In view of the increasing interest in the biochemistry of fish and particularly fish enzymes, a study has been made of some of the glycolytic enzymes of fish muscle. Some important properties of two enzymes of the Embden-Meyerhof pathway, phosphoglucomutase and phosphoglucose isomerase, and one enzyme of the "hexosemonophosphate shunt", phosphoribomutase, are reported. The procedure for purifying these enzymes by ammonium sulfate precipitation, heating or chromatography on diethylaminoethyl cellulose, is given. However, most of the experiments were carried out with crude extracts, since the partially purified enzymes were not very stable under the usual conditions of storage of enzymes. The following properties of phosphoglucomutase and of phosphoribomutase are reported: equilibrium of the reaction, optimum pH, requirement for magnesium and cysteine, and the effect of glucose-1,6-diphosphate, ribose-1,5-diphosphate and deoxyribose-1,5-diphosphate on its activity. A study of the equilibrium and optimum pH of the phosphoglucose isomerase reaction is also reported. The similarity between the properties of these fish enzymes and the corresponding ituunmalian enzymes is discussed. The question of identity or non-identity of phosphoglucomutase and phosphoribomutase is also examined. / Science, Faculty of / Zoology, Department of / Graduate

Group II introns insert site-specifically into DNA target sites through a process termed retrohoming. They consist of a structured, catalytically active intron RNA and its encoded protein. The protein contains several domains, including a reverse transcriptase domain and a DNA endonuclease domain used for bottom-strand cleavage. Recently, the thermophile Thermosynechococcus elongatus BP-1 was found to contain eight functional group II intron-encoded proteins. The proteins are thermostable and active at temperatures up to 65°C. The intron-encoded protein, TeI4c displays the greatest reverse transcriptase activity of these eight proteins, as well as high fidelity and processivity; ideal qualities for a commercial reverse transcriptase. This work explores the possibility of using TeI4c for biotechnology applications, and specifically examines the C-terminal endonuclease domain of TeI4c and its effect on reverse transcription. Additionally, this work investigates the retrohoming activity of a TeI4c truncation that deletes the endonuclease domain. / text

Synechococcus elongatus PCC 7942 is a model organism used to study the circadian rhythm, a process that is driven by an endogenous biological clock that can be modulated by external cues such as light and temperature. Some proteins have been identified that are involved in circadian signal transduction in S. elongatus. Of them, KaiA, KaiB and KaiC comprise the central oscillator components, which are essential for internal timekeeping. SasA is an important protein in the output pathway, which passes the information from central oscillator to downstream components, and thus controls metabolic and behavioral processes. CikA is a major component in the input pathway, which maintains synchrony of the oscillator with the environment. CikA is an unusual phytochrome-like histidine protein kinase. It has a pseudo receiver domain which can not accept a phosphoryl group. CikA is thought to be located at the poles of the cell through interaction between PsR and some protein or protein complex that is also localized at the poles. One of the potential CikA-interacting proteins identified through a yeast two hybrid screen is called hypothetical protein 1. It specifically recognizes a PsR bait in a yeast two hybrid assay. A bioinformatics analysis showed that there are predicted signal peptide and transmembrane domains at the N-terminal and a cytochrome C homolog domain at the C-terminal of Hyp1. Elucidating the features and function of Hyp1 will provide us with useful information to understand the function and working mechanism of CikA, and therefore will help us to clarify the signal transduction in the clock. In this research, I used genetic, cell biological and biochemical approaches to study the features and function of this newly identified clock component Hyp1. To confirm the interaction between PsR and Hyp1 and complement the yeast two hybrid data, I truncated Hyp1 (Thyp1) and purified soluble Thyp1. At the same time, I obtained purified PsR. I tried to copurify the PsR and 6-histidine-tagged Hyp1 on a nickel affinity column. However, PsR non-specifically bound to the column, which eliminated the utility of this approach to study their interaction. In addition to using a biochemical approach to study Hyp1, I constructed three hyp1 overexpression alleles for genetic analysis and two hyp1-yfp overexpression fusion alleles for subcellular localization studies. All of them will help us to understand the features and function of Hyp1.

Bochum, Univ., Diss., 2003. / Computerdatei im Fernzugriff.

Bochum, Universiẗat, Diss., 2003.

Techn. University, Diss., 2004--Berlin.

Techn. University Diss., 2005--Berlin.

Cyanobacteria are photosynthetic prokaryotes that live in aquatic environments. The cyanobacterium Synechococcus elongatus PCC 7942, (hereafter S. elongatus) coordinates its day and night behaviors via a circadian clock. The clock is entrained by light/dark cycles but continues to run in constant light conditions. The core circadian clock in S. elongatus is encoded by post-translational modifications of three Kai proteins, but the extent and mechanism of circadian gene expression are unknown. We provide the first unbiased characterization of circadian gene expression in S. elongatus, demonstrating that \(\sim 65\%\) of genes display oscillation in continuous light conditions, with some genes peaking in expression at subjective dawn and others at subjective dusk. We next sought to identify the mechanism by which such a large fraction of the genome could be rhythmically controlled. Through bioinformatic, correlative, and perturbation experiments, we find that circadian changes in chromosome topology/supercoiling are sufficient to drive rhythmic expression (Chapter 2). To further investigate how chromosome topology can control gene expression we performed a high resolution characterization of transcripts and RNA polymerase across the S. elongatus genome (Chapter 3). Bioinformatic analysis of transcription start sites suggests that the AT/GC content a particular region of the promoter is informative in defining the phase at which a transcript is maximally expressed. We find that these sequences are sufficient to drive circadian gene expression at a particular phase and that mutation of single nucleotides in this region can reverse the expression phase of a transcript (Chapter 4). To understand the role of chromosome dynamics in circadian gene expression and cyanobacterial physiology, we tagged and followed chromosomes over multiple cell divisions. We find that S. elongatus cells harbor multiple ordered copies of a single chromosome, and the organization of chromosomes in the cytoplasm facilitates equal segregation of chromosomes to daughter cells (Chapter 5).

No description available.

Las cianobacterias, microorganismos que realizan la fotosíntesis oxigénica, tienen que adaptar su metabolismo a los distintos retos ambientales a los que se enfrentan, como la limitación de nutrientes o los ciclos de luz oscuridad. Para ello, han desarrolado una serie de mecanismos de gran complejidad y alta regulación que les permiten adaptarse y sobrevivir. En este contexto, PipX, una pequeña proteína exclusiva de cianobacterias, descubierta por el grupo de investigación de genética cianobacteriana de la Universidad de Alicante, actúa como conexión, dependiente del estatus carbono/nitrógeno, entre la proteína de transducción de señales, PII, y el regulador transcripcional, NtcA. Recientemente se han descubierto otras parejas de interacción de PipX, entre las que destaca PipY, miembro de la familia de proteínas de unión a piridoxal fosfato (PLPBP) que forma un operón con PipX en la mayoría de cianobacterias, y la GTPasa de ensamblaje de ribosomas, EngA. La mayoría de estos descubrimientos se han realizado en el organismo modelo, Synechococcus elongatus PCC7942, el cual ha sido el principal objeto de estudio en esta Tesis, centrada fundamentalmente en PipX y sus parejas de interacción. Entre las aportaciones novedosas de estas Tesis se encuentran: 1. La propuesta del empleo de PipY como modelo para el estudio de miembros de la familia PLPBP/COG325; 2. La caracterización de los fenotipos de sobreexpresión de PipX y PipY, dando lugar al descubrimiento de nuevas funciones (formación de polifosfatos) y conexiones entre ellas; 3. La demostración de la existencia de interacción funcional entre EngA y PipX, y la descripción de la función de EngA en el estrés redox en cianobacterias; 4. El avance en el estudio de los terminantes moleculares de la toxicidad de PipX en ausencia de PII y el papel de esta última en el mantenimiento de los niveles intracelulares de PipX. En conclusión, esta Tesis amplía el conocimiento sobre la compleja regulación de los sistemas cianobacterianos en respuesta a distintos estímulos ambientales y, en concreto, las conexiones y el papel de PipX junto a sus antiguas y nuevas parejas de interacción.