Structural organization of virulence-associated plasmids of Yersinia pestis.

The complete nucleotide sequence and gene organization of the three virulence plasmids from Yersinia pestis KIM5 were determined. Plasmid pPCP1 (9,610 bp) has a GC content of 45.3% and encodes two previously known virulence factors, an associated protein, and a single copy of IS100. Plasmid pCD1 (70,504 bp) has a GC content of 44.8%. It is known to encode a number of essential virulence determinants, regulatory functions, and a multiprotein secretory system comprising the low-calcium response stimulation that is shared with the other two Yersinia species pathogenic for humans (Y. pseudotuberculosis and Y. enterocolitica). A new pseudogene, which occurs as an intact gene in the Y. enterocolitica and Y. pseudotuberculosis-derived analogues, was found in pCD1. It corresponds to that encoding the lipoprotein YlpA. Several intact and partial insertion sequences and/or transposons were also found in pCD1, as well as six putative structural genes with high homology to proteins of unknown function in other yersiniae. The sequences of the genes involved in the replication of pCD1 are highly homologous to those of the cognate plasmids in Y. pseudotuberculosis and Y. enterocolitica, but their localization within the plasmid differs markedly from those of the latter. Plasmid pMT1 (100,984 bp) has a GC content of 50.2%. It possesses two copies of IS100, which are located 25 kb apart and in opposite orientations. Adjacent to one of these IS100 inserts is a partial copy of IS285. A single copy of an IS200-like element (recently named IS1541) was also located in pMT1. In addition to 5 previously described genes, such as murine toxin, capsule antigen, capsule anchoring protein, etc., 30 homologues to genes of several bacterial species were found in this plasmid, and another 44 open reading frames without homology to any known or hypothetical protein in the databases were predicted.

Isolation of chromosome 18-specific brain transcripts as positional candidates for bipolar disorder.

Several studies have proposed the existence of susceptibility loci for bipolar disorder on chromosome 18. To identify possible candidate genes for this disease, we isolated brain-expressed transcripts by direct cDNA selection on chromosome 18-specific biotinylated cosmid clones. Longer cognate cDNA clones of the selected cDNAs were isolated from a normalized infant brain cDNA library. Physical mapping by PCR on a panel of somatic cell hybrids was conducted by the use of primers derived from partial sequences on either the 5' or 3' ends of the clones. In our initial analysis, 48 cDNA clones were found to be chromosome 18-specific, mapping to different subchromosomal regions. Sequence redundancy among these clones yielded 30 unique transcripts, five of which were represented in previously known genes. Further sequencing of the remaining 25 unique cDNA clones confirmed the absence of significant homology to known genes, indicating that these transcripts represented novel genes. Mapping with the use of a radiation hybrid panel positioned the brain cDNAs to within = 100 to 1100 kb from reference sequence tag sites (STSs) and assembled them into six high resolution linkage groups. The majority of the transcripts were found to cluster to discrete locations on 18p and 18q, previously hypothesized as susceptibility regions for bipolar disorder, identifying them as positional candidate genes.

Construction and characterization of human chromosome 2-specific cosmid, fosmid, and PAC clone libraries.

Three human chromosome 2-specific clone libraries were constructed and characterized. Chromosome 2-specific cosmid and fosmid clone libraries were constructed using flow-sorted DNA from the monochromosomal hybrid cell line GM10826. The cosmid and fosmid libraries consist of 38,496 and 26,400 arrayed clones, respectively, with an average size of 40 kb. Colony hybridization of a representative number of clones with both human and hamster genomic DNA probes demonstrates that between 58 and 66% of the clones in the flow-sorted libraries contain human inserts. Approximately 5% of the cosmid and fosmid clones are nonrecombinants. A chromosome 2-specific PAC library was also produced from the hybrid cell line GM10826. DNA from the hybrid cell line was cloned, and the human chromosome 2-specific clones were identified by colony hybridization. Approximately 5800 chromosome 2-specific PAC clones with an average insert size of approximately 85 kb were arrayed. Based on the size of the clones, the cosmid, fosmid, and PAC libraries are approximately 3.6x, approximately 2.5x, and approximately 1.9x, respectively in chromosomal coverage. The chromosome 2 coverage of each of the three libraries was further determined by PCR screening clone pools with 82 chromosome 2-specific STSs. The average number of clones identified for each STS in the library indicates the cosmid, fosmid, and PAC libraries to be approximately 3.2x, approximately 2.1x, and approximately 1.5x, respectively, in chromosome coverage. All except one of the 82 STSs were represented in the portions of the libraries screened.

Generation and mapping of human chromosome 2 microdissection clone-derived STSs.

Twenty-nine new sequence-tagged sites (STSs) were derived from DNA sequences of clones from two human chromosome 2 microdissection libraries. The specificity of the STSs for human chromosome 2 was first demonstrated by PCR amplification of DNA from genomic human and hamster cells and a human chromosome 2-containing human x hamster hybrid cell line. The STSs were then mapped to chromosome 2 by two different approaches. In the first attempt, 12 of the STSs were shown to PCR amplify YAC clones associated with genetic markers on the chromosome. In the second approach, 27 of the STSs were localized to chromosome bands by FISH using cosmid or PAC clones encoding the STSs. The specific STSs mapped to chromosome 2 by these two approaches tie together the genetic and cytogenetic maps of the chromosome at the two termini. The distribution of these STSs further defines the region of the chromosome present in the two microdissection libraries.

An integrated metric physical map of human chromosome 19.

A metric physical map of human chromosome 19 has been generated. The foundation of the map is sets of overlapping cosmids (contigs) generated by automated fingerprinting spanning over 95% of the euchromatin, about 50 megabases (Mb). Distances between selected cosmid clones were estimated using fluorescence in situ hybridization in sperm pronuclei, providing both order and distance between contigs. An average inter-marker separation of 230 kb has been obtained across the non-centromeric portion of the chromosome. Various types of larger insert clones were used to span gaps between contigs. Currently, the map consists of 51 'islands' containing multiple clone types, whose size, order and relative distance are known. Over 450 genes, genetic markers, sequence tagged sites (STSs), anonymous cDNAs, and other markers have been localized. In addition, EcoRI restriction maps have been generated for > 41 Mb (approximately 83%) of the chromosome.

Construction and utility of a human chromosome 22-specific Fosmid library.

We have previously demonstrated the capability of the Fosmid vector based on Escherichia coli F-factor replicon to stably propagate cosmid-sized human genomic DNA fragments. Using the Fosmid vector, we have constructed and arrayed a 10 x human chromosome 22-specific library, partly by picking human positive clones from a total Fosmid library constructed using DNA from human-hamster hybrid cell line containing human chromosome 22, and partly by using flow-sorted chromosomal DNA. The clones and physical contig maps of the clones in the library will serve as a valuable resource for detailed analysis of the chromosome by providing reliable materials for high resolution mapping and sequencing. In order to efficiently build physical maps for the chromosomal regions of interest spanning several hundred kilobases to a megabase, it is necessary to rapidly identify subsets of the Fosmid clones from the library that cover such regions. In this report, we describe a method of using random amplification products derived from YAC clones to rapidly identify a subset of Fosmid clones that cover a specific genomic subregion.

A new bacteriophage P1-derived vector for the propagation of large human DNA fragments.

We have designed a P1 vector (pCYPAC-1) for the introduction of recombinant DNA into E. coli using electroporation procedures. The new cloning system, P1-derived artificial chromosomes (PACs), was used to establish an initial 15,000 clone library with an average insert size of 130-150 kilobase pairs (kb). No chimaerism has been observed in 34 clones, by fluorescence in situ hybridization. Similarly, no insert instability has been observed after extended culturing, for 20 clones. We conclude that the PAC cloning system will be useful in the mapping and detailed analysis of complex genomes.