Manipulation of adenovirus early region 1 rescue plasmids by homologous recombination in Saccharomyces cerevisiae

The manipulation of large (>10 kb) plasmid systems amplifies problems common to traditional cloning strategies. Unique or rare restriction enzyme recognition sequences are uncommon and very rarely located in opportunistic locations. Making site-specific deletions and insertions in larger plasmids consequently leads to multiple step cloning strategies that are often limited by time-consuming, low efficiency linker insertions or blunt-end cloning strategies. Manipulation ofthe adenovirus genome and the genomes ofother viruses as bacterial plasmids are systems that typify such situations. Recombinational cloning techniques based on homologous recombination in Saccharomyces cerevisiae that circumvent many ofthese common problems have been developed. However, these techniques are rarely realistic options for such large plasmid systems due to the above mentioned difficulties associated with the addition ofrequired yeast DNA replication, partitioning and selectable marker sequences. To determine ifrecombinational cloning techniques could be modified to simplify the manipulation of such a large plasmid system, a recombinational cloning system for the creation of human adenovirus EI-deletion rescue plasmids was developed. Here we report for the first time that the 1,456 bp TRP1/ARS fragment ofYRp7 is alone sufficient to foster successful recombinational cloning without additional partitioning sequences, using only slight modifications of existing protocols. In addition, we describe conditions for efficient recombinational cloning involving simultaneous deletion of large segments ofDNA (>4.2 kb) and insertion of donor fragment DNA using only a single non-unique restriction site. The discovery that recombinational cloning can foster large deletions has been used to develop a novel recombiliational cloillng technique, selectable inarker 'kilockouf" recombinational cloning, that uses deletion of a yeast selectable marker coupled with simultaneous negative and positive selection to reduce background transformants to undetectable levels. The modification of existing protocols as described in this report facilitates the use of recombinational cloning strategies that are otherwise difficult or impractical for use with large plasmid systems. Improvement of general recombinational cloning strategies and strategies specific to the manipulation ofthe adenovirus genome are considered in light of data presented herein.

Behavioural and neural correlates of emotion recognition as a function of psychopathic personality traits

Psychopathy is associated with well-known characteristics such as a lack of empathy and impulsive behaviour, but it has also been associated with impaired recognition of emotional facial expressions. The use of event-related potentials (ERPs) to examine this phenomenon could shed light on the specific time course and neural activation associated with emotion recognition processes as they relate to psychopathic traits. In the current study we examined the PI , N170, and vertex positive potential (VPP) ERP components and behavioural performance with respect to scores on the Self-Report Psychopathy (SRP-III) questionnaire. Thirty undergraduates completed two tasks, the first of which required the recognition and categorization of affective face stimuli under varying presentation conditions. Happy, angry or fearful faces were presented under with attention directed to the mouth, nose or eye region and varied stimulus exposure duration (30, 75, or 150 ms). We found that behavioural performance to be unrelated to psychopathic personality traits in all conditions, but there was a trend for the Nl70 to peak later in response to fearful and happy facial expressions for individuals high in psychopathic traits. However, the amplitude of the VPP was significantly negatively associated with psychopathic traits, but only in response to stimuli presented under a nose-level fixation. Finally, psychopathic traits were found to be associated with longer N170 latencies in response to stimuli presented under the 30 ms exposure duration. In the second task, participants were required to inhibit processing of irrelevant affective and scrambled face distractors while categorizing unrelated word stimuli as living or nonliving. Psychopathic traits were hypothesized to be positively associated with behavioural performance, as it was proposed that individuals high in psychopathic traits would be less likely to automatically attend to task-irrelevant affective distractors, facilitating word categorization. Thus, decreased interference would be reflected in smaller N170 components, indicating less neural activity associated with processing of distractor faces. We found that overall performance decreased in the presence of angry and fearful distractor faces as psychopathic traits increased. In addition, the amplitude of the N170 decreased and the latency increased in response to affective distractor faces for individuals with higher levels of psychopathic traits. Although we failed to find the predicted behavioural deficit in emotion recognition in Task 1 and facilitation effect in Task 2, the findings of increased N170 and VPP latencies in response to emotional faces are consistent wi th the proposition that abnormal emotion recognition processes may in fact be inherent to psychopathy as a continuous personality trait.

Polynuclear Ni(II) Complexes with Schiff Bases as Bridging Ligands: A Molecular Approach to Nanoscience

The initial employment of N-salicylidene-2-amino-5-chlorobenzoic acid (sacbH2) as bridging/chelating ligand in metal cluster chemistry has provided access to five new polynuclear NiII complexes with large nuclearities, unprecedented metal core topologies, and interesting magnetic properties. The obtained results are presented in two projects. The first project includes the investigation of the general Ni2+/RCO2-/sacbH2 reaction system (where R- = CH3-, But-, ButCH2-) in which the nature of the carboxylic acid was found to be of crucial importance, affecting enormously the nuclearity of the resulting complexes. The second project deals with the study of the general Ni2+/X-/sacbH2 reaction system (where X- = inorganic anions) under basic conditions, yielding new cluster compounds with molecular chain-like structures and ferromagnetic exchange interactions between the metal centers.