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.     

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.     

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.     

Rapid screening of a YAC library by pulsed-field gel Southern blot analysis of pooled YAC clones

A new method for screening of YAC libraries is described. Individual YACs were pooled into groups of 384 clones and prepared as samples suitable for pulsed-field gel electrophoresis. A five hit human YAC library (Brownstein et al., 1989) containing approximately 60,000 clones was condensed into 150 such pools and chromosomal DNAs in each sample were separated on three pulsed field gels containing 50 samples each. Southern blots prepared from these gels were hybridized with probes of interest to identify pools containing homologous YACs. Further purification was performed using standard colony hybridization procedures. Twenty-one probes used thus far have identified 47 positive pools and corresponding YACs have been purified from 28 of these. Some significant advantages of this method include avoidance of DNA sequence analysis and primer generation prior to YAC screening and the ability to handle the entire library on three filters. The screening approach described here permits rapid isolation of YACs corresponding to unsequenced loci and will accelerate establishment of YAC contigs for large chromosomal segments.     

Characterization of four dispersed repetitive DNA sequences from Zea mays and their use in constructing contiguous DNA fragments using YAC clones.

We have isolated four repetitive DNA fragments from maize DNA. Only one of these sequences showed homology to sequences within the EMBL database, despite each having an estimated copy number of between 3 x 104 and 5 x 104 per haploid genome. Hybridization of the four repeats to maize mitotic chromosomes showed that the sequences are evenly dispersed throughout most, but not all, of the maize genome, whereas hybridization to yeast colonies containing random maize DNA fragments inserted into yeast artificial chromosomes (YACs) indicated that there was considerable clustering of the repeats at a local level. We have exploited the distribution of the repeats to produce repetitive sequence fingerprints of individual YAC clones. These fingerprints not only provide information about the occurrence and organization of the repetitive sequences within the maize genome, but they can also be used to determine the organization of overlapping maize YAC clones within a contiguous fragment (contigs). Key words : maize, repetitive DNA, YACs.     

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.     

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.     

In situ hybridization to cytogenetic bands of yeast artificial chromosomes covering 50% of human Xq24-Xq28 DNA

From the collection described by Abidi et al., 102 yeast artificial chromosomes (YACs) with human DNA inserts more than 300 kb in length were assigned to chromosomal band positions on early metaphase chromosomes by in situ hybridization using the biotin-avidin method. All the YACs hybridized within the Xq24-Xqter region, supporting the origin of the vast majority of the YACs from single human X-chromosomal sites. With assignments precise to +/- 0.5 bands, YACs were distributed among cytogenetic bands to roughly equal extents. Thus, there is no gross bias in the cloning of DNA from different bands into large YACs. To test band assignments further, hybridizations were carried out blind, and band positions were then compared with (1) probe localizations in cases in which a reported location was present in one of the YACs; (2) cross-hybridization of a labeled YAC with others in the collection; and (3) hybridization to a panel of DNAs from a series of hybrid cells containing Xq DNA truncated at various regions. Of 31 cases in which YACs contained a probe with a previously reported location, 28 in situ assignments were in agreement, and 14 other assignments, including one of the three discordant with probe localization, were confirmed by YAC cross-hybridization studies. Results with a group of nine YACs were further confirmed with a panel of somatic cell hybrid DNAs from that region. Five YACs hybridized both to Xq25 and to a second site (four in Xq27 and one in Xq28), suggestive of some duplication of DNA of the hybrid cell and perhaps in normal X chromosomes. The in situ assignments are thus sufficient to place YACs easily and systematically within bins of about 7-10 Mb and to detect some possible anomalies. Furthermore, on the basis of expectations for random cloning of DNA in YACs, the assigned YACs probably cover more than 50% of the total Xq24-Xq28 region. This provides one way to initiate the assembly of YAC contigs over extended chromosomal regions.     

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.     

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.     

Physical mapping and cloning of a translocation in sugar beet (Beta vulgaris L) carrying a gene for nematode (Heterodera schachtii) resistance from B. procumbens

Two diploid (2n=18) sugar beet (Beta vulgaris L.) lines which carry monogenic traits for nematode (Heterodera schachtii Schm.) resistance located on translocations from the wild beet species Beta procumbens were investigated. Short interspersed repetitive DNA elements exclusively hybridizing with wild beet DNA were found to be dispersed around the translocations. The banding pattern as revealed by genomic Southern hybridization was highly conserved among translocation lines of different origins indicating that the translocations are not affected by recombination events with sugar beet chromosomes. Physical mapping revealed that the entire translocation is represented by a single Sal I fragment 300 kb in size. A representative YAC (yeast artifical chromosome) library consisting of approximately 13,000 recombinant clones (2.2 genome equivalents) with insert sizes ranging between 50 and 450 kb and an average of 130kb has been constructed from the resistant line A906001. Three recombinant YACs were isolated from this library using the wild beet-specific repetitive elements as probes for screening. Colinearity between YAC inserts and donor DNA was confirmed by DNA fingerprinting utilizing these repetitive probes. The YACs were arranged into two contigs with a total size of 215 kb; these represent a minimum of 72% of the translocation.     

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.     

Analysis of the occurrence and nature of repeated DNA in an 850 kb region of Arabidopsis thaliana chromosome 4

The occurrence and nature of repeated DNA sequences has been analysed within an 850 kb YAC contig on Arabidopsis thaliana chromosome 4. Hybridization analysis with seven RFLP markers, six cosmid contigs, 29 YAC end probes and eight YAC clones showed that a least 585 kb of the 850 kb contained only low-copy sequences. One YAC end probe, EG15C8LE, hybridized to multiple genomic fragments and contained a sequence with predicted protein homology to cytochrome P450 monooxygenases. Another one, EG11B7RE, was found to be non-contiguous with the other YAC clones and contained a dispersed repetitive sequence associated with centromeric regions     

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.     

Application of restriction fragment fingerprinting with a rice microsatellite sequence to assembling rice YAC clones.

To refine the current physical map of rice, we have established a restriction fragment fingerprinting method for identifying overlap between pairs of rice yeast artificial chromosome (YAC) clones and defining the physical arrangement of YACs within contiguous fragments (contigs). In this method, Southern blots of rice YAC DNAs digested with a restriction endonuclease are probed with a rice microsatellite probe, (GGC)5. The probe produces a unique fingerprint profile characteristic of each YAC clone. The profile is then digitized, processed in a computer, and a statistic that represents the degree of overlap between two YACs is calculated. The statistics have been used to detect overlaps among YAC clones, thereby filling a gap between two neighbouring contigs and organizing overlapping rice YAC clones into contiguous fragments. We applied this method to rearranging YACs that had previously been assigned to rice chromosome 6 by anchoring with RFLP markers.     

Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification.

Alu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size. The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments. Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in greater than or equal to 90% of diploid nuclei. Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes. This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle.     

Identification of a deletion hotspot on distal mouse chromosome 4 by YAC fingerprinting

Using repetitive elements as probes, genomic DNA fingerprints of four randomly selected yeast artificial chromosome (YAC) clones (two human and two mouse-derived YAC) were analyzed to determine the mutation level following X-ray exposure. Because the repetitive probes were derived from the mammalian host DNA, most of the fingerprint bands originated from the artificial chromosomes and not from the yeast genome. For none of the YAC clones was the mutation frequency elevated following X-ray exposure. However, for one mouse-derived YAC, the mutation level was unusually high (7%; 42 mutants of 607 clones analyzed), whereas for the other three YACs, the mutation level was nearly 0%. Surprisingly, 40 of the 42 mutations were deletions occurring only at three of the 20 mouse specific fingerprint bands. One of the frequently deleted fragments was cloned, sequenced and mapped to distal mouse chromosome 4, which has been repeatedly reported to be the most unstable region of the whole mouse genome, associated with various tumors. Deletion mapping of six YAC mutants revealed this fragment to be completely deleted in four YACs. In the other two mutants, recombination occurred within the fragment, in each case initiated at the same LINE-1 element. In conclusion, the presented YAC fingerprint is a useful tool for detecting and characterizing unstable regions in mammalian genomes.     

Construction and characterization of a rice YAC library for physical mapping

Genomic libraries of rice,Oryza sativa L. cv. Nipponbare, in yeast artificial chromosomes were prepared for construction of a rice physical map. High-molecular-weight genomic DNA was extracted from cultured suspension cells embedded in agarose plugs. After size fractionation of theEco RI- andNot I-digested DNA fragments, they were ligated with pYAC4 and pYAC55, respectively, and used to transformSaccharomyces cerevisiae AB1380. A total of 6932 clones were obtained containing on average ca. 350 kb DNA. The YAC library was estimated to contain six haploid genome equivalents. The YACs were examined for their chimerism by mapping both ends on an RFLP linkage map. Most YACs withEco RI fragments below 400 kb were intact colinear clones. About 40% of clones were chimeric. Genetic mapping of end clones from large size YACs revealed that the physical distance corresponding to 1 cM genetic distance varies from 120 to 1000 kb, depending on the chromosome region. To select and order YAC clones for making contig maps, high-density colony hybridization using ECL was applied. With several probes, at least one and at most ten YAC clones could be selected in this library. The library size and clone insert size indicate that this YAC library is suitable for physical map construction and map-based cloning.     

Analysis of pericentromeric chromosome 21 specific YAC clones by FISH: Identification of new markers for molecular-cytogenetic application

Fluorescence in situ hybridization (FISH) of chromosome 21 specific yeast artificial chromosome (YAC) clones after Alu-PCR (polymerase chain reaction) amplification has been used to find new region-specific DNA probes for the heterochromatic region of chromosome 21. Six overlapping YAC clones from a pericentromeric contig map (region 21cen-21q11) were analyzed. Four YAC clones were characterized as hybridizing to several chromosomal locations. They are, therefore, either chimeric or shared by different chromosomes. Two of them containing alphoid satellite DNA, are localized at the centromeric regions of chromosomes 13 and 21 (clone 243A11), and on 13cen, 21cen and 1q3 (clone 781G5); the two others are localized at both 21q11 and 13q2 (clone 759D3), and at 18p (clone 770B3). Two YACs were strongly specific for chromosome 21q11 only (clones 124A7 and 881D2). These YACs were used effectively as probes for identifications of chromosome 21 during metaphase and interphase analysis of 12 individuals, including three families with Down syndrome offspring, and 6 amniocyte samples. The location of YAC clones on 21q11 close to the centromeric region allows the application of these clones as molecular probes for the analysis of marker chromosomes with partial deletions of the long arm as well as for pre- and postnatal diagnosis of trisomy 21 when alphoid or more distal region-specific DNA probes are uninformative. Overlapping YAC clones covering human chromosome 21q may be systematically used to detect a set of band-specific DNA probes for molecular-cytogenetic application.