Physical maps of the genomes of three Bacillus cereus strains.

NotI restriction maps of the chromosomes from Bacillus cereus ATCC 10876, ATCC 11778, and the B. cereus type strain ATCC 14579 have been established and compared with the previously established map of B. cereus ATCC 10987. Between 10 and 14 NotI fragments were observed, ranging from 15 to 1,300 kb, in digests of DNA from the various strains. The sizes of the genomes varied between 5.4 and 6.3 Mb. The maps were constructed by hybridization of 42 random probes, prepared from B. cereus ATCC 10987 libraries, to fragments from partial and complete NotI digests, separated by pulsed-field gel electrophoresis. Nine probes were specific for ATCC 10987 only. Probes for five B. subtilis and five B. cereus genes were also used. The NotI restriction fragment patterns of the four strains were strikingly different.     

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.     

Scattering of the rRNA genes on the physical map of the circular chromosome of Leptospira interrogans serovar icterohaemorrhagiae.

Leptospira interrogans is a pathogenic bacterium with a low G+C content (34 to 39%). The restriction enzymes NotI, AscI, and SrfI cut the chromosome of L. interrogans serovar icterohaemorrhagiae into 13, 3, and 5 fragments separable by one- and two-dimensional pulsed-field gel electrophoresis (PFGE). The genome is composed of a circular 4.6-Mbp chromosome and a 0.35-Mbp extrachromosomal element. A physical map of the chromosome was constructed for NotI, AscI, and SrfI by using single and double digests, or partial NotI digests obtained at random or by cross-protection of NotI sites by FnuDII methylase, and linking clones. rRNA genes were found to be widely scattered on the chromosome.     

Physical map of the Listeria monocytogenes chromosome.

The circular physical map of the pathogenic bacterium Listeria monocytogenes LO28 (serovar 1/2c) was established by using pulsed-field gel electrophoresis. The L. monocytogenes chromosome contains eight NotI fragments of 1,100, 940, 400, 335, 280, 45, 30, and 20 kb in size and eight Sse8387I fragments of 860, 680, 680, 370, 335, 130, 70, and 25 kb. Therefore, the total length of the genome is 3,150 kb. To order the NotI fragments on the chromosome, we used a strategy which can be of general use. We first cloned chromosomal HindIII or EcoRI fragments in pBR322. DNA extracted from the total libraries was digested by NotI and ligated to a NotI-kanamycin resistance cassette obtained by cutting Tn5 with NotI. After transformation in Escherichia coli, kanamycin-resistant clones originating from NotI-containing EcoRI or HindIII fragments were isolated. The two EcoRI-NotI or HindIII-NotI fragments of each recombinant plasmid were isolated and used as probes on Southern blot hybridizations to identify and link the corresponding NotI fragments. Seven NotI fragments were ordered in this way. The last junction was demonstrated by partial digest analysis. All L. monocytogenes genes identified so far as well as the six rRNA operons were localized on the NotI map. Regions homologous to genes from closely related bacteria were also detected and localized. Southern blot analysis of simple Sse8387I digests or double Sse8387I-NotI digests probed with the various NotI probes allowed us to align the Sse8387I fragments and localize the single SfiI site, resulting in the establishment of the first genetic and physical map of the L. monocytogenes chromosome.     

Size and physical map of the chromosome of Haemophilus influenzae.

A variation of pulse-field electrophoresis, field-inversion gel electrophoresis, was used to determine the size and physical map of the chromosome of Haemophilus influenzae. The DNA of H. influenzae had a low G + C content (39%) and no restriction sites for the enzymes NotI or SfiI. However, a number of restriction enzymes (SmaI, ApaI, NaeI, and SacII) that recognized 6-base-pair sequences containing only G and C nucleotides were found to generate a reasonable number of DNA fragments that were separable in agarose gels by field-inversion gel electrophoresis. The sizes of the DNA fragments were calibrated with a lambda DNA ladder and lambda DNA restriction fragments. The sum of fragment sizes obtained with restriction digests yielded a value for the chromosome of 1,980 kilobase pairs. Hybridization of a labeled fragment with two or more fragments from a digest with a different restriction enzyme provided the information needed to construct a circular map of the H. influenzae chromosome.     

Physical genome map of the unicellular cyanobacterium Synechococcus sp. strain PCC 7002.

A physical restriction map of the genome of the cyanobacterium Synechococcus sp. strain PCC 7002 was assembled from AscI, NotI, SalI, and SfiI digests of intact genomic DNA separated on a contour-clamped homogeneous electric field pulsed-field gel electrophoresis system. An average genome size of 2.7 x 10(6) bp was calculated from 21 NotI, 37 SalI, or 27 SfiI fragments obtained by the digestions. The genomic map was assembled by using three different strategies: linking clone analysis, pulsed-field fragment hybridization, and individual clone hybridization to singly and doubly restriction-digested large DNA fragments. The relative positions of 21 genes or operons were determined, and these data suggest that the gene order is not highly conserved between Synechococcus sp. strain PCC 7002 and Anabaena sp. strain PCC 7120.     

Physical and genetic map of the Listeria monocytogenes EGD serotype 1/2a chromosome

Listeria monocytogenes is a facultative intracellular pathogen responsible for both invasive and non-invasive food-borne illness in animals and humans. In this study, macrorestriction analysis following pulsed-field gel electrophoresis was used to show that Listeria monocytogenes serovar 1/2a strain EGD has a single chromosome containing eight NotI fragments of 1100, 850, 365, 320, 275, 40, 30 and 20 kb in size and 11 AscI fragments of 860, 470, 410, 360, 320, 250, 110, 80, 50, 30 and 20 kb. The total genome therefore comprises 3000 +/- 50 kb. The creation of a physical and genetic map of the Listeria genome was achieved by generating NotI linking clones and their use in subsequent hybridisation analysis. Using isogenic mutants harbouring additional artificial NotI restriction sites, we were able to precisely map the positions of all currently known virulence genes on the chromosome.     

The size and a physical map of the chromosome of Haemophilus parainfluenzae

The physical map of the Haemophilus parainfluenzae chromosome is circular and approx. 2340 kb in circumference. The size of the map was determined by digesting agarose-immobilized chromosomes with the restriction enzymes, NotI (GCGGCCGC), RsrII (CGGATCCG) and ApaI (GGGCCC), and using field-inversion gel electrophoresis to resolve the resulting fragments. The enzymes digest the H. parainfluenzae genome into 7, 10, and 18 fragments, respectively. The map order of the fragments was obtained by using Southern-blot hybridization to establish overlapping regions.     

Shotgun polymerase chain reaction: construction of clone libraries specific to a NotI fragment of flow-sorted human chromosome 22.

We have developed the "shotgun polymerase chain reaction," a method for obtaining a large number of DNA markers specific to a giant DNA fragment, which facilitates analysis of a particular chromosomal region. We applied this method to a giant NotI fragment which carries the immunoglobulin lambda constant region on chromosome 22. NotI digests of chromosome 22 flow-sorted from human B-lymphoblastoid cell line GM130B were size fractionated by pulsed-field gel electrophoresis. Preliminary Southern hybridization analysis revealed that the immunoglobulin lambda constant region was conveyed on 1.4- and 1.3-Mb NotI fragments in this cell line. The agarose gel corresponding to 1.2 to 1.5 Mb in size was excised into slices and subjected to polymerase chain reaction to identify gel slices containing NotI fragments carrying Ke-Oz+, a subtype of the immunoglobulin lambda constant region. From the NotI fragment thus identified, a large number of small DNA segments were amplified through the ligation-mediated random polymerase chain reaction method. The amplified products were cloned and analyzed for chromosomal origin and localization to particular NotI fragments. Seven of eighteen clones originated from the 1.4-Mb NotI fragment of chromosome 22 in GM130B cells, which appears to be exactly the same as detected by a probe for the immunoglobulin lambda constant region.     

Development of a physical map for three Frankia strains and a partial genetic map for Frankia EuI1c

Physical maps for Frankia strains CcI3, EAN1pec and EuI1c chromosomes were constructed by the use of macrorestriction analysis and pulsed-field gel electrophoresis (PFGE). The restriction enzymes Ase I, Pme I, Swa I and Ssp I were used to cut the Frankia chromosome into a limited number of large fragments and for double digestions. The genomes sizes, as determined by the addition of the estimated fragment sizes, were 5430 ± 35 kb, 9101 ± 109 kb and 8105 ± 842 kb for strains CcI3, EAN1pec and EuI1c, respectively. A complete physical map was achieved by the analysis of PFGE for the single and double digestions and by two-dimensional PFGE to determine doublets and overlapping fragments. For strain EuI1c, a partial genetic map was also constructed by positioning the 16S rRNA, gln II, gln A and hbo O genes on the physical map. PFGE analysis of DNA with and without proteinase K treatment together with the other results suggested a circular genome.     

A physical map of the Salmonella typhimurium LT2 genome made by using XbaI analysis.

XbaI digestion and pulsed-field gel electrophoresis of the genome of Salmonella typhimurium LT2 yields 24 fragments: 23 fragments (total size, 4,807 kb) are from the chromosome, and one fragment (90 kb) is from the virulence plasmid pSLT. Some of the 23 fragments from the chromosome were located on the linkage map by the use of cloned genes as probes and by analysis of strains which gain an XbaI site from the insertion of Tn10. Twenty-one of the fragments were arranged as a circular physical map by the use of linking probes from a set of 41 lysogens in which Mud-P22 was stably inserted at different sites of the chromosome; fragment W (6.6 kb) and fragment X (6.4 kb) were not located on the physical map. XbaI digestion of strains with Tn10 insertions allowed the physical locations of specific genes along the chromosome to be determined on the basis of analysis of new-fragment sizes. There is good agreement between the order of genes on the linkage map, which is based primarily on P22 joint transduction and F-mediated conjugation, and the physical map, but there are frequently differences in the length of the interval from the two methods. These analyses allowed the measurement of the amount of DNA packaged in phage P22 heads by Mud-P22 lysogens following induction; this varies from ca. 100 kb (2 min) to 240 kb (5 min) in different parts of the chromosome.     

Human chromosome 17 NotI linking clones and their use in long-range restriction mapping of the Miller-Dieker chromosome region (MDCR) in 17p13.3

A NotI linking library constructed from flow-sorted human chromosome 17 material was screened to aid in construction of a long-range restriction map of the Miller-Dieker chromosome region (MDCR) in 17p13.3. A total of 66 clones were mapped to one of eight regions of chromosome 17 using a somatic cell hybrid panel, and 44/66 (67%) of these clones cross-hybridized to rodent DNA on Southern blots. Of these, 24 clones were tested and all mapped to mouse chromosome 11, the homolog of human chromosome 17. Four linking clones mapped to 17p13.3 and were used for pulsed-field gel electrophoresis studies along with six other anonymous probes previously mapped to this region. Clone L132 was found to be deleted in all Miller-Dieker patients tested (n = 15) and therefore lies within the critical region for this disorder. It detects two NotI fragments (180 and 320 kb), one of which (320 kb) was shared by YNZ22 and YNH37, two probes previously shown to be co-deleted in all patients with the Miller-Dieker syndrome (MDS). These results indicate that all MDS patients share a minimum deletion region of greater than 370 kb. Two other NotI clones, L53 and L125, mapped telomeric to the MDS critical region and share a 600-kb MluI fragment with each other and with YNZ22/YNH37. This provides a 930-kb MluI map that encompasses the distal boundary of the MDS critical region but does not include the proximal boundary. A total of over 2 Mbp is represented in the MluI fragments by probes in subband p13.3, a cytogenetic region estimated to be 3-4 Mbp.     

Definition of the limits of the Wilms tumor locus on human chromosome 11p13

In a previous report, we described a contiguous restriction map of chromosome band 11p13 that localized the Wilms tumor locus to a small group of NotI fragments. In an effort to identify and isolate the 11p13-associated sporadic Wilms tumor locus, we developed a panel of NotI fragment-specific DNA probes. These probes were selected from genomic libraries constructed using the Chinese hamster ovary-human somatic cell hybrid carrying only human 11p. The libraries were prepared from NotI-digested DNA after size selection by pulsed-field gel electrophoresis. The selected NotI fragments had been previously targeted on the basis of deletion mapping as having a high probability of containing the Wilms tumor locus. We used these newly identified 11p13-specific probes to improve the resolution of the restriction map spanning the Wilms tumor locus. The locus has been defined by a homozygous deletion in a sporadic Wilms tumor. Using these probes, the region of homozygous deletion in this tumor and presumably all or part of the Wilms tumor gene have been confined to two small SfiI fragments spanning less than 350 kb.     

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.     

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 the genome of Planctomyces limnophilus, a representative of the phylogenetically distinct planctomycete lineage.

A physical map of the chromosome of Planctomyces limnophilus DSM 3776T was constructed by pulsed-field gel electrophoresis techniques. A total of 32 cleavage sites for the rare-cutting restriction endonucleases PacI, PmeI, and SwaI were located on the chromosome, which was shown to be circular and approximately 5.2 Mbp in size. An extrachromosomal element was detected but was found not to be cleaved by any of the enzymes used in the analysis of the chromosome. The order of the fragments on the chromosome was determined by hybridization of excised, labelled restriction fragments to Southern blots of pulsed-field gel electrophoresis-separated restriction digests. Seven genetic markers, rrs, rrl, atpD, tuf, gyrB, rpoD, and dnaK, on the chromosome were located by hybridization. Probes for all genetic markers were obtained by PCR. For five of these markers, probes were constructed by PCR with degenerate primers targeting conserved sequences. The arrangement of the genetic markers was compared with that found in other bacteria.     

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.     

Several regions of the repeat domain of the Staphylococcus caprae autolysin,AtlC, are involved in fibronectin binding

The chromosome of Bacteroides fragilis strain YCH46 was shown to be a single circular DNA molecule of about 5.3 Mb having 16 NotI, seven AscI, and six I-CeuI sites. A physical map of the chromosome was constructed by four independent experimental approaches: linking clone analysis, cross-Southern hybridization, partial restriction digestion, and two-dimensional pulsed-field gel electrophoresis. Six rRNA operons and 10 known genes were localized on the physical map.     

Physical map of the chromosome of the apple proliferation phytoplasma

A physical map of the apple proliferation phytoplasma strain AT chromosome was constructed from genomic DNA extracted from diseased tobacco plants. The map was generated with single and double digestions of the chromosome with BssHII, SmaI, MluI, and ApaI restriction endonucleases and resolving the fragments by pulsed-field gel electrophoresis. Partial digestion and Southern blot analysis were used to assist in the arrangement of the 14 contiguous restriction fragments obtained. From the restriction fragments generated by double digestions, the size of the circular chromosome was calculated to be approximately 645 kb. Locations of the two rRNA operons, the operon including the fus and tuf genes, and three other genes were placed on the map. Genome sizes and BssHII restriction profiles of apple proliferation strain AP15 and the pear decline and European stone fruit yellows phytoplasmas were different from that of strain AT.     

Construction of the temperature-sensitive vectors pLUCH80 and pLUCH88 for delivery of Tn917::NotI/SmaI and use of these vectors to derive a circular map of Listeria monocytogenes Scott A, a serotype 4b isolate.

A physical map of Listeria monocytogenes Scott A was generated by the pulsed-field technique of contour-clamped-homogeneous-electric-field (CHEF) electrophoresis. The circular genome of this serotype 4b strain contains 12 AscI fragments (38 to 790 kb), 5 NotI fragments (55 to 1,400 kb), 3 SrfI fragments (110, 1,110, and 2,000 kb), and 2 SfiI fragments (1,320 and 1,920 kb). Summation of individually sized fragments derived by digestion of Scott A genomic DNA with each of these four enzymes provided an average estimated genome length of 3,210 +/- 60 kb. Efforts to assemble the macrorestriction map benefited greatly from the construction and use of pLUCH80 and pLUCH88, temperature-sensitive vectors for delivering transposon Tn917::NotI/SmaI to the chromosome of Scott A. As another component of this study, the positions of four known virulence genes (inlA, mpl, hly, and prf) and three L. monocytogenes-specific sequences (lisM44, lisM51, and lisM52) were localized on the physical map of Scott A by hybridization. Probes prepared from lisM44, lisM51, and the four virulence genes hybridized within a cluster on a 150-kb fragment of the Scott A genome that overlaps part of the NotI-B and AscI-D fragments. The lisM52 probe hybridized with the AscI-F2 (120-kb) fragment of Scott A, which is separated from the NotI-B-AscI-D region by about 300 kb. These results established the first physical and genetic map of a serotype 4b strain of L. monocytogenes and provided further insight on this important food-borne pathogen at the genome level.