Chloroplast genomes of land plants show conservation of structure and gene arrangement. The spinach chloroplast genome is comprised of a covalently closed. circular DNA molecule of 150 kilobases and is typical of these plants. Approximately 20% of the proteins found in the spinach chloroplast are encoded by the chloroplast genome and translated on chloroplast ribosomes. The remainder are encoded on chromosomes in the nucleus, translated on cytoplasmic ribosomes and transported into the chloroplast. Spinach chloroplast DNA was isolated from crude 2 chloroplast preparations by a new method. Chloroplasts were lysed with alkaline sodium dodecyl sulfate, contaminating macromolecules precipitated with acidified potassium acetate and plastid DNA was purified by phenol:chloroform extraction and ethanol:ammonium acetate precipitation. The yield was approximately 50 ug chloroplast DNA per 100 grams leaf material. The DNA consisted of 10% circular molecules and 90% linear molecules. The chloroplast DNA was digested with restriction enzyme PstI and the fragments were cloned into the plasmid vector pUC9. Several recombinant plasmids were isolated and the chloroplast DNA inserts identified. The recombinant plasmid pRD105 containing the PstI #5 fragment was subjected to further investigation. The ClaI restriction sites of the PstI #5 fragment were mapped and the insert was subcloned into the plasmid vector pGEM4, which bears bacteriophage SP6 and T7 RNA polymerase promoter sequences.

DNA barcoding is a valuable tool for fish species identification by food regulators, however, it does not perform well when multiple species are present within the same food product. PCR cloning has high potential to be used in combination with DNA barcoding to overcome this challenge. The objective of this study was to examine the use of PCR cloning combined with DNA barcoding to identify fish in a mixed-species product that cannot be identified with standard DNA barcoding. A total of 15 fish ball mixtures were prepared with known amounts of Nile tilapia (Oreochromis niloticus), Pacific cod (Gadus macrocephalus), and walleye pollock (Gadus chalcogrammus). The fish balls underwent DNA extraction in triplicate, followed by DNA barcoding across the full barcode (655 bp) and SH-E mini-barcode (226 bp) of the cytochrome c oxidase subunit 1 (CO1) region. Samples that did not pass sequencing according to regulatory standards were further analyzed with PCR cloning. Full barcoding enabled identification of at least one species in 80% of the fish ball mixtures compared to 51% for minibarcoding. The results of PCR cloning with samples that did not pass DNA barcoding showed identification success rates of 61% for clones (54 of 90) that underwent full barcoding and 51% for clones (111 of 220) that underwent mini-barcoding. All fish balls made of just one species tested positive for that species (i.e., tilapia, cod, or pollock).. The combination of standard full barcoding and PCR cloning enabled identification of Nile tilapia in all 12 mixed-species fish balls and Pacific cod in 6 of 12 (50%) of mixed-species fish balls. In comparison, the combination of standard mini-barcoding and PCR cloning enabled identification of Nile tilapia in all 12 mixed-species fish balls and Pacific cod in 9 of 12 (75%) of mixed-species fish balls. Overall, the results of this study show that PCR cloning may be an effective method to identify certain fish in mixed-species products when standard DNA barcoding fails. However, additional research is needed to understand the limitations associated with primer bias.

Abstract not available

Copy of author's previously published work inserted. Bibliography: leaves 139-160.

Copy of author's previously published work inserted. / Bibliography: leaves 139-160. / 160, [10] leaves, [41] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001?

Interest in the mechanisms of homeo domain specificity led to a screen that identified Drosophila proteins able to bind a consensus homeo domain site. One clone isolated in this screen produced no homeo domain and was selected for further characterisation as a protein with an unknown DNA binding domain and the potential to interact with homeo domain proteins on the DNA. This thesis describes the characterisation of the Drosophila gene dead ringer ( dri ) corresponding to this clone. Isolation of overlapping cDNA clones and sequence analysis allowed the identification of a complete open reading frame in the dri message that gave a predicted protein of 901 amino acids. Database searches and multiple sequence alignment revealed a widely conserved motif in the Dri sequence that is found in proteins from organisms as diverse as yeast, nematodes, flies and humans. Biochemical analysis of the properties of this conserved motif revealed that it could function as a DNA binding domain when expressed in a fusion protein. The in vitro specificity of the Dri DNA binding domain was determined by selection and sequencing of target sites. The Dri consensus site obtained was strikingly similar to that of the Qfo class of homeo domains, although the sequence and predicted secondary structure of the Dri DNA binding domain do not resemble a homeo domain. Analysis of the developmental expression pattern of dri showed a ubiquitous maternal deposit gradually refined to localisation in the mesoderm at germ band extension, then further restriction to a diverse set of tissues including the salivary gland ducts, parts of the gut and a subset of the central nervous system. The phenotype of P - element insertion and deletion mutations of dri were identified as causing embryonic lethality preceded by a disruption of the hindgut and loss of Dri expression in the ring gland. The identification of the novel, conserved DNA - binding domain in Dead ringer offers an explanation for the regulatory activity of several important related proteins and presents an opportunity to use the advantages of the Drosophila model system to clarify the role of these proteins in transcriptional control. / Thesis (Ph.D.)--Departments of Biochemistry and Genetics, 1996.

Le démarrage de la traduction est un processus central dans toute cellule. L'étude des protéines assistant le ribosome pour réaliser cette étape, les facteurs de démarrage (Initiation Factors Ifs), permet d'obtenir des informations sur les mécanismes moléculaires complexes assurant la fidélité et l'efficacité du démarrage. La comparaison des jeux de facteurs protéiques dans les trois règnes du monde vivant a permis de mettre en évidence la présence de trois facteurs universellement conservés. Parmi ceux-ci, le facteur eucaryotique/archéen e/aIF5B, homologue au facteur bactérien IF2, stimule l'association des sous-unités ribosomales au même titre que chez les Bactéries. Néanmoins, l'universalité du facteur est limitée par l'absence d'interaction reportée entre le facteur e/aIF5B et l'ARNt initiateur alors que cette liaison est parfaitement caractérisée chez les Bactéries. Une partie de ce travail de thèse a permis d'étendre la similitude fonctionnelle entre les facteurs en mettant en évidence une liaison de l'ARNt initiateur méthionylé par le facteur e/aIF5B. Cette liaison présente des caractéristiques identiques à celle de l'ARNt initiateur méthionylé et formylé par le facteur bactérien IF2. Une deuxième partie du travail de thèse a concerné le facteur eIF3, le plus complexe du système de démarrage chez les Eucaryotes. Ce complexe de 13 sous-unités chez l'humain et de 5 sous-unités chez la levure n'a pas d'équivalent dans les autres domaines du vivant bien qu'il joue un rôle central et essentiel chez les Eucaryotes. La compréhension de ses fonctions est néanmoins fortement limitée par le manque d'information à l'échelle moléculaire sur les interactions entre les sous-unités le composant et avec ses autres facteurs partenaires. De plus, le facteur s'avère être impliqué dans de nombreux cancers, ce qui étend l'intérêt de son étude. Mon travail a permis de développer une bibliothèque de vecteurs permettant de coexprimer les différentes sous-unités ou des formes stabilisées des sous-unités du facteur eIF3 de levure chez la Bactérie Escherichia coli. La purification des sous-unités isolées et de différents sous-complexes nous permet d'envisager la résolution de la structure du facteur et de son organisation par une approche alliant la cristallographie et la microscopie électronique.

<p>ABSTRACT</p><p>Nontypable Haemophilus influenzae is a common cause of otitis, sinusitis and conjunctivitis. It is the most common bacterial pathogen associated with chronic obstructive pulmonary disease (COPD). Studies have shown that nonpathogenic Haemophilus haemolyticus are often mistaken for Haemophilus influenzae due to an absent hemolytic reaction on blood agar. Distinguishing H. haemolyticus from H. influenzae is important to prevent unnecessary antibiotic use, and to understand the role of H. influenzae in clinical infections. In this study, PCR-primers for amplifying 16S rDNA sequences were used to set up a method for distinguishing H. haemolyticus from H. influenzae. The aim was to use the method for analyzing apparent H. influenzae strains, to investigate if some strains were in fact H. haemolyticus. However, because of problems with unspecific primerannealing,no conclusions could be drawn regarding misclassification of H. haemolyticus.</p><p>Moraxella catarrhalis is the second most common bacterial pathogen associated with COPD. It also causes otitis and sinusitis. An important virulence factor of M. catarrhalis is the outer membrane protein Moraxella catarrhalis IgD-binding protein (MID). One part of the protein; MID764-913 , has been shown to function as an adhesin, and this part has been fragmented to further investigate its adhesive properties. The aim of this second, independent study, was to express some of these proteinfragments by cloning in E. coli. The time spent on this project was too short, and no proteins could be expressed duing this period.</p>

Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, June 1999. / Includes bibliographical references. Also available on the Internet.

Rat hepatocytes have previously been reported to possess prostaglandin E₂ receptors of the EP₃-type (EP₃-receptors) that inhibit glucagonstimulated glycogenolysis by decreasing cAMP. Here, the isolation of a functional EP₃ϐ receptor cDNA clone from a rat hepatocyte cDNA library is reported. This clone can be translated into a 362-amino-acid protein, that displays over 95% homology to the EP₃ϐ receptor from mouse mastocytoma. The amino- and carboxy-terminal region of the protein are least conserved. Transiently transfected HEK 293 cells expressed a single binding site for PGE₂ with an apparent Kd of 15 nM. PGE₂ > PGF₂α > PGD₂ competed for [³H]PGE₂ binding sites as did the EP₃ receptor agonists M&B 28767 = sulprostone > misoprostol but not the EP₁ receptor antagonist SC 19220. In stably transfected CHO cells M&B 28767 > sulprostone = PGE₂ > misoprostol > PGF₂α inhibited the forskolin-elicited cAMP formation. Thus, the characteristics of the EP₃ϐ receptor of rat hepatocytes closely resemble those of the EP₃ϐ receptor of mouse mastocytoma.