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.     

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.     

A YAC-based binning strategy facilitating the rapid assembly of cosmid contigs: 1.6 Mb of overlapping cosmids in Xp22

We have applied a yeast artificial chromosome (YAC)-based cosmidisolation and binning strategy to convert a YAC contig in Xp22into 1.6 Mb of overlapping cosmids. This strategy is based onthe screening of a high-density arrayed X chromosome-specificcosmid library with large YAC-derived restriction fragmentsand entire YAC probes. Cosmids selected in this way were griddedon dot blots and further mapped into bins defined by the overlapintervals of the YACs and YAC fragments. This rapid binningof cosmids simplified the subsequent assembly of cosmid contigsby restriction fingerprint hybridization. In total, we identified139 cosmids spanning the entire 1.6 Mb region with a minimaloverlap set of 53 clones. These cosmids were assigned to 17bins and 9 contigs. One of the contigs is 665 kb in length andis one of the largest uninterrupted cosmid contigs in humansreported to date. The gaps between the contigs are minor and,together, they represent less than 7% of the region covered.Two previously identified genes are contained in these cosmids,the gene for amelogenin (AMG) and the recently isolated putativechloride channel gene CICN4. In addition, two disease loci havebeen mapped to this region: X-linked ocular albinism type 1(OA1) and the microphthalmia with linear skin defects (MLS)syndrome. The assembly of the cosmid maps allowed us to determinethe size of the deletion intervals for these two loci, whichwere estimated to be 110 kb for OA1 and 570 kb for MLS. Thesecosmid contigs will greatly facilitate the positional cloningof the OA1 and MLS disease genes. Together with the Huntingtondisease gene region on chromosome 4, this region in Xp22 representsone of the best characterized large regions in the human genome.     

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.     

Characterization of a 1.0 Mb YAC contig spanning two chromosome breakpoints related to Menkes disease.

Menkes disease, an X-linked recessive disorder of copper metabolism, has recently been mapped to Xq13.3 by two Menkes patients carrying chromosome rearrangements within this region. The breakpoints have been investigated by nonisotopic in situ suppression hybridization using YACs isolated from this region with the flanking markers DXS56 and PGK1. Three YACs were extending over the breakpoints at Xq13.3 and were shown to be overlapping by partial digest restriction maps, IRS-PCR fingerprinting and by the presence of common cosmid clones. These cosmids were subcloned and one of the single copy probes detected both breakpoints using rare-cutting restriction enzyme digests of the patients. All the results together localize the breakpoints to about 100 kb within the overlapping region of the YACs. Mapping of both breakpoints in a 1 Mb YAC contig implies that these YACs contain at least partially, the gene responsible for Menkes disease.     

Genetic and physical characterization of the early-onset Alzheimer's disease AD3 locus on chromosome 14q24.3

Genetic linkage studies have provided significant evidence thata major gene defect, AD3, for familial early-onset Alzheimer'sdisease (EOAD) is located at chromosome 14q24.3, between theshort tandem repeat (STR) markers D14S52 and D14S53 defininga genetic size of 22.7 cM for the AD3 candidate region. We constructeda physical map of the AD3 region using yeast artificial chromosomes(YACs) selected from both the CEPH and megaCEPH YAC librariesusing the AD3 linked STR markers as well as new sequence-taggedsites (STSs) designed based on YAC terminal sequences. The YACmap is contiguous in the region between D14S258 and D14S53,a region of 8.2 cM, and has an estimated physical size of 4–8Mb. The YAC contig map was used as a framework to localize threeknown genes, a pseudogene and two brain expressed sequence tags(ESTs). Linkage analysis studies in two Belgian chromosome 14EOAD families AD/A and AD/B, identified obligate recombinantsin family AD/A with D14S289 and D14S61 reducing the geneticsize of the candidate AD3 region substantially. The minimalAD3 candidate region measured 6.4 cM on the genetic map andis contained within six overlapping megaCEPH YACs that covereda physical distance estimated between 2 and 6 Mb. These YACsas well as other YACs in the YAC contig map are valuable resourcesin gene cloning efforts or genomic sequencing experiments aimingat isolating the AD3 gene.     

An integrated physical map for the short arm of human chromosome 5

The short arm of human chromosome 5 contains approximately 48 Mb of DNA and comprises 1.5% of the genome. We have constructed a mega-YAC/ STS map of this region that includes 436 YACs anchored by 216 STSs. By combining and integrating our map with the 5p maps of other groups using the same recombinant DNA library, a comprehensive map was constructed that includes 552 YACs and 504 markers. The YAC map covers >94% of 5p in four YAC contigs, bridges the centromere, and includes an additional 5 Mb of 5q DNA. The average marker density is 95 kb. This integrated 5p map will serve as a resource for the continuing localization of genes on the short arm of human chromosome 5 and as a framework for both generating and aligning the DNA sequence of this region.     

A high-resolution physical map of human chromosome 21p using yeast artificial chromosomes

The short arm of human chromosome 21 (21p) contains many different types of repetitive sequences and is highly homologous to the short arms of other acrocentric chromosomes. Owing to its repetitive nature and the lack of chromosome 21p-specific molecular markers, most physical maps of chromosome 21 exclude this region. We constructed a physical map of chromosome 21p using sequence tagged site (STS) content mapping of yeast artificial chromosomes (YACs). To this end, 39 STSs located on the short arm or near the centromere of chromosome 21 were constructed, including four polymorphic simple tandem repeats (STRs) and two expressed sequence tags (ESTs). Thirty YACs were selected from the St. Louis YAC library, the chromosome 21-enriched ICRF YAC library, and the CEPH YAC and megaYAC libraries. These were assembled in a YAC contig map ranging from the centromere to the rDNA gene cluster at 21p12. The total size of the region covered by YACs is estimated between 2.9 and 5 Mb. The integrity of the YAC contig was confirmed by restriction enzyme fingerprinting and fluorescence in situ hybridization (FISH). One gap with an estimated size of 400 kb remained near the telomeric end of the contig. This YAC contig map of the short arm of human chromosome 21 constitutes a basic framework for further structural and functional studies of chromosome 21p.     

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.     

Physical mapping of a 670-kb region of chromosomes XVI and XVII from the human protozoan parasite Trypanosoma cruzi encompassing the genes for two immunodominant antigens

As part of the Trypanosoma cruzi Genome Initiative, we have mapped a large portion of the chromosomal bands XVI (2.3 Mb) and XVII (2.6 Mb) containing the highly repetitive and immunodominant antigenic gene families h49 and jl8. Restriction mapping of the isolated chromosomal bands and hybridization with chromosome specific gene probes showed that genes h49 and jl8 are located in a pair of size-polymorphic homologous chromosomes. To construct the integrated map of the chromosomes harboring the h49 and jl8 loci, we used YAC, cosmid, and lambda phage overlapping clones, and long range restriction analysis using a variety of probes (i.e., known gene sequences, ESTs, polymorphic repetitive sequences, anonymous sequences, STSs generated from the YAC ends). The total length covered by the YAC contig was approximately 670 kb, and its map agreed and was complementary to the one obtained by long-range restriction fragment analysis. Average genetic marker spacing in a 105 kb region around h49 and jl8 genes was estimated to be 6.2 kb/marker. We have detected some polymorphism in the H49/JL8 antigens-encoding chromosomes, affecting also the coding regions. The physical map of this region, together with the isolation of specific chromosome markers, will contribute in the global effort to sequence the nuclear genome of this parasite.     

The utrophin and dystrophin genes share similarities in genomic structure

Utrophin and dystrophin are highly homologous proteins whichare reciprocally expressed in DMD (Duchenne muscular dystrophy)muscle. The remarkable similarity of these proteins suggeststhat they may play a similar cellular role in some circumstances;If this were the case then utrophin may be capable of replacingdystrophin in DMD patients. In this paper we show that the genomicstructure of the utrophin gene is similar to the dystrophingene, further exemplifying the relatedness of the two genesand their gene products. We have constructed a 1.25Mb contigof eight yeast artificial chromosome (YAC) clones covering theutrophin gene located on chromosome 6q24. Utrophin is encodedby multiple small exons spanning approximately 900kb. The distributionof exons within the genomic DNA has similarities to that ofthe dystrophin gene. In contrast to dystrophin, the utrophingene has a long 5' untranslated region composed of two exonsand a cluster of unmethylated, rare-cutting restriction enzymesites at the 5' end of the gene. Similarities between the genomicstructure suggest that utrophin and dystrophin arose throughan ancient duplication event involving a large region of genomicDNA.     

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.     

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.     

Introduction of YACs containing a putative mammalian replication origin into mammalian cells can generate structures that replicate autonomously

Yeast artificial chromosomes (YACs) containing or lacking a biochemically defined DNA replication origin were transferred from yeast to mammalian cells in order to determine whether origin-dependent autonomous replication would occur. A specialized YAC vector was designed to enable selection for YACs in mammalian cells and for monitoring YAC abundance in individual mammalian cells. All of eight clones made with linear and circularized YACs lacking the origin and seven of nine clones made with linear and circularized YACs containing the origin region contained single copies of the transfected YAC, along with various amounts of yeast DNA, integrated into single but different chromosomal sites. By contrast, two transformants derived from circularized YACs containing the putative replication origin showed very heterogeneous YAC copy number and numerous integration sites when analyzed after many generations of in vitro propagation. Analysis of both clones at an early time after fusion revealed variously sized extrachromosomal YAC/yeast structures reminiscent of the extrachromosomal elements found in some cells harboring amplified genes. The data are consistent with the interpretation that YACs containing a biochemically defined origin of replication can initially replicate autonomously, followed by integration into multiple chromosomal locations, as has been reported to occur in many examples of gene amplification in mammalian cells.     

The isolation of cDNAs within the Huntington disease region by hybridisation of yeast artificial chromosomes to a cDNA library

Flanking recombination events have defined the Huntington Diseasegene candidate region to between D4S10 and D4S98, about 2.2Mb.Because of the large size of the candidate region and the likelylarge number of genes within it we decided to screen cDNA librarieswith probes generated from whole Yeast Artificial Chromosomes(YACs) containing parts of this region. We have thus far used4 YACs ranging in size from 180kb to 600kb covering 880kb andhave isolate 13 cDNA clones, 7 of which are unique. Three ofthe 13 clones contain parts of the 3' untranslated region ofthe     

The central part of the mouse immunoglobulin kappa locus.

At the present state of analysis the central part of the kappa locus comprises four contigs of together 1.2 Mb and contains 55 Vkappa genes. It is flanked by the 3' part of the locus with 22 Vkappa genes in 0.4 Mb (T. Kirschbaum et al., Eur. J. Immunol. 1998. 28: 1458-1466) and the 5' part with 63 Vkappa genes in six contigs of together 1.5 Mb (F. R?schenthaler et al., accompanying report). The 5' and the central regions have one large contig in common. A part of the central region is linked to the 3' region resulting in a 1.1-Mb contig. The structure of the contigs was established mainly by the analysis of overlapping cosmid clones derived from genomic DNA and yeast and bacterial artificial chromosomes (YACs and BACs) and by PCR techniques. Pulsed-field gel electrophoresis of YAC digests indicated that three gaps between the contigs of the central region are 10-40 kb in size, comprising together about 90 kb. Internal duplications in this part of the locus and rearranged YACs were the major problems of the structural work. Structural details are to be found on the Internet at http://www.med.uni-muenchen.de/biochemie/zach au/kappa.htm. In a concluding section of the report the mouse kappa locus is compared to the human one and some aspects of the evolution of the kappa locus are discussed.     

Chromosome 18 breakpoint in t(11;18)(q21;q21) translocation associated with MALT lymphoma is proximal to BCL2 and distal to DCC

The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnormality in a subset of extranodal marginal zone B‐cell lymphoma, a low‐grade lymphoma of mucosa‐associated lymphoid tissue (MALT). Neither the 11q21 nor the 18q21 breakpoints have been characterized by molecular genetic analysis. As a prelude to isolation of the gene(s) involved in this translocation, we have mapped the 18q21 breakpoint region by fluorescence in situ hybridization (FISH) of YAC and PAC clones. We mapped 37 YACs assigned to a 29‐cM region within the chromosomal band 18q21. Using nine of these YACs in single‐ and/or dual‐color FISH to analyze three cases of MALT lymphomas with the t(11;18)(q21;q21) translocation, we localized the breakpoints within a 1.6‐Mb nonchimeric YAC (938E1). This YAC is useful for the detection of the translocation in metaphase and in interphase cells. A nonchimeric YAC contig of an 8‐cM region around the breakpoint comprising nine YACs and a PAC contig of YAC 938E1 were constructed, which enabled the refinement of the breakpoint region in the proximal region of the YAC within a <820‐kb segment. This breakpoint is proximal to the BCL2 locus and distal to DCC and DPC4 loci in chromosomal band 18q21. Genes Chromosomes Cancer 24:156–159, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis

A multiplex PCR assay was developed for the rapid analysis of deletion size at the hypoxanthine phosphoribosyltransferase (hprt) locus. The DNA sequence of mapped DNA segments flanking thehprt gene was determined. These cloned DNAs were derived from the ends of a set of overlapping yeast artificial chromosomes (YAC) defining a contig of 8 Mb at Xq26 and includinghprt. We used bubble PCR to isolate an additional YAC end-clone. Seven primer pairs were derived from DNA sequence analysis of the clones and incorporated into a multiplex PCR assay. These primer pairs define loci located approximately 750 kb and 350 kb upstream ofhprt and 300 kb, 540 kb, 900 kb, 1260 kb, and 1400 kb downstream ofhprt. A primer pair for an unlinked and unselected gene sequence (K-ras) was also included in the multiplex reaction to serve as an internal positive control. Using this new assay,hprt mutant DNAs can be screened to determine the extent of deletion. Deletions larger than 2 Mb have been identified and show that large deletions can be tolerated at this hemizygous locus.