Browsing by Author "Rebecca Boston, Committee Member"

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    Genomic and Molecular Analyses of the Core DNA Replication Machinery in Plants
    (2008-04-04) Shultz, Randall William; Rebecca Boston, Committee Member; Jeffrey Thorne, Committee Member; William Thompson, Committee Co-Chair; George Allen, Committee Co-Chair; Steven Spiker, Committee Member
    Accurate and complete DNA replication is essential for maintaining the integrity of the genome. In eukaryotes, this process requires the coordinated action of numerous molecular machines. Based on yeast and animal model systems, we defined a set of fifty-one "core DNA replication proteins" that are integral to the initiation, DNA synthesis, and Okazaki fragment maturation functions of DNA replication. We used computational analyses to identify putative homologs in the genomes of two plants, Arabidopsis thaliana (Arabidopsis) and Oryza sativa (rice), providing the first comprehensive view of the core DNA replication machinery in plants. Our results indicated that the overall composition of this apparatus is conserved, but plants are unique in that multiple DNA replication genes exist as small gene families. Fourteen of the genes we annotated in this study have not been previously reported in the literature, and we have provided revised gene models for seventeen plant proteins. To better understand how the DNA replication machinery functions in plants, we cloned multiple subunits of the pre-replication complex (pre-RC) from Arabidopsis and generated antibodies against four key components of this complex — AtORC1, AtORC2, AtMCM5, and AtMCM7. We demonstrated that the pre-RC is developmentally regulated in Arabidopsis and, consistent with a role in DNA replication, is abundant in proliferating tissues. We used immunocytochemical and biochemical methods to characterize MCM7 in plants. We observed two distinct localization patterns for plant MCM7 proteins. In most cells, MCM7 was nuclear and colocalized with DNA. In a small fraction of cells, MCM7 was dispersed throughout the cytoplasmic compartment. Biochemical analysis confirmed that MCM7 binds to chromatin and that it is present in the nucleus at least during the G1, S and G2 cell cycle stages. Together, these analyses support a model where the MCM complex is loaded onto DNA in late M and early G1, released into the nucleoplasm during S phase followed by a brief dispersion into the cytoplasmic compartment concurrent with nuclear envelope breakdown in mitosis.
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    Sinks Above and Sinks Below - Physical Mapping of Fruit-derived ESTs in Peach, a Model Species for Prunus and Rosaceae Structural Genomics, and Identification of Amplified Fragment Length Polymorphism (AFLP) Markers Associated with Internal Heat Necrosis (IHN) in 4x-2x Solanum tuberosum x S. phureja-S. stenotomum hybrids
    (2005-07-26) McCord, Per Hilding; Craig Yencho, Committee Co-Chair; Bryon Sosinski, Committee Co-Chair; Rebecca Boston, Committee Member
    Peach (Prunus persica (L.) Batsch) has been proposed as a model organism for structural genomics in the family Rosaceae. Several resources have been or are currently being developed to study the structural genomics of the species. A highly saturated genetic linkage map has been developed from an interspecific 'Texas' almond X 'Earligold' peach cross (T X E), and two peach BAC libraries from the cultivars 'Nemared' and 'Lovell' have been generated for the creation of a physical mapping resource. A framework map has been generated by the hybridization of markers from the T X E and other Prunus maps to the 'Nemared' library. In addition, peach and almond unigenes are being hybridized to the library in order to generate an anchored transcript map, identify contigs for overall map construction, and provide information on genome organization. A subgroup of the Prunus unigene set comprising 942 ESTs derived from peach fruit mesocarp was hybridized to the 44,150-clone 'Nemared' library. Eight hundred ninety-nine of these ESTs successfully hybridized to the library, identifying 3475 BACs, of which 2725 were unique. Seventy-six of these ESTs are now anchored to the reference map by hybridization to genetically anchored BAC clones or contigs. Twenty-six of these ESTs mapped near loci for important agronomic traits, including flesh acidity, fruit skin pubescence, and almond shell hardness. Unanchored ESTs generated contigs of 2 to 44 clones, which are being fingerprinted and integrated into the growing peach physical map in preparation for sequencing of the peach genome. Internal heat necrosis (IHN) is a physiological disorder of potato tubers resulting in the discoloration of parenchymal tissue. Environmental conditions and the large acreage of the IHN-susceptible cultivar 'Atlantic' result in significant losses due to the disorder in the mid-Atlantic states and Florida. A combination of bulked segregant analysis and AFLP marker technology was used to search for molecular markers for IHN in a population of tetraploid 4x-2x S. tuberosum x S. phureja-S. stenotomum (tub x phu-stn) hybrids. These clones are being used to breed an IHN-resistant, high specific-gravity replacement for 'Atlantic'. One marker, potPCR13-HindIII-R, was identified in two small test populations and was strongly associated with resistance to IHN, explaining 69.9% and 64 % of the observed variation for IHN incidence and severity, respectively. This marker showed strong sequence homology to calcium-dependent protein kinases (CDPKs), reinforcing prior evidence of the importance of calcium to the manifestation of IHN. When tested on a different set of clones from a combining ability study for IHN, this marker was no longer significant. However, a second putative marker from the BSA-AFLP screen (potPCR31-A) that was not statistically significant in the small populations, was significantly associated with IHN susceptibility in this second population, and the regression models of potPCR-31-A on IHN severity and incidence explained 17.8% and 18.6% of the phenotypic variation, respectively. This marker showed homology to plastid terminal oxidases (PTOXs), which are involved in desaturation of carotenoids, and may have a role in protecting biomolecules under oxidative stress. The small sample sizes used in marker development and initial testing may have resulted in these markers being significant predictors only in certain populations. It is also possible that, given the quantitative nature of IHN, only a subset of all IHN-associated loci may be necessary for the disorder to be manifest. This research is the first molecular marker study of the genetics of IHN, and has set a foundation for future studies.