A cre-lox recombination system for the targeted integration of circular yeast artificial chromosomes into embryonic stem cells

The ability to produce embryonic stem (ES) cell lines containing different yeast artificial chromosomes (YACs) integrated into the same location in the genome provides a system for comparing the bio-logical effects of YAC transgenes without the confounding influences of integration site and copy number. A targeting system was developed for the directed integration of circular YACs into mouse ES cells. The system combines Cre-lox recombination technology, specifically a positive-selection integration system, with circular YAC lipofection technology to achieve single copy targeted integration of a transgene. Three independent germline competent ES cell lines [lox-containing ES lines (designated LES)] were created that contain a '-neo-lox' cassette integrated at different sites within the ES genome. A plasmid containing YAC vector sequences and a complementary '-neo-lox' cassette was used to circularize two linear YACs containing genomic DNA from human chromosome 21. The circularized YACs were then targeted to the lox sites of the LES cell lines. Polymerase chain reaction and Southern analysis demonstrated that 21% (5 of 24) of lox-recombinants contain a full-length intact YAC. This system will make the study of YAC transgenic mice more reliable and reproducible, allowing the potential for direct comparison of different transgenes expressed from the same site within the genome.     

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 functionhave been Investigated by re-Introducing human YAC clones containingeither centromeric or non-centromeric sequences Into hamsterand human cells. All YACs integrated into the host chromosomes.In most cell lines produced by spheroplast fusion into hamstercells, intact copies of the YAC and a large amount of yeastDNA were found. Cell lines produced by lipofection Into humancells usually contained simple structures without yeast DNA.YACs containing Y alphoid DNA reformed several of the propertiesof a centromere, Including a cytogenetlcally visible constriction,CREST antiserum binding and disruption of anaphase chromosomemovement. In contrast, YACs containing non-centromeric sequencesproduced none of these results. This work suggests that a fewhundred kb of alphoid DNA is sufficient to reconstitute severalImportant features of a centromere.     

Transfer of the human HPRT and GART genes from yeast to mammalian cells by microinjection of YAC DNA

DNA of two yeast artificial chromosomes (YACs) containing selectable human genes was transferred by microinjection to rodent cells in tissue culture. The human hypoxanthine phosphoribosyltransferase (HPRT) gene, spanning 45 kb, is contained on the 660-kb YAC yHPRT as described elsewhere. The human phosphoribosylglycinamide formyltransferase (GART) gene, spanning approximately 40 kb, is contained on the 590-kb YAC yGART2 as described previously. YAC DNA was isolated from pulsed-field gels and microinjected into mammalian cells in which the human HPRT and GART genes can be selected. The cell lines that were selected contain the entire human genes. Some of the cell lines contain multiple copies of the genes integrated at the same chromosomal position. The YAC yGART2 could not be purified away from natural yeast chromosomes of similar size, and the cell lines into which the human GART gene was introduced contain variable amounts of yeast DNA in addition to the human DNA.     

An integrated physical and genetic map of a 35 Mb region on chromosome Xp22.3-Xp21.3

We have constructed a detailed physical map of the 35 Mb regionspanning human chromosome Xp22.3–Xp21.3. The backboneof the map is represented by a single oriented contiguous stretchof 585 overlapping yeast artificial chromosome (YAC) clonescovering the entire region. The map is formatted with 615 mapobjects that include 324 YACs, 185 sequence tagged sites, 28genes, 85 chromosomal breakpoints and 37 highly polymorphicmarkers. Physical mapping was both guided and confirmed using183 bins defined by chromosomal breakpoints and by overlappingregions of YAC clones. The localization of polymorphic markersin the physical map permits the integration of physical andgenetic data across the region. These data establish chromosomeXp22.3–Xp21.3 as one of the best characterized large regionsin the human genome. The map should greatly facilitate finerscale mapping and sequencing as well as the identification ofdisease genes from this portion of the human genome.     

New vector for transfer of yeast artificial chromosomes to mammalian cells

A modification vector has been constructed to facilitate the transfer of yeast artificial chromosomes (YACs) to mammalian cells in culture by targeting a dominant selectable marker (G418 resistance) to the right arm of pYAC4 clones. The ADE2 gene is used for yeast selection with consequent disruption of the URA3 gene, allowing direct modification of YACs within the common host strain AB1380, and providing a simple test for correct targeting. This vector has been tested by modification of a 550-kb YAC containing part of the human MHC class II region and transfer to CHO cells by protoplast fusion. Analysis of 15 independent G418-resistant CHO lines obtained following fusion suggests the majority contain a complete YAC with moderate amplification in some lines.     

Engineering mammalian chromosomes

Construction of a mammalian artificial chromosome (MAC) will develop our understanding of the requirements for normal chromosome maintenance, replication and segregation while offering the capacity for introducing genes into cells. Construction of MACs with telomere, centromere and replication function has been approached by two methods. The 'top down' strategy uses artificially induced chromosome truncations as a means to define a minimal chromosome that retains the mitotic properties of a normal chromosome. The 'build up' approach has focused on attempts to assemble MAC vectors containing functionally defined telomere repeats together with candidate centromere and replication origin sequences. Here we report on significant advances in both areas, with particular emphasis on two reports showing that stable, low copy number MACs containing a functional centromere can be produced following transfection of naked DNA into the human HT1080 cell line. One approach used a transfection mixture of cloned synthetic alpha-satellite arrays up to 1 Mb in length and unlinked telomeric DNA, in either the presence or absence of random human genomic DNA fragments. In the second approach, MACs were formed from a defined yeast artificial chromosome (YAC) DNA molecule containing 100 kb of highly homo- geneous alphoid DNA retrofitted with human telomere repeats. These results demonstrate for the first time that alpha- satellite DNA can seed de novo centromeres in human cells, indicating that this repetitive sequence family plays an important role in centromere function. The stability of these MACs suggests that they have potential to be developed as gene delivery vectors.      

Cloning of the Huntington disease region in yeast artificial chromosomes.

The gene responsible for Huntington disease has been localized to a 2.5 million base pair (Mb) region between the loci D4S10 and D4S168 on the short arm of chromosome 4. As part of a strategy to clone the HD gene on the basis of its chromosomal location, we isolated genomic DNA from the HD region as a set of overlapping yeast artificial chromosome (YAC) clones. Twenty-eight YAC clones were identified by screening human YAC libraries with twelve PCR-based sequence-tagged sites (STSs) from the region. We assembled the YAC clones into overlapping sets by hybridizing them to a large number of DNA probes from the HD region, including the STSs. In addition, we isolated the ends of the human DNA inserts of most of the YAC clones to assist in the construction of the contig. Although almost half of the YACs appear to contain chimeric inserts and several contain internal deletions or other rearrangements, we were able to obtain over 2.2 Mb of the HD region in YACs, including one continuous segment of 2.0 Mb covering the region that most likely contains the HD gene. Ten of the twenty eight YAC clones comprise a minimal set spanning the 2.2 Mb. These clones provide reagents for the complete characterization of this region of the genome and for the eventual isolation of the HD gene.     

Fish mapping of YAC clones at human chromosomal band 7q31.2: Identification of YACS spanning FRA7G within the common region of LOH in breast and prostate cancer

Loss of DNA sequences within human chromosomal band 7q31.2 is frequently observed in a number of different solid tumors including breast, prostate, and ovarian cancer. This chromosomal band also contains the common fragile site, FRA7G. Many of the common fragile sites occur within chromosomal regions that are frequently deleted during tumor formation but their precise position, relative to the chromosome breakpoints and deletions, has not been defined for the majority of the fragile sites. Because the frequency of expression of FRA7G is low, we analyzed the expression of FRA7G in a chromosome 7-only somatic cell hybrid (hamster-human). YAC clones defining a contig spanning 7q31.2 were then used as FISH probes against metaphase spreads prepared from the hybrid cells after aphidicolin induction. This analysis quickly revealed whether a specific YAC clone mapped proximal, distal, or actually spanned the region of decondensation/breakage of FRA7G. By using this approach, we have identified several overlapping YAC clones that clearly span FRA7G. Interestingly, these clones map precisely to the common region of LOH in breast cancer and prostate cancer. In addition, the MET oncogene is contained within the three YACs that span FRA7G. Genes Chromosomes Cancer 21:152–159, 1998. © 1998 Wiley-Liss, Inc.     

Integration of physical, breakpoint and genetic maps of chromosome 22. Localization of 587 yeast artificial chromosomes with 238 mapped markers

Detailed physical maps of the human genome are important resourcesfor the Identification and isolation of disease genes and forstudying the structure and function of the genome. We used datafrom STS content mapping of YACs and natural and induced chromosomalbreakpoints to anchor contigs of overlapping yeast artificialchromosome (YAC) clones spanning extensive regions of humanchromosome 22. The STSs were assigned to specific regions (bins)on the chromosome using cell lines from a somatic hybrid mappingpanel defining a maximum of 25 intervals. YAC libraries werescreened by PCR amplification of hierarchical pools of yeastDNA with 238 markers, and a total of 587 YAC clones were identified.These YACs were assembled into contigs based upon their sharedSTS content using a simulated annealing algorithm. Fifteen contigs,containing between 2 and 74 STSs were assembled, and orderedalong the chromosome based upon the cytogenetic breakpoint,meiotic and PFG maps. Additional singleton YACs were assignedto unique chromosomal bins. These ordered YAC contigs will beuseful for identifying disease genes and chromosomal breakpointsby positional cloning and will provide the foundation for higherresolution physical maps for large scale sequencing of the chromosome.     

Addition of functional human telomeres to YACs

Linear mammalian artificial chromosomes (MACs) will requirefunctional telomeres, a centromere and the abllity to repllcateautonomously. We are investigating the possibility of developingMACs from yeast artificial chromosomes (YACs). Retrofittingvectors have been constructed to replace YAC telomeres withcloned human telomeric DNA. A modified YAC was introduced intomammalian cells by spheroplast tusion and the frequency withwhich the retrofitted human telomeric DNA seeded the formationof a new telomere was determined by Bal31 digestion and cytogeneticanalysis. The telomere adjacent to the selectable marker genewas functional in 5/46 clones (11%) while the telomere 200 kbaway at the other end of the YAC was functional in 1/46 clones(2%). These results indicate that despite the in vivo modificationof the end of the telomere by the addition of yeast sequences,human telomeres will function at a high enough frequency toallow the construction of MACs by this route.     

Chromosome integrity in Saccharomyces cerevisiae: the interplay of DNA replication initiation factors,elongation factors,and origins

The integrity of chromosomes during cell division is ensured by both trans-acting factors and cis-acting chromosomal sites. Failure of either these chromosome integrity determinants (CIDs) can cause chromosomes to be broken and subsequently misrepaired to form gross chromosomal rearrangements (GCRs). We developed a simple and rapid assay for GCRs, exploiting yeast artificial chromosomes (YACs) in Saccharomyces cerevisiae. We used this assay to screen a genome-wide pool of mutants for elevated rates of GCR. The analyses of these mutants define new CIDs (Orc3p, Orc5p, and Ycs4p) and new pathways required for chromosome integrity in DNA replication elongation (Dpb11p), DNA replication initiation (Orc3p and Orc5p), and mitotic condensation (Ycs4p). We show that the chromosome integrity function of Orc5p is associated with its ATP-binding motif and is distinct from its function in controlling the efficiency of initiation of DNA replication. Finally, we used our YAC assay to assess the interplay of trans and cis factors in chromosome integrity. Increasing the number of origins on a YAC suppresses GCR formation in our dpb11 mutant but enhances it in our orc mutants. This result provides potential insights into the counterbalancing selective pressures necessary for the evolution of origin density on chromosomes.     

Rescue of Targeted Regions of Mammalian Chromosomes by in Vivo Recombination in Yeast

In contrast to other animal cell lines, the chicken pre-B cell lymphoma line, DT40, exhibits a high level of homologous recombination, which can be exploited to generate site-specific alterations in defined target genes or regions. In addition, the ability to generate human/chicken monochromosomal hybrids in the DT40 cell line opens a way for specific targeting of human genes. Here we describe a new strategy for direct isolation of a human chromosomal region that is based on targeting of the chromosome with a vector containing a yeast selectable marker, centromere, and an ARS element. This procedure allows rescue of the targeted region by transfection of total genomic DNA into yeast spheroplasts. Selection for the yeast marker results in isolation of chromosome sequences in the form of large circular yeast artificial chromosomes (YACs) up to 170 kb in size containing the targeted region. These YACs are generated by homologous recombination in yeast between common repeated sequences in the targeted chromosomal fragment. Alternatively, the targeted region can be rescued as a linear YACs when a YAC fragmentation vector is included in the yeast transformation mixture. Because the entire isolation procedure of the chromosomal region, once a target insertion is obtained, can be accomplished in ~1 week, the new method greatly expands the utility of the homologous recombinationproficient DT40 chicken cell system.     

Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination.

The human dystrophin gene, mutations of which cause Duchenne and Becker muscular dystrophy, measures 2.4 Mb. This size seriously limits its cloning as a single DNA fragment and subsequent in-vitro expression studies. We have used stepwise in-vivo recombination between overlapping yeast artificial chromosomes (YACs) to reconstruct the dystrophin gene. The recombinant YACs are mitotically stable upon propagation in haploid yeast cells. In contrast, specific combinations of YACs display a remarkable mitotic and meiotic instability in diploid cells. Non-disjunction is rare for overlapping YACs, but increases upon sporulation of diploid cells containing non-overlapping molecules. We have exploited this feature in a three-point recombination to bridge a 280 kb gap between two non-overlapping YACs for which no YAC of proper polarity existed. Our largest recombinant YAC measures 2.3 Mb and contains the entire muscle specific DMD-gene with the exception of a 100 kb region containing the in-frame exon 60. The latter segment has a high tendency to undergo deletions in multi-molecular interactions, probably due to the presence of as yet unidentified instability-enhancing sequences. Fluorescent in situ hybridizations confirmed that the 2.3 Mb DMD YAC contained Xp21-sequences only and indicated a compact tertiary structure of the DMD-gene in interphase lymphocyte nuclei. We conclude that the yeast system is a flexible, efficient and generally applicable tool to reconstruct or build genomic regions from overlapping YAC constituents. Its application to the human dystrophin gene has provided many possibilities for future studies.     

A yeast artificial chromosome contig spanning the Charcot-Marie-Tooth disease type 1A duplication region.

A contiguous set of 43 overlapping yeast artificial chromosome (YAC) clones has been developed for the Charcot-Marie-Tooth disease type 1A (CMT1A) duplication region of chromosome 17p11.2. The contig spans approximately 2.0 Mb and can be represented in a minimum of five overlapping YACs. The YAC clones were isolated from two total human genomic YAC libraries and from YAC libraries made from rodent-human hybrid cell lines. YAC clones were isolated from the libraries by polymerase chain reaction (PCR) technique. Localization to chromosome 17p11.2 was confirmed by fluorescence in situ hybridization. Overlap between the YAC clones was detected by inter-Alu PCR amplification of the YACs and by cross hybridization of the YACs with YAC insert ends obtained by Vectorette PCR. This YAC contig is a useful resource for analyzing and mapping all the genes contained within the CMT1A duplication.     

Physical localisation of the breakpoints of a constitutional translocation t(5;6)(q21;q21) in a child with bilateral Wilms'' tumour.

A 6 month old boy presented with bilateral Wilms' tumour. Cytogenetic analysis of the lymphocytes from the patient showed a de novo balanced translocation t(5;6)(q21;q21), which was also present in the tumour material as the sole cytogenetic abnormality. To facilitate the identification of the translocation breakpoints, we have established a lymphoblastoid cell line (MA214L) from the patient which maintains the translocation in culture. We have used Genethon microsatellite markers as sequence tagged sites (STSs) to isolate yeast artificial chromosome (YAC) clones to 5q and 6q from human genomic libraries. Using fluorescence in situ hybridisation (FISH) on metaphase preparations of MA214L, we have physically defined the translocation breakpoints between YAC clones on each chromosome arm. The genetic distance separating the flanking YACs on 6q21 is 3 cM, while that on 5q21 is 4 cM. To date this is the first report of these chromosomal regions being implicated in Wilms' tumourigenesis.     

FRA3B extends over a broad region and contains a spontaneous HPV16 integration site: direct evidence for the coincidence of viral integration sites and fragile sites

The common fragile site at 3p14.2 (FRA3B) is the most sensitive site on normal human chromosomes for the formation of gaps and breaks when DNA replication is perturbed by aphidicolin or folate stress. Although rare fragile sites are known to arise through the expansion of CCG repeats, the mechanism responsible for common fragile sites is unknown. Beyond being a basic component of chromosome structure, no biological effects of common fragile sites have been convincingly shown, although suggestions have been made that breakage and recombination at these sites may sometimes be mechanistically involved in deletions observed in many tumors and in constitutional deletions. In an observation related to the high rate of recombination at fragile sites, a number of studies have shown a statistical association between the integration of transforming DNA viruses and chromosomal fragile sites. Using FISH analysis we recently identified a 1.3 Mb YAC spanning both FRA3B and the t(3;8) translocation associated with hereditary RCC. Here we report the further localization of FRA3B within this YAC. Using lambda subclones of the YAC as FISH probes, gaps and breaks were found to occur over a broad region of at least 50 kb. Neither CCG nor CAG repeats were found in this region suggesting a different mechanism for fragility than seen with rare fragile sites. We further show that an area of frequent gaps and breaks within FRA3B, defined by a lambda contig, coincides with a previously characterized site of HPV16 integration in a primary cervical carcinoma. The HPV16 integration event gave rise to a short chromosomal deletion limited to the local FRA3B region within 3p14.2. Interestingly, 3p14.2 lies within the smallest commonly deleted region of 3p in cervical cancers, which are often HPV16 associated. To our knowledge this is the first molecular characterization of an in vivo viral integration event within a confirmed fragile site region, supporting previous cytogenetic observations linking viral integration sites and fragile sites.      

Mapping of murine YACs containing the genesCea2 andCea4 after B1-PCR amplification and FISH-analysis

PCR with primers specific for the murine B1 consensus sequence allows amplification of DNA from murine sources. We have used B1-PCR for amplifying yeast artificial chromosome (YAC) DNA which can be used to localize single YACs by fluorescencein situ hybridization. The genes for the pregnancy-specific glycoproteins Cea2 and Cea4, both belonging to the large carcinoembryonic antigen gene family, were localized by chromosomalin situ suppression hybridization of three YAC clones to murine chromosome 7A2-A3. This was facilitated by the use of the mouse lymphoma cell line WMP/WMP which contains nine pairs of Robertsonian fusion chromosomes.