The repeatability of stand measurements derived from LiDAR data was tested in east-central Mississippi. Data collected from LiDAR missions and from ground plots were analyzed to estimate stand parameters. Two independent LiDAR missions were flown in approximate orthogonal directions. Field plots were generated where the missions overlapped, and tree data were taken in these plots. LiDAR data found 86-100% of mature pine trees, 64-81% of immature pine trees, and 63-72% of mature hardwood trees. Immature and mature pine tree heights measured from LiDAR were found to be significantly different (α= 0.05) than field measured heights. Individual tree volumes and plot volume for mature pines were precisely predicted in both flight directions. The results of this study showed that LiDAR repeatability in mature pines can be accurately achieved. But immature pine and hardwood plots were unable to match the repeatability of the mature pine plots.

No description available.

Timely completion of DNA replication in each cell cycle is crucial for maintaining genomic integrity. This is often challenged by the presence of various replication fork barriers (RFBs). On collision with a RFB, the fate of the replication fork remains uncertain. In some cases, the integrity of the fork is maintained until the barrier is removed or the fork is rescued by merging with the incoming fork. However, fork stalling can cause dissociation of all of the associated replication proteins (fork collapse). If this occurs, the cell's recombination machinery can intervene to help restart replication in a process called recombination-dependent replication (RDR). Programmed protein-DNA barriers like the Replication Terminator Sequence-1 (RTS1) have been used to demonstrate that replication fork blockage can induce recombination. However, it remains unclear how efficiently this recombination gives rise to replication restart and whether the restarted replication fork exhibits the same fidelity as an origin-derived fork. It is also unknown whether accidental replication barriers induce recombination in the same manner as programmed barriers. In this study, I introduce recombination reporters at various sites downstream of RTS1 to obtain information on both the fidelity and efficiency of replication restart. I find that unlike break induced replication (BIR), the restarted fork gives rise to hyper-recombination at least 75 kb downstream of the barrier. Surprisingly, fork convergence, rather than inducing recombination, acts to prevent or curtail genetic instability associated with RDR. I also investigate a number of genetic factors that have a role in either preventing or promoting genome instability associated with the progression of the restarted fork. To compare RTS1 with an accidental protein-DNA barrier, a novel site-specific barrier system (called MarBl) was established based on the human mariner transposase, Hsmar1, binding to its transposon end. Replication fork blockage at MarBl strongly induces recombination, more so than at RTS1. This appears to be a general feature of accidental barriers as introduction of the E. coli TusB-TerB site-specific barrier in S. pombe gives rise to a similar effect. Here, I compare and contrast accidental barriers with programmed barriers. I observe that there is very little replication restart, if any, at MarBl measured by direct repeat recombination downstream. This points to the fact that accidental barriers do not trigger fork collapse in the same way as programmed RFBs and that the increased recombination that they cause may be a consequence of the inability of replication forks to terminate correctly, owing to the bi-directional nature of the barrier. Several genetic factors are assessed for their impact on MarBl-induced recombination, which further highlights both similarities and differences with RTS1-induced recombination.

Replication Protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism including, but not limited to, DNA replication, DNA repair and recombination. The `canonical' RPA is composed of three subunits (RPA1, RPA2, and RPA3). In addition to the three canonical subunits, there is a human homolog to the RPA2 subunit, termed RPA4, which can substitute for RPA2 in complex formation. The resulting RPA complex has been termed `alternative' RPA (aRPA). The normal function of aRPA is not known; however, previous studies have shown that it does not support S-phase progression in vivo. The goal of this thesis was to characterize the function of aRPA in DNA replication, DNA repair and recombination and profile its expression in human tissues. The studies presented in this thesis show that the aRPA complex has solution and DNA binding properties indistinguishable from the canonical RPA complex as determined by gel mobility shift assays. However, aRPA was unable to support DNA replication and inhibited canonical RPA function in a cell-free simian virus 40 system. aRPA inhibited both initiation and elongation of DNA synthesis in the SV40 system. Two regions of RPA4, the putative L34 loop and the C-terminal winged helix domain, were responsible for inhibiting SV40 DNA replication. The mechanism of SV40 DNA replication inhibition during initiation and elongation was characterized using assays for DNA polymerase α and DNA polymerase δ. aRPA was shown to have reduced interaction with DNA polymerase α and was not able to efficiently stimulate DNA synthesis by DNA polymerase α on aRPA coated single-stranded DNA. However, aRPA stimulated DNA synthesis by DNA polymerase δ in the presence of PCNA and RFC even though a reduced interaction was observed between aRPA and polymerase δ. The role of aRPA in DNA repair was also investigated. aRPA interacted with both Rad52 and Rad51 but had a reduced interaction with Rad51. However, aRPA was still able to stimulate Rad51-dependent strand exchange. aRPA also supported the dual incision/excision reaction of nucleotide excision repair. aRPA was less efficient in nucleotide excision repair than canonical RPA and this reduction was attributed to reduced interactions with the repair factor XPA. In contrast, aRPA exhibited higher affinity for damaged DNA than canonical RPA. The expression of RPA4 and RPA2 was determined by quantitative PCR in established cell lines, human normal tissues and human tumor tissue. RPA4 was shown to be expressed in all normal tissues examined but the level of expression was tissue specific. Additionally, RPA4 expression was decreased in all tumor tissues examined and was at the limit of detection in established cell lines. Taken together, the results presented in this thesis suggest that aRPA is a `non-proliferative' form of RPA that functions to maintain the genomic stability of non-dividing cells.

The circular, single-stranded (ss) DNA genomes of plant viruses in the families Geminiviridae and Nanoviridae are replicated within the nucleus of a host cell by a mechanism called rolling circle replication (RCR). Although this process relies almost exclusively on the replication machinery of the host cell, initiation occurs via the interaction of the viral replication initiation protein (Rep) with regulatory DNA sequences within the viral genome. The use of a virus-based episomal amplification technology as a plant bioreactor platform exploits the process of Rep-mediated RCR for the high-level amplification of virus-based episomes in plants and subsequent expression of heterologous proteins; such an approach offers advantages over existing gene expression technologies. This PhD thesis describes research towards the development of a virus-based episomal amplification system for use in sugarcane. Such a crop is ideally suited for a plant bioreactor system due to the efficient high-level production of plant biomass and the existence of established production, harvesting and processing infrastructure. In order to rapidly assess the potential of a virus-based episomal amplification system in sugarcane, a transient assay system was established. Sugarcane callus was identified as the most suitable cell preparation; providing rapid cell regeneration, uniform experimental samples and upon isolation, total DNA suitable for Southern analysis. This assay system once established, proved effective in rapidly identifying virus-based episomes capable of undergoing RCR within sugarcane host cells. This transient assay system was then used to test the functionality of a virus-based episomal amplification system based on the ssDNA virus, Banana bunchy top virus (BBTV) in sugarcane. BBTV-based episomal amplification vectors were constructed with a reporter gene expression cassette flanked by two copies of the BBTV regulatory DNA sequences. The episomal amplification vectors were bombarded into sugarcane and banana host cells in various combinations and evidence of RCR was assessed through Southern blot analysis. RCR products were identified in banana host cells bombarded with the BBTV-based episomal amplification vectors in combination with vectors encoding BBTV Master-Rep (M Rep). RCR products were not identified within sugarcane cells bombarded with the same construct combinations. Integrated InPAct (In Plant Activation) episomal vectors based on BBTV were then employed to confirm the transient results, in addition, the functionality of an InPAct vector based on an alternate virus, Tobacco yellow dwarf virus (TYDV) was also assessed. InPAct vectors based on BBTV were constructed with an untranslatable expression cassette for integration within the sugarcane genome. Transient experiments were performed to assess the ability of BBTV M-Rep and TYDV Rep to initiate RCR of their respective InPAct vectors. Visual observation of GFP expression indicated that BBTV M-Rep was capable of initiating RCR of the BBTVbased InPAct vectors within banana host cells but no evidence was observed in sugarcane host cells. TYDV Rep was capable of initiating RCR of the TYDV-based InPAct vector within sugarcane host cells with a 100-fold increase in the number of fluorescent foci compared to cells bombarded with the TYDV InPAct vector alone. The BBTV-based InPAct vector was stably integrated within the sugarcane genome and the ability for BBTV M-Rep to initiate episome formation and RCR was assessed by Southern blot analysis. Evidence of BBTV M-Rep mediated RCR was not detected within the transgenic sugarcane bombarded with BBTV M-Rep. Transgenic sugarcane containing the TYDV-based InPAct vectors was assessed for the ability to be activated by TYDV Rep and undergo RCR. Southern blot analysis demonstrated that TYDV Rep was capable of recognising the integrated TYDVbased InPAct vector and RCR was detected within the transgenic sugarcane. The observation that episomal vectors based on TYDV were functional within sugarcane host cells and BBTV-based vectors were not, was unexpected. It had been hypothesised that an episomal vector based on a monocot-infecting virus would replicate in an alternate monocot host, while an episomal vector based on a dicot infecting virus would not. Virus replication is thought to be host-specific however most host range studies have been conducted with full length infectious clones and not deconstructed virus-based episomes. The implication that viral Reps may be functional in plant cells of non-host species was then investigated. The ability for viral Reps to recognise their cognate IR and initiate RCR of virus-based episomes in different host cells was assessed through cross-replication experiments. Four ssDNA plant viruses; BBTV, TYDV, Chloris striate mosaic virus (CSMV) and Tomato leaf curl virus - Australia (ToLCV-Au) were assessed via Southern blot analysis for their ability to initiate both autonomous replication of infectious clones and episomal amplification within three different plant hosts; tobacco, sugarcane and banana. Results from cross replication studies indicated a complex interaction between viral and host replication components. BBTV infectious clones and episomal vectors were restricted to replication within banana host cells providing a clear indication that episomal amplification vectors based on BBTV are restricted to Musa spp. BBTV M-Rep was unable to recognise the viral regulatory DNA sequences of the other three ssDNA viruses. TYDV infectious clones and episomal vectors were capable of replicating within all three host cells tested, indicating that TYDV is capable of undergoing RCR within a broad range of plant hosts. TYDV Rep was also capable of recognising the viral regulatory DNA sequences of both CSMV and BBTV given favourable conditions within specific plant hosts. Replication of the CSMV infectious clone was not detected in any of the three host cells, although fidelity of this clone requires further confirmation. CSMV episomal vectors were functional within banana host cells only, indicating that although closely related to TYDV, episomal amplification vectors based on CSMV have a restricted host range. CSMV Rep could not initiate RCR of episomal amplification vectors containing the viral regulatory DNA regions of the other three viruses in any of the plant host cells. ToLCV-Au infectious clones were capable of replicating within banana and tobacco host cells. Episomal amplification vectors based on ToLCV-Au extended the host range to sugarcane. ToLCV-Au Rep was unable to recognise the viral regulatory DNA sequences of the other three viruses in any of the plant host cells. The ability for a viral Rep to recognise its own cognate regulatory DNA sequences within alternate plant host cells is variable. Episomal amplification vectors based on TYDV and ToLCV-Au appear to be the most suitable for the further development of a virusbased bioreactor system in sugarcane. This study details the initial steps taken towards the development of a virus-based episomal amplification system in sugarcane. In doing so, fundamental knowledge into the mechanisms involved in Rep recognition of viral regulatory DNA sequences has been gathered. These research findings will provide a solid foundation for the further development of a sugarcane-based bioreactor.

No description available.

The involvement of topoisomerase in DNA replication in a prokaryotic and a eukaryotic system was examined. The mechanism of in vitro DNA replication by an isolated replicative enzyme complex was also investigated. In bacteriophage T4 there is evidence that the type II topoisomerase coded for by the phage is involved in the initiation of replicative growing points. We have looked at the topological structure of the replicating T4 nucleoid by sedimentation of the DNA in neutral sucrose gradients containing various amounts of ethidium bromide. It was determined that at no time after infection does the replicating T4 DNA contain any large amount of negative superhelicity. The absence of the phage topoisomerase did not affect the topology of the nucleoid. It was therefore concluded that the role of the T4 topoisomerase in initiating DNA synthesis in T4 was not exerted at the level of the general topology of the replicating T4 DNA. An isolation procedure for the T4 topoisomerase for pursuance of further studies was also described. New mammalian topoisomerases were shown to be stimulated by epidermal growth factor (EGF) in two cultured fibroblast cell lines. Topoisomerase activity was seen first in the cell cytoplasm and subsequently in the nucleus. The peak of topoisomerase activity in the nucleus corresponded to the peak of EGF-induced DNA synthesis in the cells. At least a part of the topoisomerase activity stimulated by EGF was shown to be due to a type II topoisomerase by the ATP-dependence of the activity. The topoisomerase activity in the cytoplasm was shown to exist in a non-soluble, sedimentable form. Further characterization of the topoisomerase containing complex isolated from the cytoplasm was carried out. The complex was seen to be non-membrane bound and complex. DNA polymerase and nucleoside diphosphate kinase activities were also demonstrated to be contained within the complex. It was shown that this cytoplasmic complex was capable of in vitro DNA replication. Many parameters of the in vitro DNA replication reaction were examined. The process was seen to mimic in vivo replication in several ways. The complex was shown to not only be able to elongate DNA but to initiate replication through the creation of a replication bubble.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

No description available.

No description available.