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.

Dinocyst biochronology and palynofacies : inferred systems tract character of Miocene sequences from New Jersey mid-Atlantic transect (ODP legs 174A and 174AX) /

One of the main objectives of the mid-Atlantic transect is to improve dating resolution of sequences and unconfonnity surfaces. Dinoflagellate cysts from two Ocean Drilling Program boreholes, the onshore Leg 174AX Ocean View Site and Leg 174A continental shelf Site 1071, are used to provide age estimates for sequences and unconfonnities fonned on the New Jersey continental margin during the Miocene epoch. Despite the occasional lack of dinocysts in barren and oxidized sections, dinocyst biochronology still offers greater age control than that provided by other microfossils in marginal marine environments. An early Miocene to late Miocene chronology based on ages detennined for the two study sites is presented. In addition, .palynofacies are used to unravel the systems tract character of the Miocene sequences and provide insight into the effects of taphonomy and preservation of palynomorphs in marginal marine and shelf environments under different ~ea level conditions. More precise placement of maximum flooding surfaces is possible through the identification of condensed sections and palynofacies shifts can also reveal subaerially exposed sections and surfaces not apparent in seismic or lithological analyses. The problems with the application of the pollen record in the interpretation of Miocene climate are also discussed. Palynomorphs provide evidence for a second-order lowering of sea level during the Miocene, onto which higher order sea level fluctuations are super-imposed. Correlation of sequences and unconfonnities is attempted between onshore boreholes and from the onshore Ocean View borehole to offshore Site 1071.

Buddhism and the earth : environmental thought in early Buddhist philosophy

Introduction Fundamental to the philosophy of Buddhism, is the insight that there is "unsatisfactohness" (dukkha) in the world and that it can be eliminated through the practice of the Noble Eight Fold Path. Buddhism also maintains that the world as we experience and entities that exist are bereft of any substantiality. Instead existence is manifest through dependent origination. All things are conditional; nothing is permanent. However, inherent in this dependent existence is the interconnectedness of all beings and their subjection to the cosmic law of karma. Part of cultivating the Eight Fold path includes a deep compassion for all other living things, 'trapped' within this cycle of dependent origination. This compassion or empathy (karuna) is crucial to the Buddhist path to enlightenment. It is this emphasis on karuna that shows itself in Mahayana Buddhism with respect to the theory of the boddhisatva (or Buddha-to-be) since the boddhisatva willingly postpones his/her own enlightenment to help others on the same path. One of the ramifications of the theory of dependent origination is that there is no anthropocentric bias placed on humans over the natural world. Paradoxically the doctrine of non-self becomes an ontology within Buddhism, culminating in the Mayahana realization that a common boundary exists between samsara and nirvana. Essential to this ontology is the life of dharma or a moral life. Ethics is not separated from ontology. As my thesis will show, this basic outlook of Buddhism has implications toward our understanding of the Buddhist world-view with respect to the current human predicament concerning the environment. While humans are the only ones who can 4 attain "Buddhahood", it is because of our ability to understand what it means to follow the Eight fold path and act accordingly. Because of the interconnectedness of all entities {dharmas), there is an ontological necessity to eliminate suffering and 'save the earth' because if we allow the earth to suffer, we ALL suffer. This can be understood as an ethical outlook which can be applied to our interaction with and treatment of the natural environment or environment in the broadest sense, not just trees plants rocks etc. It is an approach to samsara and all within it. It has been argued that there is no ontology in Buddhism due to its doctrine of "non-self". However, it is a goal of this thesis to argue that there does exist an original ontology in Buddhism; that according to it, the nature of Being is essentially neither "Being nor non-being nor not non-being" as illustrated by Nagarjuna. Within this ontology is engrained an ethic or 'right path' (samma marga) that is fundamental to our being and this includes a compassionate relationship to our environment. In this dissertation I endeavour to trace the implications that the Buddhist worldview has for the environmental issues that assail us in our age of technology. I will explore questions such as: can the Buddhist way of thinking help us comprehend and possibly resolve the environmental problems of our day and age? Are there any current environmental theories which are comparable to or share common ground with the classical Buddhist doctrines? I will elucidate some fundamental doctrines of early Buddhism from an environmental perspective as well as identify some comparable modern environmental theories such as deep ecology and general systems theory, that seem to share in the wisdom of classical Buddhism and have much to gain from a deeper appreciation of Buddhism.

Identification of novel retinoid receptors and their roles in vertebrate and invertebrate nervous systems

In vertebrates, signaling by retinoic acid (RA) is known to play an important role in embryonic development, as well as organ homeostasis in the adult. In organisms such as adult axolotls and newts, RA is also important for regeneration of the CNS, limb, tail, and many other organ systems. RA mediates many of its effects in development and regeneration through nuclear receptors, known as retinoic acid receptors (RARs) and retinoid X receptors (RXRs). This study provides evidence for an important role of the RA receptor, RAR~2, in ,( '. regeneration ofthe spinal cord and tail of the adult newt. It has previously been proposed that the ability of the nervous system to regenerate might depend on the presence or absence of this RAR~2 isoform. Here, I show for the very first time, that the regenerating spinal cord of the adult newt expresses this ~2 receptor isoform, and inhibition of retinoid signaling through this specific receptor with a selective antagonist inhibits tail and spinal cord regeneration. This provides the first evidence for a role of this receptor in this process. Another species capable of CNS ~~generation in the adult is the invertebrate, " Lymnaea stagnalis. Although RA has been detected in a small number of invertebrates (including Lymnaea), the existence and functional roles of the retinoid receptors in most invertebrate non-chordates, have not been previously studied. It has been widely believed, however, that invertebrate non-chordates only possess the RXR class of retinoid receptors, but not the RARs. In this study, a full-length RXR cDNA has been cloned, which was the first retinoid receptor to be discovered in Lymnaea. I then went on to clone the very first full-length RAR eDNA from any non-chordate, invertebrate species. The functional role of these receptors was examined, and it was shown that normal molluscan development was altered, to varying degrees, by the presence of various RXR and RAR agonists or antagonists. The resulting disruptions in embryogenesis ranged from eye and shell defects, to complete lysis of the early embryo. These studies strongly suggest an important role for both the RXR and RAR in non-chordate development. The molluscan RXR and RAR were also shown to be expressed in the adult, nonregenerating eNS, as well as in individual motor neurons regenerating in culture. More specifically, their expression displayed a non-nuclear distfibution, suggesting a possible non-genomic role for these 'nuclear' receptors. It was shown that immunoreactivity for the RXR was present in almost all regenerating growth cones, and (together with N. Farrar) it was shown that this RXR played a novel, non-genomic role in mediating growth cone turning toward retinoic acid. Immunoreactivity for the novel invertebrate RAR was also found in the regenerating growth cones, but future work will be required to determine its functional role in nerve cell regeneration. Taken together, these data provide evidence for the importance of these novel '. retinoid receptors in development and regeneration, particularly in the adult nervous system, and the conservation of their effects in mediating RA signaling from invertebrates to vertebrates.