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1.  Stable maintenance of de novo assembled human artificial chromosomes in embryonic stem cells and their differentiated progeny in mice 
Cell Cycle  2015;14(8):1268-1273.
De novo assembled alphoidtetO-type human artificial chromosomes (HACs) represent a novel promising generation of high capacity episomal vectors. Their function and persistence, and any adverse effects, in various cell types in live animals, have not, however, been explored. In this study we transferred the alphoidtetO-HAC into mouse ES cells and assessed whether the presence of this extra chromosome affects their pluripotent properties. AlphoidtetO-HAC-bearing ES cells were indistinguishable from their wild-type counterparts: they retained self-renewal potential and full capacity for multilineage differentiation during mouse development, whereas the HAC itself was mitotically and transcriptionally stable during this process. Our data provide the first example of fully synthetic DNA behaving like a normal chromosome in cells of living animals. It also opens a new perspective into functional genetic studies in laboratory animals as well as stem cell-based regenerative medicine.
PMCID: PMC4614155  PMID: 25695642
embryonic stem cells; human artificial chromosomes; gene therapy
2.  A bi-HAC vector system towards gene and cell therapy 
ACS synthetic biology  2014;3(2):83-90.
Genetic manipulations with mammalian cells often require introduction of two or more genes that have to be in trans-configuration. However, conventional gene delivery vectors have several limitations, including a limited cloning capacity and a risk of insertional mutagenesis. In this paper, we describe a novel gene expression system that consists of two differently marked HAC vectors containing unique gene loading sites. One HAC, 21HAC, is stably propagated during cell divisions, therefore, it is suitable for complementation of a gene deficiency. The other HAC, tet-O HAC, can be eliminated, providing a unique opportunity for transient gene expression, e.g. for cell reprogramming. Efficiency and accuracy of a novel bi-HAC vector system have been evaluated after loading of two different transgenes into these HACs. Based on analysis of transgenes expression and HACs stability in the proof of principle experiments, the combination of two HAC vectors may provide a powerful tool towards gene and cell therapy.
PMCID: PMC4137782  PMID: 25101815
human artificial chromosome vector; gene expression; gene and cell therapy
3.  Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette 
Nucleic Acids Research  2015;43(9):e57.
Human artificial chromosome (HAC)-based vectors represent an alternative technology for gene delivery and expression with a potential to overcome the problems caused by virus-based vectors. The recently developed alphoidtetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of chromatin modifiers, tTA or tTS, to its centromeric tetO sequences. This provides a unique control for phenotypes induced by genes loaded into the HAC. The alphoidtetO-HAC elimination is highly efficient when a high level of chromatin modifiers as tetR fusion proteins is achieved following transfection of cells by a retrovirus vector. However, such vectors are potentially mutagenic and might want to be avoided under some circumstances. Here, we describe a novel system that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step. We demonstrated that a single copy of tTAVP64 carrying four tandem repeats of the VP16 domain constitutively expressed from the HAC is capable to generate chromatin changes in the HAC kinetochore that are not compatible with its function. To adopt the alphoidtetO-HAC for routine gene function studies, we constructed a new TAR-BRV- tTAVP64 cloning vector that allows a selective isolation of a gene of interest from genomic DNA in yeast followed by its direct transfer to bacterial cells and subsequent loading into the loxP site of the alphoidtetO-HAC in hamster CHO cells from where the HAC may be MMCT-transferred to the recipient human cells.
PMCID: PMC4482055  PMID: 25712097
4.  Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast 
Nucleic Acids Research  2015;43(8):e55.
Transformation-associated recombination (TAR) protocol allowing the selective isolation of full-length genes complete with their distal enhancer regions and entire genomic loci with sizes up to 250 kb from complex genomes in yeast S. cerevisiae has been developed more than a decade ago. However, its wide spread usage has been impeded by a low efficiency (0.5–2%) of chromosomal region capture during yeast transformants which in turn requires a time-consuming screen of hundreds of colonies. Here, we demonstrate that pre-treatment of genomic DNA with CRISPR-Cas9 nucleases to generate double-strand breaks near the targeted genomic region results in a dramatic increase in the fraction of gene-positive colonies (up to 32%). As only a dozen or less yeast transformants need to be screened to obtain a clone with the desired chromosomal region, extensive experience with yeast is no longer required. A TAR-CRISPR protocol may help to create a bank of human genes, each represented by a genomic copy containing its native regulatory elements, that would lead to a significant advance in functional, structural and comparative genomics, in diagnostics, gene replacement, generation of animal models for human diseases and has a potential for gene therapy.
PMCID: PMC4417148  PMID: 25690893
5.  Recent advances in chromosome engineering 
Chromosome Research  2015;23(1):1-5.
PMCID: PMC4365175  PMID: 25682170
Artificial chromosome; CRISPR; Gene therapy; Kinetochore; Stem cell; Synthetic biology; TALEN; Transgenic; ZFN
6.  A portable BRCA1-HAC (human artificial chromosome) module for analysis of BRCA1 tumor suppressor function 
Nucleic Acids Research  2014;42(21):e164.
BRCA1 is involved in many disparate cellular functions, including DNA damage repair, cell-cycle checkpoint activation, gene transcriptional regulation, DNA replication, centrosome function and others. The majority of evidence strongly favors the maintenance of genomic integrity as a principal tumor suppressor activity of BRCA1. At the same time some functional aspects of BRCA1 are not fully understood. Here, a HAC (human artificial chromosome) module with a regulated centromere was constructed for delivery and expression of the 90 kb genomic copy of the BRCA1 gene into BRCA1-deficient human cells. A battery of functional tests was carried out to demonstrate functionality of the exogenous BRCA1. In separate experiments, we investigated the role of BRCA1 in maintenance of heterochromatin integrity within a human functional kinetochore. We demonstrated that BRCA1 deficiency results in a specific activation of transcription of higher-order alpha-satellite repeats (HORs) assembled into heterochromatin domains flanking the kinetochore. At the same time no detectable elevation of transcription was observed within HORs assembled into centrochromatin domains. Thus, we demonstrated a link between BRCA1 deficiency and kinetochore dysfunction and extended previous observations that BRCA1 is required to silence transcription in heterochromatin in specific genomic loci. This supports the hypothesis that epigenetic alterations of the kinetochore initiated in the absence of BRCA1 may contribute to cellular transformation.
PMCID: PMC4245969  PMID: 25260588
7.  Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome 
Nucleic Acids Research  2014;42(18):11502-11516.
In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed Human Artificial Chromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, suggesting that CENP-B may have a role in regulating the replication of centromeric regions. Mapping of replication initiation sites in the HAC revealed that replication preferentially initiated in transcriptionally active regions.
PMCID: PMC4191410  PMID: 25228468
8.  A new generation of human artificial chromosomes for functional genomics and gene therapy 
Since their description in the late 1990s, human artificial chromosomes (HACs) carrying a functional kinetochore were considered as a promising system for gene delivery and expression with a potential to overcome many problems caused by the use of viral-based gene transfer systems. Indeed, HACs avoid the limited cloning capacity, lack of copy number control and insertional mutagenesis due to integration into host chromosomes that plague viral vectors. Nevertheless, until recently, HACs have not been widely recognized because of uncertainties of their structure and the absence of a unique gene acceptor site. The situation changed a few years ago after engineering of HACs with a single loxP gene adopter site and a defined structure. In this review, we summarize recent progress made in HAC technology and concentrate on details of two of the most advanced HACs, 21HAC generated by truncation of human chromosome 21 and alphoidtetO-HAC generated de novo using a synthetic tetO-alphoid DNA array. Multiple potential applications of the HAC vectors are discussed, specifically the unique features of two of the most advanced HAC cloning systems.
PMCID: PMC3522797  PMID: 22907415
Human artificial chromosomes; HAC; Gene expression; Gene delivery vector
9.  Organization of Synthetic Alphoid DNA Array in Human Artificial Chromosome (HAC) with a Conditional Centromere 
ACS synthetic biology  2012;1(12):590-601.
Human artificial chromosomes (HACs) represent a novel promising episomal system for functional genomics, gene therapy and synthetic biology. HACs are engineered from natural and synthetic alphoid DNA arrays upon transfection into human cells. The use of HACs for gene expression studies requires the knowledge of their structural organization. However, none of de novo HACs constructed so far has been physically mapped in detail. Recently we constructed a synthetic alphoidtetO-HAC that was successfully used for expression of full-length genes to correct genetic deficiencies in human cells. The HAC can be easily eliminated from cell populations by inactivation of its conditional kinetochore. This unique feature provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This work describes organization of a megabase-size synthetic alphoid DNA array in the alphoidtetO-HAC that has been formed from a ~50 kb synthetic alphoidtetO-construct. Our analysis showed that this array represents a 1.1 Mb continuous sequence assembled from multiple copies of input DNA, a significant part of which was rearranged before assembling. The tandem and inverted alphoid DNA repeats in the HAC range in size from 25 to 150 kb. In addition, we demonstrated that the structure and functional domains of the HAC remains unchanged after several rounds of its transfer into different host cells. The knowledge of the alphoidtetO-HAC structure provides a tool to control HAC integrity during different manipulations. Our results also shed light on a mechanism for de novo HAC formation in human cells.
PMCID: PMC3568986  PMID: 23411994
human artificial chromosome; HAC; gene delivery; TAR cloning
10.  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.
PMCID: PMC3412920  PMID: 22733720
Xq27; hereditary prostate cancer; HPCX1; SPANXL; CDR1; TAR cloning
11.  A new assay for measuring chromosome instability (CIN) and identification of drugs that elevate CIN in cancer cells 
BMC Cancer  2013;13:252.
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.
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.
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.
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.
PMCID: PMC3671967  PMID: 23694679
Human artificial chromosome; HAC; Chromosome instability; CIN; Drug treatment
12.  Protecting a transgene expression from the HAC-based vector by different chromatin insulators 
Cellular and Molecular Life Sciences  2013;70(19):3723-3737.
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.
PMCID: PMC3771377  PMID: 23677492
Insulator; tDNA-gamma-satellite; cHS4; Human artificial chromosome-HAC
13.  Re-engineering an alphoidtetO-HAC-based vector to enable high-throughput analyses of gene function 
Nucleic Acids Research  2013;41(10):e107.
Human artificial chromosome (HAC)-based vectors represent an alternative technology for gene delivery and expression with a potential to overcome the problems caused by the use of viral-based vectors. The recently developed alphoidtetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of tTS chromatin modifiers to its centromeric tetO sequences. This provides unique control for phenotypes induced by genes loaded into the alphoidtetO-HAC. However, inactivation of the HAC kinetochore requires transfection of cells by a retrovirus vector, a step that is potentially mutagenic. Here, we describe an approach to re-engineering the alphoidtetO-HAC that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step. In the new HAC vector, a tTS-EYFP cassette is inserted into a gene-loading site along with a gene of interest. Expression of the tTS generates a self-regulating fluctuating heterochromatin on the alphoidtetO-HAC that induces fast silencing of the genes on the HAC without significant effects on HAC segregation. This silencing of the HAC-encoded genes can be readily recovered by adding doxycycline. The newly modified alphoidtetO-HAC-based system has multiple applications in gene function studies.
PMCID: PMC3664798  PMID: 23558748
14.  tRNA genes protect a reporter gene from epigenetic silencing in mouse cells 
Cell Cycle  2011;10(16):2779-2791.
It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.
PMCID: PMC3219543  PMID: 21822054
barrier elements; tRNA genes; Pol III-transcribed genes
15.  Breaking the HAC Barrier: Histone H3K9 acetyl/methyl balance regulates CENP-A assembly 
The EMBO Journal  2012;31(10):2391-2402.
Establishment of Human Artificial Chromosomes (HACs) depends on an interplay of H3 lysine 9 modifications at centromeres, providing insights into the pathways that control incorporation of the kinetochore-specificing histone H3 variant CENP-A.
The kinetochore is responsible for accurate chromosome segregation. However, the mechanism by which kinetochores assemble and are maintained remains unclear. Here we report that de novo CENP-A assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl balance. Tethering of histone acetyltransferases (HATs) to alphoid DNA arrays breaks a cell type-specific barrier for de novo stable CENP-A assembly and induces assembly of other kinetochore proteins at the ectopic alphoid site. Similar results are obtained following tethering of CENP-A deposition factors hMis18α or HJURP. HAT tethering bypasses the need for hMis18α, but HJURP is still required for de novo kinetochore assembly. In contrast, H3K9 methylation following tethering of H3K9 tri-methylase (Suv39h1) to the array prevents de novo CENP-A assembly and kinetochore formation. CENP-A arrays assembled de novo by this mechanism can form human artificial chromosomes (HACs) that are propagated indefinitely in human cells.
PMCID: PMC3364751  PMID: 22473132
CENP-A; centromeres; chromosomes; epigenetic regulation; heterochromatin
16.  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.
PMCID: PMC3200152  PMID: 21982381
17.  Human Artificial Chromosome with a Conditional Centromere for Gene Delivery and Gene Expression 
Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoidtet-O (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy.
PMCID: PMC2955713  PMID: 20798231
human artificial chromosome; conditional centromere; gene delivery
18.  Sperm-derived SPANX-B is a clinically relevant tumor antigen that is expressed in human tumors and readily recognized by human CD4+ and CD8+ T cells 
The sperm-derived SPANX family proteins can be found expressed in human tumors. Here, we aimed to perform a comprehensive study to evaluate immunotherapeutic relevance of one of its members, SPANX-B. We wanted to test its expression pattern in human tumors; and to evaluate CD4+ and CD8+ T cell responses in healthy humans after in vitro immunizations.
The Experimental Design
Expression of SPANX-B in human malignancies, including a multi-tumor tissue array of 145 primary tumors, was assessed utilizing RT/PCR, western blotting and immunohistochemical analysis. T cell immunogenicity and immunodominant epitopes of SPANX-B were studied using in vitro immunizations of healthy human donor-derived leukocytes.
SPANX-B was abundantly expressed in melanoma and carcinomas of lung, ovary, colon and breast. In melanoma, tissue array data indicated that it was expressed in advanced and metastatic disease. Unlike most tumor-associated antigens, SPANX-B was an immunogenic antigen that was recognized by circulating T cell precursors in healthy humans. Importantly, these T cells were readily expanded to generate SPANX-B –specific helper CD4+ and cytolytic CD8+ T cells that recognized unique immunodominant epitopes: at least one HLA-DR-restricted Pep-9 epitope (SPANX-B12–23) and two HLA-A2-restricted Pep-2 and Pep-4 epitopes (respectively, SPANX-B23–31 and SPANX-B57–65). The CD8+ T cells were fully functional to recognize and lyse HLA-A2-expressing tumors, including primary human melanomas.
SPANX-B is an immunogenic sperm-derived antigen that is expressed in a number of human tumors. SPANX-B is also efficiently recognized by the human T cell immune arm, indicating its significant value for the development of protective and therapeutic cancer vaccines.
PMCID: PMC2667247  PMID: 19276289
SPANX; immunogenicity of TAA; antitumor CTL; MHC epitope
19.  Evolutionary Diversification of SPANX-N Sperm Protein Gene Structure and Expression 
PLoS ONE  2007;2(4):e359.
The sperm protein associated with nucleus in the X chromosome (SPANX) genes cluster at Xq27 in two subfamilies, SPANX-A/D and SPANX-N. SPANX-A/D is specific for hominoids and is fairly well characterized. The SPANX-N gave rise to SPANX-A/D in the hominoid lineage ∼7 MYA. Given the proposed role of SPANX genes in spermatogenesis, we have extended studies to SPANX-N gene evolution, variation, regulation of expression, and intra-sperm localization. By immunofluorescence analysis, SPANX-N proteins are localized in post-meiotic spermatids exclusively, like SPANX-A/D. But in contrast to SPANX-A/D, SPANX-N are found in all ejaculated spermatozoa rather than only in a subpopulation, are localized in the acrosome rather than in the nuclear envelope, and are expressed at a low level in several nongametogenic adult tissues as well as many cancers. Presence of a binding site for CTCF and its testis-specific paralogue BORIS in the SPANX promoters suggests, by analogy to MAGE-A1 and NY-ESO-1, that their activation in spermatogenesis is mediated by the programmed replacement of CTCF by BORIS. Based on the relative density of CpG, the more extended expression of SPANX-N compared to SPANX-A/D in nongametogenic tissues is likely attributed to differences in promoter methylation. Our findings suggest that the recent duplication of SPANX genes in hominoids was accompanied by different localization of SPANX-N proteins in post-meiotic sperm and additional expression in several nongonadal tissues. This suggests a corresponding functional diversification of SPANX gene families in hominoids. SPANX proteins thus provide unique targets to investigate their roles in the function of spermatozoa, selected malignancies, and for SPANX-N, in other tissues as well.
PMCID: PMC1831492  PMID: 17406683
20.  Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation 
Nucleic Acids Research  2005;33(15):e130.
Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. An investigation into the structural and the functional relationship in centromeric tandem repeats in HACs requires the ability to manipulate repeat substructure efficiently. We describe here a new method to rapidly amplify human alphoid tandem repeats of a few hundred base pairs into long DNA arrays up to 120 kb. The method includes rolling-circle amplification (RCA) of repeats in vitro and assembly of the RCA products by in vivo recombination in yeast. The synthetic arrays are competent in HAC formation when transformed into human cells. As short multimers can be easily modified before amplification, this new technique can identify repeat monomer regions critical for kinetochore seeding. The method may have more general application in elucidating the role of other tandem repeats in chromosome organization and dynamics.
PMCID: PMC1197135  PMID: 16141190
21.  A general cloning system to selectively isolate any eukaryotic or prokaryotic genomic region in yeast 
BMC Genomics  2003;4:16.
Transformation-associated recombination (TAR) cloning in yeast is a unique method for selective isolation of large chromosomal fragments or entire genes from complex genomes. The technique involves homologous recombination, during yeast spheroplast transformation, between genomic DNA and a TAR vector that has short (~ 60 bp) 5' and 3' gene targeting sequences (hooks).
TAR cloning requires that the cloned DNA fragment carry at least one autonomously replicating sequence (ARS) that can function as the origin of replication in yeast, which prevents wide application of the method. In this paper, we describe a novel TAR cloning system that allows isolation of genomic regions lacking yeast ARS-like sequences. ARS is inserted into the TAR vector along with URA3 as a counter-selectable marker. The hooks are placed between the TATA box and the transcription initiation site of URA3. Insertion of any sequence between hooks results in inactivation of URA3 expression. That inactivation confers resistance to 5-fluoroorotic acid, allowing selection of TAR cloning events against background vector recircularization events.
The new system greatly expands the area of application of TAR cloning by allowing isolation of any chromosomal region from eukaryotic and prokaryotic genomes regardless of the presence of autonomously replicating sequences.
PMCID: PMC156606  PMID: 12720573
transformation-associated recombination cloning; gene isolation; counter-selection
22.  Optimum conditions for selective isolation of genes from complex genomes by transformation-associated recombination cloning 
Nucleic Acids Research  2003;31(6):e29.
Transformation-associated recombination (TAR) cloning in yeast is used to isolate a desired chromosomal region or gene from a complex genome without construction of a genomic library. The technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector containing short 5′ and 3′ gene-specific targeting hooks. Efficient gene capture requires a high yield of transformants, and we demonstrate here that the transformant yield increases ∼10-fold when the genomic DNA is sheared to 100–200 kb before being presented to the spheroplasts. Here we determine the most effective concentration of genomic DNA, and also show that the targeted sequences recombine much more efficiently with the vector’s targeting hooks when they are located at the ends of the genomic DNA fragment. We demonstrate that the yield of gene-positive clones increases ∼20-fold after endonuclease digestion of genomic DNA, which caused double strand breaks near the targeted sequences. These findings have led to a greatly improved protocol.
PMCID: PMC152883  PMID: 12626728
23.  A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast 
Nucleic Acids Research  2002;30(2):e8.
Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5′ and 3′ gene targeting sequences (hooks). Typically, TAR cloning produces positive YAC recombinants at a frequency of ∼0.5%; the positive clones are identified by PCR or colony hybridization. This paper describes a novel TAR cloning procedure that selects positive clones by positive and negative genetic selection. This system utilizes a TAR vector with two targeting hooks, HIS3 as a positive selectable marker, URA3 as a negative selectable marker and a gene-specific sequence called a loop sequence. The loop sequence lies distal to a targeting hook sequence in the chromosomal target, but proximal to the targeting hook and URA3 in the TAR vector. When this vector recombines with chromosomal DNA at the gene-specific targeting hook, the recombinant YAC product carries two copies of the loop sequence, therefore, the URA3 negative selectable marker becomes mitotically unstable and is lost at high frequency by direct repeat recombination involving the loop sequence. Positive clones are identified by selecting against URA3. This method produces positive YAC recombinants at a frequency of ∼40%. This novel TAR cloning method provides a powerful tool for structural and functional analysis of complex genomes.
PMCID: PMC99847  PMID: 11788734
24.  Recovery and potential utility of YACs as circular YACs/BACs 
Nucleic Acids Research  2000;28(17):e81.
A method has been established to convert pYAC4-based linear yeast artificial chromosomes (YACs) into circular chromosomes that can also be propagated in Escherichia coli cells as bacterial artificial chromosomes (BACs). The circularization is based on use of a vector that contains a yeast dominant selectable marker (G418R), a BAC cassette and short targeting sequences adjacent to the edges of the insert in the pYAC4 vector. When it is introduced into yeast, the vector recombines with the YAC target sequences to form a circular molecule, retaining the insert but discarding most of the sequences of the YAC telomeric arms. YACs up to 670 kb can be efficiently circularized using this vector. Re-isolation of megabase-size YAC inserts as a set of overlapping circular YAC/BACs, based on the use of an Alu-containing targeting vector, is also described. We have shown that circular DNA molecules up to 250 kb can be efficiently and accurately transferred into E.coli cells by electroporation. Larger circular DNAs cannot be moved into bacterial cells, but can be purified away from linear yeast chromosomes. We propose that the described system for generation of circular YAC derivatives can facilitate sequencing as well as functional analysis of genomic regions.
PMCID: PMC110718  PMID: 10954614
25.  Accelerated Evolution of the ASPM Gene Controlling Brain Size Begins Prior to Human Brain Expansion 
PLoS Biology  2004;2(5):e126.
Primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by global reduction in cerebral cortical volume. The microcephalic brain has a volume comparable to that of early hominids, raising the possibility that some MCPH genes may have been evolutionary targets in the expansion of the cerebral cortex in mammals and especially primates. Mutations in ASPM, which encodes the human homologue of a fly protein essential for spindle function, are the most common known cause of MCPH. Here we have isolated large genomic clones containing the complete ASPM gene, including promoter regions and introns, from chimpanzee, gorilla, orangutan, and rhesus macaque by transformation-associated recombination cloning in yeast. We have sequenced these clones and show that whereas much of the sequence of ASPM is substantially conserved among primates, specific segments are subject to high Ka/Ks ratios (nonsynonymous/synonymous DNA changes) consistent with strong positive selection for evolutionary change. The ASPM gene sequence shows accelerated evolution in the African hominoid clade, and this precedes hominid brain expansion by several million years. Gorilla and human lineages show particularly accelerated evolution in the IQ domain of ASPM. Moreover, ASPM regions under positive selection in primates are also the most highly diverged regions between primates and nonprimate mammals. We report the first direct application of TAR cloning technology to the study of human evolution. Our data suggest that evolutionary selection of specific segments of the ASPM sequence strongly relates to differences in cerebral cortical size.
Mutation of the ASPM gene is associated with abnormally small brain size. Comparison of the ASPM gene from several primate species suggests it as a target of evolutionary selection for increased brain size
PMCID: PMC374243  PMID: 15045028

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