Physical Mapping of Rice Chromosomes 8 and 9 with YAC Clones

First efforts for physical mapping of rice chromosomes 8 and9 were carried out by ordering YAC clones of a rice genomicDNA library covering six genome equivalents with mapped DNAmarkers. A total of 79 and 74 markers from chromosomes 8 and9, respectively, were analyzed by YAC colony and Southern hybridizationusing RFLP markers of cDNA and genomic clones, and by polymerasechain reaction (PCR) screening using PCR-derived and sequence-taggedsite (STS) markers. As a result, 252 YAC clones were confirmedto contain the mapped DNA fragments on both chromosomes. A contigmap was constructed by ordering these YAC clones and about 53%and 43% genome coverage was obtained for chromosomes 8 and 9,respectively, assuming a YAC clone size of 350 kb and overlapbetween neighboring YACs of 50%. A continuous array of YAC cloneswith minimum overlap gave a total size of 18.9 Mb for chromosome8 and 15.6 Mb for chromosome 9, which are close to previousestimates. These contig maps may provide valuable informationthat can be useful in understanding chromosome structure andisolating specific genes by map-based cloning.     

The application of AFLP fingerprinting to construct a YAC contig containing ADH2 and MTP on sheep chromosome 6.

The low density of genetic markers on livestock maps limits progress in positional cloning projects. We demonstrate a strategy of combining comparative mapping with AFLP fingerprinting to develop physical maps in a defined region of the sheep genome. Sequence tagged sites for alcohol dehydrogenase 2 (ADH2) and microsomal triglyceride transfer protein (MTP) were developed and used to screen a sheep yeast artificial chromosome (YAC) library. Nine YACs were identified containing the microsatellite marker BM1329 and either ADH2 or MTP. Additional markers in the region were not available, and AFLP analysis was developed to identify sheep-specific bands within the YACs to determine their degree of overlap. Fourteen bands common to more than one YAC were analysed and provided the markers necessary to develop a YAC contig containing the three STS markers. One YAC (yac260B5) containing all three markers (ADH2, MTP, and BM1329) was mapped to sheep chromosome 6q1.6-->q1.8 by FISH analysis.     

Rapid and reliable screening of a tomato YAC library exclusively based on PCR

An improved procedure is presented to select clones from a tomato yeast artificial chromosome (YAC) library. The procedure is based exlcusively on the polymerase chain reaction (PCR). We combined DNA from approximately 36,000 YAC clones in pools containing 96-single YAC clones from one master plate and further in super pools representing 10 master plates. This pooling strategy allows the selection of single YAC clones homologous to a target sequence after three rounds of PCR using super pools, single pools, and single YAC clones as a template. Single YAC clones were spheroplasted prior to the third PCR round in order to omit the conventional radioactive colony hybridization step. To date, we applied this PCR-based selection strategy to isolate clones homologousto ten different sequence-tagged sites (STS) that are linked to genes targeted for map-based cloning. The selection of YAC clones can be readily accomplished within three days. The PCR-based screening strategy is easy to set up and contributes to a further acceleration of the construction of YAC contigs.     

Automatable screening of yeast artificial-chromosome libraries based on the oligonucleotide-ligation assay.

The systematic screening of yeast artificial-chromosome (YAC) libraries is the limiting step in many physical mapping projects. To improve the screening throughput for a human YAC library, we designed an automatable strategy to identify YAC clones containing a specific segment of DNA. Our approach combines amplification of the target sequence from pooled YAC DNA by the polymerase chain reaction (PCR) with detection of the sequence by an ELISA-based oligonucleotide-ligation assay (OLA). The PCR-OLA approach eliminates the use of radioactive isotopes and gel electrophoresis, two of the major obstacles to automated YAC screening. Furthermore, the use of the OLA to test for the presence of sequences internal to PCR primers provides an additional level of sensitivity and specificity in comparison to methods that rely solely on the PCR.     

Physical mapping in large genomes: accelerating anchoring of BAC contigs to genetic maps through in silico analysis

Anchored physical maps represent essential frameworks for map-based cloning, comparative genomics studies, and genome sequencing projects. High throughput anchoring can be achieved by polymerase chain reaction (PCR) screening of bacterial artificial chromosome (BAC) library pools with molecular markers. However, for large genomes such as wheat, the development of high dimension pools and the number of reactions that need to be performed can be extremely large making the screening laborious and costly. To improve the cost efficiency of anchoring in such large genomes, we have developed a new software named Elephant (electronic physical map anchoring tool) that combines BAC contig information generated by FingerPrinted Contig with results of BAC library pools screening to identify BAC addresses with a minimal amount of PCR reactions. Elephant was evaluated during the construction of a physical map of chromosome 3B of hexaploid wheat. Results show that a one dimensional pool screening can be sufficient to anchor a BAC contig while reducing the number of PCR by 384-fold thereby demonstrating that Elephant is an efficient and cost-effective tool to support physical mapping in large genomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. E. Paux and F. Legeai contributed equally to this work.     

HOSEpipE—a WWW-hosted data management and analysis system for STS content mapping projects

We have developed a data management system, `HOSEpipe' (High Output STS Evaluation pipeline) to aid sample tracking and data analysis in STS content mapping projects. The system is based around a World Wide Web (WWW) server that provides a number of pages including forms for sample processing and data entry accessible via a standard WWW browser application. The system is split into two main modules: firstly, a sequence evaluation and annotation module that takes de novo sequence for a potential STS, screens it against existing STSs and DNA sequence databases, followed by appropriate primer sequence design; secondly, a module that handles YAC library STS screening and includes facilities for both sample tracking and experimental data analysis. We present the design and rationale of the HOSEpipe system and its development to support a whole chromosomal physical mapping project. This software and design approach is potentially applicable to physical mapping projects of varying sizes and resolution and to similar projects, such as sample sequencing and the construction of sequence-ready maps. Received: 18 November 1996 / Accepted: 19 March 1997     

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.     

A YAC Contig of Approximately 3 Mb from Human Chromosome 5q31 → q33

The human chromosome 5q31-q33 region contains an interesting cluster of growth factor and receptor genes. In addition, several genetic disease loci have been localized within this region, but have not as yet been isolated as molecular clones. These include those loci involved in autosomal dominant limb-girdle muscular dystrophy, diastrophic dysplasia. Treacher Collins syndrome, and myeloid disorders associated with the 5q- syndrome. A yeast artificial chromosome (YAC) contig of this region would assist in the further localization and isolation of these genes. We have used YACs isolated from the Washington University and Centre d'Etude du Polymorphisme Humain YAC libraries, including YACs from the large insert (mega) YAC library to build a contig greater than 3 Mb in size. An STS content strategy coupled with limited walking from YAC ends was used to isolate 22 overlapping YACs with as much as sixfold coverage. A total of 20 STSs, derived from genes, anonymous sequences, and vector Alu-PCR or inverse PCR products, were used to compile this contig. The order of loci, centromere-GRL-D5S207-D5S70-D5S545-D5S546-D5S547-D5S68-D5S548-D5S210-D5S549-D5S686-ADRB2-D5S559-CSF1R-D5S551-RPS14-D5S519-SPARC-telomere, was derived from the overlapping clones. This contig and clones derived from it will be useful substrates in selecting candidate cDNAs for the disease loci in this interval.     

A two-dimensional YAC pooling strategy for library screening via STS and Alu-PCR methods.

We have developed a simple two-dimensional YAC pooling strategy to facilitate YAC library screening via STS and Alu-PCR approaches. The method has been implemented using the human total genomic YAC library of Olson and coworkers, and its validity tested by isolation of many chromosomes 19- and 21-specific YACs. The Alu-PCR approach is notable in that it is hybridization-based, such that PCR primer pairs do not need to be repeatedly synthesized and tested for each screening step.     

Application of in situ PCR to yeast cells for screening YAC libraries.

We have used in situ PCR technology in yeast cells with the ultimate goal of cloning and screening genomic yeast artificial chromosome (YAC) libraries. The target sequences in YAC clones were amplified "in situ" in yeast cells by the same set of microsatellite primers used in solution-based PCR screening. The method is fast and sensitive and obviates the steps required for individual isolation of DNAs from hundreds to thousands of YAC clones and thus has an advantage over conventional solution-based PCR screening. This approach can conceivably be applied to the products of automated robotic workstations.     

The development and application of automated gridding for efficient screening of yeast and bacterial ordered libraries.

An automated gridding procedure for the inoculation of yeast and bacterial clones in high-density arrays has been developed. A 96-pin inoculating tool compatible with the standard microtiter plate format and an eight-position tablet have been designed to fit the Biomek 1000 programmable robotic workstation (Beckman Instruments). The system is used to inoculate six copies of 80 x 120-mm filters representing a total of approximately 20,000 individual clones in approximately 3 h. High-density arrays of yeast artificial chromosome (YAC) and cosmid clones have been used for rapid large-scale hybridization screens of ordered libraries. In addition, an improved PCR library screening strategy has been developed using strips cut from the high-density arrays to prepare row and column DNA pools for PCR analysis. This strategy eliminates the final hybridization step and allows identification of a single clone by PCR in 2 days. The development of automated gridding technology will have a significant impact on the establishment of fully versatile screening of ordered library resources for genomic studies.     

Use of interspersed repetitive sequences-PCR products for cDNA selection

In order to increase the efficiency of cDNA selection approaches, we describe the use of interspersed repetitive sequences-PCR (IRS-PCR) products to isolate genes from large-insert genomic clones. IRS-PCR is conducted on total yeast DNA containing a YAC of interest so that there is no need to purify the starting genomic clone. This enables the production of large amounts of genomic substrate for cDNA selection and allows the use of unstable YAC clones. Moreover, the hybridization of the IRS-PCR product to the cDNA clones after selection introduces a positive selection step. We tested these PCR products from YACs for the presence of exons, using cDNAs originating from seven different genes. In each case, at least one exon was present in the IRS-PCR product. We have applied this strategy to four YAC clones originating from the human X Chromosome (Chr). All the selected cDNAs, strongly positive with the IRS-PCR product, did indeed originate from a gene in the region covered by the YAC. In all cases, the previously known genes contained in the genomic clones have been isolated. In addition, we have isolated human genes that have already been described but not assigned to any chromosomal region.     

Systematic generation of sequence-tagged sites for physical mapping of human chromosomes: application to the mapping of human chromosome 7 using yeast artificial chromosomes.

Basic to the development of long-range physical maps of DNA are the detection and localization of landmarks within recombinant clones. Sequence-tagged sites (STSs), which are short stretches of DNA that can be specifically detected by the polymerase chain reaction (PCR), can be used as such landmarks. Our interest is to construct physical maps of whole human chromosomes by localizing STSs within yeast artificial chromosome (YAC) clones. Here we describe a generalized strategy for the systematic generation of large numbers of STSs specific for human chromosome 7. These STSs can be detected by PCR assays developed following the sequencing of anonymous pieces of chromosome 7 DNA, which was derived from flow-sorted chromosomes or from lambda clones made from DNA of a human-hamster hybrid cell line. Our approach for STS generation is tailored for the development of PCR assays capable of screening a large YAC library. In this study, we report the generation of 100 new STSs specific to human chromosome 7.     

"PCR-karyotype" of human chromosomes in somatic cell hybrids

Amplification of human DNA sequences in 16 monochromosomal somatic cell hybrids containing different human chromosomes were performed by the polymerase chain reaction (PCR) using primer directed at human-specific regions of Alu or L1, the two major classes of interspersed repetitive sequences (IRS-PCR). A chromosome-specific pattern of amplification products was observed on agarose gels run with ethidium bromide, producing a "PCR-karyotype." This simple gel analysis provides a rapid method for identifying and monitoring the human chromosomal content of monochromosomal somatic cell hybrids without conventional cytogenetic analysis. Hybrids containing multiple human chromosome produce complex gel patterns, but identification of chromosome content can be achieved by hybridization of PCR products against a reference panel of monochromosomal or highly reduced hybrids representing each human chromosome. This dot-blot method also enables identification of human marker chromosomes or translocated pieces in hybrids that are not identifiable by cytogenetic methods. These IRS-PCR methods should greatly reduce the need for more laborious cytogenetic, isozyme, and Southern blot characterizations of human-rodent cell hybrids.     

ComboScreen facilitates the multiplex hybridization-based screening of high-density clone arrays

MOTIVATION: The construction of physical maps based on bacterial clones [e.g. bacterial artificial chromosomes (BACs)] is valuable for a number of molecular genetics applications, including the high-resolution mapping of genomic regions of interest and the identification of clones suitable for systematic sequencing. A common approach for large-scale screening of bacterial clone libraries involves the hybridization of high-density arrays of immobilized, lysed colonies with collections of DNA probes. The use of a multiplex hybridization screening strategy, whereby pooled probes are analysed en masse, simplifies the effort by reducing the total number of parallel experiments required. However, this approach generates large amounts of hybridization-based data that must be carefully analysed, assimilated, and disambiguated in a careful but efficient manner. RESULTS: To facilitate the screening of high-density clone arrays by a multiplex hybridization approach, we have written a program called ComboScreen. This program provides an organizational framework and analytical tools required for the high-throughput hybridization screening of clone arrays with pools of probes. We have used this program extensively for constructing mouse sequence-ready BAC contig maps.     

A 1.5-Mb contig within the cat eye syndrome critical region at human chromosome 22q11.2

We have constructed a 1.5-Mb contig spanning the distal half of the critical region for cat eye syndrome on human chromosome 22 from D22S543 to D22S181. The contig consists of 20 P1 artificial chromosome (PAC) clones and 11 bacterial artificial chromosome (BAC) clones screened from 2 BAC and 2 PAC libraries. Continuous overlap between the clones was confirmed using vectorette PCR and riboprobes. Despite the instability of this region in a previous YAC contig, only 1 BAC showed a minor instability and then in only one isolation. This contig is now providing the basis for genomic sequencing and gene identification in the cat eye syndrome critical region.     

Analysis of the monomeric alphoid sequences in the pericentromeric region of human chromosome 7

To further define the structure of the pericentromeric region of human chromosome 7, we have identified and characterized a YAC clone (YAC 311.H5) containing the D7S1480 locus, which maps to the short arm near the centromere of this chromosome, by linkage in CEPH families and radiation hybrid analysis. This YAC contains two new blocks of alphoid DNA (named Z5 and Z6). Both Z5 and Z6 show monomeric structures and a lack of higher-order repeats, and, therefore, belong to suprachromosomal family type 4 (M1). The orientation of the two blocks and the physical distances over the region were defined by pulsed-field gel electrophoresis (PFGE) and fluorescence in situ hybridization on chromatin fibers (FiberFISH). A YAC contig spanning the centromeric region has been developed by STS content.     

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.