Physical and genetic chromosomal map of an M type 1 strain of Streptococcus pyogenes.

A physical map of the chromosome of an M type 1 strain of Streptococcus pyogenes was constructed following digestion with three different restriction enzymes, SmaI, SfiI, and SgrAI, and separation and analysis of fragments by pulsed-field gel electrophoresis. The genome size of this strain was estimated to be 1,920 kb. By employing Southern hybridization and PCR analysis, 36 genes were located on the map.     

Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome.

A physical map of the Methanobacterium thermoautotrophicum Marburg chromosome was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by NotI, PmeI, and NheI. The order of the fragments was deduced from Southern blot hybridization of NotI fragment probes to various restriction digests and from partial digests. The derived map is circular, and the genome size was estimated to be 1,623 kb. Several cloned genes were hybridized to restriction fragments to locate their positions on the map. Genes coding for proteins involved in the methanogenic pathway were located on the same segment of the circular chromosome. In addition, the genomes of a variety of thermophilic Methanobacterium strains were treated with restriction enzymes and analyzed by pulsed-field gel electrophoresis. The sums of the fragment sizes varied from 1,600 to 1,728 kb among the strains, and widely different macrorestriction patterns were observed.     

Genomic restriction map of the extremely thermophilic bacterium Thermus thermophilus HB8.

A physical map of the chromosome of the extremely thermophilic eubacterium Thermus thermophilus HB8 has been constructed by using pulsed-field gel electrophoresis techniques. A total of 26 cleavage sites for the rarely cutting restriction endonucleases HpaI, MunI, and NdeI were located on the genome. On the basis of the sizes of the restriction fragments generated, the genome size was estimated to be 1.74 Mbp, which is significantly smaller than the chromosomes of Escherichia coli and other mesophiles. Partial digestion experiments revealed the order of the six HpaI bands on the chromosome. Hybridization of isolated restriction fragments to pulsed-field gel-separated restriction digestions confirmed the deduced order of the HpaI fragments and allowed ordering and alignment of the NdeI and MunI fragments. In addition, 16 genes or gene clusters cloned from several different Thermus strains were located on the T. thermophilus HB8 chromosomal map by hybridization of gene probes to pulsed-field gel-resolved restriction digestions.     

Physical and genetic map of the Methanobacterium wolfei genome and its comparison with the updated genomic map of Methanobacterium thermoautotrophicum Marburg

A physical and genetic map of the chromosome of Methanobacterium wolfei was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by digestion with NotI and NheI. The chromosome was found to be circular and 1,729 kb in size. Twenty-eight genes were mapped to specific restriction enzyme fragments by performing hybridization experiments with gene probes from various Methanobacterium strains. The genomic map obtained was compared with the updated genomic map of Methanobacterium thermoautotrophicum Marburg. In spite of major restriction pattern dissimilarities, the overall genetic organization seemed to be conserved between the genomes of the two strains. In addition, the two rRNA operons of strain Marburg were precisely mapped on the chromosome, and it was shown that they are transcribed in the same direction.     

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.     

Physical map of Campylobacter jejuni TGH9011 and localization of 10 genetic markers by use of pulsed-field gel electrophoresis.

The physical map of Campylobacter jejuni TGH9011 (ATCC 43430) was constructed by mapping the three restriction enzyme sites SacII (CCGCGG), SalI (GTCGAC), and SmaI (CCCGGG) on the genome of C. jejuni by using pulsed-field gel electrophoresis and Southern hybridization. A total of 25 restriction enzyme sites were mapped onto the C. jejuni chromosome. The size of the genome was reevaluated and was shown to be 1,812.5 kb. Ten C. jejuni genetic markers that have been isolated in our laboratory were mapped to specific restriction enzyme fragments. Furthermore, we have accurately mapped one of the three rRNA operons (rrnA) and have demonstrated a separation of the 16S and 23S rRNA-encoding sequences in one of the rRNA operons.     

Physical and genetic maps of the genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

A restriction map of the chromosome of the cyanobacterium Anabaena sp. strain PCC 7120 was generated by the determination of the order of restriction fragments of the infrequently cleaving restriction endonucleases AvrII, SalI, and PstI. These restriction fragments were resolved by the pulsed homogeneous orthogonal field gel electrophoresis system of pulsed-field gel electrophoresis (I. Bancroft and C. P. Wolk, Nucleic Acids Res. 16:7405-7418, 1988). Other infrequently cutting restriction endonucleases (AhaII, Asp718, AsuII, BanII, BglII, BssHII, FspI, NcoI, NruI, SphI, SplI, SstII, and StuI) were identified that could prove useful for higher-resolution mapping. The chromosome was found to be 6.4 megabases in size and circular. Three apparently circular large plasmids (410, 190, and 110 kilobases) were also identified. A genetic map was constructed by hybridization with gene-specific probes. Genes encoding components of the photosynthetic electron transport chain were not within a single tight cluster.     

Construction of a chromosome map for the phage group II Staphylococcus aureus Ps55.

The genome size and a partial physical and genetic map have been defined for the phage group II Staphylococcus aureus Ps55. The genome size was estimated to be 2,771 kb by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes SmaI, CspI, and SgrAI. The Ps55 chromosome map was constructed by transduction of auxotrophic and cryptic transposon insertions, with known genetic and physical locations in S. aureus NCTC 8325, into the Ps55 background. PFGE and DNA hybridization analysis were used to detect the location of the transposon in Ps55. Ps55 restriction fragments were then ordered on the basis of genetic conservation between the two strains. Cloned DNA probes containing the lactose operon (lac) and genes encoding staphylococcal protein A (spa), gamma hemolysin (hlg), and coagulase (coa) were also located on the map by PFGE and hybridization analysis. This methodology enabled a direct comparison of chromosomal organization between NCTC 8325 and Ps55 strains. The chromosome size, gene order, and some of the restriction sites are conserved between the two phage group strains.     

Physical and genetic map of the Spiroplasma kunkelii CR2-3x chromosome

Spiroplasma kunkelii (class Mollicutes) is the characteristically helical, wall-less bacterium that causes corn stunt disease. A combination of restriction enzyme analysis, pulsed-field gel electrophoresis (PFGE), and Southern hybridization analysis was used to construct a physical and genetic map of the S. kunkelii CR2-3x chromosome. The order of restriction fragments on the map was determined by analyses of reciprocal endonuclease double digests employing I-CeuI, AscI, ApaI, EagI, SmaI, BssHII, BglI, and SalI; adjacent fragments were identified on two-dimensional pulsed-field electrophoresis gels. The size of the chromosome was estimated at 1550 kb. Oligonucleotide pairs were designed to prime the amplification of 26 S. kunkelii gene sequences in the polymerase chain reaction (PCR). Using PCR amplicons as probes, the locations of 27 S. kunkelii putative single-copy genes were positioned on the map by Southern hybridization analyses of chromosomal fragments separated in PFGE. The nucleotide sequence of the single ribosomal RNA operon was determined and its location mapped to a chromosomal segment bearing recognition sites for SalI, SmaI, EagI, and I-CeuI.     

Genome map of Campylobacter fetus subsp. fetus ATCC 27374

Abstract A physical map of the chromosome of Campylobacter fetus subsp. fetus was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by Sal I, Sma I and Not I. Digestion of the type strain ATCC 27374 with these restriction endonucleases resulted in generating 4–14 fragments. The order of the fragments was deduced from hybridization of these restriction fragments to Southern blots of pulsed-field gel electrophoresis gels generated by the other two enzymes. The estimated genome size was 1160 kb. The position of several homologous and heterologous genes was determined on the circular map. These included the 2.8-kb sapA gene, encoding the 97-kDa surface array protein. Three copies of ribosomal RNA genes for which the 16S, 23S and 5S rRNA appeared to be located in close proximity in each of the three regions. The RNA polymerase genes rpoA , rpoB , and rpoD were mapped and appeared to be situated close together in one region. The flagellin genes ( flaAB ) of C. jejuni and the gyrase genes gyrA and gyrB of C. perfringens and Bacillus subtilis , respectively, were used to identify the locations of flaAB , the gyrA and the gyrB genes on the ATCC 27374 chromosome.     

A physical and genetic map of the Stigmatella aurantiaca DW4/3.1 chromosome

A physical map of the myxobacterium Stigmatella aurantiaca DW4/3.1 chromosome was constructed by pulsed-field gel (PFG) long-range mapping. One-and two-dimensional pulsed-field gel analyses were used together with reciprocal double-restriction, cross-hybridization and hybridization fingerprint analysis. These PFG results were confirmed by Smith-Birnstiel analysis, by Southern hybridization using linking clones and clones of a λ genomic library for the determination of adjacent restriction fragments and by transposon insertion mapping using defined genomic sequences for hybridization. It was thus possible to construct a circular restriction map of the single 9.35 Mbp chromosome of S. aurantiaca based on the endonucleases Asel and Spel. Genetic loci as well as the replication origin were located on the physical map by Southern hybridization using heterologous (derived from Myxococcus xanthus, Escherichia coli and Streptomyces lividans) and homologous probes that are mainly involved in development and ceil motility.     

A physical and genetic map of Neisseria meningitidis B1940

A physical map of the chromosome of Neisseria meningitidis B1940 has been constructed by one- and two-dimensional pulsed-field gel electrophoresis techniques. Complete macrorestriction maps for the enzymes Nhe I (16 sites), Sgf I (13 sites), Sfi I (11 sites) and I-Ceu I (4 sites), as well as a partial restriction map for the restriction enzyme Spe I (15 of c. 28 sites) could be established. Altogether, 59 restriction sites were mapped on a single circular chromosome of 2.3 Mbp. By restriction endonuclease digestion and Southern hybridization of cloned genetic markers, 39 genetic loci were assigned to this map. Comparison with the metabolic maps of Neisseria gonorrhoeae MS11-N198 and FA1090 revealed a high degree of conservation in the arrangement of gene loci among these two species, although four out of 24 genetic loci are located at different chromosomal positions, indicating several genomic rearrangements.     

Physical map of the Streptomyces lividans 66 genome and comparison with that of the related strain Streptomyces coelicolor A3(2).

A physical map of the chromosome of Streptomyces lividans 66 ZX7 was constructed by ordering the macrorestriction fragments generated from the genomic DNA with the restriction enzymes AseI and DraI. AseI and DraI linking cosmids (i.e., recombinant cosmids including either AseI or DraI sites) were isolated from a gene bank and used as hybridization probes against Southern transfers of pulsed-field gel electrophoresis (PFGE) restriction patterns. The DraI sites were precisely mapped by PFGE analyses of AseI-DraI double digests and hybridization with the AseI junctions. The 16 AseI and 7 DraI fragments were aligned as a single chromosome of about 8,000 kb. The data supported the interpretation that the chromosome is a linear structure. The related strain Streptomyces coelicolor A3(2) M145, recently mapped by H. Kieser, T. Kieser, and D. A. Hopwood (J. Bacteriol. 174:5496-5507, 1992), was compared with S. lividans at the level of the genomic structure by hybridizing the linking cosmids to Southern transfers of PFGE patterns. In spite of little apparent similarity in their restriction patterns, the comparison of the physical maps revealed a common structure with an identical ordering of the cosmid sequences. This conservation of the map order was further confirmed by assigning genetic markers (i.e., cloned genes and DNA elements relevant to the unstable region) to the AseI fragments.     

Development of a physical and genetic map of the virulent Wolbachia strain wMelPop

We report here the construction of a physical and genetic map of the virulent Wolbachia strain, wMelPop. This map was determined by ordering 28 chromosome fragments that resulted from digestion with the restriction endonucleases FseI, ApaI, SmaI, and AscI and were resolved by pulsed-field gel electrophoresis. Southern hybridization was done with 53 Wolbachia-specific genes as probes in order to determine the relative positions of these restriction fragments and use them to serve as markers. Comparison of the resulting map with the whole genome sequence of the closely related benign Wolbachia strain, wMel, shows that the two genomes are largely conserved in gene organization with the exception of a single inversion in the chromosome.     

Physical and genetic map of the chromosome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803.

A combined physical and genetic map of the cyanobacterium Synechocystis sp. strain PCC 6803 chromosome was constructed. An estimated genome size of 3.82 Mb was obtained by summing the sizes of 25 MluI or 40 NotI fragments seen by pulsed-field electrophoresis. The order of the restriction fragments was determined by using two independent experimental approaches: pulsed-field fragment hybridization and linking clone analysis. The relative positions of 30 known genes or gene clusters were localized.     

Physical map of the Bartonella bacilliformis genome.

The genome of Bartonella bacilliformis was shown to be a single circular DNA molecule of about 1,600 kbp having six NotI, four SfiI, and two CeuI sites. A physical map of the DNA was constructed by contour-clamped homogeneous electric field pulsed-field gel electrophoresis of DNA restriction fragments. rRNA operons, the invasion-associated locus, and a flagellin gene were located on the map by hybridization.     

A global response to sulfur starvation in Pseudomonas putida and its relationship to the expression of low-sulfur-content proteins

Staphylococcus xylosus is a ubiquitous bacterium frequently isolated from mammalian skin and occurring naturally on meat and dairy products. A physical and genetic map of the S. xylosus C2a chromosome was constructed by pulsed-field gel electrophoresis analysis after digestion with AscI, ApaI, I-CeuI, SfiI and SmaI enzymes and hybridization analysis. The chromosome size was estimated to be 2868+/-10 kb. Thirty-three genetic markers were mapped. The probable origin of replication (oriC) was positioned. Six rrn loci were located, and their orientation was determined. The chromosomes of six additional S. xylosus strains were also analysed by I-CeuI digestion, and an intraspecies diversity of the chromosome size and the number of rrn operons was shown.     

Saturating the region of the polycystic kidney disease gene with NotI linking clones.

A NotI-linking library was constructed from a radiation hybrid containing fragments of human chromosome 16. The clones were mapped on a panel of somatic cell hybrids, and 10 different NotI site-containing clones were localized close to and between genetic markers flanking the PKD1 locus. With pulsed-field gel analysis the clones were shown to be distributed over four adjacent ClaI fragments covering 1,200 kb.     

Chromosome map of the thermophilic archaebacterium Thermococcus celer.

A physical map for the chromosome of the thermophilic archaebacterium Thermococcus celer Vu13 has been constructed. Thirty-four restriction endonucleases were tested for their ability to generate large restriction fragments from the chromosome of T. celer. Of these, the enzymes NheI, SpeI, and XbaI yielded the fewest fragments when analyzed by pulsed-field electrophoresis. NheI and SpeI each gave 5 fragments, while XbaI gave 12. The size of the T. celer chromosome was determined from the sum of the apparent sizes of restriction fragments derived from single and double digests by using these enzymes and was found to be 1,890 +/- 27 kilobase pairs. Partial and complete digests allowed the order of all but three small (less than 15 kilobase pairs) fragments to be deduced. These three fragments were assigned positions by using hybridization probes derived from these restriction fragments. The positions of the other fragments were confirmed by using hybridization probes derived in the same manner. The positions of the 5S, 16S, and 23S rRNA genes as well as the 7S RNA gene were located on this map by using cloned portions of these genes as hybridization probes. The 5S rRNA gene was localized 48 to 196 kilobases from the 5' end of the 16S gene. The 7S RNA gene was localized 190 to 504 kilobases from the 3' end of the 23S gene. These analyses demonstrated that the chromosome of T. celer is a single, circular DNA molecule. This is the first such demonstration of the structure of an archaebacterial chromosome.