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PubMed1989

Entamoeba histolytica ribosomal RNA genes are carried on palindromic circular DNA molecules.

Highly abundant DNA fragments obtained after restriction enzyme digests of nuclear DNA of Entamoeba histolytica strain HM-1:IMSS have been cloned and characterized. Northern blot hybridization to E. histolytica rRNA and sequence analysis identified the abundant DNAs as ribosomal DNA containing species. Several overlapping clones containing these abundant DNAs were isolated from 4 different genomic libraries of E. histolytica. Alignment of the restriction maps was consistent with a circular molecule, about 24.6 kilobase pairs (kb) in size. Nuclease BA131 digestion provided additional evidence for the circular nature of this DNA. The ribosomal DNA molecule contains two large inverted repeat-regions, each at least 5.2 kb in length. Sequence analysis of clone R715 revealed homology to the large rRNA units of various eukaryotic organisms. This clone was located in both inverted repeats, suggesting two rRNA cistrons per molecule. The inverted repeats are flanked by stretches of DNA which contain tandemly reiterated sequences. Southern blot analysis of E. histolytica nuclear DNA revealed the presence of two populations of molecules. These molecules have identical arrangements of restriction sites, but differ in size (0.7 kb) in a fragment containing tandemly reiterated sequences. Analysis of E. histolytica nuclear DNA by electron microscopy also revealed circular molecules. These molecules are about 26.6 kb +/- 0.5 kb in size and contain structural features predicted by the restriction map of the extrachromosomal ribosomal DNA of E. histolytica.

Animals↗

PubMed1974

Plasmids controlling synthesis of hemolysin in Escherichia coli: molecular properties.

Covalently closed extrachromosomal deoxyribonucleic acid (DNA) was isolated from alpha-hemolytic wild-type strains of Escherichia coli. Most strains examined were able to transfer the hemolytic property with varying frequencies to nonhemolytic recipient strains. Out of eight naturally isolated alphahemolytic E. coli strains, four contained a set of three different supercoiled DNAs with sedimentation coefficients of 76S (plasmid A), 63S (plasmid B), and 55S (plasmid C). The sedimentation coefficients and the contour lengths of the isolated molecules correspond to molecular weights of 65 x 10(6), 41 x 10(6), and 32 x 10(6). Three alpha-hemolytic wild-type strains carried only one plasmid with a molecular weight of 41 x 10(6), and one strain harbored two plasmids with molecular weights of 41 x 10(6) and 32 x 10(6). Alpha-hemolytic transconjugants were obtained by conjugation of E. coli K-12 with the hemolytic wild-type strains. A detailed examination revealed that plasmids with the same sizes as plasmids B and C of the wild-type strains can be transferred separately or together to the recipients. Both plasmids possess the hemolytic determinant and transfer properties. Plasmid A appears to be, at least in one wild-type strain, an additional transfer factor without a hemolytic determinant. In one case a hemolytic factor was isolated, after conjugation, that is larger in size than plasmid A and appears to be a recombinant of both plasmids B and C.

Centrifugation, Density Gradient↗

PubMed1976

Isolation, by tetracycline selection, of small plasmids derived from R-factor R12 in Escherichia coli K-12.

The examination, by agarose gel electrophoresis, of tetracycline-resistant colonies of Escherichia coli K-12 carrying R-factor R12 reveals the presence of smaller plasmid deoxyribonucleic acids (DNAs), incompatible with R12, in many of the clones. These plasmids are demonstrated to be homologous with R12 DNA by electron microscope heteroduplex experiments and by the production of consistent fragment patterns upon digestion with various restriction endonucleases. These autonomously replicating plasmids form a related series of covalently closed circular DNA molecules ranging in size from 3.6 X 10(6) to 61 X 10(6) daltons. Plasmids of molecular weight between 3.6 X 10(6) and 37 X 10(6) confer no antibiotic resistances, but when jointly present with R12 by nonetheless enhance the expression of the tetracycline resistance associated with this latter molecule.

DNA Restriction Enzymes↗

PubMed1977

Aminoglycoside-modifying enzyme of an antibiotic-producing bacterium acts as a determinant of antibiotic resistance in Escherichia coli.

Bacillus circulans NRRL B-3312, a nonpathogenic bacterium that produces the aminoglycoside antibiotic butirosin, is known to contain an aminoglycoside phosphotransferase that is similar to the neomycin phosphotransferases of clinically isolated antibiotic-resistant bacteria. Purified DNAs from B. circulans and the plasmid ColE1-ApR were digested with EcoRI endonuclease and the resulting fragments covalently joined with polynucleotide ligase. The recombined DNA was used to transform E. coli and ampicillin-neomycin resistant colonies were selected. Analysis of several clones indicated that neomycin resistance in the E. coli transformants was due to the presence of the B. circulans phosphotransferase gene. This observation is consistent with the notion that anitbiotic-modifying enzymes from antibiotic-producing organisms may be the sources of antibiotic resistance in plasmid-containing bacteria.

Bacillus↗

PubMed1977

Characterization of a plasmid from Streptomyces coelicolor A3(2).

Covalently closed circular deoxyribonucleic acid (DNA) with a molecular weight of 20 X 10(6) was identified in strains of Streptomyces coelicolor A3(2) of various fertility types. Hybridization studies and digestion by various restriction endonucleases indicated that the circular DNAs (pSH1) were identical regardless of the fertility type (UF, IF, or NF) of the strain from which it was isolated. The pSH1 DNA was cleaved to many fragments by the endonucleases HincII, SmaI, and SalI and to three or four fragments by BamHI and PstI. Plasmid pSH1 carries single sites for each of the two restriction enzymes, EcoRI and HindIII. These sites are 7.6 X 10(6) daltons apart. Attempts to isolate the fertility factor SCP1 as covalently closed circular DNA were unsuccessful. These data suggest that the biochemically isolated plasmid pSH1 is not identical to the genetically characterized fertility factor SCP1, which has been identified in an autonomous state in IF-type strains and in an integrated state in NF-type strains.

Bacteriocins↗

PubMed1977

Analysis of chromosomal integration and deletions of yeast plasmids.

Plasmid DNAs from six strains of Saccharomyces cerevisiae were compared. Three different plasmids were found, designated Scp 1, Scp 2 and Scp 3, with monomer lengths of 6.19, 6.06 and 5.97 kilobases as referenced to sequenced phiX174 DNA. DNA from each of the plasmids was inserted into a lambda vector DNA. Hybrid phage containing inserted DNA of the desired size were enriched by genetic selection and their DNAs analysed by rapid techniques. All three plasmids share the same organization, two unique sequences separated by two inverted repeats, and share basically the same DNA sequences. Scp 2 and Scp 3 differ from Scp 1 by missing a unique HpaI site and by having small overlapping deletions in the same region. The HpaI site in Scp 1 is, therefore, in a nonessential region and suitable for insertion of foreign DNA in the potential use of the yeast plasmid as a vector. Hybridization of labelled cloned plasmid DNA to restriction fragments of linear yeast DNA separated on agarose gels showed that the plasmid DNA was not stably integrated into the yeast chromosomal DNA.

Chromosomes↗

PubMed1976

The relative positions of sea urchin histone genes on the chimeric plasmids pSp2 and pSp17 as studied by electronmicroscopy.

The relative positions of the sea urchin histone genes and the spacer regions on the chimeric plasmids pS p2 and pSp17 have been mapped by hybridizing total histonemessenger RNA to single strands of the plasmid DNAs. The lengths and spacing between the several RNA:DNA duplex regions on the single strands of DNA were measured by the gene 32-ethidium bromide electron microscope mapping method. We find that the genes are interdigitated with spacer sequences of different lengths; that there are three coding sequences on pSp2, all on the same strand, with the relative order H1, H4, and B4; and that there are two coding sequences on pSp17, both on the same strand, corresponding to the messages denoted B1 and B2-B3, where B4, B1, and B2-3 are electrophoretically resolved components of histone mRNA, all of size intermediate between the larger H1 and the smaller H4 message.

Chimera↗

PubMed1988

The H circles of Leishmania tarentolae are a unique amplifiable system of oligomeric DNAs associated with drug resistance.

We have induced drug resistance against methotrexate, an inhibitor of dihydrofolate reductase, and CB3717, an inhibitor of thymidylate synthetase in a strain of Leishmania tarentolae. The drug-resistant strains contain extrachromosomal DNA circles of 68 kilobases with a 30-kilobase inverted duplication flanked by 4- and 5 kilobase unique segments. We show that these circles are highly homologous to the drug-induced H circles of L. tropica (1). All three L. tarentolae strains analyzed contain a chromosomal copy of the H region without duplication, but two of the three strains contain extrachromosomal H circles as well, predominantly present as H circle dimers in one strain and as tetramers in the other. After induction of methotrexate resistance, monomeric circles, presumably derived from the oligomers, become the major type of circle. Our results indicate that the H region represents a genomic region that can be copied at very low frequency to yield circles by a precise, but unusual mechanism under natural conditions in wild-type cells. Although superficially analogous to the episomes of bacteria, the system is without precedent in nature.

Animals↗

PubMed1975

Biochemical construction and selection of hybrid plasmids containing specific segments of the Escherichia coli genome.

Using a poly(dA-dT) "connector" method, a population of annealed hybrid circular DNAs was constructed in vitro; each hybrid DNA circle containing one full-length molecule of poly(dT)-tailed DNA from E1 colicinogenic factor (Col E1) fragmented by EcoRI endonuclease annealed to any one of a collection of poly(dA)-tailed linear DNA fragments of the entire E. coli genome. This annealed, but unligated, hybrid DNA was used to transform several different auxotrophic mutants of E. coli, and by direct selection, bacterial clones were isolated which contained specific hybrid plasmids. In this manner, bacterial strains containing Col E1 hybrid plasmids carrying the entire tryptophan operon or the arabinsoe and leucine operons were isolated. The methods described should allow the molecular cloning of any portion of the E. coli genome by selection from a pool of DNA molecules containing at least several hundred different hybrids representing the entire bacterial genome.

Arabinose↗

PubMed1986

[Isolation and properties of recombinant DNA from a plasmid and filamentous phage].

In the course of studying extrachromosomal DNA with composite replicons, a hybrid has been constructed by the in vitro recombination of the filamentous phage M13mp2 DNA (RF) and plasmid pUR222 (ApR). Both parental DNAs contain a fragment of lac-operon (ca. 800 bp), which includes the distal end of lacI gene, lacPO segments, and the lacZ gene proximal region coding for 145 N-terminal amino acid residues of beta-galactosidase and thus providing for alpha-complementation, the effect being cancelled with a polynucleotide insertion at the unique EcoRI site in the lacZ gene segment. E. coli BMH71-18 cells were transformed with the ligated mixture of EcoRI restricts of both DNAs. A phage-like nucleoprotein was isolated from colourless plaques (on the Xgal- and IPTG-supplemented medium); its deproteinization yielded a DNA which contains the ApR-determinant and, according to PAGE, structurally specific staining, restriction analysis, sequencing by the Sanger procedure, and electron microscopy data, is a linear double-stranded molecule comprising the phage and plasmid genomes in an equimolar ratio. Since the hybrid DNA does not display the alpha-complementation effect, both bacterial inserts are in the opposite orientation. Transformation of both phage (F+) and plasmid (F-) hosts with the hybrid DNA led to cultures which, after precipitation of the nucleoprotein from the extracellular medium and deproteinization, afforded the same composite DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Mapping↗

PubMed1977

Functional expression of cloned yeast DNA in Escherichia coli.

A collection of hybrid circular DNAs was constructed in vitro using the poly(dA-dT) "connector" method: each hybrid circle contained one molecule of poly(dT)-tailed DNA of plasmid ColE1 (made linear by digestion with EcoRI endonuclease) annealed to a poly(dA)-tailed fragment of yeast (Saccharomyces cerevisiae) DNA, produced originally by shearing total yeast DNA to an average size of 8 X 10(6) daltons. This DNA preparation was used to transform E. coli cells, selecting colicin-E1-resistant clones that contain hybrid ColE1-yeast DNA plasmids. Sufficient numbers of transformant clones were obtained to ensure that the hybrid plasmid population was representative of the entire yeast genome. Various hybrid ColE1-yeast DNA plasmids capable of complementing E. coli auxotrophic mutations were selected from this population. Plasmid pYeleu 10 complements several different point or deletion mutations in the E. coli or S. typhimurium leuB gene (beta-isopropylmalate dehydrogenase); plasmids pYeleu11, pYeleu12, and pYeleu17 are specific suppressors of the leuB6 mutation in E. coli C600. Plasmid pYehis2 complements a deletion in the E. coli hisB gene (imidazole glycerol phosphate dehydratase). Complementation of bacterial mutations by yeast DNA segments does not appear to be a rare phenomenon.

Alleles↗

PubMed1993

Unidirectional dominance of cytoplasmic inheritance in two genetic crosses of Plasmodium falciparum.

Malarial parasites have two highly conserved cytoplasmic DNA molecules: a 6-kb tandemly arrayed DNA that has characteristics of a mitochondrial genome, and a 35-kb circular DNA that encodes functions commonly found in chloroplasts. We examined the inheritance pattern of these elements in two genetic crosses of Plasmodium falciparum clones. Parent-specific oligonucleotide probes and single-strand conformation polymorphism analysis identified single nucleotide changes that distinguished the parental 6- and 35-kb DNA molecules in the progeny. In all 16 independent recombinant progeny of a cross between a Central American clone, HB3, and a Southeast Asian clone, Dd2, the 6- and 35-kb DNAs were inherited from the Dd2 parent. In all nine independent recombinant progeny of a cross between clone HB3 and a likely African clone, 3D7, the 6-kb DNA was inherited from the 3D7 parent. Inheritance of cytoplasmic genomes of the Dd2 and 3D7 parents was, therefore, dominant over that of the HB3 parent. Cytoplasmic DNA molecules were found almost exclusively in the female gametes of malarial parasites; hence, clone HB3 did not appear to have served as a maternal parent for the progeny of two crosses. Defective differentiation into male gametes by clone Dd2 is likely to be a reason for the cytoplasmic inheritance pattern seen in the HB3 x Dd2 cross. However, incompetence of male or female gametes is unlikely to explain the uniparental dominance in recombinant progeny of the HB3 x 3D7 cross, since both parents readily self-fertilized and completed the malaria life cycle on their own. Instead, the data suggest unidirectional parental incompatibility in cross-fertilization of these malarial parasites, where a usually cosexual parental clone can participate only as a male or as a female. Such an incompatibility may be speculated as indicating an early phase of reproductive isolation of P. falciparum clones from different geographical regions.

Animals↗

PubMed1976

A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome.

Using the poly(dA-dT) "connector" method (Lobbanand Kaiser, 1973), a population of annealed hybrid circular DNAs was constructed in vitro; each hybrid DNA circle contained one molecule of poly(dT)-tailed Col El-DNA (LRI) annealed to any one of a collection of poly(dA)-tailed linear DNA fragments, produced originally by shearing total E. coli DNA to an average size of 8.5 x 10(6) daltons. This annealed DNA preparation (12 mug) was used to transform an F+ recA E. coli strain (JA200), selecting transformants by their resistance to colicin El. A collection or "bank" pf pver 2000 colicin El-resistant clones was thereby obtained, 70% of which were shown to contain hybrid Col El DNA (E. coli) plasmids. This colony bank is large enough to include hybrid plasmids representative of the entire E. coli genome. Individual plasmids have been readily identified by replica mating the collection onto plates seeded with cultures of various F- auxotrophic recipients, selecting for complementation of the auxotrophic markers by F-mediated transfer of hybrid plasmids to the F- recipients. In this manner, over 80 hybrid Col El-DNA (E. coli), plasmid-bearing clones have been identified in the colony bank, and about 40 known E. coli genes have been tentatively assigned to these various plasmids. The hybrid plasmids are transferred efficiently from F+ donors to appropriate F- recipients. The use of this method to establish similar colony banks in E. coli containing hybrid plasmids representative of various simple eucaryotic genomes is discussed.

Chromosome Mapping↗

PubMed1975

Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid.

A composite plasmid has been constructed in vitro from colicin E1 factor (mass of 4.2 megadaltons [Md]) and nontransmissible resistance factor RSF 1010 (mass, 5.5. Md) deoxyribonucleic acids (DNAs) by the sequential action of Escherichia coli endonuclease (RI (Eco RI) and T4 phage DNA ligase on the covalently closed circular forms of the constituents. The composite plasmid was selected and amplified in vivo by sequential transformation of E. coli C600 with the ligated mixture and selection of transformants in medium containing streptomycin plus colicin E1, followed by amplification in the presence of chloramphenicol and purification of the extracted plasmid by dye-buoyant density gradient centrifugation in ethidium bromide-cesium chloride solution. Treatment of the composite plasmid with Eco RI yielded two fragments with mobilities corresponding to the linear forms of the parental plasmids, whereas Serratia marscesens endonuclease R (SmaR), which introduces a single scission in the colicin E1 factor but not in RSF 1010, convErted the composite plasmid to a single linear molecule (mass, 9.7 Md). Sequential degradation of colicin E1 factor with Sma R and Eco RI produced two fragments with masses of 3.5 and 0.7 Md; sequential degradation of RSF 1010 produced only one fragment (due to the cleavage with Eco RI), and sequential degradation of the composite plasmid produced the expected three fragments--an RSF 1010 Eco RI linear and the two expected products from the colicin E1 factor moiety. The composite plasmid conferred on the host cell resistance to streptomycin, sulfonamides, and colicin E1, but colicin E1 itself was not synthesized. In contrast, colicin E1 was synthesized by cells containing simultaneously both colicin E1 factor and RSF 1010 as separate entities. In the presence of chloramphenicol, the composite plasmid continued to replicate for 6 h. whereas replication of RSF 1010 and chromosomal DNA stopped within 2 h. Continued replication in the presence of chloramphenicol suggests that the replicator of the colicin E1 factor is functional in the composite plasmid.

Cell Fractionation↗