A physical map of the genome of ethanol fermentative bacterium Zymomonas mobilis ZM4 and localization of genes on the map

A physical map of the Zymomonas mobilis ZM4 genome has been constructed from the results of reciprocal Southern hybridization with PmeI, PacI, and NotI-digested genomic DNA fragments and linking cosmid clones. Restriction enzyme-digested Z. mobilis ZM4 genome was electrophoresed with phage lambda DNA concatemers as a size standard in a Bio-Rad CHEF-DRII pulsed-field gel electrophoresis (PFGE) system. The restriction enzyme PmeI generated 15 fragments (3–625 kb), and PacI produced 19 fragments (7–525 kb). Each size of restriction fragment was calculated by comparison to the size of phage lambda DNA concatemers, and the genome size of Z. mobilis ZM4 was estimated to be 2085.5 kb. The 19 known genes and three rrn operons were localized on the map.     

A physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome

A combined physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome was constructed using pulsed-field gel electrophoresis (PFGE) and hybridizations with cloned gene probes. Total genomic DNA was digested with the meganucleasesSwaI (5′-ATTTAAAT-3′),PacI (5′-TTAATTAA-3′), andPmeI (5′-GTTTAAAC-3′) yielding 26, 27, and 23 fragments, respectively. The chromosomal restriction fragments were then separated by PFGE. By summing up the lengths of the fragments generated with each of the three enzymes, a genome size of 3082 +/- 20 kb was determined. To identify adjacentSwaI fragments, a genomic cosmid library ofC. glutamicum was screened for chromosomal inserts containingSwaI sites. Southern blots of the PFGE gels were hybridized with these linking clones to connect theSwaI fragments in their natural order. By this method, about 90% of the genome could be ordered into three contigs. Two of the remaining gaps were closed by cross-hybridization of blottedSwaI digests using as probesPacI andPmeI fragments isolated from PFGE gels. The last gap in the chromosomal map was closed by hybridization experiments using partialSwaI digestions, thereby proving the circularity of the chromosome. By hybridization of gene probes toSwaI fragments separated by PFGE about 30 genes, including rRNA operons, IS element and transposon insertions were localized on the physical map.     

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.     

Genetic heterogeneity among keto-acid-producing strains of Gluconobacter oxydans

The genomes of four keto-acid-producing Gluconobacter oxydans strains (ATCC9937, IFO3293, IFO12258 and DSM2343) were analysed by pulse-field gel electrophoresis (PFGE). PFGE of undigested DNA allowed the detection of plasmids in the following strains: ATCC9937 (3 plasmids; 8, 27, 31 kb), IFO3293 (9 kb), DSM2343 (21 kb). The three plasmids in ATCC9937 showed no homology to each other or to plasmids in the other strains. Seventeen restriction enzymes were tested for use in PFGE analysis of the G. oxydans strains and XbaI was chosen for restriction fragment analysis of the genomes. Fairly good resolution of restriction fragments at all size ranges was achieved by using three different pulse–time programs. The genome sizes of the four strains were estimated to be between 2240 kb and 3787 kb. The XbaI restriction patterns of the four strains showed no similarities to each other. Ten random cosmid clones of ATCC9937 were used as hybridization probes against the four strains, but, with the exception of one clone, hybridization signals were only observed with ATCC9937 itself. These data show that the four strains are not closely related.     

Restriction fragment fingerprint and genome sizes of Staphylococcus species using pulsed-field gel electrophoresis and infrequent cleaving enzymes.

Large restriction fragments of genomic DNA from Staphylococcus species were separated by pulsed-field gel electrophoresis (PFGE). Five different strains of S. aureus (ISP8, SAU3A, PS96, ATCC 6538, ATCC 15564) and three representative strains of S. haemolyticus SM102, S. warneri MCS4, S. cohnii LK478 from human hosts, and one strain of S. aureus (ATCC 8432) from an avian host were used in this study. Since Staphylococcus is A + T rich (approximately 67%), restriction fragments were obtained by digesting chromosomal DNA with endonucleases that recognize GC-rich sequences. Five enzymes Csp I, Sma I, Ecl XI, Ksp I, or Sac II were used for generation of few (7 to 16) distinctly separated fragments, with average sizes in the range of 200-300 kb. The size distribution of restriction fragments for each enzyme for each strain produced a strain-identifying fingerprint, and the genome size of each strain was determined from such restriction fragments separated by PFGE.     

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.     

Cosmid cloning and sample sequencing of the genome of the uncultivable mollicute,Western X-disease phytoplasma,using DNA purified by pulsed-field gel electrophoresis

Western X-disease (WX) phytoplasma is an uncultivable, intracellular pathogen of plants and insects with an AT-rich genome of 670 kb. As part of the genome sequencing project of WX phytoplasma, we have cloned approximately 50% of its genome into the pcosRW2 cosmid vector using DNA purified from pulsed-field gels. One of the cosmid clones with an insert of 24.6 kb was sequenced, which along with the cosmid end sequences, represents 60 kb of unique sequence from the WX phytoplasma genome. The putative genes identified in this sequence represent a wide variety of functions and many had not been previously identified in a phytoplasma.     

氧化葡萄糖酸杆菌SCB329基因组的大小与结构的研究

收集维生素C产生菌氧化葡萄糖酸杆菌GluconobacteroxydansSCB32 9的纯培养对数期的菌体 ,采用凝胶包埋法制备完整染色体 ,用稀有酶切位点的限制性内切酶和脉冲场电泳技术对SCB32 9的基因组进行了分析 ,SpeⅠ (5′ ACTAGT)酶切有 2 4个片段 ,其大小从 1 0kb到32 0kb,用XbaⅠ (5′ TCTAGA)酶切产生 40个片段 ,其大小从 4kb到 2 0 0kb,综合两种限制酶酶切片段长度的总和结果 ,SCB32 9基因组大小为 2 70 0kb,SCB32 9基因组由一条 2 50 0kb的染色体和一个 2 4 5kb的质粒组成。通过用脱氧核糖核酸酶Ⅰ和S1核酸酶处理其基因组后电泳证实SCB32 9的染色体和质粒的拓扑学结构均为环状     

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.     

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.     

Use of pulsed-field-gradient gel electrophoresis to construct a physical map of the Caulobacter crescentus genome

The restriction enzyme DraI cleaves the Caulobacter crescentus genome into at least 35 fragments which have been resolved in agarose gels using pulsed-field-gradient gel electrophoresis (PFGE). When digests were performed using DNA from strains containing Tn5 insertion mutations, altered band migrations were observed. Using PFGE with the appropriate pulse times, size differences as small as 2% could be resolved in large fragments. Using this approach, we have constructed a partial physical map of the genome which correlates well with the C. crescentus genetic map and have shown the size of the genome to be approx. 3800 kb. Using hybridization with cloned genes, we have determined the map locations of five previously unmapped genes. In addition, we have shown that PFGE can be used to rapidly determine the map locations of new insertion mutations or the sizes of deletion mutations.     

Chromosomal heterogeneity of various Mycoplasma hyopneumoniae field strains.

Restriction enzyme digestion and field inversion gel electrophoresis were used to analyze the chromosomes of strains of Mycoplasma hyopneumoniae and the related organism Mycoplasma flocculare. The chromosome size for the M. hyopneumoniae type strain was calculated from individual fragments to be 1,011.3 +/- 32.9 kbp. The chromosomes of M. hyopneumoniae field strains were approximately the same size. The restriction patterns obtained for the chromosomes of phenotypically similar M. hyopneumoniae strains were quite different. Therefore, the species M. hyopneumoniae seems to be very heterogeneous. A field inversion gel electrophoresis analysis of the entire chromosomes allowed us to distinguish M. hyopneumoniae strains easily and hence to characterize further the species M. hyopneumoniae. The chromosome size for M. flocculare was calculated to be 988.3 +/- 39.5 kbp. Restriction enzyme XhoI, which statistically should cut the M. hyopneumoniae chromosome frequently, did not cut the DNA of any of the M. hyopneumoniae strains but did digest M. flocculare DNA, indicating that there is a site-specific modification at CTCGAG which probably belongs to a restriction modification system in M. hyopneumoniae and is absent in M. flocculare.     

Mitochondrial genome structure of rice suspension culture from cytoplasmic male-sterile line (A-58CMS): reappraisal of the master circle

Summary The mitochondrial DNA (mtDNA) from the cultured cells of a cytoplasmic male-sterile line (A-58CMS) of rice (Oryza sativa) was cloned and its physical map was constructed. There was structural alteration on the mitochondrial genome during the cell culture. Detailed restriction analysis of cosmid clones having mtDNA fragments suggested either that the master genome has a 100-kb duplication (the genome size becomes 450 kb) or that a master circle is not present in the genome (the net structural complexity becomes 350 kb). The physical map of plant mitochondrial genomes thus far reported is illustrated in a single circle, namely a master circle. However, no circular DNA molecule corresponding to a master circle has yet been proved. In the present report, representation of plant mitochondrial genomes and a possibility for mitochondrial genome without a master circle are discussed.     

The human immunoglobulin κ locus on yeast artificial chromosomes (YACs)

The human immunoglobulin κ locus is a duplicated structure. Contigs of 600 kb with 40 Vκ genes and 440 kb with 36 Vκ genes had been established for the Cκ proximal (p) and distal (d) copies, respectively. In addition the human genome contains more than 24 dispersed Vκ genes, called orphons. In the present study, 22 κ-locus derived YACs were analyzed in detail, while 30 orphon-derived YACs were characterized only with respect to some parameters. The κ-locus derived YACs allowed three gaps to be closed which previously could not be bridged by cosmid and phage λ cloning. At the 5′ side, the p contig was extended in the YACs by 50 kb and the d contig by 16 kb. At the 3′side, the d contig was extended by 11.5 kb. Beyond the 3′ end of the d contig a new Vκ gene was found, which is located, according to pulsed field gel electrophoresis (PFGE) experiments, at a distance of at least 140 kb from the last Vκ gene of the contig. This Vκ gene, which was termed Z0, occurred on three YACs, albeit at distances smaller than 140 kb; this was probably due to deletions in the YACs caused by abundant repetitive sequences at the borders of the locus. According to its sequence and to the restriction map of its surroundings, Z0 is an orphon gene of the so-called Z family, of which several members are known to be dispersed throughout the genome. The possibility that Z0 has been the parent of the other Z orphons is discussed.     

Physical mapping of the Mycoplasma gallisepticum S6 genome with localization of selected genes.

We report the construction of a physical map of the Mycoplasma gallisepticum S6 genome by field-inversion gel electrophoresis of DNA fragments generated by digestion of genomic DNA with rare-cutting restriction endonucleases. The size of the M. gallisepticum S6 genome was calculated to be approximately 1,054 kb. The loci of several genes have been assigned to the map by Southern hybridization utilizing specific gene probes.     

Mapping of the Cryptococcus neoformans MATalpha locus: presence of mating type-specific mitogen-activated protein kinase cascade homologs

In this study we investigated the relationship between the MATalpha locus of Cryptococcus neoformans and several MATalpha-specific mitogen-activated protein (MAP) kinase signal transduction cascade genes, including STE12alpha, STE11alpha, and STE20alpha. To resolve the location of the genes, we screened a cosmid library of the MATalpha strain B-4500 (JEC21), which was chosen for the C. neoformans genome project. We isolated several overlapping cosmids spanning a region of about 71 kb covering the entire MATalpha locus. It was found that STE12alpha, STE11alpha, and STE20alpha are imbedded within the locus rather than closely linked to the locus. Furthermore, three copies of MFalpha, the mating type alpha-pheromone gene, a MATalpha-specific myosin gene, and a pheromone receptor (CPRalpha) were identified within the locus. We created a physical map, based on the restriction enzyme BamHI, and identified both borders of the MATalpha locus. The MATalpha locus of C. neoformans is approximately 50 kb in size and is one of the largest mating type loci reported among fungi with a one-locus, two-allele mating system.     

Streptomyces coelicolor A3(2) lacks a genomic island present in the chromosome of Streptomyces lividans 66

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage phiHAU3 resistance (phiHAU3r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.     

Complete genome sequence of Mycoplasma hyopneumoniae strain 168

Mycoplasma hyopneumoniae strain 168, a pathogenic strain prevalent in China, was isolated in 1974. Although this strain has been widespread for a long time, the genome sequence had not been determined. Here, we announce the complete genome sequence of M. hyopneumoniae strain 168.     

Long range genome structure around the human alpha-globin complex analysed by PFGE

A map encompassing 300 kilobases (kb) in and around the human alpha-globin gene complex shows features with important implications for understanding the structure and function of the human genome. In contrast to other segments of the mammalian genome that have been analysed by pulsed field gradient electrophoresis (PFGE), this region contains an unusually high density of sites for infrequently cutting restriction enzymes that recognise GC rich motifs including the under-represented CpG doublet. This suggests that the 26 kilobase (kb) stretch of DNA containing the alpha-globin gene family, which is known from sequence analysis to be 60% GC rich, is itself embedded within a region of high GC content. This long-range structure, identified by PFGE, corresponds to a class of GC rich isochores that are thought to represent early replicating DNA present in Giemsa negative chromosomal bands. The identification of such regions by PFGE will be of value in understanding the organisation of human chromosomes and will influence the strategies used to construct a physical map of the genome.     

The sugar beet mitochondrial genome: A complex organisation generated by homologous recombination

Summary The complete physical map of the mitochondrial genome of the Owen cytoplasm of sugar beet has been determined from overlapping cosmid clones. The genome is 386 kb in size and has a multicircular organisation generated by homologous recombination across repeated DNA elements. The location of the rRNA genes and several polypeptide genes has been determined. In addition the mitochondrial genome was found to contain a sequence of chloroplast DNA including part of the 16 S rRNA gene.