Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

Molecular cloning restriction enzyme analysis, bovine adenovirus type 2 genome

Adenoviruses are non-enveloped icosahedral-shaped particles which possess a double-stranded DNA genome. Currently, nearly 100 serotypes of adenoviruses have been identified, 48 of which are of human origin. Bovine adenoviruses (BAVs), causing both mild respiratory and/or enteral diseases in cattle, have been reported in many countries all over the world. Currently, nine serotypes of SAVs have been isolated which have been placed into two subgroups based on a number of characteristics which include complement fixation tests as well as the ability to replicate in various cell lines. Bovine adenovirus type 2 (BAV2), belonging to subgroup I, is able to cause pneumonia as well as pneumonic-like symptoms in calves. In this study, the genome of BAV2 (strain No. 19) was subcloned into the plasmid vector pUC19. In total, 16 plasmids were constructed; three carry internal San fragments (spanning 3.1 to 65.2% ), and 10 carry internal Pstl fragments (spanning 4.9 to 97.4%), of the viral genome. Each of these plasmids was analyzed using twelve restriction endonucleases; BamHI, CiaI, EcoRl, HiOOlll, Kpnl, Noll, NS(N, Ps~, Pvul, Saj, Xbal, and Xhol. Terminal end fragments were also cloned and analyzed, sUbsequent to the removal of the 5' terminal protein, in the form of 2 BamHI B fragments, cloned in opposite orientations (spanning 0 to 18.1°k), and one Pstll fragment (spanning 97.4 to 1000/0). These cloned fragments, along with two other plasmids previously constructed carrying internal EcoRI fragments (spanning 20.6 to 90.5%), were then used to construct a detailed physical restriction map using the twelve restriction endonucleases, as well as to estimate the size of the genome for BAV2(32.5 Kbp). The DNA sequences of the early region 1 (E1) and hexon-associated gene (protein IX) have also been determined. The amino acid sequences of four open reading frames (ORFs) have been compared to those of the E1 proteins and protein IX from other Ads.

Construction of bovine adenovirus type 3 E1 and E3, substitution plasmids and the sequencing analysis of DNA pol and pTP /

The relative ease to concentrate and purify adenoviruses, their well characterized mid-sized genome, and the ability to delete non-essential regions from their genome to accommodate foreign gene, made adenoviruses a suitable candidate for the construction of vectors. The use of adenoviral vectors in gene therapy, vaccination, and as a general vector system for expressing foreign genes have been documented for some time. In this study, the objective was to rescue a BAV3 E1 or E3 recombinant vector carrying the kanamycin resistant gene, a dominant selectable marker with useful applications in studying vectored gene expression in mammalian cells. To accomplish the objective of this study, more information about BAV3 DNA sequences was required in order to make the manipulation of the virus genome accessible. Therefore, sequencing of the BAV3 genome from 1 1 .7% to 30.8% was carried out. Analysis of the determined sequences revealed the primary structure of important viral gene products coded by E2 including BAV3 DNA pol and precursor to terminal protein. Comparative analysis of these proteins with their counterparts from human and non human adenoviruses revealed important insights as to the evolutionary lineage of BAV3. In order to insert the kanamycin resistance gene in either E1 or E3, it was necessary to delete BAV3 sequences to accommodate the foreign gene so as not to exceed the limit of the packaging capacity of the virus. To construct a recombinant BAV3 in which a foreign gene was inserted in the deleted E1 region, an E1 shuttle vector was constructed. This involved the deletion from the viral sequences a region between 1.3% to 9% and inserting the kanamycin resistance gene to replace the deletion. The E1 shuttle vector contained the left (0%- 53.9%) segment of the genome and was expected to generate BAV3 recombinants that can be grown and propagated in cells that can complement the missing E1 functions. To construct a similar shuttle vector for E3 deletion, DNA sequences extending from 78.9% to 82.5% (1281 bp) were deleted from within the E3 region that had been cloned into a plasmid vector. The deleted region corresponds to those that have been shown to be non-essential for viral replication in cell culture. The resulting plasmid was used to construct another recombinant plasmid with BAV3 DNA sequences extending from 37.1% to 100% and with a deletion of E3 sequences that were replaced by kanamycin resistance gene. This shuttle plasmid was used in cotransfections with digested viral DNA in an attempt to rescue a recombinant BAV3 carrying the kanamycin resistance gene to replace the deleted E3. In spite of repeated attempts of transfection, El or E3 recombinant BAV3 were not isolated. It seems that other approaches should be applied to make a final conclusion on BAV3 infectivity.

Engineering an improved adenovirus packaging cell line

Recombinant human adenovirus (Ad) vectors are being extensively explored for their use in gene therapy and recombinant vaccines. Ad vectors are attractive for many reasons, including the fact that (1) they are relatively safe, based on their use as live oral vaccines, (2) they can accept large transgene inserts, (3) they can infect dividing and postmitotic cells, and (4) they can be produced to high titers. However, there are also a number of major problems associated with Ad vectors, including transient foreign gene expression due to host cellular immune responses, problems with humoral immunity, and the creation of replication competent adenoviruses (RCA). Most Ad vectors contain deletions in the E1 region that allow for insertion of a transgene. However, the E1 gene products are required for replication and thus must be supplied in trans by a helper ceillille that will allow for the growth and packaging of the defective virus. For this purpose the 293 cell line (Graham et al., 1977) is used most often; however, homologous recombination between the vector and the cell line often results in the generation of RCA. The presence of RCA in batches of adenoviral vectors for clinical use is a safety risk because tlley . may result in the mobilization and spread of the replication-defective vector viruses, and in significant tissue damage and pathogenicity. The present research focused on the alteration of the 293 cell line such that RCA formation can be eliminated. The strategy to modify the 293 cells involved the removal of the first 380 bp of the adenovirus genome through the process of homologous recombination. The first step towards this goal involved identifying and cloning the left-end cellular-viral jUl1ction from 293 cells to assemble sequences required for homologous recombination. Polymerase chain reaction (PCR) was performed to clone the junction, and the clone was verified through sequencing. The plasn1id PAM2 was then constructed, which served as the targeting cassette used to modify the 293 cells. The cassette consisted of (1) the cellular-viral junction as the left-end region of homology, (2) the neo gene to use for positive selection upon tranfection into 293 cells, (3) the adenoviral genome from bp 380 to bp 3438 as the right-end region of homology, and (4) the HSV-tk gene to use for negative selection. The plasmid PAM2 was linearized to produce a double strand break outside the region of homology, and transfected into 293 cells using the calcium-phosphate technique. Cells were first selected for their resistance to the drug G418, and subsequently for their resistance to the drug Gancyclovir (GANC). From 17 transfections, 100 pools of G418f and GANCf cells were picked using cloning lings and expanded for screening. Genomic DNA was isolated from the pools and screened for the presence of the 380 bps using PCR. Ten of the most promising pools were diluted to single cells and expanded in order to isolate homogeneous cell lines. From these, an additional 100 G41Sf and GANef foci were screened. These preliminary screening results appear promising for the detection of the desired cell line. Future work would include further cloning and purification of the promising cell lines that have potentially undergone homologous recombination, in order to isolate a homogeneous cell line of interest.

Locating and sequencing of the E3 and neighbouring regions in bovine advenovirus type 2

Adenoviruses are nonenveloped icosahedral shaped particles. The double stranded DNA viral genome is divided into 5 major early transcription units, designated E1 A, E1 B, and E2 to E4, which are expressed in a regulated manner soon after infection. The gene products of the early region 3 (E3), shown to be nonessential for viral replication in vitro, are believed to be involved in counteracting host immunosurveillance. In order to sequence the E3 region of Bovine adenovirus type 2 (BAV2) it was necessary to determine the restriction map for the plasmid pEA48. A physical restriction endonuclease map for BamHl, Clal, Eco RI, Hindlll, Kpnl, Pstt, Sail, and Xbal was constructed. The DNA insert in pEA48 was determined to be viral in origin using Southern hybridization. A human adenovirus type 5 recombinant plasmid, containing partial DNA fragments of the two transcription units L4 and L5 that lie just outside the E3, was used to localize this region. The recombinant plasmid pEA was subcloned to facilitate sequencing. The DNA sequences between 74.8 and 90.5 map units containing the E3, the hexon associated protein (pVIII), and the fibre gene were determined. Homology comparison revealed that the genes for the hexon associated pV11I and the fibre protein are conserved. The last 70 amino acids of the BAV2 pV11I were the most conserved, showing a similarity of 87 percent with Ad2 pV1I1. A comparison between the predicted amino acid sequences of BAV2 and Ad40, Ad41 , Ad2 and AdS, revealed that they have an identical secondary structure consisting of a tail, a shaft and a knob. The shaft is composed of 22, 15 amino acid motifs, with periodic glycines and hydrophobic residues. The E3 region was found to consist of about 2.3 Kbp and to encode four proteins that were greater than 60 amino acids. However, these four open reading frames did not show significant homology to any other known adenovirus DNA or protein sequence.

Molecular cloning and restriction endonuclease analysis of bovine adenovirus type 3

Bovine adenovirus type 3 (BAV3) is a medium size DNA virus that causes respiratory and gastrointestinal disorders in cattle. The viral genome consists of a 35,000 base pair, linear, double-stranded DNA molecule with inverted terminal repeats and a 55 kilodalton protein covalently linked to each of the 5' ends. In this study, the viral genome was cloned in the form of subgenomic restriction fragments. Five EcoRI internal fragments spanning 3.4 to 89.0 % and two Xb a I internal fragments spanning 35.7 to 82.9 % of the viral genome were cloned into the EcoRI and Xbal sites of the bacterial vector pUC19. To generate overlap between cloned fragments, ten Hi n dIll internal fragments spanning 3.9 to 84.9 and 85.5 to 96% and two BAV3 BamHI internal fragments spanning 59.8 to 84.9% of the viral genome were cloned into the HindllI and BamHI sites of pUC19. The HindlII cloning strategy also resulted in six recombinant plasmids carrying two or more Hi ndII I fragments. These fragments provided valuable information on the linear orientation of the cloned fragments within the viral genome. Cloning of the terminal fragments required the removal of the residual peptides that remain attached to the 5' ends of the genome. This was accomplished by alkaline hydrolysis of the DNA-peptide bond. BamH I restriction fragments of the peptide-free DNA were cloned into pUC19 and resulted in two plasmids carrying the BAV3 Bam HI terminal fragments spanning 0 to 53.9% and 84.9 to 100% of the viral genome.

Partial characterization of the cloned dihydrofolate reductase gene of Saccharomyces cerevisiae

The cloned dihydrofolate reductase gene of Saccharomyces cerevisiae (DFR 1) is expressed in Escherichia coli. Bacterial strain JF1754 transformed with plasmids containing DFR 1 is at least 5X more resistant to inhibition by the folate antagonist trimethoprim. Expression of yeast DFR 1 in E. coli suggests it is likely that the gene lacks intervening sequences. The 1.8 kbp DNA fragment encoding yeast dhfr activity probably has its own promotor, as the gene is expressed in both orientations in E. coli. Expression of the yeast dhfr gene cloned into M13 viral vectors allowed positive selection of DFR 1 - M13 bacterial transfectants in medium supplemented with trimethoprim. A series of nested deletions generated by nuclease Bal 31 digestion and by restriction endonuclease cleavage of plasmids containing DFR 1 physically mapped the gene to a 930 bp region between the Pst 1 and Sal 1 cut sites. This is consistent with the 21,000 molecular weight attributed to yeast dhfr in previous reports. From preliminary DNA sequence analysis of the dhfr DNA fragment the 3' terminus of DFR 1 was assigned to a position 27 nucleotides from the Eco Rl cut site on the Bam Hi - Eco Rl DNA segment. Several putative yeast transcription termination consensus sequences were identified 3' to the opal stop codon. DFR 1 is expressed in yeast and it confers resistance to the antifolate methotrexate when the gene is present in 2 - 10 copies per cell. Plasmid-dependent resistance to methotrexate is also observed in a rad 6 background although the effect is somewhat less than that conferred to wild-type or rad 18 cells. Integration of DFR 1 into the yeast genome showed an intermediate sensitivity to folate antagonists. This may suggest a gene dosage effect. No change in petite induction in these yeast strains was observed in transformed cells containing yeast dhfr plasmids. The sensitivity of rad 6 , rad 18 and wild-type cell populations to trimethoprim were unaffected by the presence of DFR 1 in transformants. Moreover, trimethoprim did not induce petites in any strain tested, which normally results if dhfr is inhibited by other antifolates such as methotrexate. This may suggest that the dhfr enzyme is not the only possible target of trimethoprim in yeast. rad 6 mutants showed a very low level of spontaneous petite formation. Methotrexate failed to induce respiratory deficient mutants in this strain which suggested that rad 6 might be an obligate grande. However, ethidium bromide induced petites to a level approximately 50% of that exhibited by wild-type and rad 18 strains.

The cloning and reconstitution of a bovine adenovirus type 2 E3 deletion mutant and the sequencing and analysis of the early 4 region

Recombinant Adenoviruses (Ads) have been shown to have potential applications in three areas: gene therapy, high level protein expression and recombinant vaccines.' At least three different locations within the Ad genome can be deleted and subsequently used for the insertion of foreign sequences. These include the Early 3 (E3), Early 1 (E1) and Early 4 (E4) regions. Viral vectors of this type have been well studied in Human Ads 2 and 5, however one has not yet been constructed for Bovine Adenovirus Type 2 (BAV2). The E3 region is located between 76.6 and 86 m.u. on the r-strand and is transcribed in a rightward direction. The gene products of the Early 3 region (E3) have been shown to be non-essential for viral replication, in vitro, but are required for host immunosurveillance. This study represents the cloning and reconstitution of a BAV2 E3 deletion mutant. A deletion of 1800bp was made within the E3 region of BAV2 and the thymidine kinase gene was subsequently inserted in the deleted area . . The plasmid pdlE3-4tk1 (23.4Kbp) was constructed and used to to facilitate homologous recombination with the wild type BAV2 to produce a mutant. Southern Blotting and Hybridization results suggest the presence of a BAV2 E3 deletion mutant with thymidine kinase sequences present. The E4 region of Human Adenovirus types 2 and 5 is located at the extreme right end of the genome (91.3 map units - 99.1 map units) and is transcribed in a leftward direction giving rise to a complicated set of differentially spliced mRNAs. Essentially there are 7 open reading frames (ORFs) encoding for at least 7 polypeptides. The gene products encoded by the E4 region have been shown to be essential for the expression of late viral genes, host cell shutoff and normal viral growth. We have cloned and sequenced the right end segment between 90.5 map units and 100 map units of the BAV2 genome. The results show several open reading frames which encode polypeptides exhibiting homology to three polypeptides encoded by the E4 region of human adenovirus type 2. These include the 14kDa protein encoded by ORF1, the 34kDa protein encoded by ORF6 and the 13kDa protein encoded by ORF3. The nucleotide sequence, restriction enzyme map, and ORF map of the E4 region could be very useful in future molecular manipulation of this region and could possibly explain the slow growth rate of BAV2 in MDBK cells.

The establishment of two novel bovine cell lines by transfection with the putative transforming region of bovine adenovirus type 3

Human adenoviruses (Ads), members of the family adenoviridae, are medium-sized DNA viruses which have been used as valuable research tools for the study of RNA processing, oncogenic transformation, and for the development of viral vectors for use in gene delivery and immunization technology. The left 12% of the linear Ad genollle codes for products which are necessary for the efficient replication of the virus, as well as being responsible for the forlllation of tumors in animallllodels. The establishlllent of the 293 cell line, by immortalization of human embryonic kidney cells with th~ E1 region of Ad type S (AdS), has facilitated extensive manipulation of the Ads and the development of recombinant Ad vectors. The study of bovine adenoviruses (BAVs), which cause mild respiratory and gastrointestinal infections in cattle has, on the other hand, been limited primarily to that of infectivity, immunology and clinicallllanifestations. As a result, any potential as gene delivery vehicles has not yet been realized. Continued research into the molecular biolo~gy of BAVs and the development of recolllbinant vectors would benefit from the development of a cell line analogous to that of the 293 cells. In an attelllpt to establish such a cell line, the recombinant plaslllid pKC-neo was constructed, containing the left 0-19.7% of the BAV type 3 (BAV3) genome, and the selectable marker for resistance to the aminoglycoside G418, a neomycin derivative. The plasmid construct was then used to transfect both the Madin-Darby bovine kidney (MDBK) -iicell line and primary bovine lung cells, after which G418-resistant foci were selected for analysis. Two cell lines, E61 (MDBK) and E24 (primary lung), were subsequently selected and analysed for DNA content, revealing the presence of the pKC-neo sequences in their respective genomes. In addition, BAV3 RNA transcripts were detected in the E61 cells. Although the presence of E1 products has yet to be confirmed in both cell lines, the E24 cells exhibit a phenotype characteristic of partial transformation by E1. The apparent immortalization of the primary lung cells will permit exploitation of their ability to take up exogenous DNA at high efficiency.

Biochemical and histological investigations of viral localisation in the hypersensitive reaction of Phaselous vulgaris L. var Pinto to tobacco mosaic virus infection

STOBBS, Lorne,W ABSTRACT Biochemical and Histological Investigations of viral localisation in the hypersensitive reaction of Phaseolus vulgaris L. var Pinto to tobacco mosaic virus infection. The infection of Phaseolus vulgaris L. var Pinto with tobacco mosaic virus (TMV) results in the production of distinct necrotic lesions confining the virus to restricted areas of the leaf surface. Biochemical and histological changes in the leaf tissue as a result of infection have been described. Trace accumulations of fluorescent metabolites, detected prior to lesion expression represent metabolites produced, by the cell in response to virus infection. These substances, are considered to undergo oxidation and in diffusing into adjacent cells, react with cellular constituents causing the death of these cells. Such cellular necrosis in advance of infection effectively limits virus spread. Chromatographic studies on extracts from TMV infected Pinto bean leaf tissue suggests that a number of extra-fluorescent metabolites produced on lesion'expression represent end products of phenolic oxidation r,eactionsoccurring earlier in these cells. Inhibition of phenolic oxidation by ascorbate infiltration or elevated temperature treatment resulted in the absence of extra-fluorescent metabolites and the continued movement of virus in the absence of necrosis. Further studies with i ascorbate infiltration indicated that irreversible necrotic events were determined as early as 12 tci 18 hrs after viral inoculation. Histochemical tests indicated that callose formation was initiated at this time, and occurred in response to necrotisation. Inhibition of necrosis by either ascorbate infiltration or elevated temp8rature treatment resulted in the absence of callose deposition. Scanning electron'micrographs of infected tissue revealed severe epidermal and palisade cell damage. Histochemical tests indicated extensive callose formation in cells bordering the lesion, and suggested the role of callose iTh the blockage of intercellular connections limiting virus movement. The significance of these cellular changes is discussed. ii

Transposable Elements Are a Significant Contributor to Tandem Repeats in the Human Genome

Sequence repeats are an important phenomenon in the human genome, playing important roles in genomic alteration often with phenotypic consequences. The two major types of repeat elements in the human genome are tandem repeats (TRs) including microsatellites, minisatellites, and satellites and transposable elements (TEs). So far, very little has been known about the relationship between these two types of repeats. In this study, we identified TRs that are derived from TEs either based on sequence similarity or overlapping genomic positions. We then analyzed the distribution of these TRs among TE families/subfamilies. Our study shows that at least 7,276 TRs or 23% of all minisatellites/satellites is derived from TEs, contributing ∼0.32% of the human genome. TRs seem to be generated more likely from younger/more active TEs, and once initiated they are expanded with time via local duplication of the repeat units. The currently postulated mechanisms for origin of TRs can explain only 6% of all TE-derived TRs, indicating the presence of one or more yet to be identified mechanisms for the initiation of such repeats. Our result suggests that TEs are contributing to genome expansion and alteration not only by transposition but also by generating tandem repeats.

HUMAN GENOME VARIATIONS AND EVOLUTION WITH A FOCUS ON THE ANALYSIS OF TRANSPOSABLE ELEMENTS

Genome sequence varies in numerous ways among individuals although the gross architecture is fixed for all humans. Retrotransposons create one of the most abundant structural variants in the human genome and are divided in many families, with certain members in some families, e.g., L1, Alu, SVA, and HERV-K, remaining active for transposition. Along with other types of genomic variants, retrotransponson-derived variants contribute to the whole spectrum of genome variants in humans. With the advancement of sequencing techniques, many human genomes are being sequenced at the individual level, fueling the comparative research on these variants among individuals. In this thesis, the evolution and functional impact of structural variations is examined primarily focusing on retrotransposons in the context of human evolution. The thesis comprises of three different studies on the topics that are presented in three data chapters. First, the recent evolution of all human specific AluYb members, representing the second most active subfamily of Alus, was tracked to identify their source/master copy using a novel approach. All human-specific AluYb elements from the reference genome were extracted, aligned with one another to construct clusters of similar copies and each cluster was analyzed to generate the evolutionary relationship between the members of the cluster. The approach resulted in identification of one major driver copy of all human specific Yb8 and the source copy of the Yb9 lineage. Three new subfamilies within the AluYb family – Yb8a1, Yb10 and Yb11 were also identified, with Yb11 being the youngest and most polymorphic. Second, an attempt to construct a relation between transposable elements (TEs) and tandem repeats (TRs) was made at a genome-wide scale for the first time. Upon sequence comparison, positional cross-checking and other relevant analyses, it was observed that over 20% of all TRs are derived from TEs. This result established the first connection between these two types of repetitive elements, and extends our appreciation for the impact of TEs on genomes. Furthermore, only 6% of these TE-derived TRs follow the already postulated initiation and expansion mechanisms, suggesting that the others are likely to follow a yet-unidentified mechanism. Third, by taking a combination of multiple computational approaches involving all types of genetic variations published so far including transposable elements, the first whole genome sequence of the most recent common ancestor of all modern human populations that diverged into different populations around 125,000-100,000 years ago was constructed. The study shows that the current reference genome sequence is 8.89 million base pairs larger than our common ancestor’s genome, contributed by a whole spectrum of genetic mechanisms. The use of this ancestral reference genome to facilitate the analysis of personal genomes was demonstrated using an example genome and more insightful recent evolutionary analyses involving the Neanderthal genome. The three data chapters presented in this thesis conclude that the tandem repeats and transposable elements are not two entirely distinctly isolated elements as over 20% TRs are actually derived from TEs. Certain subfamilies of TEs themselves are still evolving with the generation of newer subfamilies. The evolutionary analyses of all TEs along with other genomic variants helped to construct the genome sequence of the most recent common ancestor to all modern human populations which provides a better alternative to human reference genome and can be a useful resource for the study of personal genomics, population genetics, human and primate evolution.

Complete Genome Sequence of Erwinia amylovora Bacteriophage

The complete genome of an Erwinia amylovora bacteriophage, vB_EamM_Ea35-70 (Ea35-70), is 271,084 bp, encodes 318 putative proteins, and contains one tRNA. Comparative analysis with other Myoviridae genomes suggests that Ea35-70 is related to the Phikzlikevirus genus within the family Myoviridae, since 26% of Ea35-70 proteins share homology to proteins in Pseudomonas phage φKZ.