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For all organisms, segregation and proper intracellular localization of DNA are essential processes in ensuring faithful inheritance of genetic material. In prokaryotes, several different mechanisms have developed for efficiently moving chromosomal DNA to proper cellular locations prior to cell division, and the same holds true for bacterial plasmids. Low-copy-number plasmids and bacterial chromosomes encode active partition systems to ensure their inheritance within a bacterial cell population. One of the well-studied models of partition is that of the P1 plasmid in E. coli. The partition system encoded by the P1 plasmid is known as parABS - ParA is the partition ATPase, ParB is the partition site binding protein and parS is the partition site. The goal of this thesis was to investigate the nucleoprotein complexes essential in the P1 plasmid partition reaction. First, I examined how a single ParB dimer can bind its complicated arrangement of recognition motifs in parS to initiate the partition reaction. I then characterized a novel ParA interaction with the host nucleoid that is critical for proper P1 plasmid dynamics in vivo. Finally, I demonstrate how ParA can act as an adaptor between the nucleoid and the partition complex; effectively allowing the plasmid to use the nucleoid as a track for its intracellular movement and localization. My thesis work provides evidence towards a model that explains the P1 plasmid partition mechanism.

Genomic DNA fragments encoding the silent type B neurotoxin gene from Clostridium botulinum NCTC 2916 have been cloned and the complete nucleotide sequence determined. The translated sequence revealed that the gene encoded a neurotoxin which was closely related to type B neurotoxin genes from Group I Clostridium botulinum. However among the nucleotide sequence differences, aG to T transition has interrupted the coding sequence with the formation of a stop codon. In addition the deletion of an adenine residue has resulted in a frame-shift mutation. Analysis of the DNA sequence contiguous with the silent type B neurotoxin gene revealed the presence of a gene encoding a Nontoxic-Nonhaemagglutinin protein which appears to share a bicistronic mRNA transcript with the type B neurotoxin gene. In the reverse orientation, the partial sequence of a gene encoding a haemagglutinin protein was found, typical of type A and B botulinal neurotoxin complexes. Separating the genes encoding the 'components of the neurotoxin complex was a gene of 178 amino acids which possessed features commonly associated with transcriptional factors. To facilitate the in vivo study of botulinal neurotoxin complex regulation, a gene transfer system using clostridial components has been developed. The minimal replicon of the cryptic plasmid pCB 102 from Clostridium butyricum NCIB 7423 was located to 1.6 kb DNA fragment by deletion analysis, enabling the identification of hitherto undiscovered putative ORFs and secondary structures, consistent with a replicative function. The replicon has been incorporated in to a number of Escherichia coli vectors resulting in a versatile series of shuttle vectors which have demonstrated high structural and segregational stabilities in a heterologous host Clostridium beyerinckii NCI NIB 8052. Gene transfer of a Group I Clostridium botulinum type A strain was demonstrated with a representative pCB 102-derived shuttle vector, pMTL540E. In addition, a 5.9 kb plasmid indigenous to C. hotulimun NCTC 2916 was cloned and the complete nucleotide sequence determined. Eight putative ORFs have been identified, including a putative replication protein and recombinase.

Fifty three Staphylococcus aureus isolates were obtained from three centres, two hospitals and a private pathology laboratory, and studied for susceptibility to bacteriophages, resistance to antimicrobial agents and plasmid contents.Results of bacteriophage typing revealed that they belonged to a variety of phage types. Eighteen were untypable by any of the International Set of Phages, 16 belonged to phage group 111, nine to group I, four to group 11, two to group IV and two to the miscellaneous group.The isolates were resistant to one or more of methicillin (Mc), benzyl penicillin (Pc), gentamicin (Gm) , kanamycin (Km) , neomycin (Nm) , streptomycin (Sm) , trimethoprim (Tp), sulphonamides (Su), tetracycline (Tc), minocycline (Mn), chloramphenicol (Cm), novobiocin (Nb) and fusidic acid (Fa). Resistance to Pc was due to the production of beta-lactamase (Bla). No resistance to vancomycin, spectinomycin and erythromycin was detected. Resistance was also found to heavy metals such as cadmium (Cd), mercury (Hg), phenyl mercuric acetate (Pma), arsenate (Asa) and to the nucleic-acid binding compounds propamidine isethionate (Pi) and ethidium bromide (Eb).All but one of the isolates harboured plasmids. The number of plasmids the isolates carried varied from one to six and their sizes ranged from < 1.0 kb to c.48 kb.Location of the resistance determinants was ascertained by curing and transfer experiments. Loss of resistance was tested after growth at 43.5°C and transfer of resistance determinants was attempted by transduction, mixed-culture transfer and conjugation. The results revealed that resistance to Mc, Gm, Tp, Mn and Fa was chromosomal in all the resistant isolates and in some isolates Bla and resistance to Sm and Cd were chromosomal as well as plasmid encoded. In the majority of cases Bla and resistance to Km, Nm, Sm, Tc, Cin, Cd, Hg, Asa, Pma, Pi and Eb was encoded ++ / by plasmids.Conjugation experiments led to the isolation of three unique conjugative plasmids which have not been found to confer resistance to antimicrobials or to produce haemolysins or diffusible pigment (Dip). The three plasmids, pWBG620, pWBG637 and pWBG661, were indistinguishable by restriction endonuclease analysis and DNA-DNA hybridisation. However pWBG620, unlike pWBG637 and pWBG661, was not detected in the cytoplasm of its host and was only detected in transconjugants after it mobilised a non-conjugative Sm-resistance (SmR) plasmid. Further analysis indicated that it is integrated into the chromosome of its host, excises during conjugation and mobilises the SmR plasmid.These plasmids were studied further using pWBG637 as a representative. It was compared with representatives of the two groups of conjugative plasmids which have been reported in the staphylococci. These are the plasmids which encode resistance to Gm, Km and Nm and those which code for the production of diffusible pigment. The three types of conjugative plasmids were compared by restriction endonuclease analysis and DNA- DNA hybridisation and were found to be different. A preliminary restriction map of pWBG637 has also been constructed.However since pWBG637 has no resistance phenotype direct selection for it was not possible in transfer experiments and for incompatibility (Inc.). To study it further it was necessary to construct resistant derivatives which could be selected for in transfer experiments. This was achieved by labelling pWBG637 with resistance transposons to generate two conjugative plasmids, pWBG636 carrying an insert of Tn3851 (Gm- resistance) and pWBG642 carrying an insert of Tn551 (hn- resistance). It was found that transposon labelling had not changed the incompatibility of pWBG637 and therefore pWBG636 and pWBG642 were used in further experiments in place of pWBG637. Inc. ++ / tests with the pWBG637 derivatives revealed that the pWBG637 type of plasmid is not only different from the other two types of conjugative plasmids but is different from any of the described staphylococcal Inc. groups and therefore the pWBG637 type of plasmids represent a new Inc. group 15. The pWBG637 type of plasmids were studied further using plasmids pWBG636 and pWBG642. They were able to transfer conjugatively to a capsulated S.aureus strain either by the polyethylene glycol method or on filter membranes. They also transferred by conjugation to S. epidermidis and Streptococcus faecalis and were able to transfer back from these strains to S.aureus indicating that they also replicate in these hosts. Consequently they have been used to mobilise non-conjugative plasmids from S.epidermidis and non phage typable S.aureus. Both plasmids failed to transfer conjugatively to Bacillus subtilis and Escherichia coli.pWBG637 transferred non-conjugative plasmids by mobilising them in a manner similar to mobilisation (donation) in E.coli or by recombining with them to form new resistance plasmids. In one case, pWBG628 which encodes Bla and resistance to Cd, Km, Nm and Sm and has no homology with pWBG637 recombined with it during conjugation to produce three new conjugative plasmids pWBG629, pWBG630 and pWBG631 carrying resistance determinants from pWBG628. One of these plasmids, pWBG629, was found to be pWBG637 which had acquired a 4.5 kb element encoding resistance to Km, Nm and Sm. This element was shown to be transposable in both rec+ and rec- backgrounds and has been designated Tn3854. It expressed Sm resistance in E.coli and differs on this account from the Gram-negative transposon Tn5 which expresses resistance to Km, Nm and Sm in non-enteric bacteria but only resistance to Km and Nm in E. coli.Where possible the non-conjugative plasmids encoding resistance to ++ / antimicrobial agents were compared with phenotypically similar plasmids isolated from other parts of the world. It was found that the Tc and Sm resistance plasmids were closely related to other plasmids with the same phenotype whereas the Cm resistance plasmids were different.Although the majority of the Bla plasmids belonged to Inc. group 1 they demonstrated significant restriction enzyme fragment length polymorphism when compared with other Bla plasmids.This study has provided the first data on the genetics of antimicrobial resistance in Nigerian S.aureus. Although many of the plasmids studied were found to be similar to those previously described the isolates also contained some unique and previously undescribed plasmids.

DNA vaccines offer unique promise as a means of generating immunity against infectious and malignant disease. Unfortunately a number of obstacles, including rapid degradation of naked plasmid DNA (pDNA), poor cellular uptake by antigen presenting cells (APCs) and subsequent low levels of gene expression have limited the ability of DNA vaccines to raise sufficient immune responses towards the target antigen. This thesis is focused on investigating the immunostimulatory potential of liposomal nanoparticulate (LN) formulations of pDNA (stabilized plasmid lipid particles; SPLP) and cytosine-guanine oligodeoxynucleotides (CpG-ODN; LN CpG-ODN), and examining their ability to act together as a non-viral DNA vaccine in attempt to address the shortcomings of current DNA vaccine approaches. One focus of this thesis concerns investigating the immunostimulatory activity of LN formulations of CpG-ODN and pDNA. It is shown that despite dramatic differences in pharmacokinetics and biodistribution of LN CpG-ODN following intravenous (i.v.) and subcutaneous (s.c.) administration the resultant immune response is very similar, which is concluded to be due to the intrinsic ability of APCs to sequester LN CpG- ODN. In addition, it is demonstrated that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA and it is observed that SPLP maintains immunostimulatory activity in Toll-like receptor 9 (TLR9) knock-out mice. Together theses findings highlight the need for DNA-based therapies to consider both TLR9-dependent and -independent immunostimulatory activities of pDNA when constructing non-viral vectors. Furthermore, a new role for SPLP as a non-viral gene delivery vehicle for the generation of a systemically administered genetic vaccine in the presence of LN CpG-ODN is introduced. The ability of vaccination with SPLP to act prophylactically, to protect mice from tumour challenge, and therapeutically, in a novel vaccination strategy where the antigen is expressed at the tumour site as a result of SPLP-mediated transfection, is explored, demonstrating that in the presence of LN CpG-ODN SPLP possesses potential as a non-viral delivery system for DNA-based cancer vaccines. In summary, this work represents a substantial advance in the understanding of the immunostimulatory potential of both SPLP and LN CpG-ODN and provides insight into their ability to work together as a non-viral DNA vaccine.

Hepatitis C virus (HCV) causes a significant health problem worldwide due to the lack of effective vaccines. It has been recognized that a rapid, vigorous, and broadly targeted cell-mediated immune response (Th1-like) is often associated with the clearance of HCV infections. DNA vaccines represent a promising means for HCV vaccination because they tend to induce a Th1-biased cell-mediated response in the host cell. Currently, the delivery of DNA vaccine for HCV in large animals as well as in humans is not as effective as in small animals. Nano delivery systems would be a promising approach to overcome this problem. Carbon nanotubes (CNTs) have been extensively studied for delivering drugs, proteins, peptides, and nucleic acids including plasmid DNA to cells and organs with varying degrees of success, but few of them have been applied to DNA vaccine for HCV.<p> This thesis presents a study of using functionalized CNTs (f-CNTs) to improve the efficacy of plasmid DNA vaccine delivery for HCV. First, CNTs were functionalized via 1,3-dipolar cycloaddition reaction with the appropriate amino acids and aldehydes. NMR and TEM results suggested that the CNTs were successfully functionalized and became soluble in water. Then plasmid DNAs which encode green fluorescence protein reporter gene, luciferase reporter gene, and HCV core protein, respectively, were delivered into human hepatoma cells via calcium phosphate precipitation method, f-CNT delivery system, and a combination of f-CNT and calcium phosphate method, respectively. The result showed that f-CNTs, in combination with the calcium phosphate method, significantly enhanced the gene expression in human hepatoma cells.<p> Consequently, this study concludes that the f-CNT can significantly enhance gene expression in liver cells conferred by a plasmid DNA when combined with calcium phosphate precipitation method. Even though the mechanisms of this enhancement await further investigation, the results of this thesis may have important implications in developing DNA vaccines for infectious diseases in general and for hepatitis C in particular.

Hepatitis C virus (HCV) causes a significant health problem worldwide due to the lack of effective vaccines. It has been recognized that a rapid, vigorous, and broadly targeted cell-mediated immune response (Th1-like) is often associated with the clearance of HCV infections. DNA vaccines represent a promising means for HCV vaccination because they tend to induce a Th1-biased cell-mediated response in the host cell. Currently, the delivery of DNA vaccine for HCV in large animals as well as in humans is not as effective as in small animals. Nano delivery systems would be a promising approach to overcome this problem. Carbon nanotubes (CNTs) have been extensively studied for delivering drugs, proteins, peptides, and nucleic acids including plasmid DNA to cells and organs with varying degrees of success, but few of them have been applied to DNA vaccine for HCV.<p> This thesis presents a study of using functionalized CNTs (f-CNTs) to improve the efficacy of plasmid DNA vaccine delivery for HCV. First, CNTs were functionalized via 1,3-dipolar cycloaddition reaction with the appropriate amino acids and aldehydes. NMR and TEM results suggested that the CNTs were successfully functionalized and became soluble in water. Then plasmid DNAs which encode green fluorescence protein reporter gene, luciferase reporter gene, and HCV core protein, respectively, were delivered into human hepatoma cells via calcium phosphate precipitation method, f-CNT delivery system, and a combination of f-CNT and calcium phosphate method, respectively. The result showed that f-CNTs, in combination with the calcium phosphate method, significantly enhanced the gene expression in human hepatoma cells.<p> Consequently, this study concludes that the f-CNT can significantly enhance gene expression in liver cells conferred by a plasmid DNA when combined with calcium phosphate precipitation method. Even though the mechanisms of this enhancement await further investigation, the results of this thesis may have important implications in developing DNA vaccines for infectious diseases in general and for hepatitis C in particular.

HDGF (hepatoma-derived growth factor) is a novel growth factor,identified from conditioned medium of hepatoma cell line. HDGF has growth stimulating activity for fibroblast and some hepatoma cells. HDGF, a novel defined growth factor with mitogenic effect, has homology protein sequence as HMG (high mobility group) protein and their three dimension structures appeared to be similar to each other. Recently, elevated HDGF expression was found in developing kidneys but less was found in adult kidney. In addition, HDGF expression was found to be correlated with angiogenic status of tissues. Thus, it is speculated that HDGF plays a role during embryonic development and angiogenesis. HDGF also plays a role in cell-cell interaction and cell migration. HDGF is a growth factor that is involved in stimulating vascular smooth muscle cells (SMCs)proliferation during development and in disease. HDGF contains a true bipartite nuclear localization sequence necessary for nuclear targeting. HDGF is sciential factor in stimulating DNA replication and cell proliferation of vascular smooth muscle cell.In this study,we used E. coli strain BL21 (DE3) to express the recombinant protein hepatoma derived growth factor(HDGF). To find out the optimal production conditions,we studied on the different temperature and fermentor to calculate all cost .

The 2 micron plasmid of Saccharomyces cerevisiae resides in the nucleus as an extra-chromosomal element with a steady state copy number of 40-60 per cell. As a benign but selfish DNA element, the plasmid utilizes a self-encoded partitioning system and an amplification system to ensure its stable, high-copy propagation. The partitioning system consists of the plasmid encoded proteins, Rep1 and Rep2 and a cis-acting partitioning locus STB. The Rep proteins, together with several host factors, assembled at STB couple plasmid segregation to chromosome segregation. A plasmid lacking an active partitioning system is subject to a ‘diffusion barrier’, which causes it to be retained in the mother cell with a strong bias (mother bias). Currently available evidence favors the hitchhiking model for plasmid segregation, in which the tethering of plasmids to chromosome provides the basis for faithful plasmid partitioning. However, direct evidence to support this hypothesis has been difficult to obtain because of the small size of the budding yeast nucleus and the poor resolution of chromosomes in live cells or in chromosome spreads. In this study, we have attempted to verify the hitchhiking model using single copy derivatives of the 2 micron plasmid as reporters. We demonstrate, using two single copy reporters present in the same nucleus, that plasmid association with chromosome spreads is authentic, and is dependent on the partitioning system. By using a strategy that forces all chromosomes to stay in either the mother or the daughter compartment, we show that plasmid segregation can be uncoupled from nuclear envelope segregation. However, plasmid segregation cannot be uncoupled from chromosome segregation under this condition. This tight coupling between plasmid and chromosome segregation is consistent with the hitchhiking model for plasmid segregation. The plasmid partitioning complex is assembled de novo at STB during each cell cycle during the G1-S window. Plasmid replication or cell cycle cues that signal cellular DNA replication appear to trigger this assembly. Furthermore, there is an apparent temporal hierarchy in the association and dissociation of protein factors at STB. When DNA replication is delayed or blocked, the dissociation of factors from STB from the previous portioning cycle and the association of factors for the new partitioning cycle are delayed or blocked, respectively. The precise role of replication in plasmid segregation has not been elucidated. We have addressed this question by blocking either plasmid replication or all cellular DNA replication. We find that replication is not required for plasmid to overcome mother bias. However, replication is critical for the equal segregation of sister plasmid copies. These results provide a refinement of the hitchhiking model by suggesting that sister plasmids tether to sister chromatids in a replication-dependent manner and hitchhike on them during chromosome segregation. Finally, we have attempted to reconstitute the 2 micron plasmid partitioning system in mammalian cells with the goal of exploiting their larger nuclear size and the considerably higher chromosome resolution they provide. In experiments completed so far, we show that Rep2 expressed in COS7 cells localizes to chromosomes, and Rep1 does so in the presence of Rep2. Furthermore, they show co-localization on sister chromatids in a symmetric fashion, implying that plasmids associated with them are likely to follow suit. These observations suggest, by extrapolation, the Rep1-Rep2 assisted association of sister plasmids with sister chromatids in yeast as well, and are consistent with the refined hitchhiking model for plasmid segregation. / text

DNA vaccines offer unique promise as a means of generating immunity against infectious and malignant disease. Unfortunately a number of obstacles, including rapid degradation of naked plasmid DNA (pDNA), poor cellular uptake by antigen presenting cells (APCs) and subsequent low levels of gene expression have limited the ability of DNA vaccines to raise sufficient immune responses towards the target antigen. This thesis is focused on investigating the immunostimulatory potential of liposomal nanoparticulate (LN) formulations of pDNA (stabilized plasmid lipid particles; SPLP) and cytosine-guanine oligodeoxynucleotides (CpG-ODN; LN CpG-ODN), and examining their ability to act together as a non-viral DNA vaccine in attempt to address the shortcomings of current DNA vaccine approaches. One focus of this thesis concerns investigating the immunostimulatory activity of LN formulations of CpG-ODN and pDNA. It is shown that despite dramatic differences in pharmacokinetics and biodistribution of LN CpG-ODN following intravenous (i.v.) and subcutaneous (s.c.) administration the resultant immune response is very similar, which is concluded to be due to the intrinsic ability of APCs to sequester LN CpG- ODN. In addition, it is demonstrated that lipid encapsulation dramatically enhances the immunostimulatory potential of pDNA and it is observed that SPLP maintains immunostimulatory activity in Toll-like receptor 9 (TLR9) knock-out mice. Together theses findings highlight the need for DNA-based therapies to consider both TLR9-dependent and -independent immunostimulatory activities of pDNA when constructing non-viral vectors. Furthermore, a new role for SPLP as a non-viral gene delivery vehicle for the generation of a systemically administered genetic vaccine in the presence of LN CpG-ODN is introduced. The ability of vaccination with SPLP to act prophylactically, to protect mice from tumour challenge, and therapeutically, in a novel vaccination strategy where the antigen is expressed at the tumour site as a result of SPLP-mediated transfection, is explored, demonstrating that in the presence of LN CpG-ODN SPLP possesses potential as a non-viral delivery system for DNA-based cancer vaccines. In summary, this work represents a substantial advance in the understanding of the immunostimulatory potential of both SPLP and LN CpG-ODN and provides insight into their ability to work together as a non-viral DNA vaccine.