Thesis (Ph. D.)--School of Biological Sciences. University of Missouri--Kansas City, 2006. / "A dissertation in molecular biology and biochemistry and cell biology and biophysics." Advisor: Lindsey M. Hutt-Fletcher. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Nov. 9, 2007. Includes bibliographical references (leaves 89-103). Online version of the print edition.

Human Artificial Chromosomes (HAC) are fascinating extrachromosomal molecules that stay independently from the host genome and are capable of segregating as efficiently as endogenous chromosomes. It has been proven that HAC are potential tools for both basic chromosome behavioural research and agents for gene therapy purposes. My DPhil project is divided into two main themes. The first theme was to develop a novel artificial chromosome in mouse embryonic stem cells. The second theme was to understand the factors affecting chromosome stability which may also affect the efficiency of artificial chromosome formation. so that our protocol for better HAC preparation can be refined. There are six results chapters in my thesis. The first three chapters described how I developed human artificial chromosomes in mouse embryonic stem cells. Initially, vectors containing a long stretch of human alphoid DNA were delivered to mouse cells using the Herpes Simplex Virus-I (HSV-l) amplicon system but the efficiency was low. Next, mouse pericentromeric and centromeric DNAs were employed for mouse artificial chromosome (MAC) via HSV-l system. However, the efficiency remained the same. Finally, I used the Microcell-Mediated Chromosome Transfer (MMCT) system to transfer HAC from HTl 080 cells into mouse ES cells and successfully established HAC in ES which were highly stable. The results obtained in this first part of my thesis suggested that to increase HAC formation efficiency it would be necessary to improve the techniques of HSV-I delivery and MMCT. Moreover, it would also be important to better characterize factors affecting chromosome behaviour. The last three results chapters focus on factors affecting chromosomes stability and improving the HSV -1 delivery system and MMCT. I undertook an in vivo study of whole cell fusion experiments with the aid of live cell irnaging system, and found that histone H2B proteins underwent a dynamic assembly/disassembly processes. Live cell imaging of MMCT suggested that the microcell delivery is a very slow process and the results may lead to a refinement of the MMCT protocol. I found it is possible to generate a single HAC using two HSV-l amplicons containing two different constructs, potentially doubling the HSV-l HAC capacity from 150 kb to 300 kb. The last chapter illustrated how the expression of non- coding centromeric satellites impaired chromosome stability in both human cultured and human embryonic stem cells. The findings revealed that non-coding centromeric RNA plays an important role on chromosome stability that might be important for artificial chromosome development.

Thesis (M.S.)--University of Delaware, 2009. / Principal faculty advisor: Mark Parcells, Dept. of Animal & Food Sciences. Includes bibliographical references.

Thesis(M.S.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references.

No description available.

Thesis (Ph.D.)--Mississippi State University. Department of Plant and Soil Sciences. / Title from title screen. Includes bibliographical references.

Yes / Rapid advances in short-read DNA sequencing technologies have revolutionized population genomic studies, but there are genomic regions where this technology reaches its limits. Limitations mostly arise due to the difficulties in assembly or alignment to genomic regions of high sequence divergence and high repeat content, which are typical characteristics for loci under strong long-term balancing selection. Studying genetic diversity at such loci therefore remains challenging. Here, we investigate the feasibility and error rates associated with targeted long-read sequencing of a locus under balancing selection. For this purpose, we generated bacterial artificial chromosomes (BACs) containing the Brassicaceae S-locus, a region under strong negative frequency-dependent selection which has previously proven difficult to assemble in its entirety using short reads. We sequence S-locus BACs with single-molecule long-read sequencing technology and conduct de novo assembly of these S-locus haplotypes. By comparing repeated assemblies resulting from independent long-read sequencing runs on the same BAC clone we do not detect any structural errors, suggesting that reliable assemblies are generated, but we estimate an indel error rate of 5.7×10−5. A similar error rate was estimated based on comparison of Illumina short-read sequences and BAC assemblies. Our results show that, until de novo assembly of multiple individuals using long-read sequencing becomes feasible, targeted long-read sequencing of loci under balancing selection is a viable option with low error rates for single nucleotide polymorphisms or structural variation. We further find that short-read sequencing is a valuable complement, allowing correction of the relatively high rate of indel errors that result from this approach. / This study was supported by a grant from the Swedish Research Council to T.S.

In the present study, the W sex-chromosome of the codling moth was studied by means of fluorescence in situ hybridization (FISH) with probes prepared from bacterial artificial chromosome (BAC), which were isolated from the codling moth BAC library. The BAC library was screened for clones derived from both the W and Z sex chromosomes using three sets of molecular markers of codling moth sex chromosomes. A total of 54 BAC clones have been obtained. In this work, only 3 W-derived BAC clones and 1 Z-derived BAC clone were further characterized by BAC-FISH mapping on chromosome preparations of pachytene oocytes; the other BAC clones have been retained for next studies. Whereas the Z-BAC probe provided a discrete hybridization signal on the Z chromosome, and surprisingly on the W chromosome, the W-BAC probes showed multiple hybridization signals distributed on the whole W chromosome, suggesting that they are mainly composed of repetitive sequences, which occur in multiple clusters on the W chromosome. The specific pattern of W-BAC hybridization signals along with the discrete signal of the Z-BAC enabled us to discriminate left/right orientation of both the W and Z chromosomes and examine specificity of W-Z pairing during meiotic prophase I.

A phylogenetic tree based on ITS1, Adh1 and ndhF grouped the species of the genus Sorghum into one distinct monophyletic group, but including two sister lineages, one with x=5, the other with x=10 as basic chromosome numbers. The goal of this study was to elucidate major patterns in Sorghum genome evolution, particularly n=5 vs. n=10 genomes. A very recent molecular cytogenetic study in our laboratory revealed striking structural karyotypic rearrangements between S. bicolor (x=10) and an x=5 Sorghum species, S. angustum; so an immediate objective here was to determine if identical or similar rearrangements exist in other wild Sorghum species. Our approach was [1] to extend similar methods to additional species, i.e., fluorescent in situ hybridization (FISH) analyses of sorghum genomic bacterial artificial chromosome clones and multi-BAC cocktail probes to mitotic chromosomes of S. angustum, S. versicolor, S. brachypodum and S. intrans; and [2] to augment the BAC-FISH findings by comparing telomeric and ribosomal DNA FISH signal distributions to x=5 and x=10 Sorghum species. Signals from in situ hybridizations of BAC-based probes were insufficiently robust and insufficiently localized to delineate FISH signal patterns akin to those discovered previously in S. angustum. Southern blots of the same BACs to restricted DNA of these species revealed relatively moderate affinity to smeared DNA, suggesting homology to non-tandemized sequences. FISH of the A-type TRS (Arabidopsis-like telomeric repeat sequence) revealed its presence is limited to terminal chromosomal regions of the Sorghum species tested, except S. brachypodum, which displayed intercalary signal on one chromosome and no detachable signal at its termini region. The hybridization of 45S and 5S rDNA revealed that the respective sites of tandemized clusters differ among species in terms of size, number and location, except S. angustum versus S. versicolor. Well localized BAC-FISH signals normally occur when signals from low-copy sequences discernibly exceed background signal, including those from hybridization of dispersed repetitive elements. The low level of signal intensity from BAC low-copy sequences relative to the background signal "noise" seems most likely due to low homology and(or) technical constraints. Extensive dispersal of low-copy sequences that are syntenic in S. bicolor seems unlikely, but possible. In conclusion, the result was a lack of clear experimental success with BAC-FISH and an inability to effectively screen for S. angustum-like rearrangements using BAC-FISH. The telomeric and rDNA FISH indicated that the x=5 genomes vary extensively. One can surmise that although the arrangements seen in S. angustum might extend to S. versicolor, they certainly do not extend to S. versicolor, they certainly do not extend to S. intrans or S. brachypodum. It is clear that S. brachypodum has telomeric repeats that are either very short or rely on some sequence other than the A-type TRS.

The development of safe and efficient gene delivery systems in pluripotent human embryonic stem cells (hESc) is essential to realising their full potential for basic and clinical research. The purpose of this study was to develop an efficient, non-integrating gene expression system in pluripotent hESc using human artificial chromosomes (HAC). Similar to endogenous chromosomes, HAC are capable of gene expression, replication and segregation during cell division. Unlike retroviral-mediated gene delivery vectors, HAC do not integrate into the host genome and can encompass large genomic regions for the delivery of multiple genes. Despite the advantages HAC offer, their use has been limited due to laborious cloning procedures and poor transfection efficiencies, and thus only studied in immortalised and tumour-derived human cell lines. In this study, the high transduction efficiency of herpes simplex virus type-1 (HSV-1) amplicons was utilised to overcome the described difficulties and delivered HAC vectors into pluripotent hESc. Analysis of stable hESc clones showed that de novo gene-expressing HAC were present at high frequencies ranging from 10-70% of metaphases analysed, without integrating into the genome. The established HAC contained an active centromere, and were stably maintained without integration or loss in the absence of selection for 90 days. Stable HAC-containing hESc clones retained their pluripotency as demonstrated by neuronal differentiation, in vitro germ layer and teratoma formation assays. HAC gene expression persisted, with some variation, post-differentiation in the various deriving cell types. This is the first report of successful de novo HAC formation in hESc for gene expression studies. These findings show potential for delivering high-capacity genomic constructs safely and efficiently into pluripotent cells for the purpose of genetic manipulation and ultimately patient-specific somatic gene therapy.