Search

In response to our report of biparental mtDNA inheritance (1), Annis et al. have conducted their own evaluation of our results (2). They disagreed with the autosomal dominant-like inheritance model we proposed as well as the idea of NUMT contamination suggested by others (3, 4). Instead, they offer a mathematical model based on mtDNA dynamics, wherein the paternal mtDNA outcompetes the maternal mtDNA to overcome its numerical disadvantage in the embryo. The phenomenon of competitive differences between mtDNA haplotypes has been previously reported (5). We have previously considered this idea and agree with the idea of a competition mechanism by which paternal mtDNA could contribute to the … [↵][1]1To whom correspondence may be addressed. Email: taosheng.huang{at}cchmc.org. [1]: #xref-corresp-1-1

The ring-shaped cohesin complex orchestrates long-range DNA interactions to mediate sister chromatid cohesion and other aspects of chromosome structure and function. In the yeast Saccharomyces cerevisiae , the complex binds discrete sites along chromosomes, including positions within and around genes. Transcriptional activity redistributes the complex to the 3′ ends of convergently oriented gene pairs. Despite the wealth of information about where cohesin binds, little is known about cohesion at individual chromosomal binding sites and how transcription affects cohesion when cohesin complexes redistribute. In this study, we generated extrachromosomal DNA circles to study cohesion in response to transcriptional induction of a model gene, URA3. Functional cohesin complexes loaded onto the locus via a poly(dA:dT) tract in the gene promoter and mediated cohesion before induction. Upon transcription, the fate of these complexes depended on whether the DNA was circular or not. When gene activation occurred before DNA circularization, cohesion was lost. When activation occurred after DNA circularization, cohesion persisted. The presence of a convergently oriented gene also prevented transcription-driven loss of functional cohesin complexes, at least in M phase-arrested cells. The results are consistent with cohesin binding chromatin in a topological embrace and with transcription mobilizing functional complexes by sliding them along DNA.

In all three domains of life, DNA replication begins at specialized loci termed replication origins. In bacteria, replication initiates from a single, clearly defined site. In contrast, eukaryotic organisms exploit a multitude of replication origins, dividing their genomes into an array of short contiguous units. Recently, the multiple replication origin paradigm has also been demonstrated within the archaeal domain of life, with the discovery that the hyperthermophilic archaeon Sulfolobus has three replication origins. However, the evolutionary mechanism driving the progression from single to multiple origin usage remains unclear. Here, we demonstrate that Aeropyrum pernix , a distant relative of Sulfolobus , has two origins. Comparison with the Sulfolobus origins provides evidence for evolution of replicon complexity by capture of extrachromosomal genetic elements. We additionally identify a previously unrecognized candidate archaeal initiator protein that is distantly related to eukaryotic Cdt1. Our data thus provide evidence that horizontal gene transfer, in addition to its well-established role in contributing to the information content of chromosomes, may fundamentally alter the manner in which the host chromosome is replicated.

The plastid genome of higher plants is a circular double-stranded DNA molecule which is present in multiple identical copies. We report here an 868-bp plastid DNA minicircle, NICE1, that formed in tobacco (Nicotiana tabacum) plastids during transformation, as an unexpected product of homologous recombination. Such extrachromosomal elements are normally absent in plastids of higher plants. We have constructed shuttle plasmids containing NICE1 sequences which are maintained extrachromosomally when reintroduced into plastids by particle bombardment. Furthermore, recombination between homologous sequences in the shuttle plasmids and the main plastid genome occurs. Recombination products were characterized after recovery of the shuttle plasmids in Escherichia coli and of recombinant plastid genomes in the progeny of transformed plants. Our findings indicate that shuttle plasmids can be used to engineer plastid genes without concomitant integration of foreign DNA and to recover plastid mutations in E. 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.

Electrophoretic analysis of EcoRI and HindIII restriction fragments of 2-mum supercoiled DNA of Saccharomyces cerevisiae indicated that this class of DNA is heterogeneous and probably consists of two types of molecules. Integration of the 2-mum yeast DNA in E. coli plasmid pCR1 directly showed that existence of two types of molecules as each of these could be individually inserted into separate bacterial plasmids. The difference between the two types of 2-mum circles is due to an inversion of about 1.6 X 10(6) daltons. The inversion is flanked by a reversed duplicated sequence of 0.45 X 10(6) daltons. Possible implications of this structure are dicussed.

The primary etiologic agent of the acquired immune deficiency syndrome (AIDS) is a human T-lymphotropic retrovirus (the AIDS virus). However, the pathogenesis of this virus suggests that other cofactors may contribute to the development of clinically overt disease. The hepatitis B virus (HBV) has been implicated as a potential cofactor because HBV and AIDS virus infections frequently coexist, striking similarities exist in their epidemiologic patterns, and recent data indicate that HBV is lymphotropic. To establish the prevalence of HBV infections in lymphoid cells from individuals with AIDS-related disorders, sera and peripheral blood mononuclear cells (PBMC) from 16 males with AIDS virus infections were examined for the presence of HBV DNA by DNA X DNA blot hybridization. Fifteen (94%) of these individuals had serologic evidence of a recent or prior HBV infection. HBV DNA was detected in the PBMC of all of these patients, regardless of existing HBV serology. Among the 36 control individuals without AIDS-related symptomatology, PBMC-associated HBV DNA was detected in 8 of 14 carriers of hepatitis B surface antigen (HBsAg) and in 3 of 10 individuals immune to HBV, but it was absent from the PBMC of 12 individuals without HBV infection. In all instances, the HBV DNA was extrachromosomal and existed as replicative intermediates or high molecular weight oligomers of the viral genome. Replicative intermediates and serum-associated HBV DNA were detected in all hepatitis B e antigen-positive carriers, regardless of their clinical status. In contrast, the high molecular weight oligomers of HBV DNA were detected in the PBMC of all of the AIDS virus-infected patients examined, but in only 33% of those in the control group who had evidence of HBV infection. This finding suggests that a unique and complex HBV-host-cell interaction exists in patients infected with the AIDS virus.

Pulse-labeled colicin E1 plasmid (Col E1) DNA in minicells was examined to characterize replicating molecules. Replication of Col E1 DNA principally occurred in covalently closed molecules and involved the synthesis of variable length single-stranded DNA fragments that were dissociable from the template DNA and ultimately incorporated into the completely replicated molecules. RNA was found to be associated with some of these newly synthesized fragments. Replicating molecules containing different size displacement loops were observed by electron microscopy.

The tetracycline (TG)-resistant Streptococcus faecalis strain DS-5Cl harbors two plasmids designated alpha and gamma with molecular masses of approximately 6 and 35 million daltons, respectively. TC-sensitive variants were derived by storing cells at 45 degrees for 2-3 weeks. Analysis of covalently closed circular DNA from five such variants (derived independently) revealed that in each variant the alpha-plasmid, which normally sediments at 28 S (supercoiled) in a sucrose density gradient, was replaced by a 22S substance. Growth of DS-5Cl in the presence of 150 mug/ml of TC (minimum inhibitory concentration is 250 mug/ml in liquid broth) for a prolonged period of time (50-60 generations) resulted in the disappearance of 28S DNA and the appearance of a heterogeneous covalently-closed circular DNA sedimenting at about 40-48 S. This phenomenon was accompanied by an increase in the level of bacterial TC-resistance, whereby tells were subsequently grown in the absence of TC for 70-80 generations, the heterogeneous DNA disappeared and a typical 28S alpha-plasmid reappeared. The cells also became less resistant to TC, i.e., the minimum inhibitory concentration returned to 250 mug/ml. These data suggest that bacterial growth in the presence of TC results in a reversible gene amplification with respect to a TC-resistant determinant residing on the alpha-plasmid.

The bakers' yeast Saccharomyces cerevisiae is a facultative anaerobe, tolerant to mutations in its mitochondrial genome. Individual cytoplasmic petite mutants retain genetic information derived from any portion of the parenteral mtDNA, prompting questions concerning distribution of the DNA replication origin(s) on the yeast mitochondrial genome. The experiments described in this paper were designated to test the possibility of using high-frequency yeast transformation as a selection for yeast mtDNA sequences conferring autonomously replicating function. A complete petite mitochondrial genome was inserted into the yeast vector YIp5, and the hybrid plasmid (YRMp1) was used to transform yeast. YRMp1 promoted high-frequency transformation of both wild-type yeast cells and petite mutant hosts lacking mtDNA and was maintained in each of these strains as a high-copy-number extrachromosomal element. The stability and copy-number properties of YRMp1 are similar to those of YRp12, a recombinant plasmid containing a yeast chromosomal autonomously replicating sequence.

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.

A series of increasingly drug-resistant cell populations were selected and cloned from C-46 murine neuroblastoma with the chemotherapeutic drugs maytansine, vincristine, adriamycin, or Baker's antifol. All clones demonstrated reciprocal cross-resistance to these structurally and functionally diverse drugs and failed to accumulate radiolabeled vincristine, colchicine, or Baker's antifol despite normal drug binding to cell homogenates. Initial isolates of drug-resistant populations were genetically unstable, rapidly reverting to a drug-sensitive phenotype when grown without drug, at 0.05 reversion per cell division. After prolonged growth in drug, this drug-resistant genotype stabilized. Mean chromosome number increased 300% in an initially isolated 20-fold maytansine-resistant clone, which also displayed numerous double-minute chromosomes. Descendants 240-fold more resistant than the parent, also unstable, possessed the wild-type complement of 80 chromosomes, but 45% of these cells possessed 24 double-minute chromosomes per cell; such chromosomes were absent from the drug-sensitive parental clone. Only 1.0 and 1.2 double-minute chromosomes per cell were seen in a 7-fold stably resistant revertant or 1200-fold stably resistant descendants, respectively. Double-minute chromosomes containing amplified genes for the drug target dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) have been reported in an unstable methotrexate-resistant R1-A sarcoma. These extrachromosomal gene copies were absent in stably resistant progeny. The presence of similar particles in unstably drug-resistant uptake mutants of neuroblastoma and their diminution in stably resistant descendants supports and extends their possible role in the rapid onset and instability of epigenetic drug resistance in cancer chemotherapy.

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.

Complementary DNA, transcribed in vitro from purified rabbit globin messenger RNA and made double-stranded, has been inserted into Escherichia coli plasmids pSC101 and pMB9 by the poly(dT)/poly(dA) "tailing" and annealing technique. E. coli transformants given by this DNA preparation have been shown to contain globin sequences by the hybridization of globin RNA to DNA from clones grown and lysed in situ on nitrocellulose filters. An estimate of the amount of inserted globin sequences has been provided by fingerprint analysis of globin mRNA sequences hybridized to the purified plasmid chimeras. Inserted sequences so far subjected to detailed analysis have been ascribed to the rabbit beta globin chain. The susceptibility of inserted beta globin, sequences to the restriction endonuclease EcoRI confirms the existence of a site already found through previous nucleotide sequence analysis.

The mitochondrial DNA (mtDNA) in rodent-human hybrid somatic cells was studied in strains that contain nucleotide sequences from both parental mtDNAs. A test for linkage of rodent to human mtDNA was devised on the basis of the density and sequence differences between these DNAs. When a mixture of rodent and human mtDNAs was banded in a CsCl gradient and each fraction hybridized with a mixture of complementary [ 3 H]RNA transcribed from human mtDNA and complementary [ 32 P]RNA transcribed from rodent mtDNA, each DNA was detected as a distinct and separate band at its expected density by specific hybridization with its complementary RNA. In some of the hybrid cell strains, the mtDNA sequences derived from the two species did not separate in the CsCl gradients. This result is interpreted as evidence for linkage between sequences from the two parental mtDNAs. While the exact nature of the linkage and the structure of the molecules containing both types of sequences are not known, the evidence supports the conclusion that the linkage is realized by a covalent bond. An event leading to the covalent bonding of these different sequences may be described as recombination. Among 18 hybrid cell strains examined, 13 contained large proportions of recombinant molecules. These molecules were found in both mouse-human and rat-human strains, and in strains containing more human or more rodent mtDNA sequences.

Cell extracts were prepared from Escherichia coli carrying colicin E1 plasmid. The DNA in extracts was almost exclusively closed-circular DNA of the plasmid. Labeled deoxyribonucleotides were incorporated into DNA in extracts. DNA of colicin E1 plasmid was the sole DNA product, and was composed of completely replicated molecules and a class of replicative intermediates. The intermediates carried an average of approximately two pieces of DNA fragments that had a sedimentation coefficient of approximately 6 S and were not covalently attached to the parental DNA strands. The replication was initiated on closed-circular molecules and the complete molecules were synthesized semiconservatively. The DNA synthesis depended on the four ribonucleoside triphosphates and was sensitive to rifampicin. A round of DNA replication, once initiated, was completed in the presence of rifampicin, indicating that RNA synthesis is involved in the initiation of replication of the plasmid DNA. Most of the replicated molecules were isolated in super-coiled structures. These results indicate that this soluble system is capable of carrying out a complete round of replication of colicin E1 plasmid DNA.

The compositions of growing bacterial cultures containing F' plasmids are developed in theoretical terms that will be helpful in designating experiments to determine the genetic and physiological parameters involved. The genetic parameter is the inheritability of the plasmid defined as the probability, h, that a daughter bacterium will inherit the plasmid and thus be F' rather than F-. The value of h determines the chance that the plasmid will survive in a clone initiated by a single F' bacterium. If 0 less than or equal to h less than 0.5, the probability of plasmid survival is zero, whereas if 0.5 less than h less than 1 the survival is (2h - 1)/h2. While clone sizes are demonstrated to be erratic, the proportion of F' bacteria does converge to an equilibrium value if log2 h greater than alpha - 1, where the physiological factor, alpha, is the ratio between the division times of F' and F- bacteria. A general expression of this equilibrium is derived. The two cases of alpha = 0, implying that only the F' bacteria multiply on a selective medium, and alpha = 1, implying a completely nonselective medium, are analyzed in detail. It is shown that the above considerations apply generally to growing cultures of cells in which irreversible mutations occur.

The drug-resistance plasmid, NR1, is a 37-micron circular DNA molecule that contains two components: the resistance transfer factor (29 micron) carrying the transfer genes and the genes for tetracycline resistance, and the r-determinant (8 micron) carrying the genes for resistance to several other antibiotics including chloramphenicol (Cm). In Proteus mirabilis, these two components are capable of independent replication, or they may replicate as a composite molecule. When cells of P. mirabilis containing NR1 are cultured in medium containing Cm at 250 microgram/ml a growth lag of 20-35 hr ensues. During this lag, Cm induces the selective amplification of the r-determinant, including the gene for resistance to Cm. The amplification results from the excision of the r-determinant from the R plasmid, the independent replication of the r-determinant to give polymeric as well as monomeric r-determinants, and the eventual reintegration of multiple tandem copies of the r-determinant with the resistance transfer factor to form a new R plasmid with multiple copies of the r-determinant. This mechanism represents a new level of control of gene expression in bacterial systems--namely, the induction of selective gene amplification.

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.

DNA associated with double minutes (dm) of the Y1-DM mouse adrenocortical tumor cell line has been cloned in Charon 4A and a preliminary characterization has been made of a recombinant clone, lambda Y1dm-1, isolated from this dm DNA library [George, D. L. & Powers, V. E. (1981) Cell 24, 117--123]. Cloned sequences in lambda Y1dm-1 are amplified in the genome of the Y1-DM cells. They are also amplified in the genome of a related Y1 subline (Y1-HSR), which has a homogeneously staining chromosomal region (HSR). Here we report that the amplified sequences complementary to lambda Y1dm-1 are localized in the HSR, as determined by in situ hybridization. In addition, we found that a population of Y1-DM cells originally containing only dm later consisted of two cell types. Some cells retained dm; others had lost dm but gained a HSR-bearing chromosome morphologically distinct from that in the Y1-HSR cell line. Subclones isolated from this mixed culture have either dm or a HSR, but not both. Southern blotting studies revealed that genomic DNA samples from subclones with a HSR, like subclones with dm, still possess amplified copies of DNA homologous to our recombinant probe. These experiments provide direct evidence that the dm and HSRs in these Y1 cells are structurally related and further support the hypothesis that these chromosomal anomalies result from a process of gene amplification.

The octane (OCT) plasmid in Pseudomonas putida derived from the ω-hydroxylase-carrying strain of Coon and coworkers is transferable to the camphor (CAM) plasmid-bearing strain by conjugation or by transduction. While the majority of the Cam + Oct + exconjugants segregate Cam + or Oct + cells, exconjugants with stable Cam + Oct + phenotype (CAM-OCT) can be detected at a low frequency. The transductants are all of the CAM-OCT phenotype. In the stable Cam + Oct + strains, the OCT plasmid resembles the CAM plasmid with respect to curing by mitomycin C, transfer in conjugation, and reaction to ts (temperature-sensitive) mutation specifically affecting CAM plasmid replication. Therefore, it is suggested that certain regions of homology exist between the CAM and OCT plasmids that enable them to recombine to form a single plasmid, and to overcome the incompatibility barrier that prevents their coexisting.

Two mutants with specific defects in cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase; EC 1.9.3.1) have been isolated from cultures of Saccharomyces cerevisiae exposed to the mutagens ethyl-methane sulfonate and Mn++. The mutations have been shown to be extranuclear by two criteria. The phenotype persists in diploids formed by a cross with a p-o strain of yeast of the opposite mating type. Tetrad analysis indicates a non-Mendelian segregation (4:0 and 0:4) of the mutations. Both mutants show a total absence of cytochrome oxidase activity and of spectral cytochromes a and as. One of the mutants has been shown to be missing a polypeptide synthesized by mitochondria. The migration of this protein on polyacrylamide gels corresponds to the highest-molecular-weight subunit of cytochrome oxidase.

A tandemly repeated DNA hexanucleotide sequence, 5'C-C-C-C-A-A3', that occurs at the termini of extrachromosomal DNA molecules coding for rRNA (rDNA) in Tetrahymena macronuclei was examined to determine whether it is packaged as nucleosomes. This repeated DNA sequence comprises the terminal few hundred base pairs at each end of the linear rDNA molecules. Digestion of macronuclei with micrococcal nuclease showed that this DNA sequence is protected from digestion but is left, following digestion, as a single but broad size class of DNA fragments several hundred base pairs long, under conditions in which bulk macronuclear DNA and rDNA were digested to fragments that were multiples of approximately 200 base pairs in length. The repeated C-C-C-C-A-A was found protected as fragments longer than the bulk macronuclear DNA digestion products at all times during digestion. Together with putative associated protein(s), this protected DNA was soluble after lysis of micrococcal nuclease-digested macronuclei at low salt concentrations but was insoluble in 0.075--0.2 M KCl, regardless of the extend of digestion. The size and solubility properties of the repeated C-C-C-C-A-A DNA nucleoprotein complex after micrococcal nuclease digestion of macronuclei are clearly distinguishable from those of nucleosomes, and it is inferred that this DNA sequence in macronuclei is packaged in chromatin by proteins other than histones.

Under permissive conditions of growth in tissue culture, the retrovirus visna multiples over the course of a few days to high titer and kills the host cell. We show that in this lytic life cycle, viral DNA is tightly associated with, but not covalently linked to, chromosomal DNA. This finding provides explanations for a number of the unusual properties of the lentivirus subfamily of retroviruses, and suggests potential mechanisms for the block in virus gene expression in vivo responsible for the slow infection in nature.

DNA fragments obtained from EcoRI endonuclease digestion of bacteriophage varphi80pt190 (trp(+)) and the plasmid ColE1 were covalently joined with polynucleotide ligase. Transformation of Escherichia coli trp(-) strains to tryptophan independence with the recombined DNA selected for reconstituted ColE1 plasmids containing the tryptophan operon and the varphi80 immunity region. Similarly, an EcoRI endonuclease generated fragment of plasmid pSC105 DNA containing the genetic determinant of kanamycin resistance was inserted into the ColE1 plasmid and recovered in E. coli. The plasmids containing the trp operon (ColE1-trp) and the kanamycin resistance gene were maintained under logarithmic growth conditions at a level of 25-30 copies per cell and accumulate to the extent of several hundred copies per cell in the presence of chloramphenicol. Cells carrying the ColE1-trp plasmid determined the production of highly elevated levels of trp operon-specific mRNA and tryptophan biosynthetic enzymes.

The genes coding for 17S and 25S rRNA in Paramecium tetraurealia were isolated. The macronuclear ribosomal DNA (rDNA) exists as relatively small, extrachromosomal molecules with both linear and circular forms. Electron microscopy and restriction endonuclease analysis revealed that the rDNA is arranged as tandem repeats with an average repeat size of 5.5 X 10(6) daltons. Some heterogeneity of repeat lengths was found both by electron microscopy and by restriction enzyme analysis. The rDNA does not snap back after denaturation. This study provides additional evidence that extrachromosomal rDNA may be a common feature among lower eukaryotes. However, in contrast to several other cases, the rDNA of Paramecium is not palindromic, but occurs as tandem repeats as in higher eukaryotes.

In this paper, we report a tandemly repeated DNA sequence found in human chromosomes. The DNA sequence, which is present at approximately 1000 copies per haploid genome, consists of a basic unit 849 base pairs (bp) long with a single specific restriction enzyme (Sau3AI) cutting site. The unit is further composed of five subunits, each approximately 170 bp long. When DNAs from various sources were examined by Southern hybridization using the repetitive DNA as a probe, a considerable degree of restriction fragment length polymorphism was observed. Furthermore, a substantial percentage (approximately 1.0%) of the same DNA sequence was also found extrachromosomally in the cultured human (HeLa) cells as monomers and oligomers of the basic unit in the form of covalently closed circular DNA. These results suggest that the repetitive DNA is unstable and prone to be excised from the chromosomes through homologous recombination.

The transfer of the OCT plasmid from Pseudomonas oleovorans to Pseudomonas putida strain PpGl results in the acquisition of three independent replicons: OCT, factor K, and the MER plasmid. OCT is a nontransmissible plasmid harboring genes that code for the enzymes responsible for the degradation of n -octane. Factor K is a transfer plasmid capable of mobilizing OCT as well as chromosomal genes but incapable of enhancing transfer frequencies of other transmissible plasmids such as CAM, SAL, or RP-1. MER is a self-transmissible plasmid which can confer resistance to high concentrations of mercury salts. While OCT and MER are incompatible with CAM, factor K is compatible with it. Transmissible plasmids such as SAL, CAM, MER, or RP-1 cannot mobilize OCT to any significant extent, and exert strong repression on factor K-mediated transfer of chromosomal genes.

The synthesis of the covalently-closed, circular DNA form of colicinogenic factor E 1 ( Col E 1 ) continues in Escherichia coli cells after the addition of chloramphenicol. A large portion of the purified supercoiled Col E 1 DNA molecules made in the presence of chloramphenicol are converted to the open circular DNA form after treatment with alkali (pH 13), RNase A, or RNase H. These treatments do not significantly affect the covalently-closed form of Col E 1 DNA isolated from normally growing E. coli cells. The open circular product resulting from treatment of supercoiled Col E 1 DNA with RNase A possesses a single break in one strand of the circular duplex. The site sensitive to RNase A occurs with equal probability in either of the complementary strands. Both synthesis of Col E 1 DNA and the formation of supercoiled ColE 1 DNA sensitive to RNase A or alkali are prevented by the inhibitor of RNA synthesis, rifampicin. These results indicate that covalently-closed Col E 1 DNA containing one or more ribonucleotides accumulates during Col E 1 replication in the presence of chloramphenicol. It is proposed that this incorporated RNA served as a primer during the initiation of synthesis of Col E 1 DNA and that its removal from the circular DNA is inhibited in cells incubated in the presence of chloramphenicol.

Low doses of rifampicin, an antibiotic that specifically inhibits RNA polymerase, cure F(+) cells from the episome. An E. coli mutant, whose RNA polymerase is resistant to rifampicin, does not show the curing. The growth of Hfr strains, which replicate the chromosome by the F-replication system, is inhibited by low doses of the antibiotic.