The unstable region of Streptomyces ambofaciens includes 210 kb terminal inverted repeats flanking the extremities of the linear chromosomal DNA

Physical maps of the chromosomes of three strains of Streptomyces ambofaciens were constructed by ordering Ase I fragments generated from the genomic DNA as a single linear chromosome of about 8 Mb. The physical maps of the three strains were very similar. For strain DSM40697, a Dra I map was obtained by positioning the Dra I sites relative to the Ase I map. Eighteen genetic markers as well as the deletable and amplifiable region were assigned to the Ase I and Dra I fragments in this strain. The resulting genetic map resembled that of Streptomyces coelicolor A3(2). The twoterminal Ase I fragments exhibited retarded pulsed-field gel electrophoresis mobility, demonstrating that proteins are covalently bound at this position. A restriction map of this region was made using four additional endonucleases. Repeated sequences present at both ends of the chromosome were mapped as long terminal inverted repeats stretching over 210 kb. This corresponds to the longest terminal inverted repeats so far characterized. The deletable region of S. ambofaciens was localized at the chromosomal extremities.     

Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: genome size, fragment identification, and gene localization.

Four restriction endonucleases, AseI (5'-ATTAAT), SpeI (5'-ACTAGT), DraI (5'-TTTAAA), and SnaBI (5'-TACGTA), generated DNA fragments of suitable size distributions for mapping the genome of Rhodobacter sphaeroides by transverse alternating field electrophoresis. AseI produced 17 fragments, ranging in size from 3 to 1,105 kilobases (kb), SpeI yielded 16 fragments (12 to 1,645 kb), DraI yielded at least 25 fragments (6 to 800 kb), and SnaBI generated 10 fragments (12 to 1,225 kb). A total genome size of approximately 4,400 +/- 112 kb was determined by summing the fragment lengths in each of the digests generated by using the different restriction endonucleases. The total genomic DNA consisted of chromosomal DNA (3,960 +/- 112 kb) and the five endogenous plasmids (approximately 450 kb total) whose cognate DNA fragments have been unambiguously identified. A number of genes have been physically mapped to the AseI-generated restriction endonuclease fragments of total genomic DNA by Southern hybridization analysis with either homologous or heterologous specific gene probes or, in the case of several auxotrophic and pigment-biosynthetic mutants apparently generated by Tn5, a Tn5-specific probe. Other genes have been mapped by a comparison with wild-type patterns of the electrophoretic banding patterns of the AseI-digested genomic DNA derived from mutants generated by the insertion of either kanamycin or spectinomycin-streptomycin resistance cartridges. The relative orientations, distance, and location of the pufBALMX, puhA, cycA, and pucBA operons have also been determined, as have been the relative orientations between prkB and hemT and between prkA and the fbc operon.     

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     

Pulsed-field gel electrophoresis-fingerprinting, genome size estimation and rrn loci number of Rhizobium galegae

I. HUBER AND S. SELENSKA-POBELL. 1994. The genomes of several Rhizobium galegae (of.) strains, which effectively nodulate Galega officinalis host, were analysed by pulsed-field gel electrophoresis (PFGE). Individual PFGE-fingerprints were obtained for every particular strain when the rarely cutting restriction endonucleases Spe I and Asn I were applied. In hybridization experiments, where a DNA fragment carrying the rrnB ribosomal RNA operon of Escherichia coli was used as a probe, the number of the resulting strain-specific Spe I and Asn I bands was reduced to three for all of the strains studied. This suggests that in Rh. galegae (of.) there are at least three rrn loci. On the basis of the lengths of the Spe I fragments, separated by PFGE, the genome size of five Rh. galegae (of.) strains was estimated to be 5852 ± 198 kbp.     

Establishment of a physical and genetic map for bacteriophage PRD1

DNA was isolated from the lipid-containing bacteriophage PRD1 and subjected to restriction endonuclease analysis. The total genome size is 14.7 kb. PRD1 DNA was resistant to cutting by fifteen restriction endonucleases with six base specificity. HaeII made thirty-seven cuts in the DNA, MboI made one cut, and MnlI made six cuts. DNA that was not treated with protease yielded two fewer fragments when treated with HaeII. Evidence is presented to indicate that the PRD1 DNA has protein at the ends of the DNA. The thirty-eight HaeII fragments were ordered using the ladder technique of Smith and Birnstiel (1976) on MboI and MnlI fragments of the genome. Clones of HaeII partial digests of PRD1 DNA in pBR322 were analyzed by HaeII digestion and were then assigned to specific regions of the genome by their HaeII fragment composition. A comparison of the marker rescue characteristics of the cloned DNA with the overall restriction fragment map generated a physical map of the genome. Some genes that have not been mapped because of a lack of mutants or leakiness at restrictive conditions were mapped by studying the in vitro protein synthesis of restriction endonuclease fragments.     

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.     

Genome analysis of Pseudomonas aeruginosa by field inversion gel electrophoresis

Abstract The genome of Pseudomonas aeruginosa was analysed by digestion with rare-cutting restriction endonucleases and subsequent field inversion gel electrophoresis (FIGE). P. aeruginosa strain PAO and the 17 IATS strains were investigated. Each strain displayed a unique pattern of restriction fragments. Digestion with Dra I and Ssp I yielded, respectively 7–11 and 2–5 fragments of more than 130 kb in size, indicating the non-random occurrence of AT-rich sequences in the P. aeruginosa genome. The genome size of P. aeruginosa PAO was estimated to be (2.2 ± 0.3) × 10⁶ bp. The applications of DNA fingerprinting for gene cloning, construction of a physical chromosome map, and epidemiological studies, are 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.     

Pathogenicity of diatraea saccharalis Densovirus to Host Insets and Characterization of its Viral Genome

Pathogenicity of the Diatraea saccharalis densovirus (DsDNV) was tested on its host larvae. The results showed that up to 4 days after inoculation, no larvae mortality was observed and the infected larvae started to exhibit the infection symptoms from the fourth day. After 5 days of infection, the cumulative mortality of infected larvae increased significantly and reached 60% after 12 days and 100% after 21 days of infection, whereas that of the control group was only 10% and 20%, respectively, after same periods of infection, suggesting that the high mortality of infected larvae groups was due to the high pathogenicity of DsDNV. The size of the DsDNA was determined by Electron microscopy visualization of viral DNA molecules and gel electrophoresis of both native and endonuclease digested DNA fragments. The total length of the native DsDNA was about 5.95 kb. The DsDNV DNA was digested with 16 restriction enzymes and a restriction map of those enzymes was constructed with 41 restriction sites. Comparison of the restriction map of the DsDNV genome with those of the genomes ofJunonia coenia densovirus (JcDNV) and Galleria mellonella densovirus (GmDNV) indicated that the three densovirus genomes were found to share many identical restriction sites. Thus, most of the restriction sites of the following endonucleases Bam H I, Hha I, Xba I, Cla I, Asp 700, Spe I, Nco I and Bcl I, were found to be conserved among the three densovirus genomes. Symmetrical cleavage sites mapped at the both ends of the genome suggested the presence of inverted terminal repeats (ITRs) whose size was estimated to be about 500 bp. The similar genome size, almost identical restriction sites and presence of an ITR of about 500 bp for these three densoviruses suggested that they belong to the same group of ambisense densoviruses.     

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.     

Restriction cleavage map of mitochonrial DNA from the yeast Saccharomyces cerevisiae.

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb.     

Construction of physical and genetic maps of Chlamydia trachomatis serovar L2 by pulsed-field gel electrophoresis.

We constructed the physical map of Chlamydia trachomatis serovar L2 by using three restriction endonucleases, NotI (GC[GGCCGC), SgrAI (C(A/G)[CCGG(T/G)G), and Sse8387I (CCTGCA[GG), and we analyzed the fragments by pulsed-field gel electrophoresis. A total of 25 restriction endonuclease sites and 13 genes and/or operons were located on the map. The genome size was determined to be 1,045 kb. Neither highly transcribed chlamydia genes nor developmental cycle-specific genes were clustered on the genome.     

Use of pulsed-field agarose gel electrophoresis to size genomes of Campylobacter species and to construct a SalI map of Campylobacter jejuni UA580.

To determine the physical length of the chromosome of Campylobacter jejuni, the genome was subjected to digestion by a series of restriction endonucleases to produce a small number of large restriction fragments. These fragments were then separated by pulsed-field gel electrophoresis with the contour-clamped homogeneous electric field system. The DNA of C. jejuni, with its low G+C content, was found to have no restriction sites for enzymes NotI and SfiI, which cut a high-G+C regions. Most of the restriction enzymes that were used resulted in DNA fragments that were either too numerous or too small for genome size determination, with the exception of the enzymes SalI (5' ... G decreases TCGAG ... 3'), SmaI (5' .... CCC decreases GGG .... 3'), and KpnI (5' ... GGTAC decreases C .... 3'). With SalI, six restriction fragments with average values of 48.5, 80, 110, 220, 280, and 980 kilobases (kb) were obtained when calibrated with both a lambda DNA ladder and yeast Saccharomyces cerevisiae chromosome markers. The sum of these fragments yielded an average genome size of 1.718 megabases (Mb). With SmaI, nine restriction fragments with average values ranging from 39 to 371 kb, which yielded an average genome size of 1.726 Mb were obtained. With KpnI, 11 restriction fragments with sizes ranging from 35 to 387.5 kb, which yielded an average genome size of 1.717 Mb were obtained. A SalI restriction map was derived by partial digestion of the C. jejuni DNA. The genome sizes of C. laridis, C. coli, and C. fetus were also determined with the contour-clamped homogeneous electric field system by SalI, SmaI, and KpnI digestion. Average genome sizes were found to be 1.714 Mb for C. coli, 1.267 Mb for C. fetus subsp. fetus, and 1.451 Mb for C. laridis.     

Bsu2413I and Bfi2411I,two new thermophilic type II restriction endonucleases from Bacillus subtilis and Bacillus firmus: isolation and partial purification – Thermophilic endonucleases from two Bacillus species

Two new thermophilic type II restriction endonucleases, which we designated as Bsu2413I and Bfi2411I, have been isolated from gram-positive thermophilic bacteria Bacillus subtilis strain 2413 and Bacillus firmus strain 2411 respectively and partially purified. The restriction endonucleases were extracted from cell extracts and purified using single step purification through phosphocellulose column chromatography. SDS-PAGE profile showed denatured molecular weights of 33 and 67 kDa for the Bsu2413I and 39 and 67 kDa for the Bfi2411I. The partially purified Bsu2413I enzyme restricted pBR322 DNA into two fragments of 3250 and 1100 bp whereas Bfi2411I enzyme restricted pBR322 DNA into two fragments of 3500 and 800 bp. The activity of both endonucleases was assayed at 55 degrees C and they required Mg+2 as cofactor like other type II restriction endonucleases.     

A NheI macrorestriction map of the Neisseria meningitidis B1940 genome

Abstract A macrorestriction map of the Neisseria meningitidis strain B1940 genome was constructed by two-dimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. Digestion of the genomic DNA with the restriction endonuclease NHe I revealed 15 fragments between 10 kb and 450 kb. The sum of the fragments and resolution of the linearized chromosome yielded a total genome size of about 2.3 Mbp. By overlapping methylation with the Alu I-methylase six Nhe I recognition sites could be blocked. Fragments were ordered by partial/complete 2D-PFGE of genomic DNA with and without prior Alu I methylation, respectively. All nine Alu I-methylase/ Nhe I and 14 Nhe I restriction sites could be mapped on a single circular chromosome. This map will serve as a useful tool for further genetic analysis of meningococci and exemplifies the power of non-radioactive 2D-PFGE techniques to construct large physical genome maps with a single restriction enzyme.     

A restriction endonuclease map of Streptomyces phage VWB

Summary A physical map of the actinophage VWB has been constructed using the restriction endonucleases BglII, ClaI, EcoRI, EcoRV, HindIII, KpnI and SphI. Phage VWB, genome size 47.3 kb, propagates on Streptomyces venezuelae, and it can also lysogenise this species. The three BglII-generated fragments of VWB DNA were cloned in pBR322, and subsequently mapped. In this manner the restriction map of the VWB phage genome was constructed.Abbreviations dam DNA adenine methylase activity - kb kilobase pairs - :: novel joint     

Pulsed-field electrophoresis indicates larger-than-expected sizes for mycoplasma genomes.

The sizes of large DNA fragments produced from genomes of members of the Mycoplasmataceae by digestion with restriction endonucleases having infrequent (1 to 3) cleavage sites within the genome were estimated from their mobility in contour-clamped homogeneous electric field (CHEF) agarose gel electrophoresis by comparison with yeast chromosomal DNA markers. The estimates of total genome size for 7 strains of 6 species ranged from approximately 900 kilo base pairs (kb) for Ureaplasma urealyticum 960T to 1330 kb for M. mycoides subsp. mycoides, GC-1176. The values derived from this new method are considerably higher than those of approximately 500 Mdaltons or 750 kb previously reported for genome sizes in members of the Mycoplasmataceae.     

Effect of anti-mycobacterial antibodies on activation of the alternative pathway of the human complement system

Abstract Genome analysis of Pseudomonas aeruginosa was performed by digestion with rare-cutting restriction endonucleases and subsequent one- and two-dimensional field inversion gel electrophoresis (FIGE). The frequency of chromosomal recognition sites increased in the order Spe I, Dra I, Xba I, Ssp I, Nhe I. The genome size of strain PAO and the 17 IATS strains varied from 4.4 × 10⁶ to 5.4 × 10⁶ base pairs. Double restriction digests and two-dimensional FIGE provide a genome fingerprint which is useful for the identification and typing of the respective strains.     

Physical and genetic map of Streptococcus thermophilus A054.

The three restriction endonucleases SfiI, BssHII, and SmaI were found to generate fragments with suitable size distributions for mapping the genome of Streptococcus thermophilus A054. A total of 5, 8, and 24 fragments were produced with SfiI, BssHII, and SmaI, respectively. An average genome size of 1,824 kb was determined by summing the total fragment sizes obtained by digestions with these three enzymes. Partial and multiple digestions of genomic DNA in conjunction with Southern hybridization were used to map SfiI, BssHII, and SmaI fragments. All restriction fragments were arranged in a unique circular chromosome. Southern hybridization analysis with specific probes allowed 23 genetic markers to be located on the restriction map. Among them, six rrn loci were precisely located. The area of the chromosome containing the ribosomal operons was further detailed by mapping some of the ApaI and SgrAI sites. Comparison of macrorestriction patterns from three clones derived from strain A054 revealed two variable regions in the chromosome. One was associated with the tandem rrnD and rrnE loci, and the other was mapped in the region of the lactose operon.     

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.