Studies of Gene Structure: I. Expression of Human α-Globin Genes in COS Cells. II. Isolation and Characterization of the Myosin Light Chain Genes from Drosophila melanogaster

The first part of this thesis describes the establishment of COS cells as an effective host for the replication and expression of genes cloned into plasmids containing the SV40 viral replication origin. The human α-globin gene was cloned into a pBR322 derivative which contained a 311 base pair SV40 fragment. These plasmids were replicated to high copy number. The α-globin gene was transcribed to produce high levels of RNA which was indistinguishable from human α-globin mRNA. Deletion mutants were generated which defined the region from 55 to 87 base pairs upstream from the mRNA cap site as necessary for high levels of transcription of this gene. This region includes the conserved sequence CCAAT found in a similar position upstream from many eukaryotic genes. The second part of this thesis describes the cloning of muscle specific genes from Drosophila melanogaster and a detailed analysis of the two myosin light chain genes. A genomic library was screened with RNA isolated from the late pupal stage of development. Recombinant DNA clones which mapped to 24 independent locations were identified. The clones containing the myosin light chain genes were identified from within this group. A cDNA library was generated from late pupal RNA. Clones homologous to each of the myosin light chains were identified and their cloned inserts were sequenced. Their classification as Drosophila myosin light chain genes was established by comparing the derived amino acid sequence to the protein sequence of the chicken myosin light chains. Each myosin light chain is single copy. The alkali myosin light chain maps to the chromosomal locus 98B. It hybrid selects RNA which may be translated in vitro to yeild several polypeptides of molecular weight 18,000 to 19,000 daltons. The gene for myosin light chain-2 maps to the chromosomal locus 99E and is shown to encode two proteins of apparent molecular weights 26,000 and 17,000 daltons. Each protein has a putative divalent cation binding domain.</p

Developmental variations in the splicing pattern of transcripts from the Drosophila gene encoding myosin alkali light chain result in different carboxyl-terminal amino acid sequences

The total sequence of the Drosophila melanogaster gene encoding the myosin light chain dissociated by alkali (MLC-ALK) has been determined. By sequence comparisons with an MLC-ALK cDNA clone and by S1 nuclease analyses, the pattern of introns and exons within the gene has been deduced. There are multiple polyadenylylation signals that can account for most of the observed heterogeneity in the lengths of mRNAs. In the 3' half of the gene, there are two alternative splicing patterns which result in mRNAs that translate to give proteins with two alternative 14 amino acid carboxyl-terminal sequences. There is developmental regulation of the selection of the above splicing sites. One splicing pattern produces an mRNA that translates into a protein used for both larval and adult musculature, whereas the other splicing pattern is used for the latter stage only

Hippocampal–anterior thalamic pathways for memory: uncovering a network of direct and indirect actions

This review charts recent advances from a variety of disciplines that create a new perspective on why the multiple hippocampal–anterior thalamic interconnections are together vital for human episodic memory and rodent event memory. Evidence has emerged for the existence of a series of parallel temporal–diencephalic pathways that function in a reciprocal manner, both directly and indirectly, between the hippocampal formation and the anterior thalamic nuclei. These extended pathways also involve the mammillary bodies, the retrosplenial cortex and parts of the prefrontal cortex. Recent neuropsychological findings reveal the disproportionate importance of these hippocampal–anterior thalamic systems for recollective rather than familiarity-based recognition, while anatomical studies highlight the precise manner in which information streams are kept separate but can also converge at key points within these pathways. These latter findings are developed further by electrophysiological stimulation studies showing how the properties of the direct hippocampal–anterior thalamic projections are often opposed by the indirect hippocampal projections via the mammillary bodies to the thalamus. Just as these hippocampal–anterior thalamic interactions reflect an interdependent system, so it is also the case that pathology in one of the component sites within this system can induce dysfunctional changes to distal sites both directly and indirectly across the system. Such distal effects challenge more traditional views of neuropathology as they reveal how extensive covert pathology might accompany localised overt pathology, and so impair memory

Mammillothalamic disconnection alters hippocampocortical oscillatory activity and microstructure: Implications for diencephalic amnesia

Diencephalic amnesia can be as debilitating as the more commonly known temporal lobe amnesia, yet the precise contribution of diencephalic structures to memory processes remains elusive. Across four cohorts of male rats, we used discrete lesions of the mammillothalamic tract to model aspects of diencephalic amnesia and assessed the impact of these lesions on multiple measures of activity and plasticity within the hippocampus and retrosplenial cortex. Lesions of the mammillothalamic tract had widespread indirect effects on hippocampo-cortical oscillatory activity within both theta and gamma bands. Both within-region oscillatory activity and cross-regional synchrony were altered. The network changes were state-dependent, displaying different profiles during locomotion and paradoxical sleep. Consistent with the associations between oscillatory activity and plasticity, complementary analyses using several convergent approaches revealed microstructural changes, which appeared to reflect a suppression of learning-induced plasticity in lesioned animals. Together, these combined findings suggest a mechanism by which damage to the medial diencephalon can impact upon learning and memory processes, highlighting an important role for the mammillary bodies in the co-ordination of hippocampo-cortical activity

Cannabidiol regulation of emotion and emotional memory processing: relevance for treating anxiety-related and substance abuse disorders

Learning to associate cues or contexts with potential threats or rewards is adaptive and enhances survival. Both aversive and appetitive memories are therefore powerful drivers of behaviour, but the inappropriate expression of conditioned responding to fear- and drug-related stimuli can develop into anxiety-related and substance abuse disorders respectively. These disorders are associated with abnormally persistent emotional memories and inadequate treatment, often leading to symptom relapse. Studies show that cannabidiol, the main non-psychotomimetic phytocannabinoid found in Cannabis sativa, reduces anxiety via 5-HT1A and (indirect) cannabinoid receptor activation in paradigms assessing innate responses to threat. There is also accumulating evidence from animal studies investigating the effects of cannabidiol on fear memory processing indicating that it reduces learned fear in paradigms that are translationally relevant to phobias and post-traumatic stress disorder. Cannabidiol does so by reducing fear expression acutely and by disrupting fear memory reconsolidation and enhancing fear extinction, both of which can result in a lasting reduction of learned fear. Recent studies have also begun to elucidate the effects of cannabidiol on drug memory expression using paradigms with translational relevance to addiction. The findings suggest that cannabidiol reduces the expression of drug memories acutely and by disrupting their reconsolidation. Here, we review the literature demonstrating the anxiolytic effects of cannabidiol before focusing on studies investigating its effects on various fear and drug memory processes. Understanding how cannabidiol regulates emotion and emotional memory processing may eventually lead to its use as a treatment for anxiety-related and substance abuse disorders

An oral health literacy intervention for Indigenous adults in a rural setting in Australia

BACKGROUND: Indigenous Australians suffer substantially poorer oral health than their non-Indigenous counterparts and new approaches are needed to address these disparities. Previous work in Port Augusta, South Australia, a regional town with a large Indigenous community, revealed associations between low oral health literacy scores and self-reported oral health outcomes. This study aims to determine if implementation of a functional, context-specific oral health literacy intervention improves oral health literacy-related outcomes measured by use of dental services, and assessment of oral health knowledge, oral health self-care and oral health- related self-efficacy. METHODS/DESIGN: This is a randomised controlled trial (RCT) that utilises a delayed intervention design. Participants are Indigenous adults, aged 18 years and older, who plan to reside in Port Augusta or a nearby community for the next two years. The intervention group will receive the intervention from the outset of the study while the control group will be offered the intervention 12 months following their enrolment in the study. The intervention consists of a series of five culturally sensitive, oral health education workshops delivered over a 12 month period by Indigenous project officers. Workshops consist of presentations, hands-on activities, interactive displays, group discussions and role plays. The themes addressed in the workshops are underpinned by oral health literacy concepts, and incorporate oral health-related self-efficacy, oral health-related fatalism, oral health knowledge, access to dental care and rights and entitlements as a patient. Data will be collected through a self-report questionnaire at baseline, at 12 months and at 24 months. The primary outcome measure is oral health literacy. Secondary outcome measures include oral health knowledge, oral health self-care, use of dental services, oral health-related self-efficacy and oral health-related fatalism. DISCUSSION: This study uses a functional, context-specific oral health literacy intervention to improve oral health literacy-related outcomes amongst rural-dwelling Indigenous adults. Outcomes of this study will have implications for policy and planning by providing evidence for the effectiveness of such interventions as well as provide a model for working with Indigenous communities.Eleanor J. Parker, Gary Misan, Alwin Chong, Helen Mills, Kaye Roberts-Thomson, Alice M. Horowitz and Lisa M Jamieso

Comparing Notes: Recording and Criticism

This chapter charts the ways in which recording has changed the nature of music criticism. It both provides an overview of the history of recording and music criticism, from the advent of Edison’s Phonograph to the present day, and examines the issues arising from this new technology and the consequent transformation of critical thought and practice

The 'long' 16th century : a key period of animal husbandry change in England

Although many historians have extensively discussed the agricultural history of England between the Late Middle Ages and the Modern Era, this period of crucial changes has received less attention by archaeologists. In this paper, zooarchaeological evidence dated between the Late Middle Ages and the Early Modern period is analysed to investigate changes in animal husbandry during the ‘long’ sixteenth century. The size and shape of the main domestic animals (cattle, sheep, pig and chicken) is explored through biometrical data and discussed in line with evidence of taxonomic frequencies, ageing and sex ratios. Data from 12 sites with relevant chronologies and located in different areas of the country are considered. The results show that, although a remarkable size increase of animals occurred in England throughout the post-medieval period, much of this improvement occurred as early as the sixteenth century. The nature and causes of such improvement are discussed, with the aim of understanding the development of Early Modern farming and the foundations of the so-called Agricultural Revolution