Binary recombinase systems for high-resolution conditional mutagenesis.

Conditional mutagenesis using Cre recombinase expressed from tissue specific promoters facilitates analyses of gene function and cell lineage tracing. Here, we describe two novel dual-promoter-driven conditional mutagenesis systems designed for greater accuracy and optimal efficiency of recombination. Co-Driver employs a recombinase cascade of Dre and Dre-respondent Cre, which processes loxP-flanked alleles only when both recombinases are expressed in a predetermined temporal sequence. This unique property makes Co-Driver ideal for sequential lineage tracing studies aimed at unraveling the relationships between cellular precursors and mature cell types. Co-InCre was designed for highly efficient intersectional conditional transgenesis. It relies on highly active trans-splicing inteins and promoters with simultaneous transcriptional activity to reconstitute Cre recombinase from two inactive precursor fragments. By generating native Cre, Co-InCre attains recombination rates that exceed all other binary SSR systems evaluated in this study. Both Co-Driver and Co-InCre significantly extend the utility of existing Cre-responsive alleles.

CHEMOSENSITIZATION OF HEPATOCELLULAR CARCINOMA TO GEMCITABINE BY NON-INVASIVE RADIOFREQUENCY FIELD-INDUCED HYPERTHERMIA

Gemcitabine is a potent nucleoside analogue against solid tumors however drug resistance rapidly emerges. Removal of gemcitabine incorporated in the DNA by repair mechanisms could potentially contribute to resistance in chemo-refractory solid tumors. In this study, we evaluated homologous recombination repair of gemcitabine-stalled replication forks as a potential mechanism contributing to resistance. We also studied the effect of hyperthermia on homologous recombination pathway to explain the previously reported synergy between gemcitabine and hyperthermia. We found that hyperthermia degrades and inhibits localization of Mre11 to gemcitabine-stalled replication forks. Furthermore, gemcitabine-treated cells that were also treated with hyperthermia demonstrate a prolonged passage through late S/ G2 phase of cell cycle in comparison to cells treated with gemcitabine alone. This coincides with inhibition of resolution of γH2AX foci. Our findings also demonstrate that thermal sensitization of human hepatocellular carcinoma cell lines to gemcitabine is mediated through an Mre11-dependent homologous recombination repair pathway. Combination of non-invasive radiofrequency field-induced hyperthermia and gemcitabine was superior to either therapy alone (p

Design, Synthesis and Development of Transporter Targeting Agents for Image-guided Therapy and Drug Delivery

The purpose of this study was to design, synthesize and develop novel transporter targeting agents for image-guided therapy and drug delivery. Two novel agents, N4-guanine (N4amG) and glycopeptide (GP) were synthesized for tumor cell proliferation assessment and cancer theranostic platform, respectively. N4amG and GP were synthesized and radiolabeled with 99mTc and 68Ga. The chemical and radiochemical purities as well as radiochemical stabilities of radiolabeled N4amG and GP were tested. In vitro stability assessment showed both 99mTc-N4amG and 99mTc-GP were stable up to 6 hours, whereas 68Ga-GP was stable up to 2 hours. Cell culture studies confirmed radiolabeled N4amG and GP could penetrate the cell membrane through nucleoside transporters and amino acid transporters, respectively. Up to 40% of intracellular 99mTc-N4amG and 99mTc-GP was found within cell nucleus following 2 hours of incubation. Flow cytometry analysis revealed 99mTc-N4amG was a cell cycle S phase-specific agent. There was a significant difference of the uptake of 99mTc-GP between pre- and post- paclitaxel-treated cells, which suggests that 99mTc-GP may be useful in chemotherapy treatment monitoring. Moreover, radiolabeled N4amG and GP were tested in vivo using tumor-bearing animal models. 99mTc-N4amG showed an increase in tumor-to-muscle count density ratios up to 5 at 4 hour imaging. Both 99mTc-labeled agents showed decreased tumor uptake after paclitaxel treatment. Immunohistochemistry analysis demonstrated that the uptake of 99mTc-N4amG was correlated with Ki-67 expression. Both 99mTc-N4amG and 99mTc-GP could differentiate between tumor and inflammation in animal studies. Furthermore, 68Ga-GP was compared to 18F-FDG in rabbit PET imaging studies. 68Ga-GP had lower tumor standardized uptake values (SUV), but similar uptake dynamics, and different biodistribution compared with 18F-FDG. Finally, to demonstrate that GP can be a potential drug carrier for cancer theranostics, several drugs, including doxorubicin, were selected to be conjugated to GP. Imaging studies demonstrated that tumor uptake of GP-drug conjugates was increased as a function of time. GP-doxorubicin (GP-DOX) showed a slow-release pattern in in vitro cytotoxicity assay and exhibited anti-cancer efficacy with reduced toxicity in in vivo tumor growth delay study. In conclusion, both N4amG and GP are transporter-based targeting agents. Radiolabeled N4amG can be used for tumor cell proliferation assessment. GP is a potential agent for image-guided therapy and drug delivery.

Proceedings of the Sixteenth Annual Biochemical Engineering Symposium

This volume represents the proceedings of the Sixteenth Annual Biochemical Engineering Symposium held at Kansas State University on April 26, 1986. Some of the papers describe the progress of ongoing projects, and others contain the results of completed projects. Only brief summaries are given of many of the papers that will be published in full elsewhere. ContentsEnd-Product Inhibition of the Acetone-Butanol Fermentation—Bob Kuhn, Colorado State University Effect of Multiple Substrates in Ethanal Fermentations from Cheese Whey—C.J. Wang, University of Missouri Extraction and Fermentation of Ensiled Sweet Sorghum—Karl Noah, Colorado State University Removal of Nucleic Acids from Bakers' Yeast—Richard M. Cordes, Iowa State University Modeling the Effects of Plasmid Replication and Product Repression on the Growth Rate of Recombinant Bacteria—William E. Bentley, University of Colorado Indirect Estimates of Cell Concentrations in Mass Cultivation of Bacterial Cells—Andrew Fisher, University of Missouri A Mathematical Model for Liquid Recirculation in Airlift Columns—C.H.Lee, Kansas State University Characterization of Imperfect Mixing of Batch Reactors by Two Compartment Model—Peter Sohn, University of Missouri First Order Breakage Model for the Degradation of Pullalan in the Batch Fermentor—Stephen A. Milligan, Kansas State University Synthesis and Nuclear Magnetic Resonance of 13C-Labeled Amylopectin and Maltooligosaccharides—Bernard Y. Tao, Iowa State University Preparation of Fungal Starter Culture in Gas Fluidized Bed Reactor—Pal Mihaltz, Colorado State University Yeast Flocculation and Sedimentation—David Szlag, University of Colorado Protein Enrichment of Extrusion Cooked Corn by Solid Substrate Fermentation—Lucas Alvarez-Martinez, Colorado State University Optimum Design of a Hollow Fiber Mammalian Cell Reactor—Thomas Chresand, Colorado State University Gas Chromatography and Nuclear Magnetic Resonance of Trifluoroacetylated Carbohydrates—Steven T. Summerfelt, Iowa State University Kinetic and Bioenergetic Considerations for Modeling Photosynthetic Microbial P~ocesses in Producing Biomass and Treating Wastewater—H. Y. Lee, Kansas State University Mathematical Modeling and Simulation of Bicarbonate-Limited Photsynthetic Growth in Continuous Culture—Craig Curless, Kansas State University Data Acquisition and Control of a Rotary Drum Solid State Fermentor—Mnasria A. Habib, Colorado State University Biodegradation of 2,4-Dichlorophenoxyacetic Acid (2,4-D)—Greg Sinton, Kansas State University

Benthic respiration and physical oceanography on two contrasting continental margins in the western Indian Ocean: the Yemen-Somali upwelling region and the margin off Kenya

During the Netherlands Indian Ocean Project (NIOP, 1992-1993) sediment community oxygen consumption (SCOC) was measured on two continental margins in the Indian Ocean with different productivity: the productive upwelling region off Yemen-Somalia and the supposedly less productive Kenyan margin, which lacks upwelling. The two margins also differ in terms of river input (Kenya) and the more severe oxygen minimum in the Arabian Sea. Simultaneously with SCOC, distributions of benthic biomass and phytodetritus were studied. Our expectation was that benthic processes in the upwelling margin of the Arabian Sea would be relatively enhanced as a result of the higher productivity. On the Kenyan margin, SCOC (range 1-36 mmol/m**2/d) showed a clear decrease with increasing water depth, and little temporal variation was detected between June and December. Highest SCOC values of this study were recorded at 50 m depth off Kenya, with a maximum of 36 mmol/m**2/d in the northernmost part. On the margin off Yemen-Somalia, SCOC was on average lower and showed little downslope variation, 1.8-5.7 mmol/m**2/d, notably during upwelling, when the zone between 70 and 1700 m was covered with low O2 water (10-50 µM). After cessation of upwelling, SCOC at 60 m depth off Yemen increased from 5.7 to 17.6 mmol/m**2/d concurrently with an increase of the near-bottom O2 concentration (from 11 to 153 µM), suggesting a close coupling between SCOC and O2 concentration. This was demonstrated in shipboard cores in which the O2 concentration in the overlying water was raised after the cores were first incubated under in situ conditions (17 µM O2). This induced an immediate and pronounced increase of SCOC. Conversely, at deeper stations permanently within the oxygen minimum zone (OMZ), SCOC showed little variation between monsoon periods. Hence, organic carbon degradation in sediments on a large part of the Yemen slope appears hampered by the oxygen deficiency of the overlying water. Macrofauna biomass and the pooled biomass of smaller organisms, estimated by the nucleic acid content of the sediment, had comparable ranges in the two areas in spite of more severe suboxic conditions in the Arabian Sea. At the Kenyan shelf, benthic fauna (macro- and meiofauna) largely followed the spatial pattern of SCOC, i.e. high values on the northern shelf-upper slope and a downslope decrease. On the Yemen-Somali margin the macrofauna distribution was more erratic. Nucleic acids displayed no clear downslope trend on either margin owing to depressed values in the OMZ, perhaps because of adverse effects of low O2 on small organisms (meiofauna and microbes). Phytodetritus distributions were different on the two margins. Whereas pigment levels decreased downslope along the Kenya margin, the upper slope off Yemen (800 m) had a distinct accumulation of mainly refractory carotenoid pigments, suggesting preservation under low 02. Because the accumulations of Corg and pigments on the Yemen slope overlap only partly, we infer a selective deposition and preservation of labile particles on the upper slope, whereas refractory material undergoes further transport downslope.

Simulación de evolución dirigida de bacteriófagos en poblaciones de bacterias en 2D

The research group is currently developing a biological computing model to be implemented with Escherichia Coli bacteria and bacteriophages M13, but it has to be modelled and simulated before any experiment in order to reduce the amount of failed attempts, time and costs. The problem that gave rise to this project is that there are no software tools which are able to simulate the biological process underlying that com- putational model, so it needs to be developed before doing any experimental implementation. There are several software tools which can simulate most of the biological processes and bacterial interactions in which this model is based, so what needs to be done is to study those available simulation tools, compare them and choose the most appropriate in order to be improved adding the desired functionality for this design. Directed evolution is a method used in biotechnology to obtain proteins or nucleic acids with properties not found in nature. It consists of three steps: 1) creating a library of mutants, 2) selecting the mutants with the desired properties, 3) replicating the variants identified in the selection step. The new software tool will be verified by simulating the selection step of a process of directed evolution applied to bacteriophages. ---ABSRACT---El grupo de investigación está desarrollando un modelo de computación biolóogica para ser implementado con bacterias Escherichia Coli y bacteriofagos M13, aunque primero tiene que ser modelizado antes de realizar cualquier experimento, de forma que los intentos fallidos y por lo tanto los costes se verán reducidos. El problema que dio lugar a este proyecto es la ausencia de herramientas software capaces de simular el proceso biológico que subyace a este modelo de computación biológica, por lo que dicha herramienta tiene que ser desarrollada antes de realizar cualquier implementación real. Existen varias herramientas software capaces de simular la mayoría de los procesos biológicos y las interacciones entre bacterias en los que se basa este modelo, por lo que este trabajo consiste en realizar un estudio de dichas herramientas de simulación, compararlas y escoger aquella más apropiada para ser mejorada añadiendo la funcionalidad deseada para este diseño. La evolución dirigida es un método utilizado en biotecnología para obtener proteínas o ácidos nucleicos con propiedades que no se encuentran en la naturaleza. Este método consiste en tres pasos: 1) crear una librería de mutantes, 2) seleccionar los mutantes con las propiedades deseadas, 3) Replicar los mutantes deseados. La nueva herramienta software será verificada mediante la simulación de la selección de mutantes de un proceso de evolución dirigida aplicado a bacteriofagos.

In vitro properties of the first ORF protein from mouse LINE-1 support its role in ribonucleoprotein particle formation during retrotransposition

LINEs are transposable elements, widely distributed among eukaryotes, that move via reverse transcription of an RNA intermediate. Mammalian LINEs have two ORFs (ORF1 and ORF2). The proteins encoded by these ORFs play important roles in the retrotransposition process. Although the predicted amino acid sequence of ORF1 is not closely related to any known proteins, it is highly basic; thus, it has long been hypothesized that ORF1 protein functions to bind LINE-1 (L1) RNA during retrotransposition. Cofractionation of ORF1 protein and L1 RNA in extracts from both mouse and human embryonal carcinoma cells indicated that ORF1 protein binds L1 RNA, forming a ribonucleoprotein particle. Based on UV crosslinking and electrophoretic mobility-shift assays using purified components, we demonstrate here that the ORF1 protein encoded by mouse L1 binds nucleic acids with a strong preference for RNA and other single-stranded nucleic acids. Furthermore, multiple copies of ORF1 protein appear to bind single-stranded nucleic acid in a manner suggesting positive cooperativity; such binding characteristics are likely to be facilitated by the protein–protein interactions detected among molecules of ORF1 polypeptide by coimmunoprecipitation. These observations are consistent with the formation of ribonucleoprotein particles containing L1 RNA and ORF1 protein and provide additional evidence for the role of ORF1 protein during retrotransposition of L1.

Enzymatic reaction with unnatural substrates: DNA photolyase (Escherichia coli) recognizes and reverses thymine [2+2] dimers in the DNA strand of a DNA/PNA hybrid duplex

Peptide nucleic acids (PNA) are mimics with normal bases connected to a pseudopeptide chain that obey Watson–Crick rules to form stable duplexes with itself and natural nucleic acids. This has focused attention on PNA as therapeutic or diagnostic reagents. Duplexes formed with PNA mirror some but not all properties of DNA. One fascinating aspect of PNA biochemistry is their reaction with enzymes. Here we show an enzyme reaction that operates effectively on a PNA/DNA hybrid duplex. A DNA oligonucleotide containing a cis, syn-thymine [2+2] dimer forms a stable duplex with PNA. The hybrid duplex is recognized by photolyase, and irradiation of the complex leads to the repair of the thymine dimer. This finding provides insight into the enzyme mechanism and provides a means for the selective repair of thymine photodimers.

Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms

A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205–2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable.

NMR scalar couplings across Watson–Crick base pair hydrogen bonds in DNA observed by transverse relaxation-optimized spectroscopy

This paper describes the NMR observation of 15N—15N and 1H—15N scalar couplings across the hydrogen bonds in Watson–Crick base pairs in a DNA duplex, hJNN and hJHN. These couplings represent new parameters of interest for both structural studies of DNA and theoretical investigations into the nature of the hydrogen bonds. Two dimensional [15N,1H]-transverse relaxation-optimized spectroscopy (TROSY) with a 15N-labeled 14-mer DNA duplex was used to measure hJNN, which is in the range 6–7 Hz, and the two-dimensional hJNN-correlation-[15N,1H]-TROSY experiment was used to correlate the chemical shifts of pairs of hydrogen bond-related 15N spins and to observe, for the first time, hJHN scalar couplings, with values in the range 2–3.6 Hz. TROSY-based studies of scalar couplings across hydrogen bonds should be applicable for large molecular sizes, including protein-bound nucleic acids.

A Role for the GSG Domain in Localizing Sam68 to Novel Nuclear Structures in Cancer Cell Lines

The GSG (GRP33, Sam68, GLD-1) domain is a protein module found in an expanding family of RNA-binding proteins. The numerous missense mutations identified genetically in the GSG domain support its physiological role. Although the exact function of the GSG domain is not known, it has been shown to be required for RNA binding and oligomerization. Here it is shown that the Sam68 GSG domain plays a role in protein localization. We show that Sam68 concentrates into novel nuclear structures that are predominantly found in transformed cells. These Sam68 nuclear bodies (SNBs) are distinct from coiled bodies, gems, and promyelocytic nuclear bodies. Electron microscopic studies show that SNBs are distinct structures that are enriched in phosphorus and nitrogen, indicating the presence of nucleic acids. A GFP-Sam68 fusion protein had a similar localization as endogenous Sam68 in HeLa cells, diffusely nuclear with two to five SNBs. Two other GSG proteins, the Sam68-like mammalian proteins SLM-1 and SLM-2, colocalized with endogenous Sam68 in SNBs. Different GSG domain missense mutations were investigated for Sam68 protein localization. Six separate classes of cellular patterns were obtained, including exclusive SNB localization and association with microtubules. These findings demonstrate that the GSG domain is involved in protein localization and define a new compartment for Sam68, SLM-1, and SLM-2 in cancer cell lines.

Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides

Abnormal expression of major histocompatibility complex (MHC) class I and class II in various tissues is associated with autoimmune disease. Autoimmune responses can be triggered by viral infections or tissue injuries. We show that the ability of a virus or a tissue injury to increase MHC gene expression is duplicated by any fragment of double-stranded (ds) DNA or dsRNA introduced into the cytoplasm of nonimmune cells. Activation is sequence-independent, is induced by ds polynucleotides as small as 25 bp in length, and is not duplicated by single-stranded polynucleotides. In addition to causing abnormal MHC expression, the ds nucleic acids increase the expression of genes necessary for antigen processing and presentation: proteasome proteins (e.g., LMP2), transporters of antigen peptides; invariant chain, HLA-DM, and the costimulatory molecule B7.1. The mechanism is different from and additive to that of γ-interferon (γIFN), i.e., ds polynucleotides increase class I much more than class II, whereas γIFN increases class II more than class I. The ds nucleic acids also induce or activate Stat1, Stat3, mitogen-activated protein kinase, NF-κB, the class II transactivator, RFX5, and the IFN regulatory factor 1 differently from γIFN. CpG residues are not responsible for this effect, and the action of the ds polynucleotides could be shown in a variety of cell types in addition to thyrocytes. We suggest that this phenomenon is a plausible mechanism that might explain how viral infection of tissues or tissue injury triggers autoimmune disease; it is potentially relevant to host immune responses induced during gene therapy.

Thermodynamic basis of the enhanced specificity of structured DNA probes

Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their fluorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformationally constrained probes.

Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry

An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.