Extrachromosomal Genetic Elements Research Articles

ヒト細胞の微小核 染色体外遺伝因子の選択的排出機構

ヒト細胞の微小核 染色体外遺伝因子の選択的排出機構

Bacterial plasmids: replication of extrachromosomal genetic elements encoding resistance to antimicrobial compounds.

Plasmids are self-replicating extrachromosomal DNA molecules found in Gram-negative and Gram-positive bacteria as well as in some yeast and other fungi. Although most of them are covalently closed circular double-stranded DNA molecules, recently linear plasmids have been isolated from different bacteria. In general, plasmids are not essential for the survival of bacteria, but they may nevertheless encode a wide variety of genetic determinants, which permit their bacterial hosts to survive better in an adverse environment or to compete better with other microorganisms occupying the same ecological niche. The medical importance of plasmids that encode for antibiotic resistance, as well as specific virulence traits has been well documented and demonstrated the important role these bacterial genetic elements play in nature. Although they encode specific molecules required for initiation of their replication, plasmids rely on host-encoded factors for their replication. Plasmid replication initiates in a predetermined cis-site called ori and can proceed either by a rolling circle or a theta replication mechanism. Some of the plasmid-encoded elements required for their replication, such antisense RNA molecules and DNA repeated sequences located close to ori, determine plasmid attributes like copy number and incompatibility.

Bacterial plasmids replication of extrachromosomal genetic elements encoding resistance to antimicrobial compounds

Bacterial plasmids replication of extrachromosomal genetic elements encoding resistance to antimicrobial compounds

Sulfolobus genome: from genomics to biology

Major progress in sequencing the genome of Sulfolobus solfataricus has been closely concerted with the characterization and sequencing of many extrachromosomal genetic elements, including viruses, cryptic plasmids and conjugative plasmids, as well as mobile archaeal introns and transposons. The latter have provided a basis for developing the first generation of vectors that are now being used to study the genetics of Sulfolobus and other Archaea.

The Genome of Staphylococcus aureus: A Review

The genome of Staphylococcus aureus consists of a single circular chromosome (2.7-2.8 mbp) plus an assortment of extrachromosomal accessory genetic elements: conjugative and nonconjugative plasmids, mobile elements (IS, Tn, Hi), prophages and other variable elements. Plasmids (1-60 kbp) are classified into 4 classes and there are 15 known incompatibility groups. Mobile elements of the genome (0.8-18 kbp) appear in the chromosome or in plasmids of classes II and III. Prophages (45-60 kbp) are integrated in the bacterial chromosome, and they are UV- or mitomycin-inducible. Temperate bacteriophages of S. aureus are members of the Siphoviridae and the serological groups A, B and F occur most frequently. In the paper presented, the characteristics of chromosome, plasmids, transposons and other genetic elements of S. aureus genome are given and an alphabetical list of known genes of this species is included.

A double-stranded RNA mycovirus in Botrytis cinerea

In wild-type Botrytis cinerea CVg25 strain we have detected the presence of extrachromosomal genetic elements corresponding to double-stranded RNA molecules. These genetic elements have been designated L, M 1 and M 2 with molecular sizes of 8.3, 2.0 and 1.4 kb, respectively. The visualization by electron microscopy of mycelium ultrathin sections from B. cinerea CVg25 showed the presence of isometric virus-like particles of about 40 nm in diameter. Linear sucrose gradient centrifugation of mycelium-free extracts was done to determine if the double-stranded RNAs were associated with virus-like particles. The gradient profile obtained at 260 and 280 nm revealed a major peak that was analyzed by both agarose-gel electrophoresis and electron microscopy. It was observed that only the L-double-stranded RNA molecule copurified with isometric virus-like particles. These virus-like particles had a similar morphology and size as those detected by electron microscopy in the mycelium sections. These results suggest that only the L-double-stranded RNA would be encapsidated.

Homologous expression and purification of mutants of an essential protein by reverse epitope-tagging.

Purification of mutant enzymes is a prime requirement of biophysical and biochemical studies. Our investigations on the essential Escherichia coli enzyme glutaminyl-tRNA synthetase demand mutant enzymes free of any wild-type protein contamination. However, as it is not possible to express noncomplementing mutant enzymes in an E. coli glnS-deletion strain, we developed a novel strategy to address these problems. Instead of following the common tactic of epitope-tagging the mutant protein of interest on an extrachromosomal genetic element, we fused a reporter epitope to the 5' end of the chromosomal glnS-gene copy: this is referred to as 'reverse epitope-tagging.' The corresponding strain, E. coli HAPPY101, displays a normal phenotype, and glutaminyl-tRNA synthetase is exclusively present as an epitope-tagged form in cell-free extracts. Here we report the use of E. coli HAPPY101 to express and purify a number of mutant glutaminyl-tRNA synthetases independently of their enzymatic activity. In this process, epitope-tagged wild-type protein is readily separated from mutant enzymes by conventional chromatographic methods. In addition, the absence of wild-type can be monitored by immunodetection using a monoclonal antibody specific for the epitope. The strategy described here for expression and purification of an essential enzyme is not restricted to glutaminyl-tRNA synthetase and should be applicable to any essential enzyme that retains sufficient activity to sustain growth following reverse epitope-tagging.

Genetic regulation of plasmid transfer

Plasmids are extrachromosomal genetic elements which in bacterial cells occur as entities physically separated from the chromosome and, as such, are capable of self-maintenance and self-replication for an infinitely long time. They are ubiquitous, being present in bacteria of nearly all systematic groups, and are also found in cells o f a number of eukaryotes. When residing in bacteria, plasmids impart to them important properties, such as drug resistance, toxin production, and a capacity to degrade organic compounds. One of the most remarkable properties of many of them (conjugative plasmids) is the ability to transfer genetic information from t h e host (donor) bacteria to other (recipient) bacteria. This property is determined by the genetic region ha, which, as has been established for the F plasmid, comprises more than 20 genes and controls the synthesis of proteins that make possible both the conjugation of donor to recipient bacteria and DNA transfer from donors to recipients. Most of these genes have been traced to the operon traY--+ traZ [31,32,34,36,37]. A positive regulator of the traY--, traZ operon in the F plasmid is the traJ gene, whose product is required for the transcription of this operon. The traJ gene, however, is itself under the negative control of the OP system's genes that prevent its expression and, consequently, the expression of the t r a Y ~ traZ operon [29,36]. Accordingly, as we noted earlier [17], the genetic regulation of F plas-

Mitochondrial DNA alterations as ageing-associated molecular events.

Mitochondrial DNA (mtDNA) is a naked double-stranded circular extrachromosomal genetic element continuously exposed to the matrix that contains great amounts of reactive oxygen species and free radicals. The age-dependent decline in the capability and capacity of mitochondria to dispose these oxy-radicals will render mtDNA more vulnerable to mutations during the ageing process. During the past 3 years, more than 10 different types of deletions have been identified in the mtDNA of various tissues of old humans. Some of them were found only in a certain tissue but some others appeared in more than one organ or tissue. The 4977-bp deletion is the most prevalent and abundant one among these deletions. Skeletal muscle is the target tissue of most ageing-associated mtDNA deletions and has often been found to carry multiple deletions. The onset age of the various deletions in mtDNA varies greatly with individual and type of the deletion. The 4977-bp deletion has been independently demonstrated to occur in the mtDNA of various tissues of the human in the early third decade of life. However, the 7436-bp deletion was only detected in the heart mtDNA of human subjects in their late thirties. The others appeared only in older humans over 40 years old. No apparent sex difference was found in the onset age of these ageing-associated mtDNA deletions. The various ageing-associated deletions could be classified into two groups. Most of the deletions belong to the first group, in which the 5'- and 3'-end breakpoints of the deletion are flanked by 4-bp or longer direct repeats. The deletion in the second group occurs less frequently and shows no distinct repeat sequences flanking the deletion sites. These two groups of mtDNA deletions may occur by different mechanisms. The first group is most probably caused by internal recombination or slippage mispairing during replication of mtDNA by the D-loop mechanism. The deleted mtDNA and the deleted DNA fragment may be further degraded or escape from the mitochondria and get translocated into the nucleus. The latter route has been substantiated by many observations of inserted mtDNA sequences in the nuclear DNA. Thus, the fragments of migrating mtDNA may change the information content and expression level of certain nuclear genes and thereby promote the ageing process or cause cancer. Similar ageing-associated alterations of mtDNA have also been observed in aged animals and plants. I suggest that mtDNA deletions and other mutations to be discovered are molecular events generally associated with the ageing process.

Recognition of the CDEl motif GTCACATG by mouse nuclear proteins and interference with the early development of the mouse embryo

We have reported previously (1) two unexpected consequences of the microinjection into fertilized mouse eggs of a recombinant plasmid designated p12B1, carrying a 343 bp insert of non-repetitive mouse DNA. Injected at very low concentrations, this plasmid could be established as an extrachromosomal genetic element. When injected in greater concentration, an early arrest of embryonic development resulted. In the present work, we have studied this toxic effect in more detail by microinjecting short synthetic oligonucleotides with sequences from the mouse insert. Lethality was associated with the nucleotide sequence GTCACATG, identical with the CDEl element of yeast centromeres. Development of injected embryos was arrested between the one-cell and the early morula stages, with abnormal structures and DNA contents. Electrophoretic mobility shift and DNAse foot-printing assays demonstrated the binding of mouse nuclear protein(s) to the CDEl-like box. Base changes within the CDEl sequence prevented both the toxic effects in embryos and the formation of protein complex in vitro, suggesting that protein binding at such sites in chromosomal DNA plays an important role in early development.

Detection of a 2 μ derivative yeast plasmid with altered properties

The Saccharomyces cerevisiae (Sacch. cerevisiae) strain RXII, like many others, harbours plasmid DNAs. and one category of them is homologous to the 2 mu plasmid of yeast. DNA-DNA hybridization experiments indicated altered structures of this species as regards the number and distribution of the restriction sites. The efforts made to clone either the whole plasmid in pBR328 or its fragments in pBR322 vectors remained unsuccessful, since deleted plasmids were isolated without insert DNA, and even the loss of vector sequences was observed. The data suggest, that the 2 mu derivative plasmid in strain RXII represent an unique category of this extrachromosomal genetic element.

Proceedings of the Eleventh Annual Mid-Atlantic Regional Extrachromosomal Genetic Elements Conference: October 16–18, 1987, Sheraton Beach Inn, Virginia Beach, Virginia

Proceedings of the Eleventh Annual Mid-Atlantic Regional Extrachromosomal Genetic Elements Conference: October 16–18, 1987, Sheraton Beach Inn, Virginia Beach, Virginia

Correlation between albicidin production and chlorosis induction by Xanthomonas albilineans, the sugarcane leaf scald pathogen

Chlorosis of emerging leaves of sugarcane plants invaded by Xanthomonas albilineans results from blocked chloroplast differentiation, apparently caused by a phytotoxin. Chlorosis inducing isolates of X. albilineans produce a family of antimicrobial compounds, including albicidin which inhibits prokaryote DNA replication. The objective of this study was to determine whether albicidin, or a related compound produced in diseased plants, causes chlorosis of uninvaded tissues characteristic of sugarcane leaf scald disease. Spontaneous loss of albicidin production occurs at a high rate but does not involve extrachromosomal genetic elements. Results with mutants and revertants in albicidin production show a close correlation between albicidin production and ability to cause chlorosis, strongly suggesting that the chlorosis inducing toxin is biosynthetically closely related to albicidin. Plasmid pJB4JI is transmitted at high frequency from Escherichia coli to X. albilineans, where it is stably maintained as a conjugative plasmid, providing a basis for further genetic analysis of X. albilineans using well-characterized P1 group plasmids.

Proceedings of the Tenth Annual Mid-Atlantic Regional Extrachromosomal Genetic Elements Conference October 17–19, 1986, Sheraton Beach Inn, Virginia Beach, Virginia: Gene fusions demonstrate a lack of scrB induction by sucrose in Streptococcus mutans

Proceedings of the Tenth Annual Mid-Atlantic Regional Extrachromosomal Genetic Elements Conference October 17–19, 1986, Sheraton Beach Inn, Virginia Beach, Virginia: Gene fusions demonstrate a lack of scrB induction by sucrose in Streptococcus mutans

Proceedings of the Ninth Annual Mid-Atlantic Regional Extrachromosomal Genetic Elements Conference: September 27–29, 1985, Sheraton Beach Inn, Virginia Beach, Virginia

Proceedings of the Ninth Annual Mid-Atlantic Regional Extrachromosomal Genetic Elements Conference: September 27–29, 1985, Sheraton Beach Inn, Virginia Beach, Virginia

Transitory derepression and the maintenance of conjugative plasmids.

It has been proposed that bacterial plasmids cannot be maintained by infectious transfer alone and that their persistence requires positive selection for plasmid-borne genes. To test this hypothesis, the population dynamics of two laboratory and five naturally occurring conjugative plasmids were examined in chemostat cultures of E. coli K-12. Both laboratory plasmids and three of the five wild plasmids failed to increase in frequency when introduced at low frequencies. However, two of the naturally occurring plasmids rapidly increased in frequency, and bacteria carrying them achieved dominance in the absence of selection for known plasmid-borne genes. Three hypotheses for the invasion and persistence of these two plasmids were examined. It is concluded that although these two extrachromosomal genetic elements are repressed for conjugative pili synthesis, as a consequence of high rates of transfer during periods of transitory derepression in newly formed transconjugants, they become established and are maintained by infectious transfer alone. The implications of these observations to the theory of plasmid maintenance and the evolution of repressible conjugative pili synthesis are discussed.

Some consideration on plasmid number in a proliferating cell

Plasmid as an extrachromosomal genetic element is an important vehicle to support the recombinant DNA technology. A picture on plasmid number per cell as a basis of expecting the gene dosage effect is worthy of drawing from the application to practice. The purpose of this communication is to present an overall and stochastic picture on the dynamics of plasmid number per cell in relation to the specific growth rate of the host cell.

Skin graft rejection caused by the maternally transmitted antigen Mta.

Mta is a medial histocompatibility antigen of the mouse. It does not stimulate a primary mixed lymphocyte response and stimulates only a very weak secondary response. Primary skin grafts are rejected with a mean survival time of 59 days by Mta- NZB recipients, and 39 days by recipients of the C57BL/6 background. Rejection is accelerated in recipients primed against Mta with a skin graft or cells, especially when these differ by multiple minor histocompatibility antigens. Mta is determined by a maternally transmitted, extrachromosomal genetic element, so backcross mice reject skin from their inbred, homozygous paternal strain. Mta, therefore, constitutes a new exception to the classic laws of transplantation.

Proceedings of the Eighth Annual Mid-Atlantic Regional Extrachromosomal Genetic Elements Conference: October 12–14, 1984, Sheraton Beach Inn, Virginia Beach, Virginia

Proceedings of the Eighth Annual Mid-Atlantic Regional Extrachromosomal Genetic Elements Conference: October 12–14, 1984, Sheraton Beach Inn, Virginia Beach, Virginia

Role of reverse transcription in the generation of extrachromosomal copia mobile genetic elements.

The Drosophila genetic element copia is one of the best studied eukaryotic transposable sequences. Copia shares structural features with a wide variety of mobile elements in Drosophila, Lepidoptera, yeast and vertebrates, the last class being the retrovirus proviruses. Furthermore, retrovirus-like particles containing copia RNA have been isolated from Drosophila cells and extrachromosomal circular copias with structures closely resembling circular retrovirus proviruses have been isolated and cloned. Therefore, copia-like elements and retroviruses may be members of a class of mobile genetic elements existing throughout the eukaryotic kingdom. Consequently, there has been speculation that retroviruses evolved from transposable elements and, conversely, that copia-like elements transpose as retroviruses or retrovirus-like particles. To date, however, there has been no demonstration that copia RNA is reverse transcribed into copia DNA. The present report describes the isolation of linear extrachromosomal copias whose structure closely resembles the analogous retrovirus provirus linears and whose synthesis is unaffected by inhibitors of the cellular DNA polymerase responsible for chromosomal DNA replication.