Human Xq24-Xq28: approaches to mapping with yeast artificial chromosomes.

One hundred twenty-seven yeast strains with artificial chromosomes containing Xq24-Xqter human DNA were obtained starting from a human/hamster somatic cell hybrid. The clones were characterized with respect to their insert size, stability, and representation of a set of Xq24-Xqter DNA probes. The inserts of the clones add up to 19.3 megabase (Mb) content, or about 0.4 genomic equivalents of that portion of the X chromosome, with a range of 40-650 kb in individual YACs. Eleven clones contained more than one YAC, the additional ones usually having hamster DNA inserts; the individual YACs could be separated by extracting the total DNA from such strains and using it to retransform yeast cells. One of the YACs, containing the probe for the DXS49 locus, was grossly unstable, throwing off smaller versions of an initial 300-kb YAC during subculture; the other YACs appeared to breed true on subculture. Of 52 probes tested, 12 found cognate YACs; the YACs included one with the glucose-6-phosphate dehydrogense gene and another containing four anonymous probe sequences (DX13, St14, cpx67, and cpx6). Xq location of YACs is being verified by in situ hybridization to metaphase chromosomes, and fingerprinting and hybridization methods are being used to detect YACs that overlap.     

Yeast artificial chromosomes containing human Xq24-Xq28 DNA: library construction and representation of probe sequences

A library of yeast artificial chromosomes (YACs) with human DNA inserts has been assembled from a human/hamster somatic cell hybrid containing Xq24-Xqter human DNA. Screening of the agar-embedded transformants for human DNA used a manifold of 3000 stainless-steel pins to transfer colonies onto the surface of media. This facilitated the recovery of the 1 in 300 clones that contained a human DNA insert (the remainder had hamster DNA and were discarded). The library described here consists of about two genomic equivalents (102 Mb) of human DNA in 467 clones: 167 were generated by EcoRI partial digestion and contain 25.5 Mb of human DNA; 252 used partial digestion with TaqI and cover 64.2 Mb; and 48 were from sheared DNA inserts and cover 11.7 Mb. Clones were screened by hybridization with 70 probes previously assigned to Xq24-Xq28. Eleven probes did not hybridize to any YACs in the library, and 16 probes hybridized to one YAC each, 23 to two, 13 to three, and 7 to four. Also, individual YACs large enough to detect features like the clustering of polymorphic sequences in subregions of Xq24-Xqter have been obtained. For example, XY58 contained five probe sequences previously independently isolated. The overall yield of YACs containing probe sequences was indistinguishable from Poisson statistical expectations for random cloning (P = 0.9). Thus, YAC libraries such as the one described here can include most, if not all, of the sequences in the source DNA from which the library is derived. These results support the possibility that YACs may provide a reliable bridge between linkage studies and conventional recombinant DNA analyses in mapping of the human genome.     

PCR amplification and analysis of yeast artificial chromosomes

A strategy for the analysis of yeast artificial chromosome (YAC) clones that relies on polymerase chain reaction (PCR) amplification of small restriction fragments from isolated YACs following adapter ligation was developed. Using this method, termed YACadapt, we have amplified several YACs from a human Xq24-qter library and have used the PCR products for physical mapping by somatic cell hybrid deletion analysis and fluorescent in situ hybridization. One YAC, RS46, was mapped to band Xq27.3, near the fragile X mutation. The PCR product is an excellent renewable source of YAC DNA for analyses involving hybridization of YAC inserts to a variety of DNA/RNA sources.     

Distribution of moderately repetitive sequences pTR5 and LF1 in Xq24-q28 human DNA and their use in assembling YAC contigs.

Xq24-q28 DNA, from a hamster/human hybrid cell containing only that portion of the human X chromosome, was found to contain 56 TaqI restriction fragments that hybridized to the moderately repetitive sequence pTR5. Using the pTR5 sequence as a probe in colony hybridization, 136 cognate yeast artificial chromosome (YAC) clones were detected among a collection of 820 containing about three genomic equivalents of the Xq24-q28 DNA. The YACs were then grouped into 48 contigs and single clones containing one or more of the TaqI fragments. Overlaps were confirmed both by fingerprinting YACs with AluI and L1 probes and by additional information. A less complete analysis was also carried out with a second moderately repetitive sequence, LF1, and some smaller contigs were merged into larger ones. Moderately repetitive sequences can thus be used as probes for multiple loci in single hybridization experiments and can help to organize and confirm YAC overlaps during the development of maps with long-range contiguity.     

1.5-Mb YAC Contig in Xq28 Formatted with Sequence-Tagged Sites and Including a Region Unstable in the Clones

A contig of 20 yeast artificial clones (YACs) has been assembled across 1.5 Mb of Xq28 and formatted with nine previously reported probes and nine STSs developed from the sequence of probes and end fragments of YACs. YAC end fragments were obtained by subcloning, Alu-vector PCR, or primer-ligation PCR methods. Eighteen of the YACs were recovered from a library specific for Xq24-q28; two that fill a gap were obtained from a second library made from total human DNA. One region, containing probes pX78c and 2A1.1, was unstable in YACs, but it was possible to generate a self-consistent map of DNA over the entire contig. Overlaps were confirmed by Southern blot analyses of YAC DNAs, and pulsed-field gel electrophoresis confirmed the extent of the contig and identified at least four CpG islands in the region.     

Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

Use of repetitive DNA probes as physical mapping strategy in Caenorhabditis elegans.

A method for linking genomic sequences cloned in yeast artificial chromosomes (YACs) has been tested using Caenorhabditis elegans as a model system. Yeast clones carrying YACs with repeated sequences were selected from a C. elegans genomic library, total DNA was digested with restriction enzymes, transferred to nylon membranes and probed with a variety of repetitive DNA probes. YAC clones that overlap share common bands with one or more repetitive DNA probes. In 159 YAC clones tested with one restriction enzyme and six probes 28 overlapping clones were detected. The advantages and limitations of this method for construction of YAC physical maps is discussed.     

Genome mapping by arbitrary amplification of yeast artificial chromosomes

Several methods have been described for using the polymerase chain reaction (PCR) to isolate fragments of DNA for genome mapping. We have developed an approach for isolating discrete fragments by amplifying DNA with single oligonucleotides (10-mers) with arbitrarity selected sequences. The method is rapid and technically simple. We isolated fragments from a contig of three yeast artificial chromosomes (YACs) from the human Xq28 chromosomal region. We purified YACs yWXD 37, yWXD348, and yWXD705 from a preparative pulsed field gel. Amplifications of each YAC were performed with single 10-mers as the PCR primers and the products were visualized on agarose gels. These fragments have been successfully used as hybridization probes against Southern blots containing the YACs and against blots containing human genomic DNA and somatic cell hybrids containing Xq28 as their only human constituent. The results have been concordant with the known order of the YACs. We have also successfully combined 10-mers with primers derived from vector arm sequences to isolate YAC ends. We discuss several uses of this method in comparative mapping and in filling in gaps in physical and genetic maps.     

Recombination during transformation as a source of chimeric mammalian artificial chromosomes in yeast (YACs).

Mammalian DNAs cloned as artificial chromosomes in yeast (YACs) frequently are chimeras formed between noncontiguous DNAs. Using pairs of human and mouse YACs we examined the contribution of recombination during transformation or subsequent mitotic growth to chimeric YAC formation. The DNA from pairs of yeast strains containing homologous or heterologous YACs was transformed into a third strain under conditions typical for the development of YAC libraries. One YAC was selected and the presence of the second was then determined. Co-penetration of large molecules, as deduced from co-transformation of markers identifying the different YACs, was > 50%. In approximately half the cells receiving two homologous YACs, the YACs had undergone recombination. Co-transformation depends on recombination since it was reduced nearly 10-fold when the YACs were heterologous. While mitotic recombination between homologous YACs is nearly 100-fold higher than for yeast chromosomes, the level is still much lower than observed during transformation. To investigate the role of commonly occurring Alu repeats in chimera formation, spheroplasts were transformed with various human YACs and an unselected DNA fragment containing an Alu at one end and a telomere at the other. When unbroken YACs were used, between 1 and 6% of the selected YACs could incorporate the fragment as compared to 49% when the YACs were broken. We propose that Alu's or other commonly occurring repeats could be an important source of chimeric YACs. Since the frequency of chimeras formed between YACs or a YAC and an Alu-containing fragment was reduced when a rad52 mutant was the recipient and since intra-YAC deletions are reduced, rad52 and possibly other recombination-deficient mutants are expected to be useful for YAC library development.     

A 1.6-Mb contig of yeast artificial chromosomes around the human factor VIII gene reveals three regions homologous to probes for the DXS115 locus and two for the DXYS64 locus.

Two yeast artificial chromosome (YAC) libraries were screened for probes in Xq28, around the gene for coagulation factor VIII (F8). A set of 30 YACs were recovered and assembled into a contig spanning at least 1.6 Mb from the DXYS64 locus to the glucose 6-phosphate dehydrogenase gene (G6PD). Overlaps among the YACs were determined by several fingerprinting techniques and by additional probes generated from YAC inserts by using Alu-vector or ligation-mediated PCR. Analysis of more than 30 probes and sequence-tagged sites (STSs) made from the region revealed the presence of several homologous genomic segments. For example, a probe for the DXYS64 locus, which maps less than 500 kb 5' of F8, detects a similar but not identical locus between F8 and G6PD. Also, a probe for the DXS115 locus detects at least three identical copies in this region, one in intron 22 of F8 and at least two more, which are upstream of the 5' end of the gene. Comparisons of genomic and YAC DNA suggest that the multiple loci are not created artifactually during cloning but reflect the structure of uncloned human DNA. On the basis of these data, the most likely order for the loci analyzed is tel-DXYS61-DXYS64-(DXS115-3-DXS115-2)-5'F8-(D XS115-1)-3'F8-G6PD.     

Construction of a human chromosome 4 YAC pool and analysis of artificial chromosome stability.

In order to construct a human chromosome 4-specific YAC library, we have utilized pYAC4 and a mouse/human hybrid cell line HA(4)A in which the only human chromosome present is chromosome 4. From this cell line, approximately 8Mb of chromosome 4 have been cloned. The library includes 65 human-specific clones that range in size from 30kb to 290kb, the average size being 108kb. In order to optimize the manipulation of YAC libraries, we have begun to investigate the stability of YACs containing human DNA in yeast cells; these studies will also determine if there are intrinsic differences in the properties of chromosomes containing higher eukaryotic DNAs. We are examining two kinds of stability: 1] mitotic stability, the ability of the YAC to replicate and segregate properly during mitosis, and 2] structural stability, the tendency of the YAC to rearrange. We have found that the majority of YACs examined are one to two orders of magnitude less stable than authentic yeast chromosomes. Interestingly, the largest YAC analyzed displayed a loss rate typical for natural yeast chromosomes. Our results also suggest that increasing the length of an artificial chromosome improves its mitotic stability. One YAC that showed a very high frequency of rearrangement by mitotic recombination proved to be a mouse/human chimera. In contrast to studies using total human DNA, the frequency of chimeras (i.e., mouse/human) in the YAC pool appeared to be low.     

Characterization of four human YAC libraries for clone size, chimerism and X chromosome sequence representation.

Four collections of human X-specific YACs, derived from human cells containing supernumerary X chromosomes or from somatic cell hybrids containing only X human DNA were characterized. In each collection, 80-85% of YAC strains contained a single X YAC. Five thousand YACs from the various libraries were sized, and cocloning was assessed in subsets by the fraction of YAC insert-ends with non-X sequences. Cocloning was substantial, ranging up to 50% for different collections; and in agreement with previous indications, in all libraries the larger the YACs, the higher the level of cocloning. In libraries made from human-hamster hybrid cells, expected numbers of clones were recovered by STS-based screening; but unexpectedly, the two collections from cells with 4 or 5 X chromosomes yielded numbers of YACs corresponding to an apparent content of only about two X equivalents. Thus it is possible that the DNA of inactive X chromosomes is poorly cloned into YACs, speculatively perhaps because of its specialized chromatin structure.     

Fine mapping and cloning of the breakpoint associated with Menkes syndrome in a female patient.

The gene responsible for Menkes syndrome has been assigned to Xq13 by a combination of comparative mapping and linkage analysis. A previous report has mapped the translocation breakpoint associated with the disease in a female patient to an interval delimited by PGK1 and a group of six more proximal Xq13 markers, including DXS56. We have characterized a number of PGK1- or DXS56-positive YACs, from which we have generated six new markers. One of them identifies a small overlap region between a PGK1-positive YAC and three DXS56-positive YACs, distal to the Menkes breakpoint. A 560-kb region covered by a DXS56-positive YAC has been restriction-mapped and subcloned, disclosing a 187-kb MluI fragment astride the breakpoint. A probe mapping distal to the rearrangement in the same interval reveals altered PGFE fragments in a hybrid constructed from the translocation patient's DNA. We describe the development of a cosmid contig extending 150 kb from a nearby CpG island across the breakpoint. This contig includes four adjacent clones displaying cross-specific hybridization.     

Direct Complementation ofChlamydomonasMutants with Amplified YAC DNA

We previously described the construction and characterization of aChlamydomonasgenomic library in yeast artificial chromosomes (YACs). Here we describe the isolation and genetic mapping of YACs at the FLA10 locus on theunichromosome as well as isolation of a YAC spanning the PF14 locus on chromosome VI. Genetic mapping of YAC end clones by RFLP analyses in interspecific crosses reveals that YACs with a physical size of 150 kb commonly span genetic intervals defined by one or two recombination events in crosses of approximately 20 tetrads. This promises to make chromosomal walking inChlamydomonasa relatively efficient enterprise. We also describe our development of a method for direct complementation of mutant genes by transformation with amplified wildtype YAC DNA. The use of positional cloning using YACs and this direct functional assay for the presence of a gene in a YAC represent powerful molecular genetic tools enabling the cloning of most anyChlamydomonasgene.     

Yeast artificial chromosome-based genome mapping: some lessons from Xq24-q28.

Yeast artificial chromosomes (YACs) have recently provided a potential route to long-range coverage of complex genomes in contiguous cloned DNA. In a pilot project for 50 Mb (1.5% of the human genome), a variety of techniques have been applied to assemble Xq24-q28 YAC contigs up to 8 Mb in length and assess their quality. The results indicate the relative strength of several approaches and support the adequacy of YAC-based methods for mapping the human genome.     

Human glucose-6-phosphate dehydrogenase gene carried on a yeast artificial chromosome encodes active enzyme in monkey cells

Yeast artificial chromosomes (YACs) permit the cloning of large tracts of human DNA. A YAC containing the human glucose-6-phosphate dehydrogenase gene is shown to encode active enzyme, supporting the inference that the YAC conserves the structure of the genomic DNA.     

Yeast artificial chromosome-based genome mapping: Some lessons from Xq24–q28

Yeast artificial chromosomes (YACs) have recently provided a potential route to long-range coverage of complex genomes in contiguous cloned DNA. In a pilot project for 50 Mb (1.5% of the human genome), a variety of techniques have been applied to assemble Xq24–q28 YAC contigs up to 8 Mb in length and assess their quality. The results indicate the relative strength of several approaches and support the adequacy of YAC-based methods for mapping the human genome.     

Region-specific YAC banding and painting probes for comparative genome mapping: implications for the evolution of human chromosome 2

To date, several hundred nonchimeric yeast artificial chromosomes (YACs) from the Centre d'Étude du Polymorphisme Humain containing polymorphic sequence-tagged sites have been mapped by fluoresence in situ hybridization (FISH) on human metaphase chromosomes. Because they carry an average of 1 Mb of human genomic DNA, CEPH YACs generate high-intensity in situ hybridization signals. The available set of cytogenetically and genetically anchored YACs, approximately one every 5–10 cM evenly spaced over almost the entire human genome, provides complex region-specific probes for molecular cytogenetics. YAC probes can be adapted with unlimited flexibility to specific FISH applications such as the study of chromosomal evolution. We have generated representational probes for YAC banding and painting of human chromosome 2 and its great ape homologs. Convergent inversions were found in the pericentric region of the gorilla and orangutan homologs of chromosome 2p.     

A physical map with yeast artificial chromosome (YAC) clones covering 63% of the 12 rice chromosomes.

A new YAC (yeast artificial chromosome) physical map of the 12 rice chromosomes was constructed utilizing the latest molecular linkage map. The 1439 DNA markers on the rice genetic map selected a total of 1892 YACs from a YAC library. A total of 675 distinct YACs were assigned to specific chromosomal locations. In all chromosomes, 297 YAC contigs and 142 YAC islands were formed. The total physical length of these contigs and islands was estimated to 270 Mb which corresponds to approximately 63% of the entire rice genome (430 Mb). Because the physical length of each YAC contig has been measured, we could then estimate the physical distance between genetic markers more precisely than previously. In the course of constructing the new physical map, the DNA markers mapped at 0.0-cM intervals were ordered accurately and the presence of potentially duplicated regions among the chromosomes was detected. The physical map combined with the genetic map will form the basis for elucidation of the rice genome structure, map-based cloning of agronomically important genes, and genome sequencing.     

Detection and characterization of "chimeric" yeast artificial chromosome clones by fluorescent in situ suppression hybridization.

"Chimeric" yeast artificial chromosomes (YACs) are clones containing two or more noncontiguous segments of DNA and represent the most common artifact found in total genomic YAC libraries currently used for large-scale genome mapping. These YACs create spurious mapping information that complicates the construction of YAC contigs and leads to erroneous maps during chromosome walks. The presence of these artifactual clones necessitates laborious and time-consuming characterization of each isolated YAC clone, either by comparison of the physical map of the YAC with the corresponding source genomic DNA, or by demonstrating discrepant chromosomal origins for the two ends of the YAC by hybridization or polymerase chain reaction (PCR). Here, we describe a rapid and sensitive method for the assessment of YAC colinearity by fluorescence in situ suppression hybridization (FISSH) by utilizing fluorescein-12-dUTP for labeling YAC clones. We have analyzed 51 YACs and found that 43% (22 out of 51) are chimeric and significantly larger (302 kb) than colinear ones (228 kb). One of the 51 YAC clones (2%) examined contains portions of three chromosomes and 2 (4%) seem to map to a chromosome different than that of the identifying STS. FISSH analysis offers a straightforward visualization of the entire YAC insert on the chromosomes and can be used to examine many YACs simultaneously in few days.