Generating subclones from large-insert genomic clones.

This unit provides a simple protocol for generating a partial-digest sublibrary of yeast DNA containing a YAC of interest. The starting material is high-molecular-weight chromosomal DNA embedded in agarose plugs. Many genome equivalents of these YAC subclones in bacteriophage or cosmid vectors can be plated and screened by hybridization with total human DNA to identify clones that originate from the human portion of the YAC.

Advances in human artificial chromosome technology.

Human artificial chromosome (HAC) technology has developed rapidly over the past four years. Recent reports show that HACs are useful gene transfer vectors in expression studies and important tools for determining human chromosome function. HACs have been used to complement gene deficiencies in human cultured cells by transfer of large genomic loci also containing the regulatory elements for appropriate expression.

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.

Functional complementation of a genetic deficiency with human artificial chromosomes.

We have shown functional complementation of a genetic deficiency in human cultured cells, using artificial chromosomes derived from cloned human genomic fragments. A 404-kb human-artificial-chromosome (HAC) vector, consisting of 220 kb of alphoid DNA from the centromere of chromosome 17, human telomeres, and the hypoxanthine guanine phosphoribosyltransferase (HPRT) genomic locus, was transferred to HPRT-deficient HT1080 fibrosarcoma cells. We generated several cell lines with low-copy-number, megabase-sized HACs containing a functional centromere and one or possibly several copies of the HPRT1 gene complementing the metabolic deficiency.

The assembly of large BACs by in vivo recombination.

We have developed a method for recombining bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) containing large genomic DNA fragments into a single vector using the Cre-lox recombination system from bacteriophage P1 in vivo. This overcomes the limitations of in vitro methods for generating large constructs based on restriction digestion, ligation, and transformation of DNA into Escherichia coli cells. We used the method to construct a human artificial chromosome vector of 404 kb encompassing long tracts of alpha satellite DNA, telomeric sequences, and the human hypoxanthine phosphoribosyltransferase gene.

Stable integration of large (>100 kb) PAC constructs in HaCaT keratinocytes using an integrin-targeting peptide delivery system.

Transfer of large DNA constructs in gene therapy studies is being recognised for its importance in maintaining the natural genomic environment of the gene of interest and providing tissue-specific regulation and control. However, methods used to deliver such constructs have been poorly studied. We used a receptor-mediated, integrin-targeting transfection system enhanced by liposomes, to deliver a 110 kb PAC (P1-based artificial chromosome) to HaCaT keratinocytes.

Transfer of YACs up to 2.3 Mb intact into human cells with polyethylenimine.

The transfer of large YAC DNA into human cells is a laborious procedure. High quality pulsed field gel purified DNA is required, which is easily sheared during manipulation before transfection or degraded in the endosome of the cell following transfection. NaCl and polyamines compact and prevent DNA from shearing, but may not consistently protect DNA after transfection.

11q23.1 and 11q25-qter YACs suppress tumour growth in vivo.

Frequent allelic deletion at chromosome 11q22-q23.1 has been described in breast cancer and a number of other malignancies, suggesting putative tumour suppressor gene(s) within the approximately 8 Mb deleted region. In addition, we recently described another locus, at the 11q25-qter region, frequently deleted in breast cancer, suggesting additional tumour suppressor gene(s) in this approximately 2 Mb deleted region.

Generation of large insert yeast artificial chromosome libraries.

The development of YAC cloning technology has directly enhanced the relationship among genetic, physical, and functional mapping of genomes. Because of their large size, YACs have enabled the rapid construction of physical maps by ordered clone mapping and contig building, and they complement other molecular approaches for mapping complex genomes. Large insert libraries are constructed by size fractionating large DNA embedded in agarose and protecting DNA from degradation with polyamines.

A method for linking yeast artificial chromosomes.

A method for linking any standard yeast artificial chromosomes (YAC) is described. YACs are introduced into the same cell and joined by mitotic recombination between the vector arms and the homologous sequence in a linking vector; several YACs can be recombined sequentially. The linking vectors also contain the beta-galactosidase gene as an expression reporter in mammalian cells.

Physical analysis of the region deleted in the tw18 allele of the mouse tcl-4 complementation group.

We have generated a YAC contig of at least 3.3 Mb from the proximal region of In(17)4 of mouse chromosome 17. This region corresponds to DNA lost in the gastrulation mutant tw18, which belongs to the tcl-4 complementation group.

Analysis of extrachromosomal structures containing human centromeric alphoid satellite DNA sequences in mouse cells.

Yeast artificial chromosomes (YACs) spanning the centromeric region of the human Y chromosome were introduced into mouse LA-9 cells by spheroplast fusion in order to determine whether they would form mammalian artificial chromosomes. In about 50% of the cell lines generated, the YAC DNA was associated with circular extrachromosomal structures. These episomes were only present in a proportion of the cells, usually at high copy number, and were lost rapidly in the absence of selection.

MHC class I gene organization in > 1.5-Mb YAC contigs from the H2-M region.

Sixteen yeast artificial chromosome (YAC) clones have been mapped to the H2-M region at the distal end of the mouse major histocompatibility complex (MHC) on chromosome 17. Analysis of the YACs with single- and multicopy probes yielded a proximal contig spanning a minimum of 800 kb and a distal contig of 700 kb. A probe for the conserved fourth exon of MHC class I genes detected 19 restriction fragments, including 6 of the 8 previously characterized H2-M class I genes, in the proximal contig.

Fluorescence in situ hybridisation of multiple probes on a single microscope slide.

We report a method to analyse multiple samples by fluorescence in situ hybridisation on a single glass microscope slide. Wells were formed in which independent hybridisation reactions could proceed by sealing a silicon rubber gasket to the slide. In the largest format tested, different probes were hybridised simultaneously by applying them directly from a 96-well microtitre dish which was inverted on a glass plate.

Addition of functional human telomeres to YACs.

Linear mammalian artificial chromosomes (MACs) will require functional telomeres, a centromere and the ability to replicate autonomously. We are investigating the possibility of developing MACs from yeast artificial chromosomes (YACs). Retrofitting vectors have been constructed to replace YAC telomeres with cloned human telomeric DNA.

YACs, BACs, PACs and MACs: artificial chromosomes as research tools.

Yeast artificial chromosomes (YACs) have become essential research tools as they enable large fragments of DNA to be cloned. In order to overcome several disadvantages of YACs, including chimaerism and instability, several complementary bacterial artificial chromosome (BAC) vectors have been developed. More recently, attempts are being made to construct artificial chromosomes in mammalian cells (MACs).

Construction of yeast artificial chromosome libraries by pulsed-field gel electrophoresis.

Yeast artificial chromosome (YAC) libraries have been constructed from a variety of organisms using different approaches. This protocol outlines in detail the construction of YAC libraries with large inserts using size fractionation of partially digested DNA by pulsed-field gel electrophoresis.

De novo formation of several features of a centromere following introduction of a Y alphoid YAC into mammalian cells.

The DNA sequence requirements for mammalian centromere function have been investigated by re-introducing human YAC clones containing either centromeric or non-centromeric sequences into hamster and human cells. All YACs integrated into the host chromosomes. In most cell lines produced by spheroplast fusion into hamster cells, intact copies of the YAC and a large amount of yeast DNA were found.

Localization of DNA sequences required for human centromere function through an analysis of rearranged Y chromosomes.

We have localized the DNA sequences required for mitotic centromere function on the human Y chromosome. Analysis of 33 rearranged Y chromosomes allowed the centromere to be placed in interval 8 of a 24-interval deletion map. Although this interval is polymorphic in size, it can be as small as approximately 500kb.

Detailed physical and genetic mapping in the region of plasminogen, D17Rp17e, and quaking.

We present here a detailed physical map encompassing over 600 kb of mouse Chromosome (Chr) 17 in the region of plasminogen, D17Rp17e, and quaking. This region is cloned in yeast artificial chromosomes (YACs). We have identified several CpG islands within this region from pulsed field gel mapping of mouse genomic DNA and YAC DNA.

A method for the generation of YAC transgenic mice by pronuclear microinjection.

Yeast artificial chromosomes (YACs) represent the latest generation of vectors which have the great advantage of large insert size. The introduction of YACs into mammalian cells and organisms has become an important goal, since it offers the potential to study the control of large and complex transcription units and identify genes by complementation. Microinjection into the nucleus is the most direct and efficient way of delivering YAC DNA into cells, but requires the purification of the YAC from the remaining yeast chromosomes.

YAC clone contigs surrounding the Zfx and Pola loci on the mouse X chromosome.

Pulsed-field mapping of a number of DNA markers in the Pola-Zfx region of the mouse X chromosome has established a genomic restriction map extending over 1.4 Mb. A number of YAC clones from the Pola-Zfx region have been isolated from three mouse YAC libraries--first, a mouse C57BL/10 partial R1 YAC library constructed in a yeast strain carrying a rad52 mutation (Chartier et al.

Physical mapping and YAC contig analysis of the region surrounding Xist on the mouse X chromosome.

The Xist sequence has been proposed as a potential candidate for the X-inactivation center based both on its localization within the candidate region for the X-inactivation center in man and mouse and on its unique pattern of expression from the inactive X chromosome. We have cloned 550 kb of DNA surrounding the mouse Xist sequence in contiguously overlapping YAC clones and have developed a long-range restriction map that spans almost 1 Mb of this region and includes this YAC contig. The detailed restriction map we have established provides a framework for the identification of expressed sequences other than Xist that may equally exhibit unusual expression characteristics associated with X inactivation.