Detailed physical map and set of overlapping clones covering the genome of the archaebacterium Haloferax volcanii DS2.

An integrated approach of "bottom up" and "top down" mapping has produced a minimal set of overlapping cosmid clones covering 96% of the 4140 kilobase-pairs (kbp) Haloferax volcanii DS2 genome and a completely closed physical map. This genome is partitioned into five replicons: a 2920 kbp chromosome and four plasmids, of 690 kbp (pHV4), 442 kbp (pHV3), 86 kbp(pHV1) and 6.4 kbp (pHV2). A restriction map for six infrequently-cutting restriction enzymes was constructed, representing a total of 903 sites in the cloned DNA. We have placed the two ribosomal RNA operons, the genes for 7 S RNA and for RNaseP RNA and 22 protein-coding genes on the map. Restriction site frequencies show significant variation in different portions of the genome. The regions of high site density correspond to halobacterial satellite or FII DNA which includes two small regions of the chromosome, the plasmids pHV1 and pHV2, and half of pHV4, but not pHV3.     

A test case for physical mapping of human genome by repetitive sequence fingerprints: construction of a physical map of a 420 kb YAC subcloned into cosmids.

A rapid and safe method of Yeast Artificial Chromosome (YAC) physical mapping by cosmid 'fingerprinting' is presented. YACs are subcloned into cosmids which are prepared without previous separation of cloned DNA from host DNA. Groups of overlapping clones are detected according to their restriction fragments size and intensity after hybridization with total human DNA. To test this approach, a cosmid library was constructed from total DNA of a yeast strain containing a 420 kb YAC. A single contig of 84 clones was obtained with a minimal detectable overlap of 60% i.e. a 9.2 fold representative library. Large scale physical mapping of YACs would take full advantage of the DNA preparation procedure employed in this work and allows to take into account restriction fragment intensities.     

Cosmid linking clones localized to the long arm of human chromosome 11.

Molecular probes that contain DNA flanking CpG-rich restriction sites are extremely valuable in the construction of physical maps of chromosomes and in the identification of genes associated with hypomethylated HTF (HpaII tiny fragment) islands. We describe a new approach to the isolation and characterization of linking clones in arrayed chromosome-specific cosmid libraries through the large-scale semiautomated restriction mapping of cosmid clones. We utilized a cosmid library representing human chromosome 11q12-11qter and carried out automated restriction enzyme analysis, followed by regional localization to chromosome 11q using high-resolution in situ suppression hybridization. Using this approach, 165 cosmid linking clones containing one or more NotI, BssHII, SfiI, or SacII sites were identified among 960 chromosome-specific cosmids. Furthermore, this analysis allowed clones containing a single site to be distinguished from those containing clusters of two or more rare sites. This analysis demonstrated that more than 75% of cosmids containing a rare restriction site also contained a second rare restriction site, suggesting a high degree of CpG-rich restriction site clustering. Thirty chromosome 11q-specific cosmids containing rare CpG-rich restriction sites were regionally localized by high-resolution fluorescence in situ suppression hybridization, demonstrating that all of the CpG-rich sites detected by this method were located in bands 11q13 and 11q23. In addition, the distribution of (CA)n repetitive sequences was determined by hybridization of the arrayed cosmid library with oligonucleotide probes, confirming a random distribution of microsatellites among CpG-rich cosmid clones. This set of reagent cosmid clones will be useful for physical linking of large restriction fragments detected by pulsed-field gel electrophoresis and will provide a new and highly efficient approach to the construction of a physical map of human chromosome 11q.     

Physical map of the genome of Rhodobacter capsulatus SB 1003.

A map of the chromosome of Rhodobacter capsulatus was constructed by overlapping the large restriction fragments generated by endonucleases AseI and XbaI. The analyses were done by hybridization of single fragments with the restriction fragments blotted from pulsed-field gels and by grouping cosmids of a genomic library of R. capsulatus into contigs, corresponding to the restriction fragments, and further overlapping of the contigs. A technical difficulty due to a repeated sequence made it necessary to use hybridization with cloned genes and prior knowledge of the genetic map in order to close the physical circle in a unique way. In all, 41 restriction sites were mapped on the 3.6-Mb circular genome and 22 genes were positioned at 26 loci of the map. Cosmid clones were grouped in about 80 subcontigs, forming two groups, one corresponding to the chromosome of R. capsulatus and the other corresponding to a 134-kb plasmid. cos site end labeling and partial digestion of cosmids were used to construct a high-resolution EcoRV map of the 134-kb plasmid. The same method can be extended to the entire chromosome. The cosmid clones derived in this work can be used as a hybridization panel for the physical mapping of new genes as soon as they are cloned.     

Strategy to sequence the genome of Corynebacterium glutamicum ATCC 13032: use of a cosmid and a bacterial artificial chromosome library

The initial strategy of the Corynebacterium glutamicum genome project was to sequence overlapping inserts of an ordered cosmid library. High-density colony grids of approximately 28 genome equivalents were used for the identification of overlapping clones by Southern hybridization. Altogether 18 contiguous genomic segments comprising 95 overlapping cosmids were assembled. Systematic shotgun sequencing of the assembled cosmid set revealed that only 2.84 Mb (86.6%) of the C. glutamicum genome were represented by the cosmid library. To obtain a complete genome coverage, a bacterial artificial chromosome (BAC) library of the C. glutamicum chromosome was constructed in pBeloBAC11 and used for genome mapping. The BAC library consists of 3168 BACs and represents a theoretical 63-fold coverage of the C. glutamicum genome (3.28 Mb). Southern screening of 2304 BAC clones with PCR-amplified chromosomal markers and subsequent insert terminal sequencing allowed the identification of 119 BACs covering the entire chromosome of C. glutamicum. The minimal set representing a 100% genome coverage contains 44 unique BAC clones with an average overlap of 22 kb. A total of 21 BACs represented linking clones between previously sequenced cosmid contigs and provided a valuable tool for completing the genome sequence of C. glutamicum.     

A minimal tiling path cosmid library for functional analysis of the Pseudomonas aeruginosa PAO1 genome

Pseudomonas aeruginosa is an important pathogenic and environmental bacterium, with the most widely studied strain being PAO1. Using the PAO1 reference cosmid library and the recently completed PAO1 genome sequence, we have mapped a minimal tiling path across the genome using a two-step strategy. First, we sequenced both ends of a set of over 500 random and previously mapped clones to create a backbone. Second, we end-sequenced a second set of cosmid clones that were identified to lie within the larger gaps using hybridization of the reference library filters with probes designed against sequences at the center of each gap. The minimal tiling path was calculated using the program Domino (http://www.bit.uq.edu.au/download/), with the overlap between adjacent clones set to 5 kb (where possible) to minimize the chance of truncating genes. This yielded a minimal tiling cosmid library (334 clones) covering 93.7% of the genome in 57 contigs. This library has reduced to a workable set the number of clones required to represent the majority of the P. aeruginosa genome and gives the precise location of each cosmid, enabling most genes of interest to be located on clones without further screening. This library should prove a useful resource to accelerate functional analysis of the P. aeruginosa genome.     

Characterization of a gene involved in histidine biosynthesis in Halobacterium (Haloferax) volcanii: isolation and rapid mapping by transformation of an auxotroph with cosmid DNA.

Techniques for the transformation of halophilic archaebacteria have been developed recently and hold much promise for the characterization of these organisms at the molecular level. In order to understand genome organization and gene regulation in halobacteria, we have begun the characterization of genes involved in amino acid biosynthesis in Halobacterium (Haloferax) volcanii. These studies are facilitated by the many auxotrophic mutants of H. volcanii that have been isolated. In this project we demonstrate that cosmid DNA prepared from Escherichia coli can be used to transform an H. volcanii histidine auxotroph to prototrophy. A set of cosmid clones covering most of the genome of H. volcanii was used to isolate the gene which is defective in H. volcanii WR256. Subcloning identified a 1.6-kilobase region responsible for transformation. DNA sequence analysis of this region revealed an open reading frame encoding a putative protein 361 amino acids in length. A search of the DNA and protein data bases revealed that this open reading frame encodes histidinol-phosphate aminotransferase (EC 2.6.1.9), the sequence of which is also known for E. coli, Bacillus subtilis, and Saccharomyces cerevisiae.     

Nested chromosomal fragmentation in yeast using the meganuclease I-Sce I: a new method for physical mapping of eukaryotic genomes.

We have developed a new method for the physical mapping of genomes and the rapid sorting of genomic libraries which is based on chromosome fragmentation by the meganuclease I-Sce I, the first available member of a new class of endonucleases with very long recognition sequences. I-Sce I allows complete cleavage at a single artificially inserted site in an entire genome. Sites can be inserted by homologous recombination using specific cassettes containing selectable markers or, at random, using transposons. This method has been applied to the physical mapping of chromosome XI (620 kb) of Saccharomyces cerevisi and to the sorting of a cosmid library. Our strategy has potential applications to various genome mapping projects. A set of transgenic yeast strains carrying the I-Sce I sites at various locations along a chromosome defines physical intervals against which new genes, DNA fragments or clones can be mapped directly by simple hybridizations.     

Restriction maps for the cottontail rabbit herpesvirus (CTHV) genome

The sites for restriction endonucleases ApaI, BamHI and PvuII in the genome of the cottontail rabbit herpesvirus were localized. The physical mapping of the 150-kb DNA was facilitated by peculiarities of the genome structure, namely the presence of repetitive DNA and of invertible segments, and by the analysis of overlapping cosmid clones.     

The mapping of chromosomes in Saccharomyces cerevisiae. I. A cosmid vector designed to establish, by cloning into cdc-mutants, numerous start loci for chromosome walking in the yeast genome

A series of vectors for cosmid cloning in yeast has been derived from cosmid pHC79. Vectors pMT4 through pMT6 contain two tandemly arranged cohesive end sites (cos) from the genome of bacteriophage lambda. Their design allows the rapid and simple preparation of cosmid arms by linearizing a vector at the unique PvuII-restriction site located between the two cos-sequences and then cutting the linearized molecule at one of its unique cloning sites for BamHI, ClaI, PvuI, SalI or ScaI. Cosmids generated with arms from the most advanced vector, pMT6, carry the origin of replication (ori) and the ApR gene from pBR322 and the TRP1/ARS1 and URA1 genes from Saccharomyces cerevisiae. A yeast genomic DNA library was established by packaging in vitro, into bacteriophage lambda preheads, of partially restricted yeast DNA fragments ligated to cosmid arms of vector pMT6. About 80% of the clones thus obtained comprise inserts of contiguous genomic DNA over 30 kb in length. Unique DNA probes for the yeast genes CDC10, CDC39, HIS4, LEU2, and PGK1 have successfully been applied when testing for completeness of this library by isolating a series of overlapping cosmid clones that carry the respective genes. The library will thus be useful for the selection of cosmid clones which carry CDC genes from yeast by complementing first, with the vectorial yeast gene URA1, the pyrimidine auxotrophy of most cdc-strains and then, with the respective CDC wild-type genes, of the temperature-sensitive mutant alleles. Most CDC clones thus obtained will provide unique DNA probes which serve as randomly distributed start sequences within the yeast genome for overlap hybridization screening in chromosome mapping studies.     

Genomic stability in the archaeae Haloferax volcanii and Haloferax mediterranei.

Through hybridization of available probes, we have added nine genes to the macrorestriction map of the Haloferax mediterranei chromosome and five genes to the contig map of Haloferax volcanii. Additionally, we hybridized 17 of the mapped cosmid clones from H. volcanii to the H. mediterranei genome. The resulting 35-point chromosomal comparison revealed only two inversions and a few translocations. Forces known to promote rearrangement, common in the haloarchaea, have been ineffective in changing global gene order throughout the nearly 10(7) years of these species' divergent evolution.     

Molecular cloning of lin-29, a heterochronic gene required for the differentiation of hypodermal cells and the cessation of molting in C.elegans.

The lin-29 gene product of C.elegans activates a temporal developmental switch for hypodermal cells. Loss-of-function lin-29 mutations result in worms that fail to execute a stage-specific pattern of hypodermal differentiation that includes exist from the cell cycle, repression of larval cuticle genes, activation of adult cuticle genes, and the cessation of molting. Combined genetic and physical mapping of restriction fragment length polymorphisms (RFLPs) was used to identify the lin-29 locus. A probe from the insertion site of a Tc1 (maP1), closely linked and to the left of lin-29 on the genetic map, was used to identify a large set of overlapping cosmid, lambda and yeast artificial chromosome (YAC) clones assembled as part of the C.elegans physical mapping project. Radiolabeled DNA from one YAC clone identified two distinct allele-specific alterations that cosegregated with the lin-29 mutant phenotype in lin-29 intragenic recombinants. lin-29 sequences were severely under-represented in all cosmid and lambda libraries tested, but were readily cloned in a YAC vector, suggesting that the lin-29 region contains sequences incompatible with standard prokaryotic cloning techniques.     

Integrated maps of the Drosophila genome: progress and prospects

A physical map of the Drosophila melanogaster genome is being assembled, consisting of ordered overlapping cosmid clones. The map is constructed in steps, separately for each chromosomal division. Gaps in this map are to be bridged with yeast artificial chromosome clones. Hybridization to previously cloned genes and extensive use of in situ hybridization to polytene chromosomes ensure that the cosmid map is firmly anchored to the wealth of available genetic and cytogenetic information. The intention is to make the physical map widely available as part of an overall, integrated genetic resource for the Drosophila research community.     

Completion of the detailed restriction map of the E. coli genome by the isolation of overlapping cosmid clones.

Ordered sets of cosmids derived from E. coli K-12 803 overlap the 6 remaining gaps left in the physical map of strain W3110. We present detailed restriction maps of the gaps and surrounding regions, thus providing a comparison of about 30% of the genome of the two E. coli strains. Our analysis shows that there is a high degree of homology between the strains, with only occasional restriction fragment differences. However, the large inversion occurring between rrnD (72.1') and rrnE (90.4') in strain W3110 is absent in strain 803. Instead, a new inversion and adjacent deletion near argF is present in strain 803. The distribution of cosmid clones at, and adjacent to, the gaps shows that all gaps except one were difficult to clone in both lambda and cosmid clones. A low copy number cosmid vector, pOU61cos, developed previously, was essential for cloning 3 of the 8 gaps.     

A combined physical and genetic map of Pseudomonas aeruginosa PAO

A combined physical and genetic map of Pseudomonas aeruginosa PAO was constructed by pulsed-field gel electrophoresis and Southern hybridization using cosmid clones from a genomic library carrying known genes. A total of 37 SpeI restriction fragments have been mapped on the 5862 kb genome, and fragment contiguity demonstrated by hybridization with clones from a SpeI junction fragment library and fragments obtained by partial SpeI digestion, both derived from the P. aeruginosa PAO chromosome.     

High-Resolution Physical Map of the Immunoglobulin λ Variant Gene Cluster Assembled by Quantitative DNA Fiber Mapping

Quantitative DNA fiber mapping (QDFM) allows rapid construction of near-kilobase-resolution physical maps by hybridizing specific probes to individual stretched DNA molecules. We evaluated the utility of QDFM for the large-scale physical mapping of a rather unstable, repeat-rich 850-kb region encompassing the immunoglobulin λ variant (IGLV) gene segments. We mapped a minimal tiling path composed of 32 cosmid clones to three partially overlapping yeast artificial chromosome (YAC) clones and determined the physical size of each clone, the extent of overlap between clones, and contig orientation, as well as the sizes of gaps between adjacent contigs. Regions of germline DNA for which we had no YAC coverage were characterized by cosmid to cosmid hybridizations. Compared to other methods commonly used for physical map assembly, QDFM is a rapid, versatile technique delivering unambiguous data necessary for map closure and preparation of sequence-ready minimal tiling paths.     

Genetic and physical mapping of two centromere-proximal regions of chromosome IV in Aspergillus nidulans

Chromosome IV is the smallest chromosome of Aspergillus nidulans. The centromere-proximal portion of the chromosome was mapped physically using overlapping clones of a cosmid genomic library. Two contiguous segments of a physical map, based on restriction mapping of cosmid clones, were generated, together covering more than 0.4 Mb DNA. A reverse genetic mapping approach was used to establish a correlation between physical and genetic maps; i.e., marker genes were integrated into physically mapped segments and subsequently mapped by mitotic and meiotic recombination. The resulting data, together with additional classical genetic mapping, lead to a substantial revision of the genetic map of the chromosome, including the position of the centromere. Comparison of physical and genetic maps indicates that meiotic recombination is low in subcentromeric DNA, its frequency being reduced from 1 crossover per 0.8 Mb to approximately 1 crossover per 5 Mb per meiosis. The portion of the chromosome containing the functional centromere was not mapped because repeat-rich regions hindered further chromosome walking. The size of the missing segment was estimated to be between 70 and 400 kb.     

Ordered Cloned DNA Map of the Genome of Vibrio cholerae 569B and Localization of Genetic Markers

By using a low-resolution macrorestriction map as the foundation (R. Majumder et al., J. Bacteriol. 176:1105–1112, 1996), an ordered cloned DNA map of the 3.2-Mb chromosome of the hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. A cosmid library the size of about 4,000 clones containing more than 120 Mb of V. cholerae genomic DNA (40-genome equivalent) was generated. By combining landmark analysis and chromosome walking, the cosmid clones were assembled into 13 contigs covering about 90% of the V. cholerae genome. A total of 92 cosmid clones were assigned to the genome and to regions defined by NotI, SfiI, and CeuI macrorestriction maps. Twenty-seven cloned genes, 9 rrn operons, and 10 copies of a repetitive DNA sequence (IS1004) have been positioned on the ordered cloned DNA map.