Physical and Genetic Map of the Obligate Intracellular Bacterium Coxiella burnetii

Pulsed-field gel electrophoresis and PCR techniques have been used to construct a NotI macrorestriction map of the obligate intracellular bacterium Coxiella burnetii Nine Mile. The size of the chromosome has been determined to be 2,103 kb comprising 29 NotI restriction fragments. The average resolution is 72.5 kb, or about 3.5% of the genome. Experimental data support the presence of a linear chromosome. Published genes were localized on the physical map by Southern hybridization. One gene, recognized as transposable element, was found to be present in at least nine sites evenly distributed over the whole chromosome. There is only one copy of a 16S rRNA gene. The putative oriC has been located on a 27.5-kb NotI fragment. Gene organization upstream the oriC is almost identical to that of Pseudomonas putida and Bacillus subtilis, whereas gene organization downstream the oriC seems to be unique among bacteria. The physical map will be helpful in investigations of the great heterogeneity in restriction fragment length polymorphism patterns of different isolates and the great variation in genome size. The genetic map will help to determine whether gene order in different isolates is conserved.     

Complete physical map of the Bacillus subtilis 168 chromosome constructed by a gene-directed mutagenesis method

All the SfiI sites and most of the NotI sites were located precisely on the chromosome of Bacillus subtilis 168 by a novel method, termed gene-directed mutagenesis. The stepwise elimination of these restriction sites by this method allowed not only the physical connection of the restriction fragments but also the accurate determination of the position of the restriction sites themselves. The resulting physical map of the 4165 x 10(3) base-pair B. subtilis chromosome has been correlated with the genetic map by determination of the exact location of known genes. The complete physical map provides a rapid and accurate way for mapping of new genes as well as analysis of large DNA rearrangements on the chromosome. The novel strategy is, in principle, applicable to the analysis of the genome of other organisms.     

The chromosome map of Bacillus thuringiensis subsp. canadensis HD224 is highly similar to that of the Bacillus cereus type strain ATCC 14579

Abstract A physical map of the Bacillus thuringiensis subsp. canadensis HD224 chromosome based on Asc I, Not I, and Sfi I restriction sites has been established. The chromosome map of 4.3 Mb was similar to a revised map of the chromosome of the B. cereus type strain ATCC 14579, except that the B. thuringiensis subsp. canadensis HD224 chromosome lacked a Not I site and had two additional Asc I sites. The positions of 27 probes were identical in the common macromap. A probe for the insecticidal toxin gene, cryIA , hybridized only to the B. thuringiensis subsp. canadensis HD224 chromosome. The Bss HII ribotype patterns were almost identical confirming the similarity between the two strains.     

Detailed physical mapping of the ribosomal RNA genes of Bacillus subtilis

Characterization of patterns of ribosomal RNA (rRNA) homology with restriction digests of Bacillus subtilis 168 chromosomal DNA and with cloned DNA sequences has resulted in the construction of a physical map of the rRNA gene sets. There are two types of gene sets which differ in the size of "spacer" DNA sequences separating the 16S and 23S rRNA determinants. It was estimated that there are ten rRNA gene sets on the B. subtilis chromosome.     

Analysis of the genome of an alkaliphilic Bacillus strain from an industrial point of view

Bacillus species and other microbes with pH optima for growth higher than pH 9 are defined as alkaliphiles. A large number of alkaliphilic Bacillus strains producing useful enzymes, have been isolated from various environments. Some of these enzymes, such as proteases and cellulases from alkaliphilic Bacillus strains, have been commercialized and have brought great advantages to industry and domestic life. To support further development of the enzyme industry, we initiated analysis of the genome of Bacillus halodurans C-125, which is 4.25 Mb in size, and constructed a physical and genetic map for comparison with the Bacillus subtilis chromosome. Systematic sequencing of the whole genome of Bacillus halodurans C-125 has been automated since the beginning of May 1998, and sequencing of 98% of the whole genome has been done so far. Through genome analysis, it became apparent that the genome organization of alkaliphilic Bacillus halodurans C-125 is totally different from that of B. subtilis orthologues. Received: July 11, 1999 / Accepted: December 27, 1999     

Physical map of chromosomal and plasmid DNA comprising the genome of Leptospira interrogans.

The size and physical structure of the Leptospira interrogans genome was characterized using contour-clamped homogenous electric field (CHEF) gel electrophoresis. The L. interrogans genome is approximately 4750 kb in size and is composed of two molecular species of DNA: a 4400 kb chromosome; and a 350 kb plasmid, pLIN1. A physical map of the chromosome was constructed with the restriction enzymes NotI and SfiI. A physical map of pLIN1 was constructed with ApaI, NotI, Sse83871, SgrAI, and SmaI. Both the L. interrogans chromosome and pLIN1 are circular.     

Integration of the Aedes aegypti mosquito genetic linkage and physical maps

Two approaches were used to correlate the Aedes aegypti genetic linkage map to the physical map. STS markers were developed for previously mapped RFLP-based genetic markers so that large genomic clones from cosmid libraries could be found and placed to the metaphase chromosome physical maps using standard FISH methods. Eight cosmids were identified that contained eight RFLP marker sequences, and these cosmids were located on the metaphase chromosomes. Twenty-one cDNAs were mapped directly to metaphase chromosomes using a FISH amplification procedure. The chromosome numbering schemes of the genetic linkage and physical maps corresponded directly and the orientations of the genetic linkage maps for chromosomes 2 and 3 were inverted relative to the physical maps. While the chromosome 2 linkage map represented essentially 100% of chromosome 2, approximately 65% of the chromosome 1 linkage map mapped to only 36% of the short p-arm and 83% of the chromosome 3 physical map contained the complete genetic linkage map. Since the genetic linkage map is a RFLP cDNA-based map, these data also provide a minimal estimate for the size of the euchromatic regions. The implications of these findings on positional cloning in A. aegypti are discussed.     

Physical and genetic map of the Serpulina hyodysenteriae B78T chromosome.

A combined physical and genetic map of the Serpulina hyodysenteriae B78T genome was constructed by using pulsed-field gel electrophoresis and DNA blot hybridizations. The S. hyodysenteriae genome is a single circular chromosome about 3.2 Mb in size. The physical map of the chromosome was constructed with the restriction enzymes BssHII, EclXI, NotI, SalI, and SmaI. The physical map was used to constructed a linkage map for genes encoding rRNA, flagellum subunit proteins, DNA gyrase, NADH oxidase, and three distinct hemolysins. Several flaB2-related loci, encoding core flagellum subunit proteins, were detected and are dispersed around the chromosome. The rRNA gene organization in S. hyodysenteriae is unusual. S. hyodysenteriae has one gene each for 5S (rrf), 16S (rrs), and 23S (rrl) rRNAs. The rrf and rrl genes are closely linked (within 5 kb), while the rrs gene is about 860 kb from the other two rRNA genes. Using a probe for the S. hyodysenteriae gyrA gene, we identified a possible location for the chromosomal replication origin. The size and genetic organization of the S. hyodysenteriae chromosome are different from those of previously characterized spirochetes.     

A genetic linkage map of human chromosome 5 with 60 RFLP loci.

A genetic map of human chromosome 5 that contains 60 restriction fragment length polymorphism (RFLP) loci in one linkage group has been constructed. Segregation data using these markers and 40 large multigenerational families supplied by the Centre d'Etude du Polymorphisme Humain have been collected. Linkage analyses were performed with the program package CRI-MAP; using odds greater than 1000:1, 30 RFLP loci could be placed on the map. This genetic map spans 289 cM sex-equal, 353 cM in females, and 244 cM in males. While the relative rate of recombination for female meioses is nearly twice that of males over much of the chromosome, several instances of statistically significant excess male recombination were observed. The order of probes on the genetic map has been confirmed by their physical order as determined by somatic cell hybrid lines containing deletions of normal chromosome 5. There is concordance between the physical positions of markers and their genetic positions. Our most distal probes on the genetic map are cytologically localized to the most distal portions of the chromosome. This suggests that our genetic map spans most of chromosome 5.     

A complete physical map of a Bacillus thuringiensis chromosome.

Bacillus thuringiensis is the source of the most widely used biological pesticide, through its production of insecticidal toxins. The toxin genes are often localized on plasmids. We have constructed a physical map of a Bacillus thuringiensis chromosome by aligning 16 fragments obtained by digestion with the restriction enzyme NotI. The fragments ranged from 15 to 1,350 kb. The size of the chromosome was 5.4 Mb. The NotI DNA fingerprint patterns of 12 different B. thuringiensis strains showed marked variation. The cryIA-type toxin gene was present on the chromosome in four strains, was extrachromosomal in four strains, and was both chromosomal and extrachromosomal in two strains. A Tn4430 transposon probe hybridized to 5 of the 10 cryIA-positive chromosomal fragments, while cryIA and the transposon often hybridized to different extrachromosomal bands. Ten of the strains were hemolytic when grown on agar plates containing human erythrocytes. Nine of the strains were positive when assayed for the presence of Bacillus cereus enterotoxin. We conclude that B. thuringiensis is very closely related to B. cereus and that the distinction between B. cereus and B. thuringiensis should be reconsidered.     

Physical and transcriptional map of a 3-Mb region of mouse chromosome 1 containing the gene for the neural tube defect mutant loop-tail (Lp)

The Lp mouse mutant provides a model for the severe human neural tube defect (NTD), cranio-rachischisis. To identify the Lp gene, a positional cloning approach has been adopted. Previously, linkage analysis in a large intraspecific backcross was used to map the Lp locus to distal mouse chromosome 1. Here we report a detailed physical map of this region. The interval surrounding Lp has been cloned in a yeast artificial chromosome (YAC) contig consisting of 63 clones spanning approximately 3.2 Mb. Fifty sequence tagged sites (STSs) have been used to construct the contig and establish marker order across the interval. Based on the high level of conserved synteny between distal mouse chromosome 1 and human 1q21-q24, many of these STSs were designed from expressed sequences identified by cross-screening human and mouse databases of expressed sequence tags. Added to other known genes in the region, a total of 29 genes were located and ordered within the contig. Seven novel polymorphisms were identified within the region, allowing refinement of the genetic map and a reduction in the size of the physical interval containing the Lp gene. The Lp interval, between D1Mit113 and Tagln2, can be spanned by two nonchimeric overlapping YACs that define a physical distance of approximately 1 Mb. Within this region, 10 potential candidate genes have been mapped. The materials and genes described here will provide a resource for the identification and further study of the mutated Lp gene that causes this severe neural tube defect and will provide candidates for other defects known to map to the homologous region on human chromosome 1q.     

Somatic cell hybrid deletion map of human chromosome 18.

The creation of a physical map of chromosome 18 will be useful for the eventual identification of specific chromosomal regions that are critical in the occurrence of Edwards syndrome, the 18q- syndrome, and the 18p- syndrome. To begin the investigation of these syndromes, a physical map has been constructed to order random DNA fragments to specific portions of chromosome 18. A set of somatic cell hybrids that retain deletions or translocations involving chromosome 18 has been isolated and characterized. Over 200 lambda phage from a chromosome 18-specific library have been localized to 11 distinct regions of chromosome 18 using the chromosomal breakpoints present in the somatic cell hybrids.     

Meiotic Recombination, Noncoding DNA and Genomic Organization in Caenorhabditis Elegans

The genetic map of each Caenorhabditis elegans chromosome has a central gene cluster (less pronounced on the X chromosome) that contains most of the mutationally defined genes. Many linkage group termini also have clusters, though involving fewer loci. We examine the factors shaping the genetic map by analyzing the rate of recombination and gene density across the genome using the positions of cloned genes and random cDNA clones from the physical map. Each chromosome has a central gene-dense region (more diffuse on the X) with discrete boundaries, flanked by gene-poor regions. Only autosomes have reduced rates of recombination in these gene-dense regions. Cluster boundaries appear discrete also by recombination rate, and the boundaries defined by recombination rate and gene density mostly, but not always, coincide. Terminal clusters have greater gene densities than the adjoining arm but similar recombination rates. Thus, unlike in other species, most exchange in C. elegans occurs in gene-poor regions. The recombination rate across each cluster is constant and similar; and cluster size and gene number per chromosome are independent of the physical size of chromosomes. We propose a model of how this genome organization arose.     

Physical map of the genome of the green phototrophic bacterium Chlorobium tepidum.

A physical restriction map of the chromosome of the green sulfur bacterium Chlorobium tepidum was generated by determining the order of the fragments obtained after digestion with the restriction endonucleases XbaI and PacI and subsequent separation of the fragments by pulsed-field gel electrophoresis. The size of the chromosome is estimated to be 2.1 Mb. Fifteen genes and operons, mainly encoding proteins involved in photosynthesis, have been placed on this map by hybridization to fragments obtained after single- and double-restriction digestions.     

The gene specifying RNase E (rne) and a gene affecting mRNA stability (ams) are the same gene

A DNA clone complementing the rne-3071 mutation has been expressed and localized in the physical map of Escherichia coli. The DNA fragment from this clone was localized to the region of the E. coli chromosome where the rne-3071 mutation has been mapped. The position of this DNA fragment in the E. coli chromosome, the size of the product directed by this DNA fragment (110,000 Da), the restriction map of this fragment, the fact that the same clone complements the ams mutation, and the observation that the rne-3071 and the ams mutations cause similar patterns of RNA synthesis, show that the rne gene--a gene specifying the processing endonuclease RNase E--and the ams gene--a gene that affects mRNA stability--are identical.     

Towards a Drosophila genome map.

A physical map of the genome of Drosophila melanogaster has been created using 965 yeast artificial chromosome (YAC) clones assigned to locations in the cytogenetic map by in situ hybridization with the polytene salivary gland chromosomes. Clones with insert sizes averaging about 200 kb, totaling 1.7 genome equivalents, have been mapped. More than 80% of the euchromatic genome is included in the mapped clones, and 75% of the euchromatic genome is included in 161 cytological contigs ranging in size up to 2.5 Mb (average size 510 kb). On the other hand, YAC coverage of the one-third of the genome constituting the heterochromatin is incomplete, and clones containing long tracts of highly repetitive simple satellite DNA sequences have not been recovered.     

A Restriction Fragment Length Polymorphism Map and Electrophoretic Karyotype of the Fungal Maize Pathogen Cochliobolus Heterostrophus

A restriction fragment length polymorphism (RFLP) map has been constructed of the nuclear genome of the plant pathogenic ascomycete Cochliobolus heterostrophus. The segregation of 128 RFLP and 4 phenotypic markers was analyzed among 91 random progeny of a single cross; linkages were detected among 126 of the markers. The intact chromosomal DNAs of the parents and certain progeny were separated using pulsed field gel electrophoresis and hybridized with probes used to detect the RFLPs. In this way, 125 markers were assigned to specific chromosomes and linkages among 120 of the markers were confirmed. These linkages totalled 941 centimorgans (cM). Several RFLPs and a reciprocal translocation were identified tightly linked to Tox1, a locus controlling host-specific virulence. Other differences in chromosome arrangement between the parents were also detected. Fourteen gaps of at least 40 cM were identified between linkage groups on the same chromosomes; the total map length was therefore estimated to be, at a minimum, 1501 cM. Fifteen A chromosomes ranging from about 1.3 megabases (Mb) to about 3.7 Mb were identified; one of the strains also has an apparent B chromosome. This chromosome appears to be completely dispensable; in some progeny, all of 15 markers that mapped to this chromosome were absent. The total genome size was estimated to be roughly 35 Mb. Based on these estimates of map length and physical genome size, the average kb/cM ratio in this cross was calculated to be approximately 23. This low ratio of physical length to map distance should make this RFLP map a useful tool for cloning genes.     

Physical map of the chromosome of Neisseria gonorrhoeae FA1090 with locations of genetic markers, including opa and pil genes.

A physical map of the chromosome of Neisseria gonorrhoeae FA1090 has been constructed. Digestion of strain FA1090 DNA with NheI, SpeI, BglII, or PacI resulted in a limited number of fragments that were resolved by contour-clamped homogeneous electric field electrophoresis. The estimated genome size was 2,219 kb. To construct the map, probes corresponding to single-copy chromosomal sequences were used in Southern blots of digested DNA separated on pulsed-field gels, to determine how the fragments from different digests overlapped. Some of the probes represented identified gonococcal genes, whereas others were anonymous cloned fragments of strain FA1090 DNA. By using this approach, a macrorestriction map of the strain FA1090 chromosome was assembled, and the locations of various genetic markers on the map were determined. Once the map was completed, the repeated gene families encoding Opa and pilin proteins were mapped. The 11 opa loci of strain FA1090 were distributed over approximately 60% of the chromosome. The pil loci were more clustered and were located in two regions separated by approximately one-fourth of the chromosome.