Thesis (Ph. D.)--University of Chicago, Dept. of Pathology. / Includes bibliographical references. Also available on the Internet.

Transfer RNA genes require "positive" 5'-flanking sequences to direct efficient transcription. In order to delimit the modulatory sequences present in the 5'-flank of a Drosophila tRNA Val₄ gene, an extensive series of deletion mutants was constructed and end-points determined by dideoxy sequencing. The mutants were transcribed in vitro in a Drosophila Schneider II cell-free extract. Twenty nucleotides of the 5'-flank immediately adjacent to the mature tRNA coding sequence were required for transcription. Negative modulatory sequences were contained between positions -20 to -30 and -45 to -70 relative to the mature coding sequence. The -45 to -70 sequence shares homology with inhibitory sequences previously described in the 5'-flank of tRNA genes, except that this sequence was significantly larger in length. Sequences contained between positions -38 and -45 act as positive modulatory sequences which enhance the level of transcription. In addition, a Transcription Modulation Element (TME) was identified between nucleotides -33 and -38. The TME was also found in the 5'-flanking sequences of various other tRNA genes and preliminary data suggests that it enhances transcription efficiency through its position relative to the D and T control regions / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Briefly, some findings in my research are as follows: (1) The genetic variants of the CA repeats in IGF1 promoter 1 can affect the activity of promoter 1, and the CA repeat showed a suppressive effect on the activity of the promoter 1 of IGF1 gene. EMSA results have shown that the CA repeats could bind to certain nuclear protein. (2) The SNPs T/C (rs5742612) and T/A (rs2288377) can also affect the activity of the promoter 1 in IGF1 gene, and the activity of C-A haplotype is significantly higher than that of T-T haplotype. EMSA results have shown that the SNP T/A (rs2288377) could bind to certain nuclear protein. (3) I developed the new dual reporter assay method to investigate the transactivation interaction between the SNP T/G (rs2071430) and C/A (rs17000900) in the MxA promorer. This new method can not only improve the detection limit for small difference between haplotypes, but also calculate the model of transactivation effect between these two SNPs. The results were better than those of traditional method, and it gave a clear-cut demonstration of the effect of interaction between these two SNPs on the activity of MxA promoter. / In addition, in the IGF1 study, the core promoter region of promoter 2 was identified through 5' deletion mutagenesis methods. Moreover, a cell-type specific mechanism of bidirectional activation of promoter was found. / Recently, more and more studies focus on gene function with the completion of the Human Genome Project. It is well known that polymorphism of human genome sequence is a common phenomenon in the human population. Specially, a lot of genetic polymorphisms, including single nucleotide polymorphisms (SNPs) and microsatellites, have been reported in the regulatory region of many genes. However, the effects of most of these genetic polymorphisms on gene expression are still unknown. The polymorphisms in the promoter can play an important role in the gene regulation. For example, some SNPs located in the transcription factor binding site (TFBS) can affect gene transcription. So, it is very necessary to directly study the effect of genetic variants on promoter transactivation activities. In this study, we studied the effect of genetic polymorphisms on gene expression through reporter gene assay, electrophoretic mobility shift assay (EMSA), and so on. And the candidate genes include insulin-like growth factor 1 (IGF1) and myxovirus resistence 1 (MxA). Some SNPs and microsatellites have been reported in the promoters of these genes. In our previous researches, we focused on the study of the association between these polymorphisms and some diseases, and it was found that a few SNPs significantly associated with relevant diseases. Based on the previous results, in my project, I developed new functional assays and also improved existing methods to analyse the functional effect of these genetic variants of promoters on transactivation activities. / by Huang, Wei. / "March 2008." / Adviser: Nelson Leung Sang Tang. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1483. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 139-145). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

In unstimulated cells, the transcription factor NF-κB is held in the cytoplasm in an inactive state by IκB inhibitor proteins. Activation of NF--KB is mediated by signal induced degradation of IκBα via the ubiquitin proteasome-dependent pathway. Targeting the proteins for ubiquitin-mediated proteolysis is an irrevocable decision, and as such, the process needs to be highly specific and tightly regulated. This task is achieved by conjugation and deconjugation enzymes that act in a dynamic and coordinated mechanism. In a yeast two hybrid screen designed to identify proteins involved in IκBα signalling Ubch9 was found to interact with the N-terminal regulatory region of IκBα. Although Ubch9 is an enzyme homologous to E2 ubiquitin conjugating enzymes we have shown that is unable to form a thioester with ubiquitin but it is capable to form a thioester with the small ubiquitin-like protein SUMO- 1. To fully characterise the SUMO-1 modification reaction we have purified the proteins and cloned the genes encoding the SUMO-1 activating enzyme (SAEl/SAE2) and shown that it is homologous to enzymes involved in the activation of ubiquitin, Smt3p, the yeast SUMO-1 homologue, and Rublp/Nedd8, another ubiquitin-like protein. SUMO-1 is conjugated to target proteins by a pathway that is distinct from, but analogous to, ubiquitin conjugation. SUMO-1 was efficiently conjugated, both in vivo and in vitro, to IκBα on lysine 21, which is also utilised for ubiquitin modification. Thus, by blocking ubiquitination SUMO-1 modification acts antagonistically to generate a pool of IκBα resistant to proteasome-mediated degradation which consequently inhibits NF-κB dependent transcription activation. In view of several lines of similarity between NF-kB and p53, the involvement of SUMO-1 modification in the metabolism of the tumour supressor p53 was investigated. We have shown that p53 is modified by SUMO-1 at a single site, lysine 386 in the C-terminus of p53. Although p53 is regulated by ubiquitination, SUMO-1 and ubiquitin modification do not compete for the same lysine in p53. However, overexpression of SUMO-1 activates the transcriptional activity of wild type p53, but not K386R p53 where the SUMO-1 acceptor site has been mutated. A consensus sequence was obtained by comparison of the sequences surrounding the SUMO-1 acceptor lysine in proteins that have been shown to be modified by SUMO-1 and revealed a possible recognition site for SUMO-1 conjugation machinery. Tagging of proteins with SUMO-1 regulates transcriptional activation, either by interfering with subcellular location or with the ubiquitination pathway. The pathway may represent a novel target for drug development.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2004. / Härtill 4 uppsatser.

Thesis (M.A.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 132-144). Also available via the World Wide Web.

Rhizobia are Gram-negative, rod-shaped, soil bacteria with the ability to fix atmospheric nitrogen into ammonia as symbiont bacteroids within nodules of leguminous plant roots. Here, resident Rhizobium plasmids were studied as possible sources of components for the construction of a cloning vector for Rhizobium species.

The central investigative concern of this thesis is the culture of genetic counselling in Canada--a specialized health service providing information and assistance to families at risk from genetic disorders. In particular, this study describes the variability that exists in the way in which genetic counsellors view their profession and explores the factors that contribute to this variability. The genetic counsellors' training, division of roles, view of their responsibility toward clients, and the historical and contemporary context in which they practise contribute to understanding the "individuals' versions of the culture of [genetic counselling]'' (Hahn 1985:53). Three means of inquiry are utilized: extensive library research, responses from a Canada-wide questionnaire sent to genetic counsellors, and ethnographic interviews. The history of genetic counselling has been described as a series of changes from research to medicine to psychosocial concerns. Current definitions of adequate genetic care, however, stress attention to both the medical genetic and psychosocial aspects of genetic disorders suggesting that both a medical and a psychosocial paradigm now exist in genetic counselling. I argue that the association between these two paradigms is unclear, thus producing tension and ambiguity for practitioners. Genetic counsellors divide themselves into three groups--MD/MD-PhDs who are the central caregivers, PhD geneticists who are primarily involved in research, and nonMDs/nonPhDs such as nurses, social workers and master's level genetic associates who are often doing clinic-oriented clerical jobs. Nevertheless, indicative of the uneasy relationship between the paradigms of genetic counselling, considerable debate exists in the field about who should be providing genetic counselling. A further example of this paradigmatic tension concerns the implementation of genetic counselling goals, especially for physician geneticists, the central caregivers. In particular, genetic counsellors face the problematic task of "doing something" for their clients, making them aware of their options, without influencing the client's decision making. The contemporary context of genetic counselling is predominantly a medical one; although psychosocial concerns are acknowledged as important for adequate genetic care, the means for dealing with them are not clear. I suggest that the response to the underlying tensions in genetic counselling is ultimately an individually-constructed response, based on the individual practitioner's experience, interpretation of the facts, and notion of the boundaries of their responsibility in medicine. / Thesis / Master of Arts (MA)

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The most common inherited sensory disorder that affects I in 1 000 children is severe hearing loss. In developed countries, about a third of cases have a genetic origin, 80% of which are autosomal recessive forms (DFNB). Before 1993 few genes causing hearing loss had been identified, but since then a large number of genes related to this problem have been identified. Studies indicate that the DFNBI locus, located at position 13q11-12, contributes to 20% of all childhood deafness and may have a carrier rate as high as 2.8%. There are two genes linked to DFNB 1, GJB2 and GJB6, which are the major genetic cause of non-syndromic autosomal recessive deafness. GJB2 and GJB6 encode the connexin proteins connexin 26 and 30 (Cx26 and Cx30), respectively. The specific aim of this study was to determine the role of GJB2 and GJB6 in deafness within the South African population, since there are no published results involving South African patients with non-syndromic autosomal recessive deafness. This study therefore involved the identification of mutations within the coding region of the GJB2 and GJB6 genes in the South African population and the determination of their specific allele frequencies. Another aim of this study was to analyse the effectiveness of three single-strand conformation polymorphic (SSCP) gel electrophoresis systems in the detection of GJB2 mutations, for use in a standardised diagnostic program. A total of 44 families were recruited and divided into either the familial or sporadic study group, which consisted of 16 and 28 families, respectively. Control samples were also screened from 50 Caucasians and 50 Mixed Ancestry individuals collected from the general population. To achieve the aims of this study, polymerase chain reaction (PCR) amplification followed by automated DNA sequencing of the coding regions of GJB2 and GJB6 was performed. The three SSCP systems that were tested for their effectiveness in detecting mutations within the coding region of GJB2 included mini polyacrylamide, SSCP-urea and two buffer gel electrophoresis systems. In total, six previously reported mutations (35delG, 312de1l4, W24X, M34T, V37I and W44X), a novel mutation (N62I), and four benign polymorphisms (V27I, A40A, R127H and V153I) were detected in GJB2. In the GJB6 gene only the S199T polymorphism was observed. It was determined that the most common mutations found within the Caucasian and Mixed Ancestry populations of South Africa were 35delG and 312de1l4 of GJB2. An overall detection rate of 35.227% was achieved in non-syndromic autosomal recessive deafness amongst this patient cohort. It was also observed that none of the SSCP gel electrophoresis systems were effective at detecting all of the GJB2 mutations. This could change if the systems were specifically optimised for the cornmon mutations that were identified. This study therefore, provides information that can be used in the formulation of a screenmg program for non-syndromic autosomal recessive deafness specific to the South African population. Further research should be conducted involving other genes, in addition other population groups of South Africa to provide a more comprehensive genetic diagnostic and counselling tool. / AFRIKAANSE OPSOMMING: Die mees algemene oorerflike sensoriese steuring wat 1 in 1 000 kinders affekteer is ernstige gehoorverlies. In ontwikkelde lande het omtrent een-derde van die gevalle 'n genetiese oorsprong, waarvan 80% outosomaal resessiewe vorms is (DFNB). Tot en met 1993 is min gene wat gehoorverlies veroorsaak geïdentifiseer, maar sedertdien is 'n groot aantal gene gelokaliseer en verskeie is ook al gekloneer. Studies toon dat die DFNB 1 loci, wat in posisie 13q 11-12 gevind word, 20% van doofheid in kinders veroorsaak, en dit het 'n draer frekwensie van so hoog as 2.8%. Twee gene wat koppeling met DFNBI toon, GJB2 en GJB6, is die vernaamste genetiese oorsaak van nie-sindromise autosomaal resessiewe doofheid. GJB2 en GJB6 koder vir die connexin proteïne 26 en 30 (Cx26 en Cx30), onderskeidelik. Die spesifieke doel van hierdie studie is om die rol van GJR2 en GJB6 in doofheid binne die Suid- Afrikaanse populasie te bepaal, aangesien daar tans nog geen gepubliseerde resultate omtrent Suid- Afrikaanse pasiënte met nie-sindromiese outosomaal resessiewe doofheid is nie. Hierdie studie handel dus oor die identifikasie van mutasies wat binne die koderende areas van die GJR2 en GJB6 gene voorkom in die Suid-Afrikaanse populasie, asook oor die bepaling van hulle spesifieke alleel frekwensies. Verder het hierdie studie ten doelom die effektiwiteit van drie enkel-string konformasie polimorfisme (SSCP) gel-elektroforese metodes in die opsporing van GJB2 mutasies te analiseer met die oog op toekomstige gebruik in 'n gestandardiseerde diagnostiese program. Altesaam 44 families is ingesamel en gekategoriseer in familiële of sporadiese studie-groepe met 16 en 28 families onderskeidelik. Kontrole monsters van 50 Kaukasiese en 50 Gemengde Herkoms individule uit die algemene populasie is ook getoets. Om die doeleindes van die studie te bereik is PKR amplifikasie en outomatiese DNS volgordebepaling van die koderende area van GJB2 en GJR6 gedoen. Die drie SSCP sisteme wat getoets is vir hulle effektiwiteit in die identifisering van mutasies in die koderende area van GJB2 sluit in mini poli-akrielamied, urea en twee-buffer gel elektroforese sisteme. In totaal is ses gerapporteerde mutasies (35delG, 312de114, W24X, M34T, V37I en W44X), 'n nuwe mutasie (N62I), en vier onskadelike polimorfismes (V27I, A40A, R127H en V153I) opgespoor in GJB2, maar in GJB6 is net die S199T polimorfisme waargeneem. Uit die resultate kon afgelei word dat 35deiG en 312de114 van GJB2 die mees algemene mutasies binne die Kaukasiese en Gemengde Herkoms bevolkings van Suid Afrika is. Die total ontdekking standaard van 35.227%· vir nie-sindromise autosomaal resessiewe doofheid tussen herdie patient kohort was bereik. Verder is waargeneem dat geen van die SSCP gel elektroforese metodes effektief was om al die mutasies van GJB2 op te spoor nie. Die situasie kan egter verander as die sisteme spesifiek geoptimiseer word vir die algemene mutasies wat gevind is. Hierdie studie verskaf dus inligting wat gebruik kan word in die verskaffing van 'n diagnostise program vir nie-sindromise outosomaal resessiewe doofheid wat spesifiek is vir die Suid- Afrikaanse populasie. Verdere navorsing wat ander gene en ander populasie groepe van Suid-Afrika insluit, behoort egter uitgevoer te word om uiteindelik 'n meer uitgebreide genetiese diagnostiese en raadgewing diens daar te stel.

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy