This study was focused on the promoter and non-coding regions of the ribosomal RNA (rrn) operon(s) of mycobacteria; namely, the leader and the intergenic spacer regions. Two clones containing the promoter sequences of M .leprae and M. tuberculosis rrn operon were sequenced, their promoter elements were identified by primer extension experiments and by comparison with E.coli consensus promoter sequences. Their function was tested in E.coli and M. smegma tis . The sequences of the leader and intergenic spacer regions from eight and six species respectively were established after amplification by means of peR. Both leader and spacer regions contain antitermination elements and RNaseIII processing sites. The sequences established for these two regions also showed greater variability than the 168 rRNA gene and are suitable for phylogenetic studies. The sequences of the two rrn operons of M.smegmatis upstream from the 168 rRNA gene were cloned and sequenced. Their sequences showed that rrnI has a Box B element which is typical of slow-growers and that rrnII does not. Primer extension studies revealed that the rrn operon of slow-growers has a single promoter. In contrast multiple promoters were identified in the faster-growing M.smegmatis. Distinctive features, which are absent from slow-growers, were identified in the intergenic spacer regions of M.smegmatis.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Cartilaginous fish are the most ancient lineage to possess an adaptive immune system however nothing is known about the processes involved in the development, maintenance or proliferation of B cells in this group. In this thesis studies were undertaken to explore whether transcription factors, cytokines and cell surface markers that are involved in B cell immune responses in mammals are also present in cartilaginous fish. Through sequence database mining forty-one genes involved in B cell immune biology were found; of these twelve were B cell transcription factors. The presence of important B cell transcription factors, such as EBF1, pax5, E2A, Blimp1, PU.1 and Bcl6 suggests that B cells in cartilaginous fish probably undergo a similar developmental pathway as those in mammals. Eight cytokines, eleven cytokine receptors and four B cell surface markers were also identified, including CD40L, BAFF and CD79α that were further studied in this thesis. I cloned CD40L and BAFF from cartilaginous fish, both members of the tumour necrosis factor family and found cartilaginous fish BAFF genes have an extra exon that forms a unique α-helix insertion, and which may impact on receptor binding. The importance of all three molecules for the adaptive immune response was indicated by relatively high expression in shark immune tissue, such as spleen, gut-associated lymphoid tissue, gill and the Leydig organ, and the fact that their expression could be induced by immunostimulants, such as pokeweed mitogen. The finding that CD79α expression significantly correlates with those of immunoglobulin heavy-chains and the co-expression of CD79α and IgM on the B cell surface indicate that as in mammals CD79α in cartilaginous fish is expressed by B cells and associates with surface-bound immunoglobulin to form the active B cell receptor. Thus CD79α may serve as a pan B cell marker in future immunological studies in cartilaginous fish.

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.

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.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Greek alphabet delta in title. Includes bibliographical references (leaves 136-155). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--West Virginia University, 2004 / Title from document title page. Document formatted into pages; contains xiii, 119 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression.

In this study first time, we have cloned and sequenced the citrate synthase gene from a thermoacidophilic archaeon Thermoplasma (Tp.) volcanium (Optimum growth temperature of Tp.volcanium is 60oC and optimum pH is 2.0.). For cloning we have followed a PCR based approach. Amplification of citrate synthase gene from chromosomal DNA of Tp.volcanium yielded a product of 1476 bp containing an open reading frame of 1161 bp comprising the structural gene. After ligation of the PCR amplicon to pDrive vector through AU complementation, recombinant plasmids were transferred into E.coli TG-1 competent cells. Out of three recombinants, E.coli pDriveCS-31 was selected for further characterization by restriction mapping and DNA sequencing. Southern Blotting and Hybridization using the membrane blot of pDriveCS-31 plasmid and DIG-labeled PCR amplified citrate synthase gene probe, also confirmed the cloning of Tp.volcanium citrate synthase gene in E.coli. Clustal W Version 1.82 was used for alignment of aminoacid sequence of Tp.volcanium citrate synthase with that of other archaeal, bacterial and eukaryotic citrate synthases. The highest sequence similarity (87%) was found between Tp.volcanium and Tp.acidophilum enzymes. Despite low sequence homology (18%) with the pig enzyme, of the 11 residues implicated in catalytic activity of the pig citrate synthase 9 were conserved in the Tp.volcanium enzyme. Heterologous expression of this citrate synthase gene in E.coli has been achieved under the control of its promoter sequences. The recombinant enzyme (386 aa) has been purified to homogeneity by affinity chromatography on Reactive Red 120 column. The subunit molecular size was estimated as 43 kDa. The purified enzyme followed classical Michaelis-Menten kinetics. The Km values of 5.15 &amp / #956 / M and 5.60 &amp / #956 / M, and Vmax values of 1.74 &amp / #956 / moles/ml/min and 1.60 &amp / #956 / moles/ml/min were calculated from Lineweaver-Burk plots for acetyl-CoA and oxaloacetate, respectively. The recombinant enzyme was thermostable and retained about 80% of the activity at 85oC after 1 hour.