Analysis of the genome of type 7 simian adenovirus using restrictases.

The effect of the restricting endonucleases R.EcoRI, R.BamI and R.SalI on the genome of type 7 simian adenovirus (SA-7) has been studied. Since the DNA has only one site of R.EcoRI recognition the enzyme cleaves SA-7 DNA into two fragments with the molecular weights 12.0 and 10.0 . 10(6). The restrictase R.BamI cleaves the SA-7 DNA at six sites producing 7 fragments with the molecular weights 6.6, 5.9, 3.8, 2.7, 1.3, 0.7 and 0.6 . 10(6). R.SalI cleavage yields 6 fragments with the molecular weights 8.1, 5.5, 4.3, 2.45, 1.2 and 0.6 . 10(6). The R.BamI and R.SalI fragments are arranged in the orders E-A-D-F-C-G-B and A-B-D-F-E-C, respectively. The only R.EcoRI recognition site is localized in the C fragment produced by R.BamI and in the B fragment produced by R.SalI.     

Molecular cloning of tobacco chloroplast ribosomal RNA genes

Summary Tobacco chloroplast ribosomal RNAs were shown to be hybridized with two EcoRI fragments of tobacco chloroplast DNA. These DNA fragments having molecular weights of 1.9x10⁶ and 2.8x10⁶ daltons were cloned using the bacterial plasmid pMB9 as a vector and E. coli HB101 as host bacteria. The recombinant plasmids containing either or both of these fragments were constructed and characterized.Abbreviations rRNA ribosomal RNA - EDTA ethylenediamine tetraacetic acid - SSC 0.15 M NaCl-0.015 M sodium citrate - EcoRI and HindIII restriction endonucleases isolated from E. coli RY13 and Haemophilus influenzae Rd, respectively     

Restriction endonuclease mapping and mutagenesis of the F sex factor replication region.

The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of beta-lactamase production. By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that approximately 1.9 X 10(6) daltons of the 6.0 X 10(6) dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.     

Physical map of the Agrobacterium rhizogenes strain 8196 virulence plasmid

Virulence of Agrobacterium rhizogenes, agent of hairy root disease, is conferred by large plasmids called Ri (root-inducing) plasmids. We have determined the BamHI fragment map of pRi8196, MW 143 Mda, principally by analysis of recombinant plasmids containing overlapping BamHI partial-digest fragments. Clones containing solitary BamHI inserts of remaining unmapped fragments were used to probe a series of Southern-blotted, pRi8196-derived EcoRI, PstI, HindIII, SalI, or SmaI digests. Continguous hybridized bands represented complements of EcoRI, PstI, HindIII, SalI, or SmaI fragments which bridged the unmapped BamHI fragments. We present, in addition, a detailed map of the core T-DNA region with respect to the restriction endonucleases SalI, EcoRI, HpaI, and HindIII.     

Isolation of plasmid deletion mutants and study of their instability

We describe a method which allows isolation of deletions within hybrid plasmids. It is based on the fact that the tetracycline resistance (Tc^R) gene of pBR322 can be inactivated by inserting foreign DNA into its HindIII site, and that the easily selectable Tc^R mutants of such plasmids are generally (>90%) due to deletions of certain hybrid plasmid sequences. We have found that Tc^R mutants are usually maintained within the cell recombined with the parental Tc^S plasmids. Such heterodimers dissociate in both Rec⁺ and in recA hosts. Parental rather than mutant plasmids are then retained by the host cell.     

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

Summary A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA)·poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid.Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such as thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec ^- recipient strain of B. subtilis was used.Abbreviations Ap^R resistance to ampicillin - Tc^R resistance to tetracycline - Cm^R resistance to chloramphenicol - CCC covalently closed circular duplex - Mdal magadalton     

Chimeric streptococcal plasmids and their use as molecular cloning vehicles in Streptococcus sanguis (Challis).

Chimeric plasmids, which were useful as cloning vehicles in a Streptococcus sanguis (Challis) host vector system, have been constructed. By using three different strategies of restriction endonuclease digestion and ligation, a deoxyribonucleic acid (DNA) fragment bearing an erythromycin resistance determinant was ligated in vitro to a phenotypially cryptic plasmid from Streptococcus ferus. Recombinant plasmids could be recovered after transformation of S. sanguis (Challis) with these preparations. Three useful chimeras were constructed. pVA680, 5.5 megadaltons in size, contained a single KpnI site into which passenger DNA may be spliced. pVA736, 5.0 megadaltons in size, contained single EcoRI, HindIII, and KpnI sites into which passenger DNA may be spliced. The EcoRI and KpnI sites of pVA736 may be used in combination with one another when ligating DNA into this plasmid. pVA738, 3.7 megadaltons in size, contained single HindIII and AvaI sites into which passenger DNA may be spliced. pVA680, pVA736, and pVA738 were stably maintained as multicopy plasmids in S. sanguis (Challis). None of them continued to replicate (amplify) in chloramphenicol-treated cells. By using pVA736 as a vector, we have cloned a chloramphenicol resistance determinant obtained from a large, conjugative streptococcal R plasmid. In addition, chromosomal DNA sequences from Streptococcus mutans have been inserted into pVA736 by using the KpnI-EcoRI site combination.     

Construction of new cloning vehicles with genes of the tryptophan operon of Escherichia coli as genetic markers

In vitro recombination techniques were used to construct a series of new cloning vehicles with genes of the tryptophan (trp) operon of Escherichia coli as selective marker. To construct these plasmids we have made a restriction cleavage map of the trp operon for the enzymes AosI, AvaI, BglI, BglII, HindIII, HpaI, PvuII, SalI, SstI and XhoI. The constructed plasmids pHP39, pEP392, pEP3921 and pEP3923 are derived from the amplifiable plasmid pBR345 and carry two or more genes of the trp operon, which are controlled by the trp regulatory elements. Plasmid pEP3921 (7.0 kb) carries intact trpE and trpA genes and contains single BglII and SstI sites in trpE, a single HindIII site located between trpE and trpA, and single EcoRI, SalI and XhoI sites located outside the trp genes. Plasmid pEP121 (12 kb) is similar to pEP3921, but has an extra selective marker conferring bacterial resistance to ampicillin. Plasmid pEP3923 (7.4 kb) comprises intact trpB and trpA genes and single BglII, HindIII, EcoRI, SalI and XhoI sites. Plasmids pHP39 (9.8 kb) and pEP392 (9.8 kb) carry an intact trp operon and have two and one EcoRI site, respectively. Plasmid pHP3 (18 kb) carries an intact trp operon and markers for tetracycline and ampicillin resistance.     

DNase-resistant transfer of chromosomal cat and tet insertions by filter mating in Pneumococcus

Genes for chloramphenicol resistance (Cm^r) and tetracycline resistance (Tc^r), which are present as heterologous insertions in the chromosomes of some clinical isolates of Streptococcus pneumoniae (pneumococcus) and derivative strains, were transferred at a low frequency to other pneumococci by a DNase-resistant filter mating process that resembles conjugation. Cotransfer of Cm^r and Tc^r was the most common event. Tetracycline resistance was transferred alone from one Tc^r strain or rarely from Cm^rTc^r donors, whereas Cm^r was never transferred alone. Neither the donor strains nor the transconjugants contained detectable plasmids. Transconjugants acted as donors for transformation and for filter mating and had properties similar to those of the parent strain. The presence of the conjugative plasmid pIP501 in the donor did not appear to influence the transfer properties of the Cm^r or Tc^r determinants. No transfer of Cm^r or Tc^r toStreptococcus faecalis JH2-2 was observed.     

Prevalence of tetracycline resistance genes in Greek seawater habitats

The presence of selected tetracycline resistance (Tc^R) genes was studied in different Greek seawater habitats, originated from wastewater treatment facilities, fishfarm, and coastal environments. The methods employed included assessment of the presence of twelve gene clusters by PCR, followed by hybridization with specific probes, in habitat extracted DNA, Tc^R bacteria, and exogenous isolated plasmids conferring Tc^R. The direct DNA-based analysis showed that tet(A) and tet(K) genes were detected in all habitats, whilst tet(C) and tet(E) were present in fishfarm and wastewater effluent samples and tet(M) was detected in fish-farm and coastal samples. Resistance genes tet(h), tet(C), tet(K), and tet(M) were detected in 60 of the 89 isolates screened. These isolates were identified by fatty acid methyl ester analysis (FAME) as Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus, and Staphylococcus strains. The presence of the Tc^R genes in 15% of the bacterial isolates coincided with the presence of IncP plasmids. A habitat-specific dissemination of IncP alpha plasmids in wastewater effluent isolates and of IncP beta plasmids in fishfarm isolates was observed. Exogenous isolation demonstrated the presence of plasmids harbouring Tc^R genes in all the habitats tested. Plasmids were shown to carry tet(h), tet(C), tet(E), and tet(K) genes. It is concluded that Tc^R genes are widespread in the seawater habitats studied and often occur on broad host range plasmids that seem to be well disseminated in the bacterial communities.     

Versatile kanamycin-resistance cartridges for vector construction in Escherichia coli

To generate polylinker sequences which can be transferred together with an adjacent selectable marker, two plasmids (pWW-84 and pWW-97) were constructed which contain a kanamycin-resistance gene (KmR) flanked by various restriction sites. From these plasmids KmR-cartridges can be obtained as EcoRI, BamHI, SalI, AccI or HincII fragments for insertion into the appropriate restriction site of any plasmid. The following restriction sites can be introduced with these cartridges: BamHI, SalI (AccI, HincII), EcoRI, SacI, SphI and KpnI (Asp718) all adjacent to KmR, XhoI and HindIII, both within KmR. If desired, KmR can be removed by PstI digestion and religation, creating a single PstI site and leaving all adjacent sites intact.     

Amplification of the respiratory NADH dehydrogenase of Escherichia coli by gene cloning.

A relatively simple method has been used to clone the gene coding for the respiratory NADH dehydrogenase (NADH-ubiquinone oxidoreductase) of Escherichia coli from unfractionated chromosomal DNA. The restriction endonucleases EcoRI, BamI and HindIII were used to construct three hybrid plasmid pools from total E. coli DNA and the amplifiable plasmids pSF2124 and pGM706. Three different restriction endonucleases were used to increase the chances of cloning the ndh gene intact. Mobilization by the plasmid F was used to transfer the hybrid plasmids into ndh mutants and selection was made for Apr and complementation of ndh. DNA fragments complementing ndh were isolated from both the EcoRI and HindIII hybrid plasmid pools. The strain carrying the hybrid plasmid constructed with EcoRI produced about 8--10 times the normal level of the respiratory NADH dehydrogenase in the cytoplasmic membrane. Treating the cells with chloramphenicol to increase the plasmid copy number allowed the level of NADH dehydrogenase in the membrane to be increased to 50--60 times the level in the wild type. The results indicate the potential of gene cloning for the specific amplification of particular proteins prior to their purification.     

Specific pattern of instability of Escherichia coli HisG gene cloned in Bacillus subtilis via the Staphylococcus aureus plasmid pCS194

The plasmid pCS194, generated in vivo by recombination of two Staphylococcus aureus plasmids, pC194 and pS194, coding, respectively, for chloramphenicol (Cm) and streptomycin (Sm) resistance, can be replicated also in Bacillus subtilis in the presence of either of the two antibiotics. In their absence, no segregation of the individual components is observed, but the whole plasmid is lost at a rate of about 10% per generation. The unique EcoRI site of pCS194 is located in the Sm^R determinant. EcoRI-cleaved pCS194 has been joined to an EcoRI-linearized Escherichia coli replicon, the in vitro recombinant pHisG plasmid, composed of the vector pBR313 plus a BglII-segment of E. coli chromosomal DNA, containing a functional hisG gene. The ligation mixture has been used to transform either E. coli or B. subtilis. Following E. coli transformation and selection for Ap^R and Cm^R (the latter is expressed in E. coli by the pC194 determinant), two his⁺ clones were picked at random and the plasmids extracted. These appear identical and contain the original segments. Conversely, after transformation of B. subtilis and selection for Cm^R, only his^? clones have been obtained. From them, deleted plasmids have been extracted. They have lost part or, more frequently, all of the E. coli DNA insert. In the latter case also most of the bracketing pS194 sequence has been lost, and the resulting plasmids are almost identical to pC194, the Cm^R parent of pCS194. When the intact recombinant plasmids, isolated from his⁺ Ap^R Cm^RE. coli clones, have been used to transform B. subtilis cells for Cm^R, again deleted plasmids almost identical to pC194 have been obtained. The events causing these rearrangements occur after in vitro ligation, during either transformation or early propagation of the plasmids, and are probably caused by a translocatable element present in pCS194. A detailed physical map of pC194, carrying the restriction sites for HindIII, HaeIII, HpaII, MboII, AluI, HhaI, and BglI, is presented.     

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.     

Structure and properties of the region of homology between plasmids pMB1 and ColE1

Summary Physical maps of the two independently isolated Escherichia coli plasmids, pMB1 and ColE1, were prepared with 13 restriction endonucleases and compared. A 5.1 kilobase continuous region covering 55% of pMB1 and 75% of ColE1 was found to have similar, but non-identical, restriction maps. The differences in the maps of this region probably arose by localized mutational events rather than by major sequence rearrangements. The F-factor was found to mobilize pMB1 efficiently for conjugal transfer. A region on pMB1 required for its F-mediated transfer was mapped. Results of our study combined with results of other investigators suggest that pMB1 and ColE1 share functional properties such as colicin production, colicin immunity, mode of replication, and mobilization by the F-factor, and that the sequences required to code these functions are contained within the 5.1 kilobase homologous region.     

Cloning the genetic determinant of alpha-hemolysin and obtaining a series of insertional mutations in this determinant by the transposon Tn1000

Functionally active genetic determinant of alpha-hemolysin was cloned. Hemolytic plasmid pHly195 was used as a donor of the determinant and pBR322 plasmid served as recipient. Cloning was done with a help of HindIII restriction endonuclease. The recombinant plasmid obtained represents pBR322 plasmid with the built-in fragment of 7.4 kb containing genes of functionally active determinant of alpha-hemolysin. Restriction map was constructed using HindIII, EcoRI, BamHI and SalI restriction endonucleases. Insertional mutagenesis was carried out with the help of the Tn1000 transposon. Plasmid DNAs were isolated from insertional mutants of Hly- phenotype and treated with EcoRI, SalI and BamHI. On the basis of the sizes of restriction fragments of the mutant plasmid DNAs localization and orientation of insertions of Tn1000 into the cloned determinant of alpha-hemolysin were determined.     

Human papillomavirus DNA: physical mapping of the cleavage sites of Bacillus amyloliquefaciens (BamI) and Haemophilus parainfluenzae (HpaII) endonucleases and evidence for partial heterogeneity.

The DNA of human papillomavirus (HPV) obtained from a pool of plantar warts is cleaved by bacillus amyloliquefaciens (BamI) and Haemophilus parainfluenzae (HpaII) restriction endonucleases at one and four specific sites, respectively. These sites were localized on the previously established cleavage map of HPV DNA, using the Hind, HindIII, HpaI, and EcoRI endonuclease restriction sites as reference. The four HpaII sites were mapped, clockwise, at 1.4, 41.1, 44.3, and 52.8% of the genome length from the unique BamI cleavage site taken as point zero. The HpaII site mapped at 1.4% of the genome length was absent in 40 to 50% of the molecules, thus showing a genetic heterogeneity of HPV DNA.     

Organization and expression of a two-gene cluster in the arginine biosynthesis of Saccharomyces cerevisiae.

Summary Using the pMB9 recombinant plasmid pMY3, which contains a functional gene for the tRNA^Try mutant Su⁺7, the EcoRI fragment containing the tRNA^Try gene is mapped and oriented with respect to the HindIII site in the tetracycline region of pMB9. Complete HpaII and HaeIII maps of the EcoRI fragment are derived. The Su⁺7 tRNA gene is placed by hybridization to these fragments, and the tRNA gene is oriented by using the restriction sites for HinfI, TaqI, and HpaII in the tRNA gene itself. A tRNA^Asp gene is shown to lie adjacent to tRNA^Try, and is also placed and oriented in the map. The RI fragment itself originates in a locus adjacent to, and transcribed in the same direction as, the ribosomal RNA genes of 80d₃.The implications of the structure of the cloned DNA for its previously measured regulatory and tRNA gene activities are discussed. In particular, the effect on the regulation of RNA synthesis is attributable to an E. coli DNA sequence, but cannot be due to the presence of a normal tRNA promoter on the plasmid.Abbreviations MD megadaltons; expressions of the form HpaII:0.075 refer to a fragment generated by the indicated restriction nuclease, having the indicated molecular weight, in MD     

Curing of Escherichia coli K12 plasmids by coumermycin

Summary Low concentrations of the antibiotic coumermycin A₁, the inhibitor of bacterial DNA gyrase, effectively eliminate pBR322, pMB9 and other ColE1 related plasmids from E. coli K12 strains. The curing action of antibiotic seems to result from the plasmid degradation and not just from the inhibition of replication.     

Genetic and physical characterization of the ColIb plasmid using ColIb-R222 hybrids

Summary The presence of the ColIb plasmid in Escherichia coli cells inhibits the growth of bacteriophages BF23 and T5 (Ibf phenotype; inhibition of BF23 and T5 growth). To understand this abortive infection, we devised a method of isolating mutants that were defective in some ColIb phenotypes including Ibf. This method consisted of transduction of the tet (Tc^r; tetracycline resistance) or cml (Cm^r; chloramphenicol resistance) gene of plasmid R222 with phage P22 into ColIb, construction of Tc^rCm^rIbf⁺ Imm⁺ (immunity to colicin Ib) Cib^- (no production of colicin Ib) recombinants by crossing between the transductants, and isolation of deletion mutants from the recombinants by phage P1 transduction. By this procedure, pKM25-2 (Tc^rCm^sIbf^-Imm^-Cib^-) and pKM25-1 (Tc^rCm^sIbf⁺Imm⁺Cib^-) were isolated. Construction of the cleavage map of the ColIb plasmid by restriction endonucleases and comparative analyses of the DNA fragments produced from the mutant plasmids revealed that the genes determining Ibf and Imm mapped on a 4.60 Mdal HindIII fragment (H-3) and the gene determining Cib on a 1.71 Mdal EcoRI fragment (E-12).These results together with other observations (Wilkins et al. 1981; Hama personal communication) also show the approximate positions of the genes for Rep (replication), Inc (incompatibility), and Sog (suppression of dnaG) as well as Ibf, Imm, and Cib phenotypes on the cleavage map of the ColIb plasmid.Preliminary data were reported in the 1979 Annual Meeting of the Japan Molecular Biology Society (Uemura and Mizobuchi, Abst Ann Mol Biol Meet 1979, p 36)