Efficiency of de novo centromere formation in human artificial chromosomes

In a comparative study, we show that human artificial chromosome (HAC) vectors based on alpha-satellite (alphoid) DNA from chromosome 17 but not the Y chromosome regularly form HACs in HT1080 human cells. We constructed four structurally similar HAC vectors, two with chromosome 17 or Y alphoid DNA (17alpha, Yalpha) and two with 17alpha or Yalpha and the hypoxanthine guanine phosphoribosyltransferase locus (HPRT1). The 17alpha HAC vectors generated artificial minichromosomes in 32-79% of the HT1080 clones screened, compared with only approximately 4% for the Yalpha HAC vectors, indicating that Yalpha is inefficient at forming a de novo centromere. The 17alpha HAC vectors produced megabase-sized, circular HACs containing multiple copies of alphoid fragments (60-250 kb) interspersed with either vector or HPRT1 DNA.The 17alpha-HPRT1 HACs were less stable than those with 17alpha only, and these results may influence the design of new HAC gene transfer vectors.     

Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes

Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome ΔYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from ΔYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.     

Sequence, higher order repeat structure, and long-range organization of alpha satellite DNA specific to human chromosome 8.

We have characterized alphoid repeat clones derived from a chromosome 8 library. These clones are specific for human chromosome 8, as demonstrated by use of a somatic cell hybrid mapping panel and by in situ hybridization. Hybridization of the clones to HindIII digests of human genomic DNA reveals a complex pattern of fragments ranging in size from 1.3 to greater than 20 kb. One clone, which corresponds in size to the most prevalent genomic HindIII fragment, appears to represent a major higher order repeat in the chromosome 8 centromere. The DNA sequence of this clone reveals a dimeric organization of alphoid monomers. Restriction analysis of two other clones indicates that they are derivatives of this same repeat unit. The chromosome 8 alphoid clones hybridize to EcoRI fragments of genomic DNA ranging up to 1000 kb in length and reveal a high degree of polymorphism between chromosomes. Distribution of higher order repeat units across the centromere was examined by two-dimensional gel electrophoresis. Repeat units of the same size class tended to cluster together in restricted regions of centromeric DNA.     

Human artificial chromosomes constructed using the bottom-up strategy are stably maintained in mitosis and efficiently transmissible to progeny mice

Human artificial chromosomes (HACs) are alternative vectors that promise to overcome problematic transgene expression often occurring with conventional vectors in mammalian cells and bodies. We have successfully generated HACs by multimerization of a cloned long alphoid stretch in a human cell line, HT1080. Furthermore, we developed technologies for cloning large genomic regions into HACs by means of co-transfection of clones with the alphoid array and clones encoding the genomic region of interest. The purpose of this study was to investigate the mitotic and meiotic stability of such HACs in mouse cells and bodies. We transferred a circular HAC containing the guanosine triphosphate cyclohydrolase I gene (GCH1-HAC) and a linear HAC containing the human globin gene cluster (globin-HAC) from HT1080 cells into mouse embryonic stem (ES) cells by microcell-mediated chromosome transfer. The HACs were stably maintained in mouse ES cells for 3 months. GCH1-HACs in every ES cell line and globin-HACs in most ES cell lines maintained their structures without detectable rearrangement or acquisition of mouse genomic DNA except one globin-HAC in an ES cell line rearranged and acquired mouse-type centromeric sequences and long telomeres. Creation of chimeric mice using ES cells containing HAC and subsequent crossing showed that both the globin-HAC that had rearranged and acquired mouse type centromeric sequences/long telomeres and GCH1-HACs were retained in tissues of mice and transmitted to progeny. These results indicate that human artificial chromosomes constructed using the bottom-up strategy based on alphoid DNA are stable in mouse bodies and are transmissible.     

Investigation of Schizosaccharomyces pombe as a cloning host for human telomere and alphoid DNA

The fission yeast Schizosaccharomyces pombe (Sch. pombe) has been proposed as a possible cloning host for both mammalian artificial chromosomes (MACs) and mammalian genomic libraries, due to the large size of its chromosomes and its similarity to higher eukaryotic cells. Here, it was investigated for its ability to form telomeres from human telomere sequence and to stably maintain long stretches of alphoid DNA. Using linear constructs terminating in the telomere repeat, T2AG3, human telomere DNA was shown to efficiently seed telomere formation in Sch. pombe. Much of the human telomeric sequence was removed on addition of Sch. pombe telomeric sequence, a process similar to that described in S. cerevisiae. To investigate the stability of alphoid DNA in fission yeast, bacterial artificial chromosomes (BACs) containing 130 and 173 kb of alphoid DNA were retrofitted with the Sch. pombe ars1 element and ura4+ marker using Cre-lox recombination. These alphoid BACs were found to be highly unstable in Sch. pombe deleting down to less than 40 kb, whilst control BACs of 96 and 202 kb, containing non-repetitive DNA, were unrearranged. Alphoid DNA has been shown to be sufficient for human centromere function, and this marked instability excludes Sch. pombe as a useful cloning host for mammalian artificial chromosomes. In addition, regions containing repetitive DNA from mammalian genomes may not be truly represented in libraries constructed in Sch. pombe.     

Human artificial chromosomes containing chromosome 17 alphoid DNA maintain an active centromere in murine cells but are not stable

Human artificial chromosomes (HACs) are autonomous molecules that can function and segregate as normal chromosomes in human cells. De novo HACs have successfully been used as gene expression vectors to complement genetic deficiencies in human cultured cells. HACs now offer the possibility of studying the regulation and expression of large genes in a variety of cell types from different tissues and correcting gene deficiencies caused by human inherited diseases. Complementary gene expression studies in mice, especially in mouse models of human genetic diseases, are also important in determining if large human transgenes can be expressed appropriately from artificial chromosomes. Toward this aim we are establishing artificial chromosomes in murine cells as novel gene expression vectors. Initially we transferred HAC vectors into murine cells, but were unable to generate de novo HACs at a reasonable frequency. We then transferred HACs previously established in human HT1080 cells to three different murine cell types by microcell fusion, followed by positive selection. We observed that the HACs in murine cells bound centromere protein C (CENP-C), a marker of active centromeres, and were detected under selection but rapidly lost when selection was removed. These results suggest that the HACs maintain at least a partially functional centromere complex in murine cells, but other factors are required for stability and segregation. Artificial chromosomes containing mouse centromeric sequences may be required for better stability and maintenance in murine cells.     

Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation

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.     

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 alphoid^tet-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.     

Localization of G6PD and HPRT to different arms of the X chromosome of the North American marsupial (Didelphis virginiana) by in situ hybridization and deletion mapping: evolutionary significance

We have mapped HPRT and G6PD loci on the X chromosome in the American opossum, Didelphis virginiana, by in situ hybridization to cells derived from two females by using genomic opossum DNA as probes. The localizations (G6PD to Xp13 and HPRT to Xq21), indicating that the two genes are separated by the centromere, were confirmed by results of hybridization to X chromosomes with deletions that include the HPRT locus and opossum-mouse cell hybrids containing the relevant fragment of the opossum X chromosome.     

Structure of the major block of alphoid satellite DNA on the human Y chromosome

Alphoid DNA is a family of tandemly repeated simple sequences found mainly at the centromeres of the chromosomes of many primates. This paper describes the structure of the alphoid DNA at the centromere of the human Y chromosome. We have used pulsedfield gradient gel electrophoresis, cosmid cloning and DNA sequencing to determine the organization of the alphoid DNA on each of the Y chromosomes present in two somatic cell hybrids. In each case there is a single major block of alphoid DNA. This is approximately 470,000 bases (475 kb) long on one chromosome and approximately 575 kb long on the other. Apart from the size difference, the structures of the two blocks and the surrounding sequences are very similar. However, one restriction enzyme, AvaII, detects two clusters of sites within one block but does not cleave the other. The alphoid DNA within each block is organized into tandemly repeating units, most of which are about 5.7 kb long. A few variant units present on one chromosome are about 6.0 kb long. These variants, like the AvaII site variants, are clustered. The 5.7 kb and 6.0 kb units themselves consist of tandemly repeating 170 base-pair subunits. The 6.0 kb unit has two more of these subunits than the 5.7 kb unit. Our results provide a basis for further structural analysis of the human Y chromosome centromeric region, and suggest that long-range structural polymorphisms of tandemly repeated sequence families may be frequent.     

Alphoid satellite DNA is tightly associated with centromere antigens in human chromosomes throughout the cell cycle

In this study, we have examined a DNA element specific to the centromere domain of human chromosomes. Purified HeLa chromosomes were digested with the restriction enzyme Sau3AI and fractionated by sedimentation through a sucrose gradient. Fractions showing antigenecity to anticentromere (kinetochore) serum obtained from a scleroderma CREST patient were used to construct a DNA library. From this library we found one clone which has specifically hybridized to the centromere domain of metaphase chromosomes using a biotinylated probe DNA and FITC-conjugated avidin. The clone contained a stretch of alphoid DNA dimer. To determine precisely the relative location of the alphoid DNA stretch and the centromere antigen, a method was developed to carry out in situ hybridization of DNA and indirect immunofluorescent staining of antigen on the same cell preparation. Using this method, we have found perfect overlapping of the alphoid DNA sites with the centromere antigen sites in both metaphase chromosomes and nuclei at various stages in the cell cycle. We have also observed this exact correlation at the attachment sites of artificially extended sister chromatids. These results suggest the possibility that alphoid DNA repeats are a key component of kinetochore structure.     

Localization of G6PD and HPRT to different arms of the X chromosome of the North American marsupial (Didelphis virginiana) by in situ hybridization and delection mapping: Evolutionary significance

We have mapped HPRT and G6PD loci on the X chromosome in the American opossum, Didelphis virginiana, by in situ hybridization to cells derived from two females by using genomic opossum DNA as probes. The localizations (G6PD to Xp13 and HPRT to Xq21), indicating that the two genes are separated by the centromere, were confirmed by results of hybridization to X chromosomes with deletions that include the HPRT locus and opossum-mouse cell hybrids containing the relevant fragment of the opossum X chromosome.     

Assay of centromere function using a human artificial chromosome

In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host. When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the α21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1–5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the α21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that α21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes. Received: 22 August 1998 / Accepted: 28 August 1998     

CENP-B interacts with CENP-C domains containing Mif2 regions responsible for centromere localization

Recently, human artificial chromosomes featuring functional centromeres have been generated efficiently from naked synthetic alphoid DNA containing CENP-B boxes as a de novo mechanism in a human cultured cell line, but not from the synthetic alphoid DNA only containing mutations within CENP-B boxes, indicating that CENP-B has some functions in assembling centromere/kinetochore components on alphoid DNA. To investigate whether any interactions exist between CENP-B and the other centromere proteins, we screened a cDNA library by yeast two-hybrid analysis. An interaction between CENP-B and CENP-C was detected, and the CENP-C domains required were determined to overlap with three Mif2 homologous regions, which were also revealed to be involved in the CENP-C assembly of centromeres by expression of truncated polypeptides in cultured cells. Overproduction of truncated CENP-B containing no CENP-C interaction domains caused abnormal duplication of CENP-C domains at G2 and cell cycle delay at metaphase. These results suggest that the interaction between CENP-B and CENP-C may be involved in the correct assembly of CENP-C on alphoid DNA. In other words, a possible molecular linkage may exist between one of the kinetochore components and human centromere DNA through CENP-B/CENP-B box interaction.     

Efficient combination of large DNA in vitro: in gel site specific recombination (IGSSR) of PAC fragments containing alpha satellite DNA and the human HPRT gene locus.

In an attempt to combine a cloned genomic copy of a selectable gene with different cloned centromeric sequences to develop mammalian artificial chromosomes (MAC) we used site specific recombination mediated by purified Cre recombinase acting on the loxP sequence in PAC vector DNA. A new method was required to purify highly concentrated, virtually 100% intact PAC DNA which could be stored for a long period. Here we show the efficient linking of linearized PACs containing alpha satellite DNA from chromosomes X and 17 with sizes of 125 and 140 kb, respectively, to a 95 kb restriction fragment derived from a 175 kb PAC containing the intact human HPRT gene locus.     

CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA

Centromere protein (CENP) B boxes, recognition sequences of CENP-B, appear at regular intervals in human centromeric alpha-satellite DNA (alphoid DNA). In this study, to determine whether information carried by the primary sequence of alphoid DNA is involved in assembly of functional human centromeres, we created four kinds of synthetic repetitive sequences: modified alphoid DNA with point mutations in all CENP-B boxes, resulting in loss of all CENP-B binding activity; unmodified alphoid DNA containing functional CENP-B boxes; and nonalphoid repetitive DNA sequences with or without functional CENP-B boxes. These four synthetic repetitive DNAs were introduced into cultured human cells (HT1080), and de novo centromere assembly was assessed using the mammalian artificial chromosome (MAC) formation assay. We found that both the CENP-B box and the alphoid DNA sequence are required for de novo MAC formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E. Using the chromatin immunoprecipitation assay, we found that direct assembly of CENP-A and CENP-B in cells with synthetic alphoid DNA required functional CENP-B boxes. To the best of our knowledge, this is the first reported evidence of a functional molecular link between a centromere-specific DNA sequence and centromeric chromatin assembly in humans.     

Homologous alpha satellite sequences on human acrocentric chromosomes with selectivity for chromosomes 13, 14 and 21: implications for recombination between nonhomologues and Robertsonian translocations.

We report a new subfamily of alpha satellite DNA (pTRA-2) which is found on all the human acrocentric chromosomes. The alphoid nature of the cloned DNA was established by partial sequencing. Southern analysis of restriction enzyme-digested DNA fragments from mouse/human hybrid cells containing only human chromosome 21 showed that the predominant higher-order repeating unit for pTRA-2 is a 3.9 kb structure. Analysis of a "consensus" in situ hybridisation profile derived from 13 normal individuals revealed the localisation of 73% of all centromeric autoradiographic grains over the five acrocentric chromosomes, with the following distribution: 20.4%, 21.5%, 17.1%, 7.3% and 6.5% on chromosomes 13, 14, 21, 15 and 22 respectively. An average of 1.4% of grains was found on the centromere of each of the remaining 19 nonacrocentric chromosomes. These results indicate the presence of a common subfamily of alpha satellite DNA on the five acrocentric chromosomes and suggest an evolutionary process consistent with recombination exchange of sequences between the nonhomologues. The results further suggests that such exchanges are more selective for chromosomes 13, 14 and 21 than for chromosomes 15 and 22. The possible role of centromeric alpha satellite DNA in the aetiology of 13q14q and 14q21q Robertsonian translocations involving the common and nonrandom association of chromosomes 13 and 14, and 14 and 21 is discussed.     

A novel human artificial chromosome gene expression system using herpes simplex virus type 1 vectors

Human artificial chromosome (HAC) vectors are an important gene transfer system for expression and complementation studies. We describe a significant advance in HAC technology using infectious herpes simplex virus type 1 (HSV-1) amplicon vectors for delivery. This highly efficient method has allowed gene-expressing HACs to be established in glioma-, kidney- and lung-derived cells. We also developed an HSV-1 hypoxanthine phosphoribosyltransferase (HPRT) HAC vector, which generated functional HPRT-expressing HACs that complemented the genetic deficiency in human cells. The transduction efficiency of the HSV-1 HAC amplicons is several orders of magnitude higher than lipofection-mediated delivery. Studies on HAC stability between cell types showed important differences that have implications for HAC development and gene expression in human cells. This is the first report of establishing gene-expressing HACs in human cells by using an efficient, high-capacity viral vector and by identifying factors that are involved in cell-type-specific HAC instability. The work is a significant advance for HAC technology and the development of HAC gene expression systems in human cells.     

Identification of two distinct subfamilies of alpha satellite DNA that are highly specific for human chromosome 15

We report the isolation of two distinct subfamilies of alpha satellite DNA (pTRA-20 and -25) from human chromosome 15. In situ hybridization experiments indicated that both subfamilies are highly specific for this chromosome. Southern analysis of a somatic hybrid cell line carrying human chromosome 15 revealed a likely higher-order genomic band of 2.5 kb for pTRA-20. Similar analysis for pTRA-25 showed multiple higher-order bands of 3.5, 4.5, and 5 kb at moderately high hybridization stringency, but a predominance of the 4.5-kb species at very high stringency. Direct comparison with human genomic DNA confirmed the authenticity of these higher-order structures and demonstrated polymorphic variations using both probes. The origin of the different alphoid subfamilies on chromosome 15 is discussed. These sequences should be useful for the construction of centromere-based genetic linkage maps for human chromosome 15 and, in conjunction with the other alphoid sequences already reported for chromosomes 13, 14, 21, and 22, should allow a concerted analysis of the evolution and the possible etiological role of these DNAs in aberrations commonly seen in these chromosomes.     

Construction of a novel human artificial chromosome vector for gene delivery

Potential problems of conventional transgenes include insertional disruption of the host genome and unpredictable, irreproducible expression of the transgene by random integration. Alternatively, human artificial chromosomes (HACs) can circumvent some of the problems. Although several HACs were generated and their mitotic stability was assessed, a practical way for introducing exogenous genes by the HACs has yet to be explored. In this study, we developed a novel HAC from sequence-ready human chromosome 21 by telomere-directed chromosome truncation and added a loxP sequence for site-specific insertion of circular DNA by the Cre/loxP system. This 21HAC vector, delivered to a human cell line HT1080 by microcell fusion, bound centromere proteins A, B, and C and was mitotically stable during long-term culture without selection. The EGFP gene inserted in the HAC vector expressed persistently. These results suggest that the HAC vector provides useful system for functional studies of genes in isogenic cell lines.