rings around single-stranded DNA. Each subunit has two lobes, so the hexamer appears to be two-tiered, with a small ring stacked on a large ring. The single-stranded DNA passes through the central hole of the hexamer, and the data exclude substantial wrapping of the DNA about or within the protein ring. Further, the hexamer binds DNA with a defined polarity as the smaller ring of the hexamer points toward the 5' end of the DNA. The similarity in three-dimensional structure of the T7 gene 4 proteins to that of the Escherichia coli RuvB helicase suggests that polar rings assembled around DNA may be a general feature of numerous hexameric helicases involved in DNA replication, transcription, recombination, and repair.

In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus.

Replication of the single-stranded DNA genome of geminiviruses occurs via a double-stranded intermediate that is subsequently used as a template for rolling-circle replication of the viral strand. Only one of the proteins encoded by the virus, here referred to as replication initiator protein (Rep protein), is indispensable for replication. We show that the Rep protein of tomato yellow leaf curl virus initiates viral-strand DNA synthesis by introducing a nick in the plus strand within the nonanucleotide 1TAATATT decreases 8AC, identical among all geminiviruses. After cleavage, the Rep protein remains bound to the 5' end of the cleaved strand. In addition, we show that the Rep protein has a joining activity, suggesting that it acts as a terminase, thus resolving the nascent viral single strand into genome-sized units.

NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides.

A procedure for the enzymatic synthesis of uniformly 13C15N-labeled DNA oligonucleotides in milligram quantities for NMR studies is described. Deoxynucleotides obtained from microorganisms grown on 13C and 15N nutrient sources are enzymatically phosphorylated to dNTPs, and the dNTPs are incorporated into oligonucleotides using a 3'-5' exonuclease-deficient mutant of Klenow fragment of DNA polymerase I and an oligonucleotide template primer designed for efficient separation of labeled product DNA from unlabeled template. The labeling strategy has been used to uniformly label one or the other oligonucleotide strand in the DNA duplex dGGCAAAACGG.dCCGTTTTGCC in order to facilitate assignment and structure determination by NMR. Application of 15N and 13C heteronuclear NMR experiments to isotopically labeled DNA is presented.

DNA recombination is sufficient for retroviral transduction.

Oncogenic retroviruses carry coding sequences that are transduced from cellular protooncogenes. Natural transduction involves two nonhomologous recombinations and is thus extremely rare. Since transduction has never been reproduced experimentally, its mechanism has been studied in terms of two hypotheses: (i) the DNA model, which postulates two DNA recombinations, and (ii) the RNA model, which postulates a 5' DNA recombination and a 3' RNA recombination occurring during reverse transcription of viral and protooncogene RNA. Here we use two viral DNA constructs to test the prediction of the DNA model that the 3' DNA recombination is achieved by conventional integration of a retroviral DNA 3' of the chromosomal protooncogene coding region. For the DNA model to be viable, such recombinant viruses must be infectious without the purportedly essential polypurine tract (ppt) that precedes the 3' long terminal repeat (LTR) of all retroviruses. Our constructs consist of a ras coding region from Harvey sarcoma virus which is naturally linked at the 5' end to a retroviral LTR and artificially linked at the 3' end either directly (construct NdN) or by a cellular sequence (construct SU) to the 5' LTR of a retrovirus. Both constructs lack the ppt, and the LTR of NdN even lacks 30 nucleotides at the 5' end. Both constructs proved to be infectious, producing viruses at titers of 10(5) focus-forming units per ml. Sequence analysis proved that both viruses were colinear with input DNAs and that NdN virus lacked a ppt and the 5' 30 nucleotides of the LTR. The results indicate that DNA recombination is sufficient for retroviral transduction and that neither the ppt nor the complete LTR is essential for retrovirus replication. DNA recombination explains the following observations by others that cannot be reconciled with the RNA model: (i) experimental transduction is independent of the packaging efficiency of viral RNA, and (ii) experimental transduction may invert sequences with respect to others, as expected for DNA recombination during transfection.

Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast.

The CDC47 gene was isolated by complementation of a cdc47 temperature-sensitive mutant in Saccharomyces cerevisiae and was shown to encode a predicted polypeptide, Cdc47, of 845 aa. Cdc47 belongs to the Cdc46/Mcm family of proteins, previously shown to be essential for initiation of DNA replication. Using indirect immunofluorescence microscopy and subcellular fractionation techniques, we show that Cdc47 undergoes cell cycle-regulated changes in its subcellular localization. At mitosis, Cdc47 enters the nucleus, where it remains until soon after the initiation of DNA replication, when it is rapidly exported back into the cytoplasm. Cdc47 protein levels do not vary with the cell cycle, but expression of CDC47 and nascent synthesis of Cdc47 occur late in the cell cycle, coinciding with mitosis. Together, these results show that Cdc47 is not only imported into the nucleus at the end of mitosis but is also exported back into the cytoplasm at the beginning of S phase. The observation that Cdc47 is exported from the nucleus at the beginning of S phase has important implications for how initiation of DNA replication is controlled.

Pollen, DNA and Vegetation records from surface sediments of lacustrine lakes along a forest tundra - tundra transect at the Taymyr peninsula, northern Siberia

Reliable information of past vegetation changes are important to project future changes, especially for areas undergoing rapid transitioning such as the boreal treeline. The application of detailed sedDNA records has the potential to enhance our understanding of vegetation changes gained mainly from pollen studies of lake sediments. This study investigates sedDNA and pollen records from 31 lakes along a gradient of increasing larch forest cover in northern Siberia (Taymyr Peninsula) and compares them with vegetation field surveys within the lake's catchment. With respect to vegetation richness, sedDNA recorded 114 taxa, about half of them to species level, while pollen analyses identified 43 pollen taxa. Both approaches exceed the 31 taxa revealed by vegetation field surveys of 400 m**2 plots. From north to south, Larix percentages increase, as is consistently recorded by all three methods. Furthermore, tundra sites are separated from forested sites in the plots of the principal component analyses. Comparison of ordination results by Procrustes and Protest analyses yields a significant fit among all compared pairs of records. Despite the overall comparability of sedDNA and pollen analyses certain idiosyncrasies in the compositional signal are observed, such as high percentages of Alnus and Betula in all pollen spectra and high percentages of Salix in all sedDNA spectra. In conclusion, our results from the treeline show that sedDNA analyses perform better than pollen in recording site-specific richness (i.e. presence/absence of certain vegetation taxa in the direct vicinity of the lake) and perform as good as pollen in tracing regional vegetation composition.

Results of an in situ stable isotope labeling study in intertidal sediment

We report the results of an in situ tracer experiment in an intertidal sediment, where bacterial carbon was tagged with stable carbon-isotope label, after the injection of 13C-glucose. The appearance of label in bacteria (based on label incorporation in bacteria-specific, phospholipid-derived fatty acids) and subsequent transfer to meiobenthos (group level) and macrobenthos (species level) was followed for 36 days. The label dynamics of benthic taxa were either fitted with a simple-isotope model or evaluated against enrichment in bacteria, to derive the importance of bacterially derived carbon for the meiobenthos and macrobenthos. Although selective uptake of bacteria was evident, as 2.4 times more bacterial carbon was grazed as expected from indiscriminate feeding, bacterial carbon accounted on average for only 0.08 and 0.11 of the carbon requirements of meiobenthic and macrobenthic taxa, respectively. Additionally, the contribution of bacterial carbon to total carbon requirements did not depend on the living/feeding depth in the sediment or organism size (evaluated over a size range of four orders of magnitude). The observed overall low contribution of bacterial carbon implies that most intertidal benthic fauna depend primarily on other carbon resources that may assert a stronger control on the structure of intertidal-sediment communities.

Giegling's Market on south end of the Northern Hotel

verso: Louie Larsen said this was Nick Waal's Market for as long as he can remember. After the market quit Aug Redman had a cigar factory in the building. Later he added a pool room to the cigar factory. Harold (Capt.) Edwardsen tore it down about in the 1930's and built a summer cottage up north from the material. The market was adjacent [to] the Northern Hotel on the south.

DISCUS : an end-to-end solution for ubiquitous broadband optical access

Fiber to the premises has promised to increase the capacity in telecommunications access networks for well over 30 years. While it is widely recognized that optical-fiber-based access networks will be a necessity in the shortto medium-term future, its large upfront cost and regulatory issues are pushing many operators to further postpone its deployment, while installing intermediate unambitious solutions such as fiber to the cabinet. Such high investment cost of both network access and core capacity upgrade often derives from poor planning strategies that do not consider the necessity to adequately modify the network architecture to fully exploit the cost benefit that a fiber-centric solution can bring. DISCUS is a European Framework 7 Integrated Project that, building on optical-centric solutions such as long-reach passive optical access and flat optical core, aims to deliver a cost-effective architecture for ubiquitous broadband services. DISCUS analyzes, designs, and demonstrates end-to-end architectures and technologies capable of saving cost and energy by reducing the number of electronic terminations in the network and sharing the deployment costs among a larger number of users compared to current fiber access systems. This article describes the network architecture and the supporting technologies behind DISCUS, giving an overview of the concepts and methodologies that will be used to deliver our end-to-end network solution. © 2013 IEEE.

DISCUS : end-to-end network design for ubiquitous high speed broadband services

Fibre-to-the-premises (FTTP) has been long sought as the ultimate solution to satisfy the demand for broadband access in the foreseeable future, and offer distance-independent data rate within access network reach. However, currently deployed FTTP networks have in most cases only replaced the transmission medium, without improving the overall architecture, resulting in deployments that are only cost efficient in densely populated areas (effectively increasing the digital divide). In addition, the large potential increase in access capacity cannot be matched by a similar increase in core capacity at competitive cost, effectively moving the bottleneck from access to core. DISCUS is a European Integrated Project that, building on optical-centric solutions such as Long-Reach Passive Optical access and flat optical core, aims to deliver a cost-effective architecture for ubiquitous broadband services. One of the key features of the project is the end-to-end approach, which promises to deliver a complete network design and a conclusive analysis of its economic viability. © 2013 IEEE.

Instability of CTG Repeats is Governed by the Position of a DNA Base Lesion through Base Excision Repair

Trinucleotide repeat (TNR) expansions and deletions are associated with human neurodegeneration and cancer. However, their underlying mechanisms remain to be elucidated. Recent studies have demonstrated that CAG repeat expansions can be initiated by oxidative DNA base damage and fulfilled by base excision repair (BER), suggesting active roles for oxidative DNA damage and BER in TNR instability. Here, we provide the first evidence that oxidative DNA damage can induce CTG repeat deletions along with limited expansions in human cells. Biochemical characterization of BER in the context of (CTG)20 repeats further revealed that repeat instability correlated with the position of a base lesion in the repeat tract. A lesion located at the 59-end of CTG repeats resulted in expansion, whereas a lesion located either in the middle or the 39-end of the repeats led to deletions only. The positioning effects appeared to be determined by the formation of hairpins at various locations on the template and the damaged strands that were bypassed by DNA polymerase b and processed by flap endonuclease 1 with different efficiency. Our study indicates that the position of a DNA base lesion governs whether TNR is expanded or deleted through BER.

Linking Old Librarianship to New: Aligning 5-Steps of The Innovator's DNA in Creating Thematic Discovery Systems for the Everglades

This poster presentation from the May 2015 Florida Library Association Conference, along with the Everglades Explorer discovery portal at http://ee.fiu.edu, demonstrates how traditional bibliographic and curatorial principles can be applied to: 1) selection, cross-walking and aggregation of metadata linking end-users to wide-spread digital resources from multiple silos; 2) harvesting of select PDFs, HTML and media for web archiving and access; 3) selection of CMS domains, sub-domains and folders for targeted searching using an API. Choosing content for this discovery portal is comparable to past scholarly practice of creating and publishing subject bibliographies, except metadata and data are housed in relational databases. This new and yet traditional capacity coincides with: Growth of bibliographic utilities (MarcEdit); Evolution of open-source discovery systems (eXtensible Catalog); Development of target-capable web crawling and archiving systems (Archive-it); and specialized search APIs (Google). At the same time, historical and technical changes – specifically the increasing fluidity and re-purposing of syndicated metadata – make this possible. It equally stems from the expansion of freely accessible digitized legacy and born-digital resources. Innovation principles helped frame the process by which the thematic Everglades discovery portal was created at Florida International University. The path -- to providing for more effective searching and co-location of digital scientific, educational and historical material related to the Everglades -- is contextualized through five concepts found within Dyer and Christensen’s “The Innovator’s DNA: Mastering the five skills of disruptive innovators (2011). The project also aligns with Ranganathan’s Laws of Library Science, especially the 4th Law -- to "save the time of the user.”

(Table 1) Individual codes, haplotype codes, GenBank accession numbers for DNA sequences and sample locality of the analysed Betamorpha fusiformis specimens from the ANDEEP III expedition

Based on our current knowledge about population genetics, phylogeography and speciation, we begin to understand that the deep sea harbours more species than suggested in the past. Deep-sea soft-sediment environment in particular hosts a diverse and highly endemic invertebrate fauna. Very little is known about evolutionary processes that generate this remarkable species richness, the genetic variability and spatial distribution of deep-sea animals. In this study, phylogeographic patterns and the genetic variability among eight populations of the abundant and widespread deep-sea isopod morphospecies Betamorpha fusiformis [Barnard, K.H., 1920. Contributions to the crustacean fauna of South Africa. 6. Further additions to the list of marine isopods. Annals of the South African Museum 17, 319-438] were examined. A fragment of the mitochondrial 16S rRNA gene of 50 specimens and the complete nuclear 18S rRNA gene of 7 specimens were sequenced. The molecular data reveal high levels of genetic variability of both genes between populations, giving evidence for distinct monophyletic groups of haplotypes with average p-distances ranging from 0.0470 to 0.1440 (d-distances: 0.0592-0.2850) of the 16S rDNA, and 18S rDNA p-distances ranging between 0.0032 and 0.0174 (d-distances: 0.0033-0.0195). Intermediate values are absent. Our results show that widely distributed benthic deep-sea organisms of a homogeneous phenotype can be differentiated into genetically highly divergent populations. Sympatry of some genotypes indicates the existence of cryptic speciation. Flocks of closely related but genetically distinct species probably exist in other widespread benthic deep-sea asellotes and other Peracarida. Based on existing data we hypothesize that many widespread morphospecies are complexes of cryptic biological species (patchwork hypothesis).

Topoisomerase 1 (Top1)-associated Genome Instability in Yeast: Effects of Persistent Cleavage Complexes or Increased Top1 Levels

Topoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. Top1 cleaves one strand of DNA, covalently associates with the 3’ end of the nick to form a Top1-cleavage complex (Top1cc), passes the intact strand through the nick and finally re-ligates the broken strand. The chemotherapeutic drug, Camptothecin, intercalates at a Top1cc and prevents the crucial re-ligation reaction that is mediated by Top1, resulting in the conversion of a nick to a toxic double-strand break during DNA replication or the accumulation of Top1cc. This mechanism of action preferentially targets rapidly dividing tumor cells, but can also affect non-tumor cells when patients undergo treatment. Additionally, Top1 is found to be elevated in numerous tumor tissues making it an attractive target for anticancer therapies. We investigated the effects of Top1 on genome stability, effects of persistent Top1-cleavage complexes and elevated Top1 levels, in Saccharomyces cerevisiae. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in reciprocal crossovers, three- to fifteen fold increase in mutagenesis and greatly increased instability within the rDNA and CUP1 tandem arrays. Increased Top1 levels resulted in a fifteen- to twenty-two fold increase in mutagenesis and increased instability in rDNA locus. These results have important implications for understanding the effects of CPT and elevated Top1 levels as a chemotherapeutic agent.