Diversity of DNA methylation pattern and total DNA restriction pattern in symbiotic Nostoc

Attempts were made to use total DNA restriction patterns and the response of purified DNA to treatment with restriction endonucleases to characterize several symbiotic Nostoc strains which had been isolated from different host plants cultivated in Italy. Among 27 restriction endonucleases tested, several did not cut any DNA and no significant variation in the susceptibility of the genomes to DNA restriction was seen among the strains. Therefore the Nostoc strains could not be separated into groups based on their different susceptibilities to the action of restriction endonucleases. However, in studies of total DNA restriction patterns, the restriction endonucleases BfrI and HpaI gave unique band patterns for each cyanobacterial isolate. Different profiles were even found in strains isolated from host plants belonging to the same species. The results do not support any definition of symbiotic Nostoc genomic groups or species and show that a tight specificity between the host plant and the cyanobacterium might not exist in the symbiotic associations involving Nostoc.     

RESTRICTION ENZYME ANALYSIS AND CLONING OF HIGH MOLECULAR WEIGHT GENOMIC DNA ISOLATED FROM CHLORELLA SOROKINIANA (CHLOROPHYTA)1

High molecular weight (50–70 kb) genomic DNA was isolated from the eukaryotic green alga, Chlorella sorokiniana spec. nov, (formerly Chlorella pyrenoidosa Chick, strain 7-11-05), for restriction endonuclease digestion studies and for preparation of a genomic DNA library. Twenty restriction endonucleases were examined for their abilities to digest this DNA. Nine of the endonucleases gave nearly complete digestion of the DNA, whereas 11 gave only partial digestion. Additional purification steps to remove possible contamination by proteins, RNAs, or polysaccharides did not improve digestion. Digestion studies with pairs of endonuclease isoschizomers, of which one member was sensitive to base methylation, suggested that 5-methylcytosine might be responsible Jor inhibition of certain endonucleases. Analysis of the DNA showed it to contain 63% GC and to have a high content (5.1 mol %) of 5-methylcytostne but no other methylated or unusual bases. Evidence indicates that this high 5-methylcytosine content, which is a characteristic of higher plant genomic DNA rather than of eukaryotic microorganisms, interfered with the cloning of restriction fragments (or fragments produced by mechanical shearing) of C. sorokiniana genomic DNA in standard bacterial host-strains. Escherichia coli strain K803, which is a permissive host for cloning highly methylated DNA from higher plants, also permitted the cloning of a complete genomic library of 15–20 kb Mbol restriction fragments inserted into the BamHI site of the γ vector, EMBL 3. This C. sorokiniana genomic library appears to be the first genomic-library constructed for any species of Chlorella.     

The mitochondrial genome of an exsymbiotic Chlorella-like green alga

Mitochondrial (mt) DNA from the unicellular, exsymbiotic Chlorella-like green alga, strain Nla was isolated and cloned. The mtDNA has a buoyant density of 1.692 g/ml in CsCl and an apparent G/C base composition of 32.5%. The genome contains approximately 76 kbp of DNA based on restriction fragment summation and electron microscopic measurements. A map of restriction endonuclease sites using Sst I, Bam I, Sal I and Xho I was generated. The genome maps as a circular molecule and appears as such under the electron microscope. Eight genes were assigned to the map by hybridization to specific restriction fragments using heterologous mt-encoded specific probes. These include the genes for subunits 6, 9, and alpha of the F₀-F₁ ATPase complex, the large and small subunit rRNAs, cytochrome oxidase subunits I and II, and apocytochrome b.     

A Computer Graphics Study of Sequence-Directed Bending in DNA

Abstract

We present theoretical results to account for the unusual physical properties of a 423 bp DNA restriction fragment isolated from the kinetoplast of the trypanosomatid Leishmania tarentolae. This fragment has an anomalously low electrophoretic mobility in Polyacrylamide gels and a rotational relaxation time smaller than that of normally-behaved control fragments of the same molecular weight. Our earlier work (Proc. Natl. Acad. Sci. USA 79, 7664, 1982) has attributed these anomalies to the highly periodic distribution of the dinucleotide ApA in the DNA sequence. As originally proposed by Trifonov and Sussman (Proc. Natl. Acad. Sci. USA 77, 3816,1980) local features of the DNA structure such as a small bend at ApA, if repeated with the periodicity of the helix, will cause systematic bending of the molecule.

Computer graphics representations of DNA chain trajectories are presented for different structural models. It is shown that the structural model of Calladine (J. Mol. Biol. 161, 343, 1982) which is based on crystallographic data, is unsuccessful in predicting the systematic bending of DNA in solution.     

Replication,maturation and physical mapping of bacteriophage MB78 genome

Bacteriophage MB78 is a virulent phage ofSalmonella typhimurium. The viral DNA is 42 kb in size and seems to be circularly permuted. We show that viral DNA replication is through concatemeric DNA formation which is subsequently converted into full length DNA through headful packaging. A restriction map of MB78 DNA for six restriction endonucleases e.g.BgIII,PvuII, ECORI, ClaI, SalI and SmaI has been constructed. The yield of certain fragments in less than molar amount is explained in terms of permutation and the headful mechanism of packaging. The packaging site (pac site) has been suggested.     

Restriction Enzyme Mapping of Carcinogen Binding Regions on pBR322

Abstract

Previous equilibrium binding experiments (S.A. Winkle and T.R. Krugh, Nucleic Acids Res. 9, 3175–3186 (1981)) suggested that the carcinogen N-hydroxy-N-acetyl-2-aminofluorene might exhibit preferential binding to a small number of sites on phiXl 74 DNA To examine whether the covalently binding analogue N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF) also possesses high affinity sites, the plasmid pBR322 was reacted with 3H labeled acetoxyAAF to give one to sixteen adducts per DNA molecule. Thus only higher affinity sites would be affected. The DNA was subsequently cleaved with either Alu I, Hae III, Hha I, Hinf I or Hpa II restriction endonuclease and the restriction fragments isolated by gel electrophoresis. Examination of the distribution of 3H acetoxyAAF among the fragments was not random but, rather, with each enzyme, the acetoxyAAF was found predominantly in a few fragments. The locations of the bands containing the acetoxyAAF for each enzyme overlap - suggesting that there are regions on pBR 322 which contain high affinity sites for acetoxyAAF binding.     

The control region of the F sex factor DNA transfer cistrons: Physical mapping by deletion analysis

Summary A technique has been developed which allows the isolation of random deletions extending from unique restriction enzyme sites in plasmid DNA molecules. The method involves transformation of E. coli cells with linear plasmid DNAs generated by restriction enzyme cleavage. We have used this technique to map DNA transfer genes in the tra control region of F sex factor DNA. Deletions within EcoRI fragment f6 of F DNA have been isolated and used to assign physical locations to tra genes by a combination of genetic complementation tests, restriction enzyme analysis, DNA heteroduplexing and the analysis of the proteins synthesised in minicells and in vitro. Deletion analysis has also allowed the identification of the traK gene product.     

Physical characterization of the genome of the Myxococcus xanthus bacteriophage MX-8

Summary We have constructed a restriction map for the genome of bacteriophage MX-8 from Myxococcus xanthus using the enzymes PvuII, MboI, and EcoRI. The phage genome size, as determined by restriction analysis, is 51.7±0.6 Kb. Double digestions, redigestions of isolated fragments, and crossed-contact hybridization of partial digestion products show that the restriction map is circular. Restriction analysis and Southern hybridization show that the phage DNA molecules are packaged sequentially from a concatemer starting from a specific site which we have mapped. The DNA molecules have an average terminal redundancy of approximately 8% and are circularly permuted over at least 40% of the genome.     

Molecular cloning and characterization of the chicken gene homologous to the transforming gene of rous sarcoma virus

Four molecular clones containing DNA homologous to the Rous sarcoma virus transforming gene (src) have been isolated from a random library of normal chicken DNA. The four clones are distinct overlapping isolates, which together span approximately 33 kb of cellular DNA. The cloned locus appears to represent the major region of chicken DNA homologous to src, since src-containing restriction fragments of this locus account for the fragments detected by hybridization of src-specific probe to restriction digests of total chicken DNA. Analysis of the cloned chicken src locus by restriction and heteroduplex mapping indicates that the locus contains 1.6-1.9 kb of DNA homologous to the viral src gene. The chicken DNA sequences homologous to viral src are interrupted by five or six nonhomologous regions, totaling approximately 6 kb, which presumably represent introns in the cellular src gene.     

Highly transformable mutants of Streptomyces fradiae defective in several restriction systems

Summary Streptomyces fradiae JS85 is a mutant defective in tylosin production and an efficient recipient for conjugal transfer of tylosin genes. JS85 was mutagenized with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and derivatives defective in restriction were isolated by sequential selection for increased transformability by several plasmid DNAs. From the number of mutation and selection cycles required to eliminate most restriction, it was estimated that wild type S. fradiae expressed at least five restriction systems. From the patterns of restriction enzyme digestion of chromosomal DNA observed in the series of mutants that became progressively less restricting, it was suggested that wild type S. fradiae normally expresses modification (and presumably restriction) systems similar or analogous to PstI, XhoI, ScaI and EcoRI. The least restricting mutant of S. fradiae was readily transformable by many plasmids, including a bifunctional cosmid vector containing a large insert of Streptomyces DNA.     

Restriction of streptomyces phage SH5 by endonuclease ShyI from Streptomyces hygroscopicus 0477

Summary DNA of the temperate Streptomyces phage SH5 (DNA molecular weight 27x10⁶) is subject to restriction-modification mediated by S. hygroscopicus 0477, S. levoris 1331 and 2340. The restriction endonuclease ShyI (isoschizomeric with SacII) isolated from S. hygroscopicus 0477 is involved in restriction of SH5·1331 and SH5·2340 DNAs in S. hygroscopicus 0477.     

Transposon-like structure of a new plasmid-encoded restriction-modification system in Rhizobium leguminosarum VF39SM

Total DNA isolated from Rhizobium leguminosarum VF39SM cells is resistant to cleavage by the restriction endonuclease PstI. Plasmid curing and transfer studies localized this phenotype to pRleVF39b, the second smallest of six plasmids found in this bacterium. In vitro selection for vector modification was employed to isolate a presumptive methylase gene (M.Rle39BI) from a plasmid gene library. Total and plasmid DNAs isolated from E. coli containing M.RleBI were resistant to digestion by PstI. Sequence data suggested that a putative restriction endonuclease (R.Rle39BI) was also encoded on the same fragment. The two genes were flanked by identical copies of a putative insertion sequence, which was also present in several copies elsewhere in the VF39SM genome. The presence of this element in other strains examined suggested that this element is indeed an insertion sequence. The differences in G/C content between the DNA coding for the R/M system and that of the IS element suggest that this DNA region may have been acquired by horizontal transfer. Received: 28 January 1997 / Accepted: 3 June 1997     

Isolation and characterization of a linear DNA plasmid from Streptomyces clavuligerus

Summary A linear DNA plasmid (pSCL) has been isolated from Streptomyces clavuligerus by a method employing high concentrations of protease. Rate-zonal sedimentation on sucrose gradients was used to purify the plasmid. The plasmid is 12 kb in length and appears to be linked to protein at its 5 termini. A restriction endonuclease map of the plasmid for ten enzymes has been determined. Evidence for terminally repeated sequences is provided by cross-hybridization analysis.     

Newly evolved highly repeated DNA sequences ofTupaia glis (Tupaiidae,Scandentia)

We have studied highly repeated DNA sequences ofTupaia glis (Tupaiidae, Scandentia) with restriction endonucleases and Southern blotting techniques. Five highly repeated DNA fragments have been isolated fromT. glis and hybridized with genomic DNAs (cleaved by different restriction enzymes) of several non-human primate species and one insectivore (E. europaeus), in order to highlight eventual differences or similarities of their highly repeated DNA sequences. Our first preliminary findings suggest that the newly isolated highly repeated DNA fragments ofT. glis are distinct from both non-human primates and insectivore, the two taxonomic groups considered most similar to the Tupaiidae.     

Identification and characterization of CbeI,a novel thermostable restriction enzyme from <Emphasis Type="Italic">Caldicellulosiruptor bescii</Emphasis> DSM 6725 and a member of a new subfamily of HaeIII-like enzymes

Potent HaeIII-like DNA restriction activity was detected in cell-free extracts of Caldicellulosiruptor bescii DSM 6725 using plasmid DNA isolated from Escherichia coli as substrate. Incubation of the plasmid DNA in vitro with HaeIII methyltransferase protected it from cleavage by HaeIII nuclease as well as cell-free extracts of C. bescii. The gene encoding the putative restriction enzyme was cloned and expressed in E. coli with a His-tag at the C-terminus. The purified protein was 38 kDa as predicted by the 981-bp nucleic acid sequence, was optimally active at temperatures between 75°C and 85°C, and was stable for more than 1 week when stored at 35°C. The cleavage sequence was determined to be 5′-GG/CC-3′, indicating that CbeI is an isoschizomer of HaeIII. A search of the C. bescii genome sequence revealed the presence of both a HaeIII-like restriction endonuclease (Athe 2438) and DNA methyltransferase (Athe 2437). Preliminary analysis of other Caldicellulosiruptor species suggested that this restriction/modification activity is widespread in this genus. A phylogenetic analysis based on sequence alignment and conserved motif searches identified features of CbeI distinct from other members of this group and classified CbeI as a member of a novel subfamily of HaeIII-like enzymes.     

Isolation and characterization of Paracoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn) DNA-modifying property

A selection scheme was devised to isolate Paracoccus denitrificans mutants with increased recipient qualities in transfer experiments, using broad host range plasmids. In some of the mutants obtained, a DNA modifying activity that prevents the activity of the restriction endonucleases BamHI and BglII on isolated P. denitrificans DNA had simultaneously been lost. From a detailed analysis of the restriction properties of the enzymes SAU3 AI, MboI and DpnI, it was concluded that a subset of GATC sequences in P. denitrificans DNA may be methylated at an unusual position. It was concluded that P. denitrificans possesses at least one potent host-dependent restriction/modification system which affects conjugation. In addition to the class of restriction-defective mutants, at least one other class of enhanced transfer mutants with unknown defect(s) was isolated. Strains, in which the two mutant classes were combined, exhibited transfer frequencies which were significantly higher than strains containing either mutation alone. Such double mutant strains appeared to be well suited for future experiments like complementation analysis, transposon mutagenesis and gene replacement by homologous recombination.     

Cloning in escherichia coli of a bi‐functional cellulase/xylanase enzyme from ruminococcus flavefaciens FD‐1

Abstract

A genomic library of Ruminococcus fl avef aciens FD‐1 DNA was constructed using the Escherichia coli bacteriophage λ vector λDASH. A recombinant phage exhibiting activity against both Ostazin brilliant red‐hydroxyethyl cellulose (OBR‐HEC) and carboxymethyl cellulose (CMC) was isolated. This clone (designated FD1‐1) was further analyzed by restriction endonuclease mapping and Southern blot analysis. Substrate specificity data shows that the cloned gene(s) encodes both endoglucanase activity and endoxylanase activity. CMC and xylan zymograms of protein(s) produced by this clone and then separated by non‐denaturing PAGE suggest that the endoglucanase/endoxylanase activities reside on the same polypeptide or protein complex. An additional xylanase product lacking CMCase activity was also detected.     

Restriction Endonuclease Sst12I from a Strain of Streptomyces Recognizing the Nucleotide Sequence 5"-CTGCAG-3"

A new restriction endonuclease Sst12I belonging to type II and recognizing the sequence 5"-CTGCAG-3" was isolated from the bacterial strain Streptomycessp. St-12. The enzyme hydrolyzes DNA between adenine and guanine residues; thus, it is a true isoschizomer of restrictase PstI. In contrast to PstI, the restriction endonuclease Sst12I hydrolyses DNA both at 37 and 55°C and remains active after long-term storage.     

Possible Molecular Detent in the DNA Structure at Regulatory Sequences

Abstract

A common feature that appears in a number of DNA sites where proteins interact is the sequence GTG/CAC. In the lac operator this sequence leads to a region with a higher imino proton exchange rate well below the optical melting temperature. It is suggested that this reflects a structural feature recognized by proteins that bind specific sites on the DNA molecule.     

Molecular characterization of new group A streptococcal bacteriophages containing the gene for streptococcal erythrogenic toxin A (speA)

Summary Bacteriophage T12 is the prototype phage carrying the streptococcal erythrogenic toxin A (speA) gene. To examine more closely the phages involved in lysogenic conversion, we examined 300 group A streptococcal strains, and identified and isolated two new phages that carry the speA gene. The molecular sizes of these phage genomes were between 32 and 40 kb, similar to that of phage T12 (35 kb). However, as ascertained by restriction analysis, the physical maps of the new phage genomes were different from phage T12 and from each other. Hybridization analysis also showed that all of these phages were only partially related to one another and the speA gene was always located close to the phage attachment site. Additionally, colony hybridization showed that whereas phage T12 or one of its close relatives is the most common phage associated with the group A streptococci, phage 49 has a much stronger association with the speA gene. A defective phage was also found following pulsed field gel electrophoresis of total phage DNA. This phage appears to be a resident of strain T25₃c and is found only following induction of a T25₃c lysogen. Restriction enzyme analysis of the isolated defective phage DNA suggests that it is the source of the submolar amounts of DNA previously found in association with phage T12 digestion patterns. Additionally, the defective phage may serve as the site of integration of the speA gene-carrying phages described above.