Rotavirus RNA replication requires a single-stranded 3' end for efficient minus-strand synthesis

The segmented double-stranded (ds) RNA genome of the rotaviruses is replicated asymmetrically, with viral mRNA serving as the template for the synthesis of minus-strand RNA. Previous studies with cell-free replication systems have shown that the highly conserved termini of rotavirus gene 8 and 9 mRNAs contain cis-acting signals that promote the synthesis of dsRNA. Based on the location of the cis-acting signals and computer modeling of their secondary structure, the ends of the gene 8 or 9 mRNAs are proposed to interact in cis to form a modified panhandle structure that promotes the synthesis of dsRNA. In this structure, the last 11 to 12 nucleotides of the RNA, including the cis-acting signal that is essential for RNA replication, extend as a single-stranded tail from the panhandled region, and the 5' untranslated region folds to form a stem-loop motif. To understand the importance of the predicted secondary structure in minus-strand synthesis, mutations were introduced into viral RNAs which affected the 3' tail and the 5' stem-loop. Analysis of the RNAs with a cell-free replication system showed that, in contrast to mutations which altered the structure of the 5' stem-loop, mutations which caused complete or near-complete complementarity between the 5' end and the 3' tail significantly inhibited (>/=10-fold) minus-strand synthesis. Likewise, incubation of wild-type RNAs with oligonucleotides which were complementary to the 3' tail inhibited replication. Despite their replication-defective phenotype, mutant RNAs with complementary 5' and 3' termini were shown to competitively interfere with the replication of wild-type mRNA and to bind the viral RNA polymerase VP1 as efficiently as wild-type RNA. These results indicate that the single-strand nature of the 3' end of rotavirus mRNA is essential for efficient dsRNA synthesis and that the specific binding of the RNA polymerase to the mRNA template is required but not sufficient for the synthesis of minus-strand RNA.     

The processing of DNA ends at double-strand breaks during homologous recombination: different roles for the two ends

We have investigated the role of DNA ends during gap repair by homologous recombination. Mouse cells were transfected with a gapped plasmid carrying distinctive ends: on one side mouse LINE-1 repetitive sequences (L1Md-A2), and on the other rat LINE-1 sequences (L1Rn-3). The gap could be repaired by homologous recombination with endogenous mouse genomic LINE-1 elements, which are on average 95% and 85% homologous to L1Md-A2 and L1Rn-3 ends, respectively. Both L1Md-A2 and L1Rn-3 ends were found to initiate gap repair with equal efficiency. However, there were two types of gap repair products--precise and imprecise--the occurrence of which appears to depend on which end had been used for initiation and thus which end was left available for subsequent steps in recombination. These results, together with sequence analysis of recombinants obtained with plasmids having either mouse or rat LINE-1 sequences flanking the gap, strongly suggest that the two DNA ends played different roles in recombinational gap repair. One end was used to initiate the gap repair process, while the other end was involved at later steps, in the resolution of the recombination event.     

Role of partner homology in DNA recombination. Complementary base pairing orients the 5'-hydroxyl for strand joining during Flp site-specific recombination

Absolute homology between partner substrates within the strand exchange region is an essential requirement for recombination mediated by the yeast site-specific recombinase Flp. Using combinations of specially designed half- and full-site Flp substrates, we demonstrate that the strand joining step of recombination is exquisitely sensitive to spacer homology. At each exchange point, 2-3 spacer nucleotides adjacent to the nick within the cleaved strand of one substrate must base pair with the corresponding segment of the un-nicked strand from the second substrate for efficient strand joining in the recombinant mode. In accordance with the "cis-activation/trans-nucleophilic attack" model for each of the two transesterification steps of Flp recombination (strand cleavage and strand joining), we propose that the limited strand pairing orients the DNA-nucleophile (5'-hydroxyl) for attack on its target diester (3'-phosphotyrosyl-Flp). During one round of recombination, 4-6 terminal base pairs of the spacer (2-3 base pairs at each spacer end) must unpair, following strand cleavage, within a DNA substrate and pair with the partner substrate prior to strand union. In this model, the extent of branch migration of the covalently closed Holliday intermediate is limited to the central core of the spacer. The templated positioning of reactive nucleic acid groups (which is central to the model) may be utilized by other recombination systems and by RNA splicing reactions.     

Vaccinia DNA topoisomerase I: evidence supporting a free rotation mechanism for DNA supercoil relaxation

The Vaccinia type I topoisomerase catalyzes site-specific DNA strand cleavage and religation by forming a transient phosphotyrosyl linkage between the DNA and Tyr-274, resulting in the release of DNA supercoils. For type I topoisomerases, two mechanisms have been proposed for supercoil release: (I) a coupled mechanism termed strand passage, in which a single supercoil is removed per cleavage/religation cycle, resulting in multiple topoisomer intermediates and late product formation, or (2) an uncoupled mechanism termed free rotation, where multiple supercoils are removed per cleavage/religation cycle, resulting in few intermediates and early product formation. To determine the mechanism, single-turnover experiments were done with supercoiled plasmid DNA under conditions in which the topoisomerase cleaves predominantly at a single site per DNA molecule. The concentrations of supercoiled substrate, intermediate topoisomers, and relaxed product vs time were measured by fluorescence imaging, and the rate constants for their interconversion were determined by kinetic simulation. Few intermediates and early product formation were observed. From these data, the rate constants for cleavage (0.3 s(-1)), religation (4 s(-1)), and the cleavage equilibrium constant on the enzyme (0.075) at 22 degrees C are in reasonable agreement with those obtained with small oligonucleotide substrates, while the rotation rate of the cleaved DNA strand is fast (approximately 20 rotations/s). Thus, the average number of supercoils removed for each cleavage event greatly exceeds unity (delta n = 5) and depends on kinetic competition between religation and supercoil release, establishing a free rotation mechanism. This free rotation mechanism for a type I topoisomerase differs from the strand passage mechanism proposed for the type II enzymes.     

A parallel DNA triplex as a model for the intermediate in homologous recombination

Parallel DNA triplexes considered to be putative intermediates in homologous recombination, are studied by means of theoretical conformational analysis. These triplexes are denoted as the R-form DNA. Two types of triplexes are analyzed: extended R-form DNA, modeling the triple standard structure, created transiently in the presence of recombination proteins (e.g. RecA); and collapsed R-form, obtained after deproteinization. These structures are stereochemically possible for any arbitrary sequence and have the following properties: (1) the third, R-strand, is parallel to the identical duplex strand and is located in the major groove of the duplex; (2) positions of all four bases in the R-strand are nearly isomorphic; (3) the proposed triplets are consistent with the chemical modification data for deproteinized DNA; we suggest, however, that they are the same in the RecA-DNA complex as well. Since the patterns of charges on each base of the R-strand are strictly complementary to the charges of the homologous Watson-Crick (WC) pair in the major groove, we propose that the selection of the homologous sequence may occur through these complementary electrostatic interactions (electrostatic recognition code). We demonstrate that in the collapsed triplex with a rise of about 3.4 A, the bases from the third R-strand can be inclined and interact with two WC base-pairs simultaneously, which could lead to recognition errors. These mispairings are unlikely in the extended triplex. Therefore, we speculate that a functional role of the extended and underwound DNA structure, transiently formed in the complex with RecA protein, is to obviate such errors and increase the stringency of recognition. In other words, RecA plays the role of a DNA chaperone facilitating the recognition of the single stranded DNA and the duplex. Finally, we show that the proposed isomorphic triplets are conformationally advantageous for strand exchange.     

Attributions for negative life events and depression: The role of perceived control.

Although perceptions of control occupied a central role in the development of learned helplessness theory, recent helplessness research has not considered controllability judgments when relating attributions to depression. Supporting the importance of this construct, the research discussed in this article found evidence that judgments of control interact with other attributions in predicting depression. Specifically, in a prospective study of stress and well-being in adolescence, internal, stable, and global attributions for negative events attributed to uncontrollable causes were found to be positively related to increases in depression (as predicted by the reformulated helplessness theory), but internal and global attributions for negative events attributed to controllable causes were found to be inversely related to increases in depression. The discussion considers the implications of the findings for understanding the nature of the relation between attributions for naturally occurring life events and depression. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Non-disjunction of chromosome 21 in maternal meiosis I: evidence for a maternal age-dependent mechanism involving reduced recombination

Over 300 cases of trisomy 21 were analyzed to characterize the causes of maternal non-disjunction and to evaluate the basis for maternal age-dependent trisomy 21. We confirmed the observation that recombination along 21q is reduced among non-disjoined chromosomes 21 and further demonstrated that the alterations in recombination are restricted to meiosis I origin. Analysis of the crossover distribution indicates that reduction in recombination is not due simply to failure of pairing and/or absence of recombination in a proportion of cases. Instead, we observed an increase in both zero- and one-exchange events among trisomy 21-generating meioses suggesting that an overall reduction in recombination may be the underlying cause of non-disjunction. Lastly, we observed an age-related reduction in recombination among the meiosis I cases, with older women having less recombination along 21q than younger women. Thus, reduced genetic recombination may be responsible, at least in part, for the association between advancing maternal age and trisomy 21.     

Homologous recombination of a flanking repeat gene cluster is a mechanism for a common contiguous gene deletion syndrome

Smith-Magenis syndrome (SMS), caused by del(17)p11.2, represents one of the most frequently observed human microdeletion syndromes. We have identified three copies of a low-copy-number repeat (SMS-REPs) located within and flanking the SMS common deletion region and show that SMS-REP represents a repeated gene cluster. We have isolated a corresponding cDNA clone that identifies a novel junction fragment from 29 unrelated SMS patients and a different-sized junction fragment from a patient with dup(17)p11.2. Our results suggest that homologous recombination of a flanking repeat gene cluster is a mechanism for this common microdeletion syndrome.     

Molecular mechanism of morphogenesis: a theory of locational DNA

A molecular mechanism of reading of positional information by cells in morphogenesis and regeneration is proposed. It permits to translate the genome information into three-dimensional form of an organism. In the mechanism, a new fraction of DNA, so called "location DNA" is used which is suggested as a substitution instead of a former "egoistic" DNA. Domains, that are formed by this DNA and packed by lipid-containing bridges, are selectively unpacking in the gradient of inductor, the concentration of which positively correlates with the production of free radicals in the cells. Free radicals induce a selective destruction of lipid bridges which are variable in their resistance to the oxidative destruction. Hence the domains of location DNA are selectively decomactizing and activiting after bridge's elimination. In this way, a reading of positional information is performed. An epigenetic memory concerning the cellular determination state, that was already achieved, is based on the so called process of triplexation, the essence of which is a triplex formation, between signal RNA molecule and nascent double stranded DNA. A triplex is formed during the lagging strand synthesis or in the course of DNA repair synthesis. A telomeric element of postmitotic neurons, so called chronomere, assists to the organism in measuring of the flow of biological time, while chronomere length is an indicator of biological age of the organism.     

Reflectivity and learning from aversive events: Toward a psychological mechanism for the syndromes of disinhibition.

E. E. Gorenstein and J. P. Newman (1980) proposed that poorly modulated responding for reward is the common diathesis underlying disinhibited behavior in several traditionally distinct person categories: psychopathy, hysteria, early onset alcoholism, childhood hyperactivity, and nonpathological impulsivity (e.g., extraversion). The authors extend this proposal by theorizing a psychological mechanism that highlights relations among disinhibition, reflection, and failure to learn from aversive feedback. The hypothesized mechanism is presented as 4 generic stages of response modulation: the dominant response set, the reaction to an aversive event, the subsequent behavioral adaptation, and the immediate and long-term consequences of reflection, or the lack thereof. The mechanism has implications for disinhibited individuals' impulsivity and provides a point of departure to study factors responsible for similarities and differences among these syndromes. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The electronic clipboard: an automated system for accurately recording events during a cardiac arrest

An electronic clipboard has been developed which is simple to used and allows for the accurate timing and recording of events during a cardiac arrest. A flowsheet made of translucent 8 1/2 inches by 11 inches paper is clipped to a plastic board. Along the left margin of the flowsheet is a list of common events and therapies which should be recorded during a cardiac arrest (ie, cardiac rhythm, defibrillation, intubation, intravenous (IV) therapy, medications, etc). Across the page next to each category are columns of boxes, illuminated from below automatically by bars of light from the clipboard at one-minute intervals. Comparison of the events recorded during the first 20 minutes of cardiac resuscitatioon in an emergency department in 18 patients using the electronic clipboard method versus 18 randomly selected, matched patients with conventional recording methods demonstrated the superiority of the newer technique. More total events were recorded with the electronic clipboard (P less than 0.005), especially in the following categories: use of cardiopulmonary resuscitation Thumper, cardiac rhythm, defibrillation, sampling of arterial blood gases, IV insertion, and use of medications. The flowsheet is a superb instrument for reconstructing events for scientific analysis, legal documentation, and education.     

Dissociative effects of elaboration on memory of enacted and non-enacted events: a case of a negative effect

One experiment compared the effect of elaboration on enacted and non-enacted events. The commands were either presented in a basic form (e.g., "wave your hands") or in an enriched form. The commands were enriched by adding statements to the commands of how to perform the actions (e.g., "wave your hands as a conductor"). Free- and cued-recall data showed elaboration to have a dissociative effect on enacted and non-enacted events. Memory for the non-enacted events benefited from enrichment, whereas simple enacted events were remembered to a higher extent than complex enacted events. Lack of benefit from elaboration on memory of enacted events is suggested to be due to enactment leading to a sufficient degree of item-specific processing, and a negative effect of elaboration is suggested to occur when the way of manipulating item complexity decreases the familiarity of the actions. Familiarity ratings of the items by two independent groups of subjects supported this interpretation.     

Mood reactivity to daily negative events in early adolescence: Relationship to risk for psychopathology.

Emotional responses to negative daily experiences in young adolescents may provide important clues to the development of psychopathology, but research is lacking. This study assessed momentary mood reactivity to daily events as a function of risk profile in a school sample, ages 11-14. High-risk (HR, n = 25) and low-risk (LR, n = 106) subgroups completed frequent self-reports of mood and events for 5 days. HR adolescents reported more negative events involving family and peers. Multilevel modeling results showed that negative events, especially if stressful, were associated with increased negative and decreased positive affects, with heightened responses in HR adolescents. HR adolescents with greater stress over the last 3 months showed additional increases in depressed mood following negative events. Altered reactivity to and dysfunctional appraisals of daily events may link adolescent risk profiles to later mental health problems. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Amnesia for discrete events in rats with fornicotomies: Effects of interference on spatial alternation performance.

12 male hooded Long-Evans rats were divided into groups for either a control operation or a lesion of the fimbria-fornix. Ss were trained in several versions of a barpress, spatial alternation task. In free operant conditions across 5 experiments, Ss with fimbria-fornix lesions showed adequate alternation performance when the bars were relatively separated by placement either at the ends of an alley or at the ends of the arms of a T-maze, but they were impaired when bars were adjacent. Lesioned Ss also failed to alternate after interpolation of either baffles or a 10-sec delay in the stem, both of which resulted in intervening turning responses before each choice. Results are accounted for by a failure to discriminate memories of relevant, discrete events from those of similar, intervening events. (41 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Memory for positive versus negative life events: Theories for the differences between happy and unhappy persons.

Three studies examined the following hypotheses for the relation of subjective well-being (SWB) with memory for positive versus negative life events: (1) differences in retrieval mood, (2) the incidence of positive and negative events, (3) the interpretation of events, and (4) frequency of rehearsal. In Studies 1 (n?=?420) and 2 (n?=?94), the partial correlation of retrieval mood with recall, controlling for SWB, was trivial, suggesting that mood had little or no effect on recall. Endorsement frequencies of positive minus negative concrete events and interpretive events on checklists in Studies 2 and 3 each correlated with SWB (ps?