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PubMed1975

Cloning, isolation, and characterization of replication regions of complex plasmid genomes.

EcoRI endonuclease-generated DNA fragments carrying replication regions of the F'lac and R6-5 plasmids have been cloned and isolated, using as a selection vehicle a nonreplicating ampicillin-resistance DNA fragment derived from a Staphylococcus aureus plasmid. Heteroduplex analysis of the constructed plasmid chimeras and the parent replicons has localized the cloned R6-5 replication region to a DNA segment between kilobase pair coordinates 1.0 and 88.0 on the R6-5 map. Physical proximity between the plasmid replication functions and the locus governing plasmid incompatibility has been shown for both parent replicons. The cloning method reported appears to be generally applicable for the identification and isolation of replication regions of a variety of complex genomes.

Centrifugation, Density Gradient↗
PubMed1975

Sulfonamide resistance mechanism in Escherichia coli: R plasmids can determine sulfonamide-resistant dihydropteroate synthases.

Several natural isolate E. coli strains highly resistant to sulfonamides and antibiotics are shown to contain a sulfonamide-resistant dihydropteroate synthase (2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine-diphosphate:4-aminobenzoate 2-amino-4-hydroxydihydropteridine-6-methenyltransferase, EC 2.5.1.15) in addition to the normal sensitive enzyme. The resistant dihydropteroate synthases examined are determined by an R plasmid and are smaller and less heat stable than the normal sulfonamide-sensitive enzyme. One synthase resistant to any sulfonamide tested, and to sulfanilic and arsanilic acids, was still inhibited by several non-sulfonamide analogs of p-aminobenzoate. Citrobacter and Klebsiella pneumoniae strains also show similar mechanisms of sulfonamide resistance.

Aminobenzoates↗
PubMed1975

Bidirectional replication of plasmid R6K DNA in Escherichia coli; correspondence between origin of replication and position of single-strand break in relaxed complex.

Replicating molecules of plasmid R6K DNA have been purified as covalently closed circular DNA forms and analyzed in the electron microscopy after cleavage with the EcoRI restriction endonuclease. It has been determined that in most cases replication proceeds bidirectionally from an origin whose position is indistinguishable from the site of the single-strand break (nick) in the open circular DNA form of the relaxation complex of R6K DNA and protein. Evidence is presented for the existence of a unique replication terminus asymmetrically placed approximately 20% of genome size from the origin. The positions of the replication forks in a majority of the molecules indicate that replication proceeds sequentially from the fixed origin first in one direction to the terminus and then progresses from the origin in the other direction.

DNA Replication↗
PubMed1975

Translocation of a plasmid DNA sequence which mediates ampicillin resistance: molecular nature and specificity of insertion.

A series of recombinant plasmids was generated in Escherichia coli in which the TEM beta-lactamase translocon (TnA) was inserted into the small plasmid RSF1010. RSF1010 is a 5.5 X 10(6) dalton nonconjugative plasmid which confers resistance to streptomycin and sulfonamide. The recombinant plasmids can be classified into three clearly defined phenotypic groups. Group I is ampicillin-, streptomycin- and sulfonamide-resistant. Group II is ampicillin- and sulfonamide-resistant but has lost streptomycin resistance. Group III is ampicillin-resistant but is sensitive to sulfonamide and shows a simultaneous 30-fold reduction in the minimal inhibitory concentration of streptomycin. It was possible to map the site of insertion of TnA within RSF1010 by electron microscope studies of DNA heteroduplexes formed between RSF1010 and recombinant plasmids. Insertions of TnA occur at, at least, 12 distinct sites in a region corresponding to one-third of the RSF1010 DNA molecule. Those insertions giving rise to particular phenotypes are clustered. Insertions of TnA-like insertion sequences (IS) appear to give rise to strongly polar mutations.

Ampicillin↗
PubMed1975

Transposition of R factor genes to bacteriophage lambda.

Transpositions of segments of R factor (antibiotic resistance plasmids) to bacteriophage lambda have been selected and characterized. Cells of Escherichia coli harboring R factors that determine kanamycin resistance were infected with phage lambda, and lambdakan transducing lines were obtained. Each of the three examined is unusual when compared to lambda transducing phages containing E. coli chromosomal genes: the kan insertions (a) occur at several sites, each well removed from the integration region POP', (b) are not associated with deletion of lambda phage DNA, and (c) are separable from the lambda genome during transduction or during lytic growth. Two insertions from the same R factor contain 1.5 kilobase sequences repeated in inverted order. The properties of the lambdakan phage suggest that R factors contain systems capable of mediating genetic exchange in the absence of extensive DNA homology. It is suggested that such systems of exchange may have played important roles in R factor evolution.

Chromosome Mapping↗
PubMed1975

Transformation of Pseudomonas putida and Escherichia coli with plasmid-linked drug-resistance factor DNA.

Conditions optimal for the transformation of Pseudomonas putida and E. coli with a drug-resistance factor (RP 1) DNA, which specifies resistance to carbenicillin, tetracycline, kanamycin, and neomycin, are described. The transformants retain all the fertility, incompatibility, and drug-resistance characteristics present in the parent. Covalently-closed circular molecules of almost identical contour lengths have been isolated from the parent and the transformants. The frequency of transformation is drastically reduced by treatment of RP 1 DNA with DNase and by denaturation or sonication. Shearing of RP 1 DNA in vitro and their subsequent introduction in P. putida cells, by transformation, produces transformants that exhibit a wide range of drug-resistant phenotypes, including those which are resistant to neomycin but sensitive to kanamycin. Isolation of such neomycin-resistant but kanamycin-sensitive transformants indicates that there might be two separate mechanisms specified by RP 1 for resistance to the two antibiotics.

Calcium↗
PubMed1976

Novel F prime factors able to replicate in Escherichia coli Hfr strains.

A novel type of F' plasmid that can replicate extrachromosomally in cells of Hfr strains has been isolated. Genetic analysis of these plasmids indicates that all carry the dnaA-bglA region of the E. coli chromosome as well as ilv+ used as a selective marker. It is suggested that a specific site, designated poh+ (permissive on Hfr), is located in this region, and is essential for these plasmids to replicate in Hfr cells. The most interesting explanation, among others, would be that the poh+ site represents the replication origin of the bacterial chromosome.

Chromosome Mapping↗
PubMed1976

Molecular cloning of DNA from F sex factor of Escherichia coli K-12.

We describe the molecular cloning of various DNA segments generated by partial EcoRI endonuclease digestion of the sex factor F. These segments have been analyzed by agarose gel electrophoresis of EcoRI digests and were arranged in a series of overlapping fragments using the EcoRI fragment map of F established by H. Ohtsubo and E. Ohtsubo. The clones isolated demonstrate one or more of the following F-specified functions: inhibition of female-specific phage (T7) multiplication, formation of F pili, surface exclusion, or immunity to lethal zygosis. These properties are discussed in terms of the EcoRI fragments of F that specify them.

Coliphages↗
PubMed1976

Amplification in Escherichia coli of enzymes involved in genetic recombination: construction of hybrid ColE1 plasmids carrying the structural gene for exonuclease I.

Endo-R-HindIII restriction endonuclease fragments obtained from F30 and pMB9 plasmid DNAs were ligated in vitro and used to transform a recB21 recC22 sbcB15 strain of E. coli K-12. The inability of this strain to stably maintain pMB9 alone permitted the isolation of transformants that carried hybrid plasmids containing the sbcB+ allele. These transformants became sensitive to ultraviolet light and recombination defieient and showed a 25-fold increase in the level of exonuclease I activity. The stability of the sbcB hybrid plasmids and their effects on exonuclease I activity have also been determined in wild-type and recA1 genetic backgrounds. The presence of the plasmids results in a 7-fold increase in the level of exonuclease I in a wild-type strain and 15-fold increase in a recA1 strain. The increased activity in the recA1 mutant appears to be a result of increased plasmid stability in this genetic background.

Base Sequence↗
PubMed1976

Biochemical construction of specific chimeric plasmids from ColE1 DNA and unfractionated Escherichia coli DNA.

A series of chimeric plasmids was constructed using colicinigenic factor E1 (ColE1) DNA as the replicon and DNA fragments carrying the galactose or tryptophan operons from E. coli. Restriction endonuclease EcoRI digests of ColE1 DNA and various DNAs containing the trp or gal operons were joined by T4 polynucleotide ligase [polynucleotide synthetase (ATP), poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming), EC 6.5.1.1]. Chimeric plasmids carrying the desired genes were selected after transformation of Trp- or Gal- cells with ligated DNA. By using this method, we constructed ColE1-gal and ColE1-trp chimeric plasmids in which the source of the bacterial gal and trp operons was an unfractionated EcoRI digest of total E. coli DNA. The frequency of recovery of such chimeric plasmids is 10 to 20 colonies per mug of ligated DNA used in the transformation step. The method utilized in this report for constructing specific chimeric plasmids from total E. coli DNA is very simple. It requires only endonuclease R-EcoRI and T4 polynucleotide ligase, both of which are commercially available. The yield of transformants suggests that this method will be useful for cloning and amplifying a wide variety of functionally defined genes from E. coli and other prokaryotic organisms.

Colicins↗
PubMed1976

A plasmid cloning vehicle allowing regulated expression of eukaryotic DNA in bacteria.

We have constructed a plasmid cloning vehicle in which transcription of inserted heterologous DNA fragments can be regulated by a defined bacterial operator and promoter. The lambda plac 5 EcoRIDNA fragment containing the operator, promoter, and beta-galactosidase gene of the lactose operon was linked to the ColE1 derivative plasmid pSF2124, creating a plasmid designated pBGP100, pBGP100 contains one EcoRI site at the lac DNA/pSF2124 DNA junction and another at the lambda DAN/pSF2124 DNA junction. We deleted the latter EcoRI site to generate a plasmid (pBGP120) retaining a single EcoRI site at the lac DNA/nSF2124 DNA junction. To determine whether DNA introduced at the EcoRI site of pBGP120 was expressed under lactose control, we inserted the EcoRI fragment containing 28S ribosomal DNA of Xenopus laevis, creating the hybrid plasmid pBGP123. RNA-DNA hybridization of pulse-labeled RNA from cells containing pBGP123 showed that induction of the lac operon increases the percentage of labeled RNA complementary to Xenopus 28S DNA about 9-fold. This vehicle may be of use for production of eukaryotic gene products in bacteria.

Animals↗
PubMed1976

Expression of the thymidylate synthetase gene of the Bacillus subtilis bacteriophage Phi-3-T in Escherichia coli.

The thymidylate synthetase gene of B. subtilis bacteriophage Phi-3-T, when cloned in plasmids pSC101 or pMB9 is expressed in E. coli. The promoter of the cloned gene is likely to originate in Phi-3-T. Rearrangements of hybrid plasmid sequences during the cloning have been noted. B. subtilis strains can be transformed with hybrid DNAs. The transformants contain sequences of Phi-3-T, but not those of plasmid vectors.

Bacillus subtilis↗
PubMed1976

Construction of plasmids carrying the cI gene of bacteriophage lambda.

By techniques of recombination in vitro, we have constructed a plasmid bearing the repressor gene (cI) of bacteriophage lambda fused to the promoter of the lac operon. Strains carrying this plasmid overproduce lambda repressor. This functional cI gene was reconstituted by joining DNA fragments bearing different parts of that gene. Flush end fusion techniques, involving no sequence overlap, were necessary for the construction; in certain cases, the abutting of the DNA molecules bearing ends generated by different restriction endonucleases creates a sequence at the junction which is recognized by one of the restriction endonucleases.

Coliphages↗
PubMed1976

Modified recombination and transmission of mitochondrial genetic markers in rho minus mutants of Saccharomyces cerevisiae.

A large number of primary petite (rho-) clones were isolated after ethidium bromide mutagenesis of various grande (rho+) strains of S. cerevisiae that contained the mitochondrial genetic markers, CR, ER, OIR (or OIIR), and PR. From the frequency of coretention of markers in the petites, we have deduced a probable circular order of the markers in the grande mitochondrial genome. From these primary clones several series of pure and stable petite clones were obtained and analyzed genetically. (a) In general, the omega allele is retained or lost together with the region carrying both CR and ER markers. (b) The petites that have retained only the CR marker fall into two classes: some have kept the omega allele of the grande strain they issued from; others exhibit a new omega expression. (c) The proportion of diploid petites in petite X grande crosses is independent of the presence of the omega allele. (d) In most cases, the coordinated transmission of markers observed so far in all grande X grande nonpolar corsses does not exist anymore in petites.

Chromosome Mapping↗
PubMed1976

Large subunit ribulosebisphosphate carboxylase messenger RNA from Euglena chloroplasts.

RNA from wild-type Euglena, aplastidic mutant cells, and purified chloroplasts was chromatographed on oligo(dT)-cellulose at 4 degrees. The poly(A)-containing and poly(A)-lacking fractions from each were then tested in a cell-free protein-synthesizing system from wheat germ for their abilities to specifically stimulate synthesis of large subunit ribulosebisphosphate carboxylase [EC 4.1.1.39; 3-phospho-D-glycerate carboxy-lyase (dimerizing)]. The large subunit polypeptide (59,000 molecular weight) was identified in the in vitro reaction by two-dimensional electrophoresis involving isoelectric focusing and size filtration on polyacrylamide gels. Template activity for the large subunit was detected in wild-type cells but not in aplastidic mutant strains; it was highly enriched in the isolated chloroplasts. This messenger was present only in the poly(A)-lacking RNA fraction, where it constituted the most prominent template species of chloroplast RNA. The large subunit message was freed of considerable non-messenger RNA contamination and localized to the 10-20S fraction by sucrose gradient centrifugation.

Carboxy-Lyases↗
PubMed1976

Variant forms of mitochondrial translation products in yeast: evidence for location of determinants on mitochondrial DNA.

Products of mitochondrial protein synthesis in yeast have been labeled in vivo with 35SO42-. More than 20 polypeptide species fulfilling the criteria of mitochondrial translation products have been detected by analysis on sodium dodecyl sulfate-exponential polyacrylamide slab gels. A comparison of mitochondrial translation products in two wild-type strains has revealed variant forms of some polypeptide species which show genetic behavior consistent with the location of their structural genes on mtDNA. Our results demonstrate the feasibility of performing genetic analysis on putative gene products of mtDNA in wild-type yeast by direct examination of the segregation and recombination behavior of specific polypeptide species.

Chloramphenicol↗
PubMed1976

Molecular cloning of DNA fragments produced by restriction endonucleases Sa1I and BamI.

The highly specific restriction endonucleases Sa1I and BamI produce DNA fragments with complementary, cohesive termini that can be covalently joined by DNA ligase. The Escherichia coli kanamycin resistance factor pML21 has one SalI site, at which DNA can be inserted without interfering with the expression of drug resistance or replication of the plasmid. A more convenient cloning vehicle can be made with the tetracycline resistance factor pSC101, since insertion of DNA either at its single site for Sa1I or at that for BamI inactivates plasmid-specified drug resistance but not replication. To take advantage of this insertional inactivation, pSC101 was joined to a Co1E1-ampicillin resistance plasmid having no Sa1I site, and to a Co1E1-kanamycin resistance plasmid having no BamI site. Chimeras formed with the resulting hybrid vehicles can be identified simply by replica plating. These three vehicles, which all replicate under relaxed control, have been used to clone and amplify Drosophila melanogaster DNA fragments.

Bacillus↗