Transformation-associated recombination (TAR) cloning and its applications for gene function; genome architecture and evolution; biotechnology and biomedicine.

Transformation-associated recombination (TAR) cloning represents a unique tool to selectively and efficiently recover a given chromosomal segment up to several hundred kb in length from complex genomes (such as animals and plants) and simple genomes (such as bacteria and viruses). The technique exploits a high level of homologous recombination in the yeast Sacharomyces cerevisiae. In this review, we summarize multiple applications of the pioneering TAR cloning technique, developed previously for complex genomes, for functional, evolutionary, and structural studies, and extended the modified TAR versions to isolate biosynthetic gene clusters (BGCs) from microbes, which are the major source of pharmacological agents and industrial compounds, and to engineer synthetic viruses with novel properties to design a new generation of vaccines. TAR cloning was adapted as a reliable method for the assembly of synthetic microbe genomes for fundamental research. In this review, we also discuss how the TAR cloning in combination with HAC (human artificial chromosome)- and CRISPR-based technologies may contribute to the future.

Actively transcribed rDNA and distal junction (DJ) sequence are involved in association of NORs with nucleoli.

Although they are organelles without a limiting membrane, nucleoli have an exclusive structure, built upon the rDNA-rich acrocentric short arms of five human chromosomes (nucleolar organizer regions or NORs). This has raised the question: what are the structural features of a chromosome required for its inclusion in a nucleolus? Previous work has suggested that sequences adjacent to the tandemly repeated rDNA repeat units (DJ, distal junction sequence) may be involved, and we have extended such studies by addressing several issues related to the requirements for the association of NORs with nucleoli. We exploited both a set of somatic cell hybrids containing individual human acrocentric chromosomes and a set of Human Artificial Chromosomes (HACs) carrying different parts of a NOR, including an rDNA unit or DJ or PJ (proximal junction) sequence. Association of NORs with nucleoli was increased when constituent rDNA was transcribed and may be also affected by the status of heterochromatin blocks formed next to the rDNA arrays. Furthermore, our data suggest that a relatively small size DJ region, highly conserved in evolution, is also involved, along with the rDNA repeats, in the localization of p-arms of acrocentric chromosomes in nucleoli. Thus, we infer a cooperative action of rDNA sequence-stimulated by its activity-and sequences distal to rDNA contributing to incorporation into nucleoli. Analysis of NOR sequences also identified LncRNA_038958 in the DJ, a candidate transcript with the region of the suggested promoter that is located close to the DJ/rDNA boundary and contains CTCF binding sites. This LncRNA may affect RNA Polymerase I and/or nucleolar activity. Our findings provide the basis for future studies to determine which RNAs and proteins interact critically with NOR sequences to organize the higher-order structure of nucleoli and their function in normal cells and pathological states.

Novel assay to measure chromosome instability identifies Punica granatum extract that elevates CIN and has a potential for tumor- suppressing therapies.

Human artificial chromosomes (HACs) have provided a useful tool to study kinetochore structure and function, gene delivery, and gene expression. The HAC propagates and segregates properly in the cells. Recently, we have developed an experimental high-throughput imaging (HTI) HAC-based assay that allows the identification of genes whose depletion leads to chromosome instability (CIN). The HAC carries a GFP transgene that facilitates quantitative measurement of CIN. The loss of HAC/GFP may be measured by flow cytometry or fluorescence scanning microscope. Therefore, CIN rate can be measured by counting the proportion of fluorescent cells. Here, the HAC/GFP-based assay has been adapted to screen anticancer compounds for possible induction or elevation of CIN. We analyzed 24 cytotoxic plant extracts. Punica granatum leaf extract (PLE) indeed sharply increases CIN rate in HT1080 fibrosarcoma cells. PLE treatment leads to cell cycle arrest, reduction of mitotic index, and the increased numbers of micronuclei (MNi) and nucleoplasmic bridges (NPBs). PLE-mediated increased CIN correlates with the induction of double-stranded breaks (DSBs). We infer that the PLE extract contains a component(s) that elevate CIN, making it a candidate for further study as a potential cancer treatment. The data also provide a proof of principle for the utility of the HAC/GFP-based system in screening for natural products and other compounds that elevate CIN in cancer cells.

Assembly of Multiple Full-Size Genes or Genomic DNA Fragments on Human Artificial Chromosomes Using the Iterative Integration System.

Human artificial chromosomes (HACs) are gene delivery vectors that have been used for decades for gene functional studies. HACs have several advantages over viral-based gene transfer systems, including stable episomal maintenance in a single copy in the cell and the ability to carry up to megabase-sized genomic DNA segments. We have previously developed the alphoidtetO -HAC, which has a single gene acceptor loxP site that allows insertion of an individual gene of interest using Chinese hamster ovary (CHO) hybrid cells. The HAC, along with a DNA segment of interest, can then be transferred from donor CHO cells to various recipient cells of interest via microcell-mediated chromosome transfer (MMCT). Here, we detail a protocol for loading multiple genomic DNA segments or genes into the alphoidtetO -HAC vector using an iterative integration system (IIS) that utilizes recombinases Cre, ΦC31, and ΦBT. This IIS-alphoidtetO -HAC can be used for either serially assembling genomic loci or fragments of a large gene, or for inserting multiple genes into the same artificial chromosome. The insertions are executed iteratively, whereby each round results in the insertion of a new DNA segment of interest. This is accompanied by changes of expression of marker fluorescent proteins, which simplifies screening of correct clones, and changes of selection and counterselection markers, which constitutes an error-proofing mechanism that removes mis-incorporated DNA segments. In addition, the IIS-alphoidtetO -HAC carrying the genes can be eliminated from the cells, offering the possibility to compare the phenotypes of human cells with and without functional copies of the genes of interest. The resulting HAC molecules may be used to investigate biomedically relevant pathways or the regulation of multiple genes, and to potentially engineer synthetic chromosomes with a specific set of genes of interest. The IIS-alphoidtetO -HAC system is expected to be beneficial in creating multiple-gene humanized models with the purpose of understanding complex multi-gene genetic disorders. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Integration of the first DNA segment of interest into the IIS-alphoidteto -HAC Basic Protocol 2: Integration of a second DNA segment of interest into the IIS-alphoidteto -HAC Basic Protocol 3: Integration of a third DNA segment of interest into the IIS-alphoidteto -HAC Support Protocol: Fluorescence in situ hybridization analysis for the circular IIS-alphoidtetO -HAC.

Highly Efficient Microcell-Mediated Transfer of HACs Containing a Genomic Region of Interest into Mammalian Cells.

Human artificial chromosomes (HACs) are considered promising tools for gene delivery, functional analyses, and gene therapy. HACs have the potential to overcome many of the problems caused by the use of viral-based gene transfer systems, such as limited cloning capacity, lack of copy number control, and insertional mutagenesis during integration into host chromosomes. The recently developed alphoidtetO -HAC has an advantage over other HAC vectors because it can be easily eliminated from dividing cells by inactivation of its conditional kinetochore. This provides a unique control mechanism to study phenotypes induced by a gene or genes carried on the HAC. The alphoidtetO -HAC has a single gene acceptor loxP site that allows insertion of an individual gene of interest or a cluster of genes of up to several Mb in size in Chinese hamster ovary (CHO) hybrid cells. The HACs carrying chromosomal copies of genes can then be transferred from these donor CHO cells to different recipient cells of interest via microcell-mediated chromosome transfer (MMCT). Here, we describe a detailed protocol for loading a gene of interest into the alphoidtetO -HAC vector and for the subsequent transfer of the HAC to recipient cells using an improved MMCT protocol. The original MMCT protocol includes treatment of donor cells with colcemid to induce micronucleation, wherein the HAC becomes surrounded with a nuclear membrane. That step is followed by disarrangement of the actin cytoskeleton using cytochalasin B to help induce microcell formation. The updated MMCT protocol, described here, features the replacement of colcemid and cytochalasin B with TN16 + griseofulvin and latrunculin B, respectively, and the use of collagen/laminin surface coating to promote attachment of metaphase cells to plates during micronuclei induction. These modifications increase the efficiency of HAC transfer to recipient cells ten fold. The improved MMCT protocol has been successfully tested on several recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Insertion of a BAC containing a gene of interest into a single loxP loading site of alphoidtetO -HAC in hamster CHO cells Basic Protocol 2: Microcell-mediated chromosome transfer from donor hamster CHO cells to mammalian cells.

Highly Selective, CRISPR/Cas9-Mediated Isolation of Genes and Genomic Loci from Complex Genomes by TAR Cloning in Yeast.

Here we describe an updated TAR cloning protocol for the selective and efficient isolation of any genomic fragment or gene of interest up to 280 kb in size from genomic DNA. The method exploits the special recombination machinery of the yeast Saccharomyces cerevisiae. TAR cloning is based on the high level of in vivo recombination that occurs between a specific genomic DNA fragment of interest and targeting sequences (hooks) in a TAR vector that are homologous to the 5' and 3' ends of the targeted region. Upon co-transformation into yeast, this results in the isolation of the chromosomal region of interest as a circular YAC molecule, which then propagates and segregates in yeast cells and can be selected for. In the updated TAR cloning protocol described here, the fraction of region-positive clones typically obtained is increased from 1% up to 35% by pre-treatment of the genomic DNA with specifically designed CRISPR/Cas9 endonucleases that create double-strand breaks (DSBs) bracketing the target genomic DNA sequence, thereby making the ends of the chromosomal region of interest highly recombinogenic. In addition, a new TAR vector was constructed that contains YAC and BAC cassettes, permitting direct transfer of a TAR-cloned DNA from yeast to bacterial cells. Once the TAR vector with the hooks is constructed and genomic DNA is prepared, the entire procedure takes 3 weeks to complete. The updated TAR protocol does not require significant yeast experience or extensively time-consuming yeast work because screening only about a dozen yeast transformants is typically enough to find a clone with the region of interest. TAR cloning of chromosomal fragments, individual genes, or gene families can be used for functional, structural, and population studies, for comparative genomics, and for long-range haplotyping, and has potential for gene therapy. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of CRISPR/Cas9-treated genomic DNA for TAR cloning Basic Protocol 2: Isolation of a gene or genomic locus by TAR cloning Basic Protocol 3: Transfer of TAR/YAC/BAC isolates from yeast to E. coli.

Terpyridine platinum compounds induce telomere dysfunction and chromosome instability in cancer cells.

Telomerase/telomere-targeting therapy is a potentially promising approach for cancer treatment because even transient telomere dysfunction can induce chromosomal instability (CIN) and may be a barrier to tumor growth. We recently developed a dual-HAC (Human Artificial Chromosome) assay that enables identification and ranking of compounds that induce CIN as a result of telomere dysfunction. This assay is based on the use of two isogenic HT1080 cell lines, one carrying a linear HAC (containing telomeres) and the other carrying a circular HAC (lacking telomeres). Disruption of telomeres in response to drug treatment results in specific destabilization of the linear HAC. Results: In this study, we used the dual-HAC assay for the analysis of the platinum-derived G4 ligand Pt-tpy and five of its derivatives: Pt-cpym, Pt-vpym, Pt-ttpy, Pt(PA)-tpy, and Pt-BisQ. Our analysis revealed four compounds, Pt-tpy, Pt-ttpy, Pt-vpym and Pt-cpym, that induce a specific loss of a linear but not a circular HAC. Increased CIN after treatment by these compounds correlates with the induction of double-stranded breaks (DSBs) predominantly localized at telomeres and reflecting telomere-associated DNA damage. Analysis of the mitotic phenotypes induced by these drugs revealed an elevated rate of chromatin bridges (CBs) in late mitosis and cytokinesis. These terpyridine platinum-derived G4 ligands are promising compounds for cancer treatment.

The genomic structure of a human chromosome 22 nucleolar organizer region determined by TAR cloning.

The rDNA clusters and flanking sequences on human chromosomes 13, 14, 15, 21 and 22 represent large gaps in the current genomic assembly. The organization and the degree of divergence of the human rDNA units within an individual nucleolar organizer region (NOR) are only partially known. To address this lacuna, we previously applied transformation-associated recombination (TAR) cloning to isolate individual rDNA units from chromosome 21. That approach revealed an unexpectedly high level of heterogeneity in human rDNA, raising the possibility of corresponding variations in ribosome dynamics. We have now applied the same strategy to analyze an entire rDNA array end-to-end from a copy of chromosome 22. Sequencing of TAR isolates provided the entire NOR sequence, including proximal and distal junctions that may be involved in nucleolar function. Comparison of the newly sequenced rDNAs to reference sequence for chromosomes 22 and 21 revealed variants that are shared in human rDNA in individuals from different ethnic groups, many of them at high frequency. Analysis infers comparable intra- and inter-individual divergence of rDNA units on the same and different chromosomes, supporting the concerted evolution of rDNA units. The results provide a route to investigate further the role of rDNA variation in nucleolar formation and in the empirical associations of nucleoli with pathology.

Analysis of Complex DNA Rearrangements during Early Stages of HAC Formation.

Human artificial chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN), and for possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC with a unique structure that cannot be precisely predicted from the structure of the HAC-seeding DNA. Although it has been reported that these rearrangements can happen, the timing and mechanism of their formation has yet to be described. Here we synthesized a HAC-seeding DNA with two distinct structural domains and introduced it into HT1080 cells. We characterized a number of HAC-containing clones and subclones to track DNA rearrangements during HAC establishment. We demonstrated that rearrangements can occur early during HAC formation. Subsequently, the established HAC genomic organization is stably maintained across many cell generations. Thus, early stages in HAC formation appear to at least occasionally involve a process of DNA shredding and shuffling that resembles chromothripsis, an important hallmark of many cancer types. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes.

CENP-B creates alternative epigenetic chromatin states permissive for CENP-A or heterochromatin assembly.

CENP-B binds to CENP-B boxes on centromeric satellite DNAs (known as alphoid DNA in humans). CENP-B maintains kinetochore function through interactions with CENP-A nucleosomes and CENP-C. CENP-B binding to transfected alphoid DNA can induce de novo CENP-A assembly, functional centromere and kinetochore formation, and subsequent human artificial chromosome (HAC) formation. Furthermore, CENP-B also facilitates H3K9 (histone H3 lysine 9) trimethylation on alphoid DNA, mediated by Suv39h1, at ectopic alphoid DNA integration sites. Excessive heterochromatin invasion into centromere chromatin suppresses CENP-A assembly. It is unclear how CENP-B controls such different chromatin states. Here, we show that the CENP-B acidic domain recruits histone chaperones and many chromatin modifiers, including the H3K36 methylase ASH1L, as well as the heterochromatin components Suv39h1 and HP1 (HP1α, ß and γ, also known as CBX5, CBX1 and CBX3, respectively). ASH1L facilitates the formation of open chromatin competent for CENP-A assembly on alphoid DNA. These results indicate that CENP-B is a nexus for histone modifiers that alternatively promote or suppress CENP-A assembly by mutually exclusive mechanisms. Besides the DNA-binding domain, the CENP-B acidic domain also facilitates CENP-A assembly de novo on transfected alphoid DNA. CENP-B therefore balances CENP-A assembly and heterochromatin formation on satellite DNA.

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice.

Human artificial chromosomes (HACs), including the de novo synthesized alphoidtetO-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoidtetO-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from FVIIIY/- mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoidtetO-HAC in hamster CHO cells. Subsequently, the therapeutic alphoidtetO-HAC-FVIII was transferred into the FVIIIY/- iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector-the alphoidtetO-HAC.

Selective isolation of large segments from individual microbial genomes and environmental DNA samples using transformation-associated recombination cloning in yeast.

Here, we describe an extension of our original transformation-associated recombination (TAR) cloning protocol, enabling selective isolation of DNA segments from microbial genomes. The technique is based on the previously described TAR cloning procedure developed for isolation of a desirable region from mammalian genomes that are enriched in autonomously replicating sequence (ARS)-like sequences, elements that function as the origin of replication in yeast. Such sequences are not common in microbial genomes. In this Protocol Extension, an ARS is inserted into the TAR vector along with a counter-selectable marker, allowing for selection of cloning events against vector circularization. Pre-treatment of microbial DNA with CRISPR-Cas9 to generate double-stranded breaks near the targeted sequences greatly increases the yield of region-positive colonies. In comparison to other available methods, this Protocol Extension allows selective isolation of any region from microbial genomes as well as from environmental DNA samples. The entire procedure can be completed in 10 d.

Human artificial chromosome (HAC) for measuring chromosome instability (CIN) and identification of genes required for proper chromosome transmission.

Chromosomal instability (CIN) is one of the characteristics of cancer inherent for tumor initiation and progression, which is defined as a persistent, high rate of gain/loss of whole chromosomes. In the vast majority of human tumors the molecular basis of CIN remains unknown. The development of a conceptually simple colony color sectoring assay that measures yeast artificial chromosome (YAC) loss provided a powerful genetic tool to assess the rate of chromosome mis-segregation and also identified 937 yeast genes involved in this process. Similarly, a human artificial chromosome (HAC)-based assay has been recently developed and applied to quantify chromosome mis-segregation events in human cells. This assay allowed identification of novel human CIN genes in the library of protein kinases. Among them are PINK1, TRIO, IRAK1, PNCK, and TAOK1. The HAC-based assay may be applied to screen siRNA, shRNA and CRISPR-based libraries to identify the complete spectrum of CIN genes. This will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.

A novel assay to screen siRNA libraries identifies protein kinases required for chromosome transmission.

One of the hallmarks of cancer is chromosome instability (CIN), which leads to aneuploidy, translocations, and other chromosome aberrations. However, in the vast majority of human tumors the molecular basis of CIN remains unknown, partly because not all genes controlling chromosome transmission have yet been identified. To address this question, we developed an experimental high-throughput imaging (HTI) siRNA assay that allows the identification of novel CIN genes. Our method uses a human artificial chromosome (HAC) expressing the GFP transgene. When this assay was applied to screen an siRNA library of protein kinases, we identified PINK1, TRIO, IRAK1, PNCK, and TAOK1 as potential novel genes whose knockdown induces various mitotic abnormalities and results in chromosome loss. The HAC-based assay can be applied for screening different siRNA libraries (cell cycle regulation, DNA damage response, epigenetics, and transcription factors) to identify additional genes involved in CIN. Identification of the complete spectrum of CIN genes will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.

TAR Cloning: Perspectives for Functional Genomics, Biomedicine, and Biotechnology.

Completion of the human genome sequence and recent advances in engineering technologies have enabled an unprecedented level of understanding of DNA variations and their contribution to human diseases and cellular functions. However, in some cases, long-read sequencing technologies do not allow determination of the genomic region carrying a specific mutation (e.g., a mutation located in large segmental duplications). Transformation-associated recombination (TAR) cloning allows selective, most accurate, efficient, and rapid isolation of a given genomic fragment or a full-length gene from simple and complex genomes. Moreover, this method is the only way to simultaneously isolate the same genomic region from multiple individuals. As such, TAR technology is currently in a leading position to create a library of the individual genes that comprise the human genome and physically characterize the sites of chromosomal alterations (copy number variations [CNVs], inversions, translocations) in the human population, associated with the predisposition to different diseases, including cancer. It is our belief that such a library and analysis of the human genome will be of great importance to the growing field of gene therapy, new drug design methods, and genomic research. In this review, we detail the motivation for TAR cloning for human genome studies, biotechnology, and biomedicine, discuss the recent progress of some TAR-based projects, and describe how TAR technology in combination with HAC (human artificial chromosome)-based and CRISPR-based technologies may contribute in the future.

Transfer of Synthetic Human Chromosome into Human Induced Pluripotent Stem Cells for Biomedical Applications.

AlphoidtetO-type human artificial chromosome (HAC) has been recently synthetized as a novel class of gene delivery vectors for induced pluripotent stem cell (iPSC)-based tissue replacement therapeutic approach. This HAC vector was designed to deliver copies of genes into patients with genetic diseases caused by the loss of a particular gene function. The alphoidtetO-HAC vector has been successfully transferred into murine embryonic stem cells (ESCs) and maintained stably as an independent chromosome during the proliferation and differentiation of these cells. Human ESCs and iPSCs have significant differences in culturing conditions and pluripotency state in comparison with the murine naïve-type ESCs and iPSCs. To date, transferring alphoidtetO-HAC vector into human iPSCs (hiPSCs) remains a challenging task. In this study, we performed the microcell-mediated chromosome transfer (MMCT) of alphoidtetO-HAC expressing the green fluorescent protein into newly generated hiPSCs. We used a recently modified MMCT method that employs an envelope protein of amphotropic murine leukemia virus as a targeting cell fusion agent. Our data provide evidence that a totally artificial vector, alphoidtetO-HAC, can be transferred and maintained in human iPSCs as an independent autonomous chromosome without affecting pluripotent properties of the cells. These data also open new perspectives for implementing alphoidtetO-HAC as a gene therapy tool in future biomedical applications.

Systematic Analysis of Compounds Specifically Targeting Telomeres and Telomerase for Clinical Implications in Cancer Therapy.

The targeting of telomerase and telomere maintenance mechanisms represents a promising therapeutic approach for various types of cancer. In this work, we designed a new protocol to screen for and rank the efficacy of compounds specifically targeting telomeres and telomerase. This approach used two isogenic cell lines containing a circular human artificial chromosome (HAC, lacking telomeres) and a linear HAC (containing telomeres) marked with the EGFP transgene; compounds that target telomerase or telomeres should preferentially induce loss of the linear HAC but not the circular HAC. Our assay allowed quantification of chromosome loss by routine flow cytometry. We applied this dual-HAC assay to rank a set of known and newly developed compounds, including G-quadruplex (G4) ligands. Among the latter group, two compounds, Cu-ttpy and Pt-ttpy, induced a high rate of linear HAC loss with no significant effect on the mitotic stability of a circular HAC. Analysis of the mitotic phenotypes induced by these drugs revealed an elevated rate of chromatin bridges in late mitosis and cytokinesis as well as UFB (ultrafine bridges). Chromosome loss after Pt-ttpy or Cu-ttpy treatment correlated with the induction of telomere-associated DNA damage. Overall, this platform enables identification and ranking of compounds that greatly increase chromosome mis-segregation rates as a result of telomere dysfunction and may expedite the development of new therapeutic strategies for cancer treatment.Significance: An assay provides a unique opportunity to screen thousands of chemical compounds for their ability to inactivate replication of telomeric ends in cancer cells and holds potential to lay the foundation for the discovery of new treatments for cancer. Cancer Res; 78(21); 6282-96. ©2018 AACR.

Human Artificial Chromosome with Regulated Centromere: A Tool for Genome and Cancer Studies.

Since their description in the late 1990s, Human Artificial Chromosomes (HACs) bearing functional kinetochores have been considered as promising systems for gene delivery and expression. More recently a HAC assembled from a synthetic alphoid DNA array has been exploited in studies of centromeric chromatin and in assessing the impact of different epigenetic modifications on kinetochore structure and function in human cells. This HAC was termed the alphoidtetO-HAC, as the synthetic monomers each contained a tetO sequence in place of the CENP-B box that can be targeted specifically with tetR-fusion proteins. Studies in which the kinetochore chromatin of the alphoidtetO-HAC was specifically modified, revealed that heterochromatin is incompatible with centromere function and that centromeric transcription is important for centromere assembly and maintenance. In addition, the alphoidtetO-HAC was modified to carry large gene inserts that are expressed in target cells under conditions that recapitulate the physiological regulation of endogenous loci. Importantly, the phenotypes arising from stable gene expression can be reversed when cells are "cured" of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. AlphoidtetO-HAC-based technology has also been used to develop new drug screening and assessment strategies to manipulate the CIN phenotype in cancer cells. In summary, the alphoidtetO-HAC is proving to be a versatile tool for studying human chromosome transactions and structure as well as for genome and cancer studies.

Analysis of the 9p21.3 sequence associated with coronary artery disease reveals a tendency for duplication in a CAD patient.

Tandem segmental duplications (SDs) greater than 10 kb are widespread in complex genomes. They provide material for gene divergence and evolutionary adaptation, while formation of specific de novo SDs is a hallmark of cancer and some human diseases. Most SDs map to distinct genomic regions termed 'duplication blocks'. SDs organization within these blocks is often poorly characterized as they are mosaics of ancestral duplicons juxtaposed with younger duplicons arising from more recent duplication events. Structural and functional analysis of SDs is further hampered as long repetitive DNA structures are underrepresented in existing BAC and YAC libraries. We applied Transformation-Associated Recombination (TAR) cloning, a versatile technique for large DNA manipulation, to selectively isolate the coronary artery disease (CAD) interval sequence within the 9p21.3 chromosome locus from a patient with coronary artery disease and normal individuals. Four tandem head-to-tail duplicons, each ∼50 kb long, were recovered in the patient but not in normal individuals. Sequence analysis revealed that the repeats varied by 10-15 SNPs between each other and by 82 SNPs between the human genome sequence (version hg19). SNPs polymorphism within the junctions between repeats allowed two junction types to be distinguished, Type 1 and Type 2, which were found at a 2:1 ratio. The junction sequences contained an Alu element, a sequence previously shown to play a role in duplication. Knowledge of structural variation in the CAD interval from more patients could help link this locus to cardiovascular diseases susceptibility, and maybe relevant to other cases of regional amplification, including cancer.