Identical Templates Research Articles

Targeting BCR/ABL-RAD51 Interaction to Prevent Unfaithful Homeologous Recombination Repair.

Abstract 3272Poster Board III-1CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) and cell lines transformed by non-mutated BCR/ABL kinase or the tyrosine kinase inhibitor (TKI)-resistant mutants contain numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic stress. In addition CD34+CD38- CML-CP and CD34+ CML-BC stem cell-enriched populations seem to display more DSBs than normal counterparts. DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. We reported that numerous ROS- and radiation- induced DSBs induce chromosomal instability implicating enhanced, but unfaithful repair in BCR/ABL-positive leukemias [Leukemia, 2008]. We show here that BCR/ABL kinase (non-mutated and TKI-resistant mutants) facilitate recombination repair (RR) of DSBs. Although recombination usually represents a faithful mechanism of DSB repair, it may generate chromosomal aberrations when similar (homeologous), but not identical (homologous) templates are employed during the repair. To study unfaithful homeologous recombination repair (HomeoRR) a reporter repair cassette containing I-SceI endonuclease-;inducible DSB site and a repair template displaying 19% divergence sequence relative to the DSB site was integrated into the genome of 32Dcl3 murine hematopoietic cells and BCR/ABL-positive counterparts. BCR/ABL kinase caused about 3-fold increase in HomeoRR activity implicating its role in accumulation of chromosomal aberrations in CML cells. RAD51, a key regulator of recombination repair, forms a complex with BCR/ABL which depends on the proline- rich (PP) regions of RAD51 and the SH3 domain and SH2-catalytic domain (SH2-CD) linker of BCR/ABL. SH3+SH2-CD domains of BCR/ABL form a pocket binding the PP regions of RAD51. Single amino acid substitutions in the BCR/ABL SH3+SH2-CD pocket, which disrupted binding to the RAD51 PP regions reduced complex formation with RAD51. 32Dcl3 murine hematopoietic cells expressing BCR/ABL SH3+SH2-CD pocket mutant displayed slow proliferation rate and responded poorly to genotoxic stress despite intact kinase activity. On the other hand, disruption of the PP regions of RAD51 by P-L amino acid substitutions (PP-LL mutants) prevented its interaction with BCR/ABL SH3+SH2-CD pocket. Interestingly, expression of RAD51 PP-LL mutant abrogated the clonogenic capability of BCR/ABL-transformed leukemia cells, without any toxic effect on normal counterparts. BCR/ABL-RAD51 complex results in direct phosphorylation of RAD51 on Y315, which is located in the vicinity of PP motifs in the C-terminal portion (aa 271–339) of RAD51. C-terminal Y315F mutant formed more abundant complex with BCR/ABL that the wild-type form, but it did not restore the lost interaction of the PP/LL mutant. Thus, BCR/ABL-mediated RAD51[Y315] phosphorylation appears to be important for disassembly of RAD51 from BCR/ABL. In concordance, RAD51[Y315F] mutant remained mostly in the cytoplasm, while the wild-type protein accumulated in the nucleus in BCR/ABL-positive cells in response to DSBs induced by genotoxic treatment. In addition to the regulation of BCR/ABL-RAD51 interaction, phospho-Y315 is located in a critical fragment of RAD51 essential for its filament formation on DSBs, implicating its direct role in recombination. To test this hypothesis we employed a peptide aptamer strategy targeting phospho-Y315 of RAD51. Peptides corresponding to the RAD51 fragment containing phospho-Y315, but not these with Y315F substitution reduced HomeoRR activity by approximately 2-fold in BCR/ABL-positive leukemia cells. Altogether, it appears that PP-regions of RAD51 interact with SH3+SH2-CD niche of BCR/ABL, which leads to phosphorylation of RAD51 on Y315 and disassembly of the complex. Phospho-Y315 stimulates abundant nuclear localization of RAD51 on DSBs, which disrupts the mechanisms responsible for preventing recombination using divergent templates resulting in unfaithful HomeoRR in BCR/ABL-positive leukemia cells. In summary, BCR/ABL-RAD51 interaction promotes survival and accumulation of chromosomal aberrations of CML cells expressing non-mutated and TKI-resistant BCR/ABL kinase. We hypothesize that targeting BCR/ABL-RAD51 interaction may prevent/delay accumulation of secondary chromosomal aberrations and CML-BC progression. Disclosures:No relevant conflicts of interest to declare.

Characterization of NS3, NS5A and NS5B of classical swine fever virus through mutation and complementation analysis

In order to get further insight into the organization of the pestiviral replication machinery, characterization of NS3, NS5A and NS5B of classical swine fever virus (CSFV) through mutation and complementation analysis was performed. Mutation analysis in genomic replicons and subgenomic replicons indicated importance of the GDD motif in NS5B, the DEYH motif in NS3 and the conserved sequence C2717-C2740-C2742-C2767 in the NS5A for CSFV recover and viral RNA synthesis. Complementation experiments were performed between subgenomic replicons, between RNA replicons or between RNA replicon and expressed nonstructural protein. Rescue of virus and recover of viral RNA synthesis were examined in these complementation experiments. Results showed that mutations within NS5A, neither NS5B nor NS3, can be trans-complemented, strongly suggesting that NS5B and NS3 function in cis mode for regulation of replication. We assumed that the necessary membrane association of CSFV NS5B and NS3 could occur only when they are being translated and originated from an identical translation template, with the exception of NS5A whose membrane association might occur post-translationally.

Sequence Divergence Impedes Crossover More Than Noncrossover Events During Mitotic Gap Repair in Yeast

Homologous recombination between dispersed repeated sequences is important in shaping eukaryotic genome structure, and such ectopic interactions are affected by repeat size and sequence identity. A transformation-based, gap-repair assay was used to examine the effect of 2% sequence divergence on the efficiency of mitotic double-strand break repair templated by chromosomal sequences in yeast. Because the repaired plasmid could either remain autonomous or integrate into the genome, the effect of sequence divergence on the crossover-noncrossover (CO-NCO) outcome was also examined. Finally, proteins important for regulating the CO-NCO outcome and for enforcing identity requirements during recombination were examined by transforming appropriate mutant strains. Results demonstrate that the basic CO-NCO outcome is regulated by the Rad1-Rad10 endonuclease and the Sgs1 and Srs2 helicases, that sequence divergence impedes CO to a much greater extent than NCO events, that an intact mismatch repair system is required for the discriminating identical and nonidentical repair templates, and that the Sgs1 and Srs2 helicases play additional, antirecombination roles when the interacting sequences are not identical.

Legionellaand the Prevention of Legionellosis

Legionella and the Prevention of Legionellosis is much less about the former and more about the latter. The book is essentially a risk-management manual for legionellosis, modeled on the World Health Organization’s framework for providing safe drinking water. Introductory chapters on the disease and the ecology of Legionella spp. and a concluding chapter on laboratory aspects of Legionella spp. serve as bookends for 7 chapters on risk management of legionellosis. The intervening chapters discuss known sources of risk for the disease: potable water; cooling towers and evaporative condensers; healthcare facilities; hotels and ships; and natural spas, hot tubs, and swimming pools. A chapter on disease surveillance and outbreak management and another on regulatory aspects complete the core. Although this book is more a manual than a text, it has much to recommend and little to criticize. An international group of 58 experts contributed to the book, assuring consensus, completeness, and accuracy. Also, unlike many multiauthored texts, which typically suffer from duplication, frequent omissions, and widely varying writing styles, the book’s careful editing has averted these common pitfalls. However, an effort to ensure uniformity in some chapters has led to too much rigid conformity to style. An identical template is used for all risk-management chapters, and frequent use of bulleted lists is not particularly engaging and may prove insufficient for some readers. The text is generously supplemented, perhaps overly so, with 33 tables, 14 figures, and 24 call-out boxes. However, the book’s front matter gives a listing of these illustrations for handy reference. Three appendixes are included: a sample water system checklist, a form for compiling relevant epidemiologic information about patients with Legionnaires’ disease, and an example of a national surveillance form. The list of references is impressive, and the glossary of terms will be valuable to many readers. Notably, this disease-specific treatise arrives at a time when public health officials in some countries are moving toward an all-hazards approach to public health preparedness. Even within this context, this text will remain an authoritative reference for many years to come, and the generic algorithm for ensuring water safety has utility beyond the immediate scope of the book. The brochureaccompanying the book recommends it to environmental and public health officials, healthcare workers, workers in the travel industry, certain researchers, and perhaps some special interest groups. I concur with that general assessment, although the book will be used more frequently by some of those groups than by others.

The role of dislocations as nonradiative recombination centers in InGaN quantum wells

InGaN multiple quantum wells (MQWs) were grown on atomically smooth c-GaN templates and identical c-GaN templates etched to reveal hexagonal pits associated with screw dislocations. We found that the room temperature internal quantum efficiency of the MQWs grown on the etched c-GaN templates is a factor of 2 higher than that of the smooth QWs. This finding is accounted for by the fact that the QWs on the nonplanar surfaces are thinner than the c-plane QWs, and thus the carriers are prevented from reaching the dislocations due to the energy barrier around each defect.

An evaluation of automated homology modelling methods at low target–template sequence similarity

There are two main areas of difficulty in homology modelling that are particularly important when sequence identity between target and template falls below 50%: sequence alignment and loop building. These problems become magnified with automatic modelling processes, as there is no human input to correct mistakes. As such we have benchmarked several stand-alone strategies that could be implemented in a workflow for automated high-throughput homology modelling. These include three new sequence-structure alignment programs: 3D-Coffee, Staccato and SAlign, plus five homology modelling programs and their respective loop building methods: Builder, Nest, Modeller, SegMod/ENCAD and Swiss-Model. The SABmark database provided 123 targets with at least five templates from the same SCOP family and sequence identities </=50%. When using Modeller as the common modelling program, 3D-Coffee outperforms Staccato and SAlign using both multiple templates and the best single template, and across the sequence identity range 20-50%. The mean model RMSD generated from 3D-Coffee using multiple templates is 15 and 28% (or using single templates, 3 and 13%) better than those generated by Staccato and Salign, respectively. 3D-Coffee gives equivalent modelling accuracy from multiple and single templates, but Staccato and SAlign are more successful with single templates, their quality deteriorating as additional lower sequence identity templates are added. Evaluating the different homology modelling programs, on average Modeller performs marginally better in overall modelling than the others tested. However, on average Nest produces the best loops with an 8% improvement by mean RMSD compared to the loops generated by Builder.

Empire, War and Nation: Heritage Management Perspectives from Canada and Malta

This article offers recent insights on contested heritage from Canada and Malta. These contrasting geographical extremes span a range of heritage dissonances but share a common historical identity as successor states to the British Empire, entailing familiar postcolonial heritage equivocations. Dissonances between colonial and indigenous heritage meanings are discussed. The principal focus of the paper is the Empire at war, as an issue of heritage management in Ottawa, the capital of Canada, and in Malta; comparative insights are generated with resonance for other imperial successor states such as Australia, New Zealand and Singapore. In Ottawa the National Capital Commission is engaged in a delicate management of heritage evolution from the imperial past to the multicultural present, involving adjustment and diversification of heritage meanings in which the indigenous peoples and Canada's wartime/military history figure prominently. Malta's time-depth generates an embarrassment of heritage resources, necessitating choices as it moves from 'blue' seacoast to 'grey' heritage tourism; while earlier eras are favoured, the British imperial and military heritage is inescapable, especially the heroic shared defence of 1940-3, generating management issues over recency, postcoloniality, the naval legacy and the problem of marketing to the former enemy populations. Questions of whose heritage, using which resources of what period, for whose benefit and how managed, elicit a different range of answers in the two cases: British colonial heritage is too diverse to be value-generalised, and there is no single, immutable colonial template for postcolonial identity. However, the particular legacy of the Empire at war is notably formative in the evolution of succeeding national identities.

The effect of the 2-amino group of 7,8-dihydro-8-oxo-2'-deoxyguanosine on translesion synthesis and duplex stability

Replication of DNA containing 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG) gives rise to G → T transversions. The syn-isomer of the lesion directs misincorporation of 2′-deoxyadenosine (dA) opposite it. We investigated the role of the 2-amino substituent on duplex thermal stability and in replication using 7,8-dihydro-8-oxo-2′-deoxyinosine (OxodI). Oligonucleotides containing OxodI at defined sites were chemically synthesized via solid phase synthesis. Translesion incorporation opposite OxodI was compared with 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG), 2′-deoxyinosine (dI) and 2′-deoxyguanosine (dG) in otherwise identical templates. The Klenow exo− fragment of Escherichia coli DNA polymerase I incorporated 2′-deoxyadenosine (dA) six times more frequently than 2′-deoxycytidine (dC) opposite OxodI. Preferential translesion incorporation of dA was unique to OxodI. UV-melting experiments revealed that DNA containing OxodI opposite dA is more stable than when the modified nucleotide is opposed by dC. These data suggest that while duplex DNA accommodates the 2-amino group in syn-OxodG, this substituent is thermally destabilizing and does not provide a kinetic inducement for replication by Klenow exo−.

Theoretical study of the ligand-CYP2B4 complexes: effect of structure on binding free energies and heme spin state.

The molecular origins of temperature-dependent ligand-binding affinities and ligand-induced heme spin state conversion have been investigated using free energy analysis and DFT calculations for substrates and inhibitors of cytochrome P450 2B4 (CYP2B4), employing models of CYP2B4 based on CYP2C5(3LVdH)/CYP2C9 crystal structures, and the results compared with experiment. DFT calculations indicate that large heme-ligand interactions (ca. -15 kcal/mol) are required for inducing a high to low spin heme transition, which is correlated with large molecular electrostatic potentials (approximately -45 kcal/mol) at the ligand heteroatom. While type II ligands often contain oxygen and nitrogen heteroatoms that ligate heme iron, DFT results indicate that BP and MF heme complexes, with weak substrate-heme interactions (ca. -2 kcal/mol), and modest MEPS minima (>-35 kcal/mol) are high spin. In contrast, heme complexes of the CYP2B4 inhibitor, 4PI, the product of benzphetamine metabolism, DMBP, and water are low spin, have substantial heme-ligand interaction energies (<-15 kcal/mol) and deep MEPS minima (<-45 kcal/mol) near their heteroatoms. MMPBSA analysis of MD trajectories were made to estimate binding free energies of these ligands at the heme binding site of CYP2B4. In order to initially assess the realism of this approach, the binding free energy of 4PI inhibitor was computed and found to be a reasonable agreement with experiment: -7.7 kcal/mol [-7.2 kcal/mol (experiment)]. BP was determined to be a good substrate [-6.3 kcal/mol (with heme-ligand water), -7.3 kcal/mol (without ligand water)/-5.8 kcal/mol (experiment)], whereas the binding of MF was negligible, with only marginal binding binding free energy of -1.7 kcal/mol with 2-MF bound [-3.8 kcal/mol (experiment)], both with and without retained heme-ligand water. Analysis of the free energy components reveal that hydrophobic/nonpolar contributions account for approximately 90% of the total binding free energy of these substrates and are the source of their differential and temperature-dependent CYP2B4 binding. The results indicate the underlying origins of the experimentally observed differential binding affinities of BP and MF, and indicate the plausibility of the use of models derived from moderate sequence identity templates in conjunction with approximate free energy methods in the estimation of ligand-P450 binding affinities.

In vitro effects of a C4'-oxidized abasic site on DNA polymerases.

Oxidative damage to DNA produces abasic sites resulting from the formal hydrolysis of the nucleotides' glycosidic bonds, along with a variety of oxidized abasic sites. The C4'-oxidized abasic site (C4-AP) is produced by several DNA-damaging agents. This lesion accounts for approximately 40% of the DNA damage produced by bleomycin. The effect of a C4'-oxidized abasic site incorporated at a defined site in a template was examined on Klenow fragments with and without 3' --> 5' exonuclease activity. Both enzymes preferentially incorporated dA > dG >> dC, T opposite C4-AP. Neither enzyme is able to extend the primer past the lesion. Experiments with regular AP sites in an otherwise identical template indicate that Klenow does not differentiate between these two disparate abasic sites. Extension of the primer by alternative polymerases pol II, pol II exo(-), pol IV, and pol V was examined. Pol II exo(-) was most efficient. Qualitative translesion synthesis experiments showed that pol II exo(-) preferentially incorporates T opposite C4-AP, followed in order by dG, dA, and dC. Thymidine incorporation opposite C4'-AP is distinct from the pol II exonuclease interaction with a regular AP site in an otherwise identical template. These in vitro experiments suggest that bypass polymerases may play a crucial role in survival of cells in which C4-AP is produced, and unlike a typical AP site, the C4-AP lesion may not follow the "A-rule". The interaction between bypass polymerases and a C4-AP lesion could explain the high levels of G:C --> T:A transversions in cells treated with bleomycin.

Mechanism of RNA Recombination in Carmo- and Tombusviruses: Evidence for Template Switching by the RNA-Dependent RNA Polymerase In Vitro

RNA recombination occurs frequently during replication of tombusviruses and carmoviruses, which are related small plus-sense RNA viruses of plants. The most common recombinants generated by these viruses are either defective interfering (DI) RNAs or chimeric satellite RNAs, which are thought to be generated by template switching of the viral RNA-dependent RNA polymerase (RdRp) during the viral replication process. To test if RNA recombination is mediated by the viral RdRp, we used either a purified recombinant RdRp of Turnip crinkle carmovirus or a partially purified RdRp preparation of Cucumber necrosis tombusvirus. We demonstrated that these RdRp preparations generated RNA recombinants in vitro. The RdRp-driven template switching events occurred between either identical templates or two different RNA templates. The template containing a replication enhancer recombined more efficiently than templates containing artificial sequences. We also observed that AU-rich sequences promote recombination more efficiently than GC-rich sequences. Cloning and sequencing of the generated recombinants revealed that the junction sites were located frequently at the ends of the templates (end-to-end template switching). We also found several recombinants that were generated by template switching involving internal positions in the RNA templates. In contrast, RNA ligation-based RNA recombination was not detected in vitro. Demonstration of the ability of carmo- and tombusvirus RdRps to switch RNA templates in vitro supports the copy-choice models of RNA recombination and DI RNA formation for these viruses.

Influence of Growth Parameters on the Deep Level Spectrum in MBE-Grown n-GaN

ABSTRACTThe impact of growth temperature and Ga/N flux ratio on deep levels in GaN grown by molecular beam epitaxy (MBE) is systematically investigated using both deep level optical spectroscopy (DLOS) and deep level transient spectroscopy (DLTS) in a study designed to map out the presence and concentration of defects over a defined region of the MBE GaN growth phase diagram. A series of Si-doped GaN films were grown to cover a substrate temperature range and a Ga/N flux ratio range that spans from the N stable to the Ga droplet regimes along both variables. Identical growth templates were used to eliminate variations in dislocations between samples so that point defect variations could be tracked. For these samples, traps are detected at EC-Et=0.25, 0.60, 0.90, 1.35, 2.40, 3.04, and 3.28 eV. The near valence bands states at EC–3.04 and EC–3.28 eV are found to be strongly dependent on Ga/N flux with decreased concentrations as a function of increasing Ga flux toward the Ga droplet regime, but with little effect from growth temperature. The EC-1.35 eV level shows a strong dependence on growth temperature and only slight dependence on Ga/N flux ratio. In contrast, the concentration of the EC-Et=0.25, 0.90 eV levels increased with increasing Ga flux toward the Ga droplet regime, while the EC-Et=0.60 shows no dependence. The variation in concentration of the EC-2.40 eV level that has been related to VGa was difficult to quantify, but tends to increase towards nitrogen rich growth. The dependencies for the detected states with respect to growth temperature and Ga/N flux ratio suggest different physical point defect sources.

DNA hybridization to mismatched templates: a chip study.

High-density oligonucleotide arrays are among the most rapidly expanding technologies in biology today. In the GeneChip system, the reconstruction of the sample mRNA concentrations depends upon the differential signal generated by hybridizing the RNA to two nearly identical templates: a perfect match probe (PM) containing the exact biological sequence; and a single mismatch (MM) differing from the PM by a single base substitution. It has been observed that a large fraction of MMs repetitively bind targets better than the PMs, against the obvious expectation of sequence specificity. We examine this problem via statistical analysis of a large set of microarray experiments. We classify the probes according to their signal to noise (S/N) ratio, defined as the eccentricity of a (PM,MM) pair's "trajectory" across many experiments. Of those probes having large S/N (>3) only a fraction behave consistently with the commonly assumed hybridization model. Our results imply that the physics of DNA hybridization in microarrays is more complex than expected, and suggest estimators for the target RNA concentration.

A regularity-based hierarchical symbolic analysis method for large-scale analog networks

This paper presents a novel hierarchical symbolic analysis method for automatically producing relationships between the parameters of an analog network and the building blocks of the network. The originality of the symbolic technique stems from exploiting regularity aspects for addressing the exponential complexity of the symbolic expressions. The regularity aspects that were identified are: 1) structural regularity: majority of the network blocks are connected in identical templates and 2) symbolic parameter regularity: parameters for a connection template require similar sets of operations. The paper discusses the three components of the proposed symbolic analysis method: 1) an efficient representation of symbolic expressions, 2) an algorithm for construction of symbolic expressions; and 3) a decomposition technique for extracting the structural regularity of a network. For large networks, the size of the symbolic models produced by our symbolic analysis method is much less than the size of the models produced by other methods such as the two-graph method. We mathematically show that the generated models are of polynomial size if the two kinds of regularity are exploited. The described symbolic technique was implemented and used successfully for synthesis and optimization of different analog systems such as filters and communication systems.

"Have you been saved?" Models of Identity for Maladies of the Soul: Evangelicalism and Feminist Performance Art

Jimmy Swaggart exhorting his audience to forsake dastardly dark sins of secular society and Karen Finley doing unmentionable things with canned yams are at opposite ends of shamanistic spectrum. The evangelist is coiffed, impeccably groomed, acutely aware of his always already converted audience. The other (literally Other) is, quite simply, messy. She smashes unboiled coloured eggs in a plastic bag, smears them all over her naked body using stuffed animals as applicators, and then sprinkles herself with glitter and confetti as her audience watches in a state of bemusement, or confusion, or disgust.1 He proclaims and displays truth of Word in a predictable narrative of salvation whereas she performs story-telling on body, in exaggerated, gross gestures which ironically bespeak her eternal damnation. Although ideologies motivating performers and salvations they offer differ wildly, evangelists and feminist performance artists use some remarkably similar techniques and even enact similar spiritual missions: they both offer solutions, in divergent models of identity, for malaise of in postmodern era. According to Julia Kristeva in New Maladies of Soul, blocked, inhibited and destroyed (8) psychic of contemporary society has contributed to displays of states (8) at root of which is the inability to (9), and which can be cured through establishing a logos of (26). Within this paradigm, models of identity presented by individual evangelical preachers such as Billy Graham and Jimmy Swaggart, and enacted at phenomenon of Toronto Blessing, can be compared to those models of identity displayed in work of feminist performance artists such as Carolee Schneemann, Karen Finley, and Shawna Dempsey and Lorri Millan. In New Maladies of Soul, Julia Kristeva pursues intersection of psychoanalytic study and spirituality by examining soul as psyche. For Kristeva, soul is a necessary part of existence: You are alive if and only if you have a psychic life (5-6). It is this psychic life, more specifically Kristeva's interpretation of Freudian psychic apparatus, which constitutes her working definition of registers representations and their meaningful values for (8). The effect of neglect of this psychic apparatus is dire: Held back by his aloofness, modern man is a narcissist-a narcissist who may suffer.... He manifests his suffering in his body and he is afflicted with somatic symptoms (7). Without a template of identity, individual is tormented: left without a sexual, subjective, or moral identity, this amphibian is a being of boundaries, a borderline, or a `false self' (7-8). Kristeva goes on to describe what she calls maladies of soul: wounded `narcissisms,' `false personalities,' `borderline states,' or `psychosomatic conditions' (9). As an example of this condition, Kristeva describes a patient named Didier, whose is manifested in his laconic speech (11), and is played out on his body. His denial of preoedipal conflicts produces somatic symptoms: the thin autoerotic layer gave way to a metaphor, symptom of dermatitis (15). In another case, Kristeva describes a patient who is indistinguishable from his mother (51). The patient manifests this borderline condition: when faced with collapse of absolute prohibition, [he] lost his means of defense and symbolization, and quite literally negated himself in a spastic, demented and aphasic jouissance (50). This state of being, as Kristeva makes clear, is a logic of inversion (the subject becomes object and vice versa) (51). The boundary between self and other is blurred. No matter what kind of borderline is displayed, common denominator, says Kristeva, is the inability to represent ... inability to symbolize (9). It is her task as a psychoanalyst to help cure patient by means of an analytical process which fosters a bridge between patient and false self or condition that is suffered. …

NusA contacts nascent RNA in Escherichia coli transcription complexes.

We have examined the interaction between NusA and the nascent RNA in Escherichia coli transcription complexes on four different templates. Photocrosslinking CTP and UTP analogs were incorporated internally and at the 3' end of the RNA. Identical templates with and without boxA sequences were compared. We found that NusA did not contact the ten nucleotides nearest to the 3' end of the RNA in complexes containing RNA up to 20 nucleotides long. Longer RNA did crosslink to NusA with all four templates examined, however. We reported that RNA 80 nucleotides long from the bacteriophage T7 A1 promoter substituted in two RNA stem-loops with photocrosslinking UMP analogs did not crosslink to NusA, even though interaction between NusA and the transcription complex were demonstrated. Here, we report that when this same RNA is substituted at CMP residues, it does crosslink to NusA. Templates containing the E. coli ribosomal RNA promoter rrnG P2, with and without a boxA sequence downstream, were compared. Long RNAs from both crosslinked to NusA, and thus boxA RNA sequences are not required for interaction with NusA. NusA did not interact with the free RNA containing boxA once released from the transcription complex, nor did it interact with RNA in a binary complex containing only RNA polymerase and RNA, without the DNA template.

NusA contacts nascent RNA in Escherichia coli transcription complexes

We have examined the interaction between NusA and the nascent RNA in Escherichia coli transcription complexes on four different templates. Photocrosslinking CTP and UTP analogs were incorporated internally and at the 3' end of the RNA. Identical templates with and without boxA sequences were compared. We found that NusA did not contact the ten nucleotides nearest to the 3' end of the RNA in complexes containing RNA up to 20 nucleotides long. Longer RNA did crosslink to NusA with all four templates examined, however. We reported that RNA 80 nucleotides long from the bacteriophage T7 A1 promoter substituted in two RNA stem-loops with photocrosslinking UMP analogs did not crosslink to NusA, even though interaction between NusA and the transcription complex were demonstrated. Here, we report that when this same RNA is substituted at CMP residues, it does crosslink to NusA. Templates containing the E. coli ribosomal RNA promoter rrnG P2, with and without a boxA sequence downstream, were compared. Long RNAs from both crosslinked to NusA, and thus boxA RNA sequences are not required for interaction with NusA. NusA did not interact with the free RNA containing boxA once released from the transcription complex, nor did it interact with RNA in a binary complex containing only RNA polymerase and RNA, without the DNA template.

TGGE in quantitative PCR of DNA and RNA.

The copy numbers of DNA sequences (templates) can be determined quantitatively by a combination of PCR and temperature gradient gel electrophoresis (TGGE; ,; Chapter 20) as illustrated in Fig. 1. Briefly, quantitation is performed by addition of a calibrated amount of DNA “standard” to the template that is subsequently amplified by PCR. The standard is identical to the template except for a single base substitution. Because of the identical priming sites and almost identical sequence composition, both template and standard are coamplified homogeneously; the relative amounts of each product type at any time point accurately reflect the original ratio prior to amplification. This holds true for the exponential amplification phase as well as for the stationary phase.

Detection and identification of diagnostic Histoplasma capsulatum precipitates by counterelectrophoresis.

Studies were carried out to develop and evaluate a counterelectrophoresis (CEP) technique for the rapid and specific identification of the diagnostically important histoplasmosis H and M precipitin bands. Well-defined and centrally located precipitin bands were produced by using a discontinuous buffer system and a gel matrix composed of agarose and ionagar no. 2. A template was devised which allowed the selective identification of the H and M precipitins. Comparative evaluations were performed with the microimmunodiffusion (ID) and complement fixation tests. In 52 sera from persons with histoplasmosis, either the H or M precipitin, or both, were identified in 42 (81%) of the cases with the CEP technique and in 43 (83%) with the ID test. With sera from 28 persons with heterologous diseases, the CEP technique, like the ID test, failed to react. The specificity of the CEP technique was dependent upon the use of the identity template. The CEP technique is recommended for routine use in laboratories testing moderate numbers of sera. It provides accurate and reproducible results within 90 min, in contrast to the ID test, which requires 18 to 24 h.

Detection and Identification of Diagnostic Histoplasma capsulatum Precipitates by Counterelectrophoresis

Studies were carried out to develop and evaluate a counterelectrophoresis (CEP) technique for the rapid and specific identification of the diagnostically important histoplasmosis H and M precipitin bands. Well-defined and centrally located precipitin bands were produced by using a discontinuous buffer system and a gel matrix composed of agarose and ionagar no. 2. A template was devised which allowed the selective identification of the H and M precipitins. Comparative evaluations were performed with the microimmunodiffusion (ID) and complement fixation tests. In 52 sera from persons with histoplasmosis, either the H or M precipitin, or both, were identified in 42 (81%) of the cases with the CEP technique and in 43 (83%) with the ID test. With sera from 28 persons with heterologous diseases, the CEP technique, like the ID test, failed to react. The specificity of the CEP technique was dependent upon the use of the identity template. The CEP technique is recommended for routine use in laboratories testing moderate numbers of sera. It provides accurate and reproducible results within 90 min, in contrast to the ID test, which requires 18 to 24 h.