Cloning and characterization ofDaphnia mitochondrial DNA

Summary The mitochondrial genome ofDaphnia pulex (Crustacea, Cladocera) was cloned as a single fragment into the plasmid vector pUC12. The genome size, estimated from restriction endonuclease fragment lengths, is 15,400±200 base pairs. The GC content, estimated from thermal denaturation studies, is 42%. The positions of 39 cleavage sites were mapped for 14 restriction enzymes. The distribution of these sites within the genome is random (P=0.44). Heterologous hybridizations withDrosophila sylvestris mitochondrial DNA (mtDNA) probes indicate that gene orders withinDaphnia andDrosophila mtDNAs are similar.     

Physical and genetic studies with restriction endonucleases on the broad host-range plasmid RK2

Summary The cleavage map of the plasmid RK2 was determined for the five restriction endonucleases EcoRI, HindIII, Bam H-I, Sal I and Hpa I. DNA has been inserted into several of these sites and cloned in Escherichia coli. Efforts to obtain derivatives of RK2 reduced in size by restriction endonuclease digestion of the plasmid were not successful and indicated that genes required for the maintenance of this plasmid in E. coli are not tightly clustered. An RK2 derivative possessing an internal molecular rearrangement was obtained by transformation with restriction endonuclease digests of the plasmid.     

Linear plasmid DNAs of the plant pathogenic fungus Rhizoctonia solani with unique terminal structures

Summary Three linear DNA plasmids were found in isolate RI-64 of anastomosis group 4 (AG-4) of Rhizoctonia solani. These plasmids, designated pRS64-1, -2, and -3, possessed the same size of 2.7 kb. Restriction mapping and Southern hybridization analysis of pRS64-1, -2, and -3 revealed the presence of homologous regions at both termini. The plasmid DNAs were resistant to both 3-exonuclease and 5-exonuclease even after treatment with proteinase K or alkali. The length of both terminal fragments that were generated by restriction endonuclease digestion was doubled under the denaturation condition, indicating that the linear plasmid DNAs have hairpin loops at both termini. Southern blotting analysis of total DNA showed the presence of two types of dimeric forms of pRS64 DNA. One is a head-to-head dimer and the other is a tail-to-tail dimer. The role of these unique DNA structures in replication of the plasmids is discussed.     

Overcoming the restriction barrier to plasmid transformation and targeted mutagenesis in Bifidobacterium breve UCC2003

In silico analysis of the Bifidobacterium breve UCC2003 genome predicted two distinct loci, which encode three different restriction/modification systems, each comprising a modification methylase and a restriction endonuclease. Based on sequence homology and observed protection against restriction we conclude that the first restriction endonuclease, designated BbrI, is an isoschizomer of BbeI, the second, BbrII, is a neoschizomer of SalI, while the third, BbrIII, is an isoschizomer of PstI. Expression of each of the B. breve UCC2003 methylase‐encoding genes in B. breve JCM 7017 established that BbrII and BbrIII are active and restrict incoming DNA. By exploiting knowledge on restriction/modification in B. breve UCC2003 we successfully increased the transformation efficiency to a level that allows the reliable generation of mutants by homologous recombination using a non‐replicative plasmid.     

Characterization of eight excision plasmids of Pseudomonas syringae pv. phaseolicola

Summary Pseudomonas syringae pv. phaseolicola strain LR719 contains a 150 kilobase pair (kb) plasmid pMC7105, stably integrated into its chromosome. Occasionally, single colony isolates of this strain contain an excision plasmid. Eight unique excision plasmids were selected and characterized by BamHI restriction endonuclease and blot hybridization analyses. These plasmids ranged in size from 35 to 270 kb; the largest contained approximately 130 kb of chromosomal DNA sequences. Restriction maps of pMC7105 were developed to deduce the site of integration and to identify the fragments in which recombination occurred to produce each excision plasmid. The eight excision plasmids were arranged into five classes based on the sites where excision occurs. A 20 kb region of pMC7105, which includes BamHI fragment 9 and portions of adjacent fragments, is present in all excision plasmids and thought to contain the origin of replication. The site of integration on pMC7105 maps within BamHI fragment 8. This fragment shows homology with seven other BamHI fragments of pMC7105 and with five chromosomal fragments identified among the excision plasmids. The data strongly suggest that the integration of pMC7105 may have occurred at a repetitive sequence present on the chromosome and on the plasmid.     

Physical mapping of a plasmid from Bacillus megaterium by restriction endonuclease cleavage.

A 4-Mdalton plasmid from Bacillus megaterium strain 216 has been physically mapped by restriction endonuclease digestion. A combination of single and double digests with seven restriction enzymes, together with a terminal labeling procedure, has produced a physical map containing 21 apparently unique cleavage sites. The data are most consistent with the view that this plasmid does not contain extensive variability in sequence.     

Restriction Fragment Length Polymorphism Analyses of the Plasmid DNAs in Strains of Xanthomonas campestris pv. vesicatoria from Different Geographic Areas

Restriction fragment length polymorphisms (RFLPs) of plasmid DNAs in Xanthomonas campestris pv. vesicatoria were analysed using 77 strains from the United states, Argentina, Australia, Taiwan, and Korea. One or more plasmids were detected in all tested strains, irrespective of geographic origin, host plant from which isolated, or chemical resistance. All Korean strains contained a few plasmids of similar high molecular weight, whereas some small plasmids occured only in strains from the United States, Argentina, and Taiwan. After digesting total plasmid DNAs with each of four restriction endonucleases, 18 fragments with sizes from about 1 to 23 kb were visualized. Seventy-seven strains of diverse geographic origins, with different levels of resistance to streptomycin and copper, were classified into the 14 RFLP groups based on the restriction endonuclease digestion patterns of their plasmid DNAs. Strains belonging to each group shared DNA fragments of identical size, suggesting the possible presence of similar plasmids in these strains. A 5.8-kb EcoRI plasmid DNA probe prepared from the United States strain 81-23 hybridized to EcoRI plasmid digests from all tested strains. Other plasmid DNA fragments of the strain81-2,3 used as probes had no homology to plasmid DNA fragments from several strains around the world. The variation in hybridization profiles of plasmid DNA was very similar to the results obtained by RFLP analysis of plasmid DNA digested by four restriction enzymes. Most of the Korean strains tested were highly sensitive to streptomycin and copper, whereas most strains from other geographic areas showed a high level of resistance to one or two of the chemicals. Cluster analysis of genetic distance between the strains based on the data obtained generated the dendrograms that separated all Korean strains from the other strains, suggesting that plasmid DNA of the Korean strains may be genetically very different from those of the others.     

Restriction endonuclease analysis of plastid DNA from tomato, potato and some of their somatic hybrids

Summary The buoyant density and endonuclease restriction patterns of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum) ptDNA were examined and compared with those of their somatic hybrids. The plastids from these plants, both of which belong to the family of Solanaceae, contain a single DNA species whose density of 1.697 gcm^-3 and size of approximately 156 kbp are similar to those of ptDNA from other higher plants. The Sal I restriction patterns were indistinguishable; however, those obtained with Kpn I, Pst I, and Eco RI disclosed that each species possesses a unique ptDNA. These observations suggest a relatively recent divergence of both species. Of the twelve hybrid lines screened, eight contained exclusively potato ptDNa and four contained only tomato ptDNA at a 0.1–3% level of detection. Rearrangements of modifications of DNA were not detected. The plastid identities of three hybrid lines that had previously been analyzed by isoelectric focusing of RuBPcase subunits (Melchers et al. 1978) agreed with those determined by restriction endonuclease analysis.Abbreviations used in the text ptDNA plastid DNA, chloroplast DNA - cDNA copy DNA - RuBPcase ribulose bisphosphate carboxylase/oxygenase - LSU large subunit of RuBPcase - kbp kilobase pairs - SDS sodium dodecyl sulfate - SSC standard saline citrate - IEF isoelectric focusing     

Physical and genetic studies with restriction endonucleases on the broad host-range plasmid RK2.

The cleavage map of the plasmid RK2 was determined for the five restriction endonucleases EcoRI, HindIII, BamH-I, SalI and HpaI. DNA has been inserted into several of these sites and cloned in Escherichia coli. Efforts to obtain derivatives of RK2 reduced in size by restriction endonuclease digestion of the plasmid were not successful and indicated that genes required for the maintenance of this plasmid in E. coli are not tightly clustered. An RK2 derivative possessing an internal molecular rearrangement was obtained by transformation with restriction endonuclease digests of the plasmid.     

Transformation-derived Neisseria gonorrhoeae plasmids with altered structure and function.

Plasmid deoxyribonucleic acid from Neisseria gonorrhoeae containing a 7.1-kilobase (kb) (4.7-megadalton) penicillinase (Pcr) plasmid transformed homogenic gonococci to penicillinase production at a low frequency. About 25% of the penicillinase-producing gonococcal transformants contained Pcr plasmids which were either larger or smaller than the 7.1 kb donor plasmid; these Pcr plasmids varied in size from 3.45 to 42 kb. Some of these altered plasmids differed from the donor plasmid in stability or in frequency of mobilization by a 36-kb (24-megadalton) conjugative plasmid. A restriction endonuclease cleavage map of the 7.1-kilobase Pcr plasmid and several of the smaller deleted plasmids was constructed. The most common size of altered Pcr plasmid was 5.1 kb (3.4 megadaltons). A Pcr plasmid isolated from a gonococcus in London, England, was identical with these 5.1-kb transformant plasmids in both size and restriction endonuclease cleavage profiles, suggesting that the 5.1-kb Pcr plasmid could have arisen from a 7.1-kb Pcr plasmid by a transformation-associated deletion in nature.     

Genetic, molecular, and functional analysis of Streptococcus faecalis R plasmid pJH1.

Streptococcus faecalis JH1 contains two conjugative plasmids, pJH1, an R plasmid that codes for resistance to kanamycin, streptomycin, erythromycin, and tetracycline, and pJH2, a hemolysin-bacteriocin plasmid. Strain JH1 was used as an antibiotic resistance donor in conjugation experiments with two plasmid-free S. faecalis recipient strains, JH2-2 and OG1-RF1. Plasmid pJH1 was purified from one transconjugant, DL77, and subjected to restriction endonuclease analyses. Five restriction enzymes, EcoRI, XbaI, BamHI, SalI, and XhoI, yielding 10, 9, 3, 2, and 2 fragments, respectively, were used to determine the size (80.7 kilobases) of pJH1 and to construct a restriction endonuclease map of the plasmid. Twenty-eight percent of the antibiotic-resistant transconjugants examined expressed only part of the resistance pattern (Kmr Smr Emr Tcr) associated with pJH1, that is, they were resistant to kanamycin, streptomycin, and erythromycin; to erythromycin and tetracycline; or to erythromycin or to tetracycline only. Most of these strains also produced hemolysin and bacteriocin, and several contained a hybrid plasmid consisting of pJH2 and specific segments of pJH1 DNA. Several of these hybrid plasmids, as well as a deletion derivative of pJH1 that coded for resistance to tetracycline but not to kanamycin, streptomycin, or erythromycin, were purified and used to confirm the arrangement of restriction endonuclease fragments on the pJH1 map and to locate the resistance determinants on this map.     

Organelle DNA compositions and isoenzyme expression in an interspecific somatic hybrid of Daucus

Summary Cell suspension cultures of Daucus carota, D. capillifolius and a somatic hybrid of these lines were analyzed to determine their chloroplast and mitochondrial DNA compositions. The plastid DNAs (pDNA) from the somatic hybrid and D. carota were identical and were different from that of D. capillifolius when analyzed on agarose electrophoretic gels after digestion by the restriction endonuclease HpaII. The endonuclease restriction patterns of the mitochondrial DNAs (mtDNA) from each cell line were different. Although the restriction pattern of the mtDNA from the somatic hybrid contained fragments in common with one or both parents, unique fragments not found in the restriction pattern of either parent were also present.The amounts and feedback regulation of aspartokinase, homoserine dehydrogenase and dihydrodipicolinic acid synthase were quantified to define the effects of somatic hybridization upon the pathway leading to the biosynthesis of lysine, threonine, methionine and isoleucine. Regulation of each enzyme by end product inhibitors was not altered in the somatic hybrid, but levels of each enzyme appeared to be increased. However, isoenzyme analysis indicated two major forms of homoserine dehydrogenase were present in the hybrid, including one unique form not present in either parent.     

Suppression of an Escherichia coli dnaA mutation by the integrated R factor R100.1: generation of small plasmids after integration.

We have observed that integration of the R plasmid R100.1 into the chromosome of Escherichia coli is associated with the formation of small, covalently closed circular elements. Contour length measurements, partial denaturation mapping, and analysis of the deoxyribonucleic acid fragments produced by digestion of one of these, pLC1, with the restriction endonuclease EcoRI indicate that it is the r-determinant element of R100.1.     

Isolation and partial characterization of plasmid DNA from Streptococcus thermophilus

Of 23 strains of Streptococcus thermophilus examined, 5 were found to contain a single small cryptic plasmid, designated pHM1 through pHM5. Through analysis by restriction endonuclease mapping and DNA-DNA hybridization, the five plasmids were found to be closely related. They were present in 4 to 18 copies per cell and ranged in size from 1.4 to 2.2 megadaltons. Plasmids pHM1 and pHM5, as well as pHM2 and pHM4, were found to be identical. Single restriction endonuclease sites were observed on each plasmid for PvuII and MboI. Plasmids pHM2 and pHM3 each had an additional single site for HhaI, and pHM1 had an additional single site for HindIII. The characteristics of these plasmids may make them useful for the development of cloning vectors for use in S. thermophilus.     

Isolation and partial characterization of plasmid DNA from Streptococcus thermophilus.

Of 23 strains of Streptococcus thermophilus examined, 5 were found to contain a single small cryptic plasmid, designated pHM1 through pHM5. Through analysis by restriction endonuclease mapping and DNA-DNA hybridization, the five plasmids were found to be closely related. They were present in 4 to 18 copies per cell and ranged in size from 1.4 to 2.2 megadaltons. Plasmids pHM1 and pHM5, as well as pHM2 and pHM4, were found to be identical. Single restriction endonuclease sites were observed on each plasmid for PvuII and MboI. Plasmids pHM2 and pHM3 each had an additional single site for HhaI, and pHM1 had an additional single site for HindIII. The characteristics of these plasmids may make them useful for the development of cloning vectors for use in S. thermophilus.     

Molecular cloning of the terminal hairpin of vaccinia virus DNA as an imperfect palindrome in an Escherichia coli plasmid

The genome of vaccinia virus is a linear duplex molecule of approximately 185 kb with hairpins at each end that link the complementary strands. The hairpins, which exist in two forms that are inverted and complementary in sequence, were isolated as XbaI restriction fragments and converted to a linear intermolecular duplex structure by denaturation and reannealing. The latter was then stably cloned as a 142-bp imperfect palindrome in an Escherichia coli plasmid. The insert was excised from the plasmid and the palindrome was extended on both sides by ligating it to the adjacent vaccinia virus DNA segment. The resulting fragment was cloned as a 278-bp imperfect palindrome. Restriction endonuclease analysis and DNA sequencing indicated the absence of any deletions or rearrangements. After excision from the plasmid, the palindrome was converted by heating and rapid cooling to the original two hairpin forms. In this manner, large quantities of vaccinia virus telomeres may be obtained for physical and biochemical studies.     

Physical characterization of plasmids isolated fromStreptosporangium

Plasmids were isolated from two species ofStreptosporangium by CsCl-ethidium bromide equilibrium density gradient centrifugation. A plasmid isolated fromS. brasiliense, designated pSgB-1, was characterized by electron microscopy and agarose gel electrophoresis. The pSgB-1 plasmid is a closed circular DNA molecule of 9.4 × 10⁶ Da. A restriction endonuclease map was generated and unique cleavage sites were found forEcoRI, ClaI, XbaI, and MstII. Another plasmid, pSgV-1, isolated fromS. viriodogriseum, has an estimatedM_r of 54 × 10⁶. The pSgB-1 plasmid is phenotypically cryptic but an unusual phenotypic trait, resembling phage plaques, may be associated with theS. viridogriseum plasmid pSgV-1.     

A method for the generation of small pre-determined deletions in plasmid DNA: deletion analysis of the tetR region of vector pBR322

A general method is described that allows precise deletion of a chosen restriction fragment(s) from a plasmid having many cleavage sites for that restriction enzyme. The DNA to be deleted is first separated from the rest of the plasmid on a larger DNA fragment contained between two different unique restriction sites. This fragment is then subdigested by the restriction endonuclease of interest, which recognises two or more tetranucleotide (cohesive end or blunt end) sequences on the fragment, and is recloned between the two original unique restriction sites. The method is rapid, efficient, and the results are predictable. Examples are given in which predetermined HpaII (9 bp, 147 bp), TaqI (141 bp) and AluI (15 bp, 403 bp) fragments have been selectively removed from the tetR region of plasmid pBR322.     

A sequence of the yeast 2 micron DNA plasmid chromosome near the origin of replication is exposed to restriction endonuclease digestion

The yeast 2 μm DNA plasmid nucleoprotein complex was subjected to restriction endonuclease digestion to ascertain whether all possible sites are equally accessible to hydrolysis. When plasmid nucleoprotein complexes which had been fixed with formaldehyde were exhaustively digested with restriction endonucleases HinfI or CfoI, only a few of the limit digest products were produced. Furthermore, the limited set of restriction endonuclease sites exposed in formaldehyde-treated plasmid chromosomes could be shown to be preferentially hydrolyzed when plasmid chromosomes which had not been treated with formaldehyde were digested with the same restriction endonucleases. Mapping of the preferred sites revealed that they mapped to the region of the plasmid near the replication origin. These results demonstrate that the protection of DNA from nuclease activity is not constant along the plasmid chromatin, and that a region near the replication origin is preferentially exposed to endonuclease hydrolysis.