Homology of plasmids in strains of unicellular Cyanobacteria.

Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron microscopy and agarose gel electrophoresis. The sizes of the various plasmid species were determined; in each of the Synechococcus strains 6301, 6707, and 6908 two plasmid species were found with molecular weights of 5.3 × 10⁶ and 32.7 × 10⁶. Synechococcus strain 7425 had two plasmids of molecular weight 5.4 × 10⁶ and 24 × 10⁶. Synechococcus strain 6312 and Synechocystis strain 7005 each contained one plasmid species with molecular weight of 15.9 × 10⁶ and 2.0 × 10⁶, respectively. Restriction enzyme analysis revealed identical cleavage patterns for the plasmids of identical molecular weight.     

Transfer of plasmid pTO1 from Escherichia coli to various representatives of the order Actinomycetales by intergeneric conjugation

Plasmid pTO1 containing the oriT fragment from RK2, the Escherichia coli replication function from pBR322, and a DNA fragment of actinophage φC31 with the attachment site was transferred from E. coli S17-1 to strains of the genera Actinomadura, Arthrobacter, Micromonospora, Nocardia, Rhodococcus, and to 16 strains of the genus Streptomyces. The frequency of conjugant formation was 1×10⁻³–1×10⁻⁵ depending on the strain. Hybridization experiments demonstrated that plasmid pTO1 integrates into chromosomes of a number of the recipient strains examined.     

Identification of cyanobacteria by polymorphisms of PCR-amplified ribosomal DNA spacer region

The 16S-23S ribosomal DNA spacer region of selected cyanobacterial strains was amplified by the polymerase chain reaction using primers to conserved flanking sequences. Single or multiple rDNA amplification products were generated depending on the strain and primer pair. Species could generally be distinguished on the basis of size heterogeneity of the products. Analysis of restriction digests of the amplified rDNAs indicated polymorphisms useful in identification. Four enzymes (HinfI, DdeI, AluI, TaqI) generated restriction fragment length patterns that could discriminate between the cyanobacteria to the taxonomic levels of genus and species. This approach should prove useful in the rapid identification of cyanobacteria.     

Physical and functional mapping of rat-liver mitochondrial DNA

Summary Rat-liver mitochondrial DNA (mtDNA) contains 2 cleavage sites of the restriction endonuclease XbaI. The molecular sizes of restriction fragments are 6.6×10⁶ and 3.7×10⁶ D. The results of partial cleavage of mtDNA with EcoRI allow the fragment F (0.32×10⁶ D) to be localized in the sequence ABCEGFHDA. The functional map of mtDNA is constructed for two genes of the ATP-ase mRNAs from rat-liver mitochondria. Molecular hybridization shows that the ATPase genes are located in fragment B and in the GEHD area of mtDNA EcoRI cleavage.     

Efficient Transformation System for Propionibacterium freudenreichii Based on a Novel Vector

A 3.6-kb endogenous plasmid was isolated from a Propionibacterium freudenreichii strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of an Escherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli and P. freudenreichii. Whereas electroporation of P. freudenreichii with vector DNA isolated from an E. coli transformant yielded 10 to 30 colonies per μg of DNA, use of vector DNA reisolated from a Propionibacterium transformant dramatically increased the efficiency of transformation (≥10⁸ colonies per μg of DNA). It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of Propionibacterium with DNA ligation mixtures.     

Characterization of pRGO1, a Plasmid from Propionibacterium acidipropionici, and Its Use for Development of a Host-Vector System in Propionibacteria

The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6,868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 × 10⁶ CFU/μg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10⁴ to 10⁷ CFU/μg of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.     

A study of sequence homologies in four satellite DNAs of man.

Satellites I, II, III and IV (Corneo et al., 1968,1970,1971) have been purified from human male placental DNA. The sequences present in these four DNA components have been characterized by analytical buoyant density, thermal denaturation, DNA reassociation, DNA hybridization and gel electrophoresis coupled with hybridization following either HaeIII or EcoRI restriction endonuclease digestion. Satellites III and IV were found to be virtually indistinguishable by a variety of criteria. Cross-satellite reassociation showed that 40% of the molecules present in satellite III contain sequences that are homologous to 10% of the molecules of either satellite I or satellite II. Reassociated satellite I melts as a single component, as do the hybrid duplexes between satellite I and satellite III. In contrast, reassociated satellites II, III and IV, and the hybrid duplexes formed between satellites II and III and between satellites II and IV, melt as two distinct components with different thermal stabilities.Digestion of satellite III with HaeIII gives rise to a series of fragments whose sizes are 2, 3, 4, 5, 6, 7, 8 and 11 times the size of the smallest 0.17 × 10³ basepair fragment, in addition to a 3.4 × 10³ base-pair male-specific fragment (Cooke, 1976) and high molecular weight material. The sequences contained in the fragments of the HaeIII ladder are diverged from each other as well as being non-homologous with those of the 3.4 × 10³ base-pair and high molecular weight fragments. The latter contain EcoRI recognition sites. Satellite II has a similar pattern of fragments to satellite III following digestion with HaeIII, although it can be distinguished from satellite III on the basis of the products of EcoRI digestion. Satellite I contains neither HaeIII nor EcoRI recognition sites. The cross-satellite homologies of the sequences present in fragments of differing sizes produced by restriction enzyme digestion have also been studied.     

Characterization of the DNA and structural proteins of entomopoxviruses from Melanoplus sanguinipes,Arphia conspirsa, and Phoetaliotes nebrascensis (Orthoptera)

Entomopoxvirus (EPV) occlusion bodies were isolated from virus infected nymphs of the grasshoppers Melanoplus sanguinipes, Arphia conspirsa, and Phoetaliotes nebrascensis. Separation of the viral structural proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave unique protein patterns for each of the three viruses. An occlusion body protein of approximately 100,000 MW was isolated from each virus. Cleavage of viral DNA with HinddIII and BamHI restriction endonucleases and separation of the fragments by agarose gel electrophoresis gave different DNA fragment patterns for each of the three entomopoxviruses. Molecular weight estimates of 120 × 10⁶ for M. sanguinipes EPV DNA, 129 × 10⁶ for A. conspirsa EPV DNA, and 125 × 10⁶ for P. nebrascensis EPV DNA were calculated from the sizes of the viral DNA fragments. Approximately 55% base sequence homology was detected by Southern hybridization of α-³²P-labeledM. sanguinipes EPV DNA with P. nebrascensis DNA. No base sequence homology was detected by Southern hybridization of labeled M. sanguinipes EPV DNA to Othnonius batesi EPV DNA (Coleoptera), Amsacta moorei EPV DNA (Lepidoptera), Euxoa auxiliaris EPV DNA (Lepidoptera), and vaccinia virus DNA fragments.     

Pannonibacter indica sp. nov., a highly arsenate-tolerant bacterium isolated from a hot spring in India

A novel aerobic bacterium, strain HT23^T, able to grow on 500 mM sodium arsenate was isolated from a hot-spring sediment sample collected from Athamallik, Orissa, India. Cells of this isolate were Gram negative. Heterotrophic growth was observed at pH 6.0–11.0 and 20–45 °C. Optimum growth was observed at 37 °C and pH 7.0–10.0. The major polar lipids are diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline and phosphatidyl monomethyl ethanolamine. The major isoprenoid quinone was Q-10. 16S rRNA gene sequence analysis indicated that the bacterium clustered with the genus Pannonibacter and showed 98.9 % similarity with Pannonibacter phragmitetus C6-19^T (DSM 14782^T) and 98 % with the P. phragmitetus group B and P. phragmitetus group E strains. Levels of DNA–DNA relatedness between the strain HT23^T and P. phragmitetus C6-19^T (DSM 14782^T) and other strains of P. phragmitetus group B and group E strains were below 55 %. On the basis of phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequence analysis and DNA–DNA hybridization data, strain HT23^T is considered to represent a novel species of the genus Pannonibacter, for which the name Pannonibacter indica sp. nov. is proposed. The type strain is HT23^T (=JCM 16851^T = DSM 23407^T = LMG 25769^T).     

Specific Origin in SV40 DNA Replication

THE oncogenic virus SV40 contains a covalently closed circular DNA molecule of 3 × 10⁶ molecular weight¹. In infected permissive cells, SV40 DNA replicates through a Cairns type intermediate² with the parental strands forming a partially twisted, covalently closed molecule³. We have used a specific bacterial restriction endonuclease⁴ to analyse SV40 DNA replication. The restriction endonuclease of H. influenzae makes double-strand breaks in DNA at specific hexanucleotide sequences^{5, 6} and splits SV40 DNA into eleven fragments, separable by Polyacrylamide gel electrophoresis, ranging in molecular weight from 6.5 × 10⁵ (about 20% of the molecule) to 7.4 × 10⁴ (about 2.5% of the molecule). The largest eight fragments are present in the digest in amounts equimolar with the starting DNA⁴. Therefore, by digesting labelled replicating SV40 DNA and newly completed DNA and measuring the relative yield of each fragment, we could determine whether a particular region of the DNA is synthesized first or last and also estimate the time needed to replicate one molecule completely.     

Electron microscopic determination of the preferential binding sites of Escherichia coli RNA polymerase to phi X174 replicative form DNA

Binding of Escherichia coli RNA polymerase to φX174 DNA replicative form (RF) has been studied by electron microscopy. Samples of the binary complexes were spread for observation upon polylysine-coated carbon films. Binding was obtained with both RFI and RFIII forms of the DNA; complexes formed with the former were treated with restriction enzyme PstI before spreading. A histogram constructed from the positions of 558 polymerase molecules bound to 181 DNA strands exhibited three prominent, sharp peaks at 3.3 × 10² nucleotides, 39.7 × 10² nucleotides and 49.0 × 10² nucleotides from the PstI cleavage point. These positions correspond closely to those of the D, A and B promoter sequences, as derived from φX174 DNA sequence data by Sanger et al. (1977).     

Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells.

Treatment of Ltk^?, mouse L cells deficient in thymidine kinase (tk), with Bam I restriction endonuclease cleaved DNA from herpes simplex virus-1 (HSV-1) produced tk⁺ clones with a frequency of 10^?6/2 μg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk⁺ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk^? phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (1–5 × 10^?3). HSV-1 DNA Bam restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Bam digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.     

Fractionation and molecular weight determination of deoxyribonucleic acid fragments using agarose column chromatography

DNA fragments of several sizes have been produced by shearing E. coli DNA under different pressures. These fragments have been used to demonstrate that column chromatography on agarose Bio-Gel A-15M can provide a rapid, inexpensive fractionation and sizing method for single-stranded nucleic acids having masses between 10⁵ and 10⁶ daltons. Both chromatographic and electrophoretic analysis of the sheared DNA indicated that discrete fragment populations were produced at each shearing pressure and that these fragments were distributed essentially symmetrically around a mean piece size. The average molecular weight of the several DNA fragment distributions was determined electrophoretically by comparison with standard DNA fragments obtained from restriction endonuclease cleavage of SV40 viral DNA. The molecular weights of the denatured, sheared fragments (single-stranded) ranged from 1.25 × 10⁵ to 7.4 × 10⁵. The single-stranded DNA fragments were chromatographed over agarose Bio-Gel A-15M and a linear relationship was found to exist between the mobilities and logarithms of the molecular weights. Readily available tRNA, 5s RNA, and φX174 single-stranded circular DNA chromatographed at the extremes of the linear relationship and could be used to calibrate the column chromatography.     

Electron microscopic studies of the binding of Escherichia coli RNA polymerase to DNA. I. Characterization of the non-specific interactions of holoenzyme with a restriction fragment of bacteriophage T7 DNA

Non-specific interactions between a 3800 base-pair restriction fragment of bacteriophage T7 DNA (MboI-C) and Escherichia coli RNA polymerase holoenzyme have been examined by electron microscopy. Holoenzyme displays a relatively weak and rapidly reversible binding to DNA that is only slightly reduced at elevated salt concentrations. As the concentration of NaCl is increased from 50 mm to 200 mm, the binding constant decreases from 2 × 10⁴m^?1to 4 × 10³m^?1. It is concluded that only 1 to 2 sodium ions are released from the DNA when holoenzyme binds non-specifically.The validity of the electron microscopic technique for determining binding constants has been investigated by varying aspects of the grid surface and by examining the non-specific interactions of lac repressor with DNA.     

Recognition of Leuconostoc oenos strains by the use of DNA restriction profiles

The chromosome of 41 Leuconostoc oenos strains obtained from collections in different countries was analysed with the aim of differentiating the strains. Pulsed field electrophoresis (TAFE) was used to separate large DNA fragments created by the restriction enzymes NotI, SfiI and ApaI, which specifically recognize guanines or cytosines. The genomic DNA of 11 strains was analysed initially with NotI and only four different restriction profiles were observed. The genome size ranged from 1.8 to 2.1 megabase pairs (Mbp). Constant field electrophoresis applied to DNA treatment with 19 different restriction enzymes showed that the size of the fragments obtained increased proportionally to the percentage G+C present at the site of restriction. EcoRI and HindIII profiles revealed that the zone between 9 and 23 kbp allowed differentiation of the strains tested. Thus, the 41 strains fell into 30 restriction groups using only two enzymes. Hybridization with a non-radioactive DNA probe coding for 16S rRNA revealed that there were two 16S genes on the chromosome. Correspondence to: C. Diviès     

Length heterogeneity in a region of the human ribosomal gene spacer is not accompanied by extensive population polymorphism

We carried out a restriction enzyme analysis of human ribosomal DNA structure on total placental DNA using the Southern (1975) method. Studies on a single individual using HindIII, PstI, HpaI and BglII revealed that a region of the non-transcribed spacer near the 3′ end of the 28 S gene was heterogeneous in size. Four fragment classes were detected in this individual. Adjacent classes differed in size from one another by about 0·8 × 10³ bases. Analysis of 19 additional placental samples and four cell lines revealed no fragment classes other than those detected in the original sample. Mixing experiments carried out with all 20 placental DNA samples provided further evidence for the discrete nature of the population polymorphism in the length of this region of the spacer. This finding contrasts sharply with the almost continuous nature of the population polymorphism in the length of a region of the non-transcribed spacer in Xenopus laevis.     

Simple and Rapid Detection of Salmonella by Direct PCR Amplification of Gene fimW

This study established a simple method of specifically detecting Salmonella species by amplifying fimW gene, which was involved in regulating Salmonella type I fimbriae expression. A pair of primers was designed to target and discriminate the 68 Salmonella strains of 23 Salmonella serovars available to us from 12 non-Salmonella strains of five different kinds of bacteria by polymerase chain reaction (PCR) amplification. Results showed that specific DNA fragment with an expected size of 477 bp was successfully amplified from all Salmonella serovars, while no target band was detected in non-Salmonella species. The sensitivity of this PCR-amplifying system reached to 1 pg DNA chromosome and 10² cfu of Salmonella enteritis strain CMCC(B) 50336. The above results demonstrated the method as a simple, sensitive, and specific way for Salmonella detection.     

Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures.

Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules.Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10⁻² μm) is smaller than the random coil segment value b (7·17 × 10⁻² μm) was observed, while circular structures of the dimer of the same molecule (12 × 10⁻² μm) were detected.     

ATP Hydrolysis by Restriction Endonuclease from <Emphasis Type="Italic">E. coli</Emphasis> K

We wish to report a puzzling ATPase activity associated with the DNA restriction endonuclease from E. coli strain K^1–3. This enzyme makes a limited number of double chain breaks in DNA molecules lacking the host-controlled modification imparted by strain K. Unmodified DNA molecules from bacteriophage λ, which serve as a convenient substrate, are broken into fragments with a weight average molecular weight of approximately 7 × 10⁶, about one-fifth the size of the intact λ chromosome. The reaction requires Mg2⁺, ATP and S-adenosylmethionine (SAM).