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1.  Exclusion of the 750-kb Genetically Unstable Region at Xq27 as a Candidate Locus for Prostate Malignancy in HPCX1-linked Families 
Genes, chromosomes & cancer  2012;51(10):933-948.
Several linkage studies provided evidence for the presence of the hereditary prostate cancer locus, HPCX1, at Xq27-q28. The strongest linkage peak of prostate cancer overlies a variable region of ~750 kb at Xq27 enriched by segmental duplications (SDs), suggesting that the predisposition to prostate cancer may be a genomic disorder caused by recombinational interaction between SDs. The large size of SDs and their sequence similarity make it difficult to examine this region for possible rearrangements using standard methods. To overcome this problem, direct isolation of a set of genomic segments by in vivo recombination in yeast (a TAR cloning technique) was used to perform a mutational analysis of the 750 kb region in X-linked families. We did not detect disease-specific rearrangements within this region. In addition, transcriptome and computational analyses were performed to search for non-annotated genes within the Xq27 region, which may be associated with genetic predisposition to prostate cancer. Two candidate genes were identified, one of which is a novel gene termed SPANXL that represents a highly diverged member of the SPANX gene family, and the previously described CDR1 gene that is expressed at a high level in both normal and malignant prostate cells, and mapped 210 kb of upstream the SPANX gene cluster. No disease-specific alterations were identified in these genes. To summarize, our results exclude the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy. Adjacent regions appear to be the most likely candidates to identify the elusive HPCX1 locus.
doi:10.1002/gcc.21977
PMCID: PMC3412920  PMID: 22733720
Xq27; hereditary prostate cancer; HPCX1; SPANXL; CDR1; TAR cloning
2.  A new assay for measuring chromosome instability (CIN) and identification of drugs that elevate CIN in cancer cells 
BMC Cancer  2013;13:252.
Background
Aneuploidy is a feature of most cancer cells that is often accompanied by an elevated rate of chromosome mis-segregation termed chromosome instability (CIN). While CIN can act as a driver of cancer genome evolution and tumor progression, recent findings point to the existence of a threshold level beyond which CIN becomes a barrier to tumor growth and therefore can be exploited therapeutically. Drugs known to increase CIN beyond the therapeutic threshold are currently few in number, and the clinical promise of targeting the CIN phenotype warrants new screening efforts. However, none of the existing methods, including the in vitro micronuclei (MNi) assay, developed to quantify CIN, is entirely satisfactory.
Methods
We have developed a new assay for measuring CIN. This quantitative assay for chromosome mis-segregation is based on the use of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene. Thus, cells that inherit the HAC display green fluorescence, while cells lacking the HAC do not. This allows the measurement of HAC loss rate by routine flow cytometry.
Results
Using the HAC-based chromosome loss assay, we have analyzed several well-known anti-mitotic, spindle-targeting compounds, all of which have been reported to induce micronuclei formation and chromosome loss. For each drug, the rate of HAC loss was accurately measured by flow cytometry as a proportion of non-fluorescent cells in the cell population which was verified by FISH analysis. Based on our estimates, despite their similar cytotoxicity, the analyzed drugs affect the rates of HAC mis-segregation during mitotic divisions differently. The highest rate of HAC mis-segregation was observed for the microtubule-stabilizing drugs, taxol and peloruside A.
Conclusion
Thus, this new and simple assay allows for a quick and efficient screen of hundreds of drugs to identify those affecting chromosome mis-segregation. It also allows ranking of compounds with the same or similar mechanism of action based on their effect on the rate of chromosome loss. The identification of new compounds that increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target the CIN phenotype in cancer cells.
doi:10.1186/1471-2407-13-252
PMCID: PMC3671967  PMID: 23694679
Human artificial chromosome; HAC; Chromosome instability; CIN; Drug treatment
3.  Protecting a transgene expression from the HAC-based vector by different chromatin insulators 
Human artificial chromosomes (HACs) are vectors that offer advantages of capacity and stability for gene delivery and expression. Several studies have even demonstrated their use for gene complementation in gene-deficient recipient cell lines and animal transgenesis. Recently, we constructed an advance HAC-based vector, alphoidtetO-HAC, with a conditional centromere. In this HAC, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While by definition this domain is permissive for transcription, there have been no long-term studies on transgene expression within centrochromatin. In this study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA, and tDNA, on the expression of an EGFP transgene inserted into the alphoidtetO-HAC vector. Insulator function was essential for stable expression of the transgene in centrochromatin. In two analyzed host cell lines, a tDNA insulator composed of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-013-1362-9) contains supplementary material, which is available to authorized users.
doi:10.1007/s00018-013-1362-9
PMCID: PMC3771377  PMID: 23677492
Insulator; tDNA-gamma-satellite; cHS4; Human artificial chromosome-HAC
4.  Rapid generation of long tandem DNA repeat arrays by homologous recombination in yeast to study their function in mammalian genomes 
We describe here a method to rapidly convert any desirable DNA fragment, as small as 100 bp, into long tandem DNA arrays up to 140 kb in size that are inserted into a microbe vector. This method includes rolling-circle phi29 amplification (RCA) of the sequence in vitro and assembly of the RCA products in vivo by homologous recombination in the yeast Saccharomyces cerevisiae. The method was successfully used for a functional analysis of centromeric and pericentromeric repeats and construction of new vehicles for gene delivery to mammalian cells. The method may have general application in elucidating the role of tandem repeats in chromosome organization and dynamics. Each cycle of the protocol takes ~ two weeks to complete.
doi:10.1186/1480-9222-13-8
PMCID: PMC3200152  PMID: 21982381

Results 1-4 (4)