(1/58) Novel generation of human satellite DNA-based artificial chromosomes in mammalian cells.
An in vivo approach has been developed for generation of artificial chromosomes, based on the induction of intrinsic, large-scale amplification mechanisms of mammalian cells. Here, we describe the successful generation of prototype human satellite DNA-based artificial chromosomes via amplification-dependent de novo chromosome formations induced by integration of exogenous DNA sequences into the centromeric/rDNA regions of human acrocentric chromosomes. Subclones with mitotically stable de novo chromosomes were established, which allowed the initial characterization and purification of these artificial chromosomes. Because of the low complexity of their DNA content, they may serve as a useful tool to study the structure and function of higher eukaryotic chromosomes. Human satellite DNA-based artificial chromosomes containing amplified satellite DNA, rDNA, and exogenous DNA sequences were heterochromatic, however, they provided a suitable chromosomal environment for the expression of the integrated exogenous genetic material. We demonstrate that induced de novo chromosome formation is a reproducible and effective methodology in generating artificial chromosomes from predictable sequences of different mammalian species. Satellite DNA-based artificial chromosomes formed by induced large-scale amplifications on the short arm of human acrocentric chromosomes may become safe or low risk vectors in gene therapy. (+info)
(2/58) Genomic structure, chromosomal localization and identification of mutations in the xeroderma pigmentosum variant (XPV) gene.
The xeroderma pigmentosum variant (XP-V) is one of the most common forms of this cancer-prone syndrome. XP groups A through G are characterized by defective nucleotide excision repair, whereas the XP-V phenotype is proficient in this pathway. The XPV gene encodes DNA polymerase eta, which catalyzes an accurate translesion synthesis, indicating that the XPV gene contributes tumor suppression in normal individuals. Here we describe the genomic structure and chromosomal localization of the XPV gene, which includes 11 exons covering the entire coding sequence, lacks a TATA sequence in the upstream region of the transcription-initiation, and is located at the chromosome band 6p21.1-6p12. Analyses of patient-derived XP-V cell lines strongly suggested that three of four cell lines carried homozygous mutations in the XPV gene. The fourth cell line, XP1RO, carried heterozygous point mutations in the XPV gene, one of which was located at the splice acceptor site of exon 2, resulting in the omission of exon 2 from the mature mRNA. These findings provide a basis for diagnosis and therapy of XP-V patients. (+info)
(3/58) Molecular and cytological analysis of a 5.5 Mb minichromosome.
Mammalian artificial chromosomes (MACs) provide a new tool for the improvement of our knowledge of chromosome structure and function. Moreover, they constitute an alternative and potentially powerful tool for gene delivery both in cultured cells and for the production of transgenic animals. In the present work we describe the molecular structure of MC1, a human minichromosome derived from chromosome 1. By means of restriction and hybridization analysis, satellite-PCR, in situ hybridization on highly extended chromatin fibres, and indirect immunofluorescence, we have established that: (i) MC1 has a size of 5.5 Mb; (ii) it consists of 1.1 Mb alphoid, 3.5 Mb Sat2 DNA, and telomeric and subtelomeric sequences at both ends; (iii) it contains an unusual region of interspersed Sat2 and alphoid DNAs at the junction of the alphoid and the Sat2 blocks; and (iv) the two alphoid blocks and the Sat2-alphoid region bind centromeric proteins suggesting that they participate in the formation of a functional kinetochore. (+info)
(4/58) Functional complementation of a genetic deficiency with human artificial chromosomes.
We have shown functional complementation of a genetic deficiency in human cultured cells, using artificial chromosomes derived from cloned human genomic fragments. A 404-kb human-artificial-chromosome (HAC) vector, consisting of 220 kb of alphoid DNA from the centromere of chromosome 17, human telomeres, and the hypoxanthine guanine phosphoribosyltransferase (HPRT) genomic locus, was transferred to HPRT-deficient HT1080 fibrosarcoma cells. We generated several cell lines with low-copy-number, megabase-sized HACs containing a functional centromere and one or possibly several copies of the HPRT1 gene complementing the metabolic deficiency. The HACs consisted of alternating alphoid and nonalphoid DNA segments derived only from the input DNA (within the sensitivity limits of FISH detection), and the largest continuous alphoid segment was 158-250 kb. The study of both the structure and mitotic stability of these HACs offers insights into the mechanisms of centromere formation in synthetic chromosomes and will further the development of this human-gene-transfer technology. (+info)
(5/58) Genomic and genetic definition of a functional human centromere.
The definition of centromeres of human chromosomes requires a complete genomic understanding of these regions. Toward this end, we report integration of physical mapping, genetic, and functional approaches, together with sequencing of selected regions, to define the centromere of the human X chromosome and to explore the evolution of sequences responsible for chromosome segregation. The transitional region between expressed sequences on the short arm of the X and the chromosome-specific alpha satellite array DXZ1 spans about 450 kilobases and is satellite-rich. At the junction between this satellite region and canonical DXZ1 repeats, diverged repeat units provide direct evidence of unequal crossover as the homogenizing force of these arrays. Results from deletion analysis of mitotically stable chromosome rearrangements and from a human artificial chromosome assay demonstrate that DXZ1 DNA is sufficient for centromere function. Evolutionary studies indicate that, while alpha satellite DNA present throughout the pericentromeric region of the X chromosome appears to be a descendant of an ancestral primate centromere, the current functional centromere based on DXZ1 sequences is the product of the much more recent concerted evolution of this satellite DNA. (+info)
(6/58) A new approach to genome mapping and sequencing: slalom libraries.
We describe here an efficient strategy for simultaneous genome mapping and sequencing. The approach is based on physically oriented, overlapping restriction fragment libraries called slalom libraries. Slalom libraries combine features of general genomic, jumping and linking libraries. Slalom libraries can be adapted to different applications and two main types of slalom libraries are described in detail. This approach was used to map and sequence (with approximately 46% coverage) two human P1-derived artificial chromosome (PAC) clones, each of approximately 100 kb. This model experiment demonstrates the feasibility of the approach and shows that the efficiency (cost-effectiveness and speed) of existing mapping/sequencing methods could be improved at least 5-10-fold. Furthermore, since the efficiency of contig assembly in the slalom approach is virtually independent of length of sequence reads, even short sequences produced by rapid, high throughput sequencing techniques would suffice to complete a physical map and a sequence scan of a small genome. (+info)
(7/58) Alpha-satellite DNA and vector composition influence rates of human artificial chromosome formation.
Human artificial chromosomes (HACs) have been proposed as a new class of potential gene transfer and gene therapy vector. HACs can be formed when bacterial cloning vectors containing alpha-satellite DNA are transfected into cultured human cells. We have compared the HAC-forming potential of different sequences to identify features critical to the efficiency of the process. Chromosome 17 or 21 alpha-satellite arrays are highly competent HAC-forming substrates in this assay. In contrast, a Y-chromosome-derived alpha-satellite sequence is inefficient, suggesting that centromere specification is at least partly dependent on DNA sequence. The length of the input array is also an important determinant, as reduction of the chromosome-17-based array from 80 kb to 35 kb reduced the frequency of HAC formation. In addition to the alpha-satellite component, vector composition also influenced HAC formation rates, size, and copy number. The data presented here have a significant impact on the design of future HAC vectors that have potential to be developed for therapeutic applications and as tools for investigating human chromosome structure and function. (+info)
(8/58) Chromosome engineering: prospects for gene therapy.
Recent advances in chromosome engineering and the potential for downstream applications in gene therapy were presented at the Artificial Chromosome Session of Genome Medicine: Gene Therapy for the Millennium in Rome, Italy in September 2001. This session concentrated primarily on the structure and function of human centromeres and the ongoing challenge of equipping human artificial chromosomes (HACs) with centromeres to ensure their mitotic stability. Advances in the 'bottom up' construction of HACs included the transfer into HT1080 cells of circular PACs containing alpha satellite DNA, and the correction of HPRT deficiency in cells using HACs. Advances in the 'top down' construction of HACs using telomere associated chromosome fragmentation in DT40 cells included the formation of HACs that are less than a megabase in size and transfer of HACs through the mouse germline. Significant progress has also been made in the use of human minichromosomes for stable trans-gene expression. While many obstacles remain towards the use of HACs for gene therapy, this session provided an optimistic outlook for future success. (+info)