Evaluation of a cosmid contig physical map of human chromosome 16.

A cosmid contig physical map of human chromosome 16 has been developed by repetitive sequence finger-printing of approximately 4000 cosmid clones obtained from a chromosome 16-specific cosmid library. The arrangement of clones in contigs is determined by (1) estimating cosmid length and determining the likelihoods for all possible pairwise clone overlaps, using the fingerprint data, and (2) using an optimization technique to fit contig maps to these estimates. Two important questions concerning this contig map are how much of chromosome 16 is covered and how accurate are the assembled contigs. Both questions can be addressed by hybridization of single-copy sequence probes to gridded arrays of the cosmids. All of the fingerprinted clones have been arrayed on nylon membranes so that any region of interest can be identified by hybridization. The hybridization experiments indicate that approximately 84% of the euchromatic arms of chromosome 16 are covered by contigs and singleton cosmids. Both grid hybridization (26 contigs) and pulsed-field gel electrophoresis experiments (11 contigs) confirmed the assembled contigs, indicating that false positive overlaps occur infrequently in the present map. Furthermore, regional localization of 93 contigs and singleton cosmids to a somatic cell hybrid mapping panel indicates that there is no bias in the coverage of the euchromatic arms.     

Physical and genetic analysis of cosmids from the vicinity of the cystic fibrosis locus.

Cosmid libraries have been constructed from DNA of somatic cell hybrid cell lines, each containing a fragment of human chromosome seven and including sequences closely linked to cystic fibrosis (CF). Cosmids containing human DNA as insert were isolated from the library. Three cosmids, when used as probes to total genomic DNA, detected polymorphic loci, each of which was shown to be in strong linkage disequilibrium with CF. Restriction endonuclease digestion of cosmid clones and use of a new, rapid method of chromosome walking based on competitive hybridisation of cosmid inserts has allowed identification of several groups of overlapping cosmids ("contigs") from the vicinity of CF.     

Towards a physical map of the Drosophila melanogaster genome: mapping of cosmid clones within defined genomic divisions.

A physical map of the D. melanogaster genome is being constructed, in the form of overlapping cosmid clones that are assigned to specific polytene chromosome sites. A master library of ca. 20,000 cosmids is screened with probes that correspond to numbered chromosomal divisions (ca. 1% of the genome); these probes are prepared by microdissection and PCR-amplification of individual chromosomes. The 120 to 250 cosmids selected by each probe are fingerprinted by Hinfl digestion and gel electrophoresis, and overlaps are detected by computer analysis of the fingerprints, permitting us to assemble sets of contiguous clones (contigs). Selected cosmids, both from contigs and unattached, are then localized by in situ hybridization to polytene chromosomes. Crosshybridization analysis using end probes links some contigs, and hybridization to previously cloned genes relates the physical to the genetic map. This approach has been used to construct a physical map of the 3.8 megabase DNA in the three distal divisions of the x chromosome. The map is represented by 181 canonical cosmids, of which 108 clones in contigs and 32 unattached clones have been mapped individually by in situ hybridization to chromosomes. Our current database of in situ hybridization results also includes the beginning of a physical map for the rest of the genome: 162 cosmids have been assigned by in situ hybridization to 129 chromosomal subdivisions elsewhere in the genome, representing 5 to 6 megabases of additional mapped DNA.     

Identification and characterization of 23 RFLP loci by screening random cosmid genomic clones.

As part of our search for polymorphic DNA probes, we have screened cosmids from a human genomic DNA library for their ability to reveal RFLPs. A total of 101 randomly isolated cosmid clones were tested in Southern hybridizations for polymorphic band patterns. Fifty-four of these clones revealed RFLPs with one or more of nine restriction enzymes. Twenty-three of these clones have been further characterized and assigned to 10 different chromosomes by linkage analysis or by hybridization to panels of human-hamster hybrid cell lines. Fifteen of the probes have heterozygosities greater than or equal to .5. The relative efficiency of RsaI and PstI restriction enzymes in detecting polymorphism was different from results obtained with libraries constructed in bacteriophage vectors. Screening randomly selected cosmid probes is an efficient method for detecting RFLPs.     

Metagenomic profiling: microarray analysis of an environmental genomic library

Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing approximately 1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.     

Metagenomic Profiling: Microarray Analysis of an Environmental Genomic Library

Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing ~1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.     

Isolation of new probes from Xq12-->q13: an example of the screening of reference libraries with Alu-PCR products from radiation hybrids.

In order to isolate new probes from the juxtacentromeric region of the long arm of the human X chromosome, we used Alu-mediated polymerase chain reaction (Alu-PCR) products as probes to directly screen a chromosome X-specific gridded cosmid library. These Alu-PCR products were synthesized from radiation hybrids containing the loci DXS159, PGK1, and PGK1P1. This approach allowed us to select 18 cosmids capable of hybridizing with at least two Alu-PCR products. Four cosmids hybridized to more than three Alu-PCR products. Three of these four cosmids were contiguous, and the fourth was independent. Two cosmids that hybridized with two Alu-PCR products were further characterized. Physical mapping indicated that all of these clones are located in the expected region on Xq, confirming the validity of our approach.     

The txtAB genes of the plant pathogen Streptomyces acidiscabies encode a peptide synthetase required for phytotoxin thaxtomin A production and pathogenicity

Four Streptomyces species have been described as the causal agents of scab disease, which affects economically important root and tuber crops worldwide. These species produce a family of cyclic dipeptides, the thaxtomins, which alone mimic disease symptomatology. Structural considerations suggest that thaxtomins are synthesized non-ribosomally. Degenerate oligonucleotide primers were used to amplify conserved portions of the acyladenylation module of peptide synthetase genes from genomic DNA of representatives of the four species. Pairwise Southern hybridizations identified a peptide synthetase acyladenylation module conserved among three species. The complete nucleotide sequences of two peptide synthetase genes (txtAB) were determined from S. acidiscabies 84.104 cosmid library clones. The organization of the deduced TxtA and TxtB peptide synthetase catalytic domains is consistent with the formation of N-methylated cyclic dipeptides such as thaxtomins. Based on high-performance liquid chromatography (HPLC) analysis, thaxtomin A production was abolished in txtA gene disruption mutants. Although the growth and morphological characteristics of the mutants were identical to those of the parent strain, txtA mutants were avirulent on potato tubers. Moreover, introduction of the thaxtomin synthetase cosmid into a txtA mutant restored both pathogenicity and thaxtomin A production, demonstrating a critical role for thaxtomins in pathogenesis.     

Isolation of a Cosmid Sublibrary for a Region of Chromosome 12 Frequently Amplified in Human Cancers Using a Complex Chromosome Microdissection Probe

Chromosome-specific cosmid libraries are an extremely useful resource for positional cloning projects. Once a particular region of interest has been identified, it would be of value to have an approach for isolating chromosome band-specific cosmids that could be assembled into a sublibrary for rapid screening. We constructed a region-specific sublibrary of 700 cosmids by screening a chromosome 12-specific cosmid library with a complex probe generated by degenerate oligonucleotide-primed PCR of a microdissected homogeneously staining region containing sequences amplified from chromosome 12q13–q15. Based on fluorescencein situhybridization, approximately 60% of the cosmids in the sublibrary were derived from the microdissected region. To demonstrate further the utility of this sublibrary, a 150-kb contig containing the SAS and CDK4 genes was constructed, as well as several additional contigs between CDK4 and MDM2. This study demonstrates the possibility of utilizing probes generated by microdissection for assembling band-specific sublibraries that are amenable to rapid screening with multiple markers.     

Four restriction fragment length polymorphisms revealed by probes from a single cosmid map to human chromosome 12q

Summary Human gene mapping would be greatly facilitated if marker loci with sufficient polymorphism information content were generally available. As a source of such markers, we have used cosmids from a human genomic library. We have used a rapid method for screening random cosmids to identify those homologous to genomic regions especially rich in restriction fragment length polymophisms (Litt and White 1985). This method allows whole cosmids to be used as probes against Southern transfers of genomic DNA; regions of cosmid probes homologous to repeated genomic sequences are rendered unable to anneal with Southern transfers by prerendered of the probes with a vast excess of non-radioactive genomic DNA. From one cosmid (C1-11) identified by this procedure, we have isolated four single-copy probes, each of which identifies a polymorphic locus. Despite the existence of some linkage disequilibrium in this system, the polymorthism information content was computed as 0.73. Using a somatic cell hybrid mapping panel, we have mapped probes from cosmid 1–11 to human chromosome 12q. Additionally, in situ hybridization of the whole cosmid to metaphase spreads allowed more precise assignment of the locus to the region 12cenq13. The locus revealed by probes from cosmid 1–11 has been designated D12S6.     

Studies on locus expansion, library representation, and chromosome walking using an efficient method to screen cosmid libraries

We have developed an efficient screening method to search for clones in cosmid libraries prepared from human genomic DNA. Genomic, cDNA, and cosmid probes have been used to isolate homologous cosmids from human chromosomes 7, 10, 16, 17 and X as part of a search for polymorphic nucleotide sequences. This method has been successfully applied to chromosome walking experiments at the interstitial retinol-binding protein locus on chromosome 10, and may be a useful tool for investigating representation of cloned sequences in cosmid libraries. Our library was prepared in the vector c2RB (Bates and Swift, 1983), but the method is applicable to any cosmid cloning system in which the inserted DNA can be separated from the vector by restriction enzyme digestion. A cosmid library containing five human genome equivalents can be rapidly screened using three to four Southern hybridization filters. This results in substantial labor saving, particularly when screening genomes of high complexity with many different probes. Another advantage of the system is that it allows for the long-term storage of the cosmids so that they can be screened whenever necessary. As a consequence, cosmid screening can be made a routine laboratory procedure.     

Isolation of cDNA clones using yeast artificial chromosome probes.

The cloning of large DNA fragments of hundreds of kilobases in Yeast artificial chromosomes, has simplified the analysis of regions of the genome previously cloned by cosmid walking. The mapping of expressed sequences within cosmid contigs has relied on the association of genes with sequence motifs defined by rare-cutting endonucleases, and the identification of sequence conservation between species. We reasoned that if the contribution of repetitive sequences to filter hybridizations could be minimised, then the use of large cloned DNAs as hybridisation probes to screen cDNA libraries would greatly simplify the characterisation of hitherto unidentified genes. In this paper we demonstrate the use of this approach by using a YAC, containing 180 kb of human genomic DNA including the aldose reductase gene, as a probe to isolate an aldose reductase cDNA from a lambda gt11 human foetal liver cDNA library.     

The genome structure of Pseudomonas putida: high-resolution mapping and microarray analysis

As part of a collaborative project aimed at sequencing and functionally analysing the entire genome of Pseudomonas putida strain KT2440, a physical clone map was produced as an initial resource. To this end, a high-coverage cosmid library was arrayed and ordered by clone hybridizations. Restriction fragments generated by rare-cutting enzymes and plasmids containing the rrn operon and 23S rDNA of Pseudomonas aeruginosa were used as probes and, parts of the cosmids were end-sequenced. This provided the information necessary for merging and comparing the macro-restriction map, cosmid clone order and sequence information, thereby assuring co-linearity of the eventual sequence assembly with the actual genome. A tiling path of clones was selected, from the shotgun clones used for sequencing, for the production of DNA microarrays that represent the entire genome including its non-coding portions.     

Isolation of region-specific cosmids from chromosome 5 by hybridization with microdissection clones.

A method is described for the isolation of chromosome region specific cosmids. The 5q35 region of the long arm of human chromosome 5 was microdissected, digested with MboI, ligated to oligonucleotide adaptors, amplified by the polymerase chain reaction and cloned into a plasmid vector. Inserts which did not contain highly repetitive sequences were used to screen a chromosome 5 cosmid library by direct hybridization. There were 33 positive cosmid clones identified with 4 microclones. Individual cosmid clones were biotinylated and used as probes for fluorescence in situ hybridization to metaphase chromosomes. Of the 33 cosmids that were mapped, 29 localized to q35 and 4 to q34, demonstrating the specificity of the microdissection library and the cosmids.     

Rapid mapping of cosmid clones on pig chromosomes by fluorescence in situ hybridization

Nineteen cosmids have been mapped to pig chromosomes by fluorescence in situ hybridization. Two kinds of cosmid clones were isolated as potential physical and genetic markers for the pig genome. Anonymous cosmids were obtained by screening a commercial cosmid library and were localized to Chromosomes (Chrs) 1, 2, 6, 7, 8, 10, 11, 12, 13, and 14. Some of these cosmids were found to reveal RFLP type DNA polymorphism. Microsatellite-containing cosmid clones were isolated by screening a pig cosmid library with a (CA)₁₀ probe and were regionally mapped to Chrs 2, 6, 7, 13, and 14. Ten of the 19 chromosomes in the pig were labeled with these probes. Two-color fluorescence in situ hybridization was used to increase the efficiency of the cosmid localizations.     

High-resolution alignment of a 1-megabase-long genome region of three strains of Rhodobacter capsulatus.

A detailed restriction map of the genome of Rhodobacter capsulatus SB1003 was constructed recently by using an ordered set of overlapping cosmids. Pulsed-field gel electrophoresis-generated restriction patterns of the chromosomes of 14 other R. capsulatus strains were compared. Two of them, St. Louis and 2.3.1, were chosen for high-resolution alignment of their genomes with that of SB1003. A 1-Mb segment of the R. capsulatus SB1003 cosmid set was used as a source of ordered probes to group cosmids from the other strains. Selected cosmids were linked into one 800-kb contig and two smaller contigs of 100 kb each. EcoRV and BamHI restriction maps of the newly ordered cosmids were constructed by using lambda terminase. Long-range gene order in the new strains was mainly conserved for the regions studied. However, one large genome rearrangement inverted a 470-kb DNA fragment of the St. Louis strain between the rrnA and rrnB operons. A 50-kb deletion covering three SB1003 probes was found in strain 2.3.1 near rrnB. Conservation of about 50% of the positions of restriction sites in all these strains and nearly 80% for the pair 2.3.1- St. Louis made it possible to produce high-resolution alignment of the contiguous 800-kb genome segment. Ten deletions of 2 to 27 kb, one 30-kb inversion, and three translocations were found in this region. Strong clustering of the positions of polymorphic restriction sites was observed. For a 50-kb size interval, two patterns of the distribution of restriction sites were found, one with about 90% and the other with 5 to 30% conservation of sites. This structure may be explained by independent acquisition of these divergent regions from other Rhodobacter strains.     

A cosmid library specific for human Chromosome regions 6p21.3 and 6q27

We have explored the potential of irradiation-fusion gene transfer (IFGT) hybrids as a source of well-defined human chromosome fragments from which probes can be derived. Extensive characterization of the IFGT hybrid 4J4 with a full panel of markers from Chromosome (Chr) 6 showed that the human DNA content derives largely from 6p21.3 and 6q27. A cosmid library has been constructed from 4J4 DNA, and 370 recombinants containing human DNA have been isolated and overlapping clones ordered into 20 contigs. Regional localization of representative clones from each contig, determined by fluorescent in situ hybridization (FISH), places 13 contigs in 6q27 and 6 in 6p21.3. Preliminary screening of cDNA libraries with selected cosmids has identified two expressed sequences. Since there are a number of medically important genes in both these regions of human Chr 6 with several disease loci linked to the HLA-A region in 6p21.3 and various tumor suppressor genes to 6q27, this library will provide a valuable resource to aid the isolation of candidate genes for these diseases. In addition, unique markers for detailed physical and genetic mapping of these regions of human Chr 6 can be easily obtained.     

Molecular analysis of the rat MHC. II. Isolation of genes that map to the RT1.E-grc region

An initial mapping analysis of growth and reproduction complex (grc) and grc+ genomic DNA identified several restriction fragment length polymorphisms specific for the grc region of the MHC. To analyze further the genomic organization and structure of the grc, a cosmid library was constructed from a grc+-bearing strain (R21). One cosmid cluster, encompassing 41.4 kb of DNA, contained four, or possibly five, class I genes that mapped to the RT1.E-grc region Two unique non-class I fragments were isolated from certain cosmids within this cluster. These fragments were hybridized to genomic DNA derived from five rat strains (BIL/2, R18, R21, R22, and BIL/1), and the results showed that grc-bearing rats have a deletion of at least 3.1 kb of DNA in the region immediately adjacent to the MHC. The loss of the genes in this region is probably the cause of the growth and reproductive defects in these animals and probably also of their increased susceptibility to chemical carcinogens.     

Use of high coverage reference libraries of Drosophila melanogaster for relational data analysis. A step towards mapping and sequencing of the genome

Three differently made, primary Drosophila cosmid libraries of 16-fold genome coverage have been generated. Also, a jumping library has been created by a new method that takes advantage of methylation differences between genomic DNA and vector. Thirdly, two cDNA libraries have been picked. All these libraries have been arrayed on high-density in situ filters, each containing 9216 clones. As a reference system, such filters are distributed and identified clones are provided. Single-copy probes have identified on average 1.4 cosmids per genome equivalent. Together with cytogenetically mapped yeast artificial chromosomes, the libraries are also being used for physically mapping the genome, mainly by oligonucleotide fingerprinting and pool hybridizations. cDNA clones are further examined by a partial sequencing analysis by oligomer hybridization.