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PubMed1996

Mycobacterium xenopi IS1395, a novel insertion sequence expanding the IS256 family.

An insertion sequence (IS) of Mycobacterium xenopi has been isolated and sequenced. This 1323 bp element, designated IS1395, is present in up to 18 copies in the M. xenopi genome and may be harboured in an M. xenopi extrachromosomal element. It encodes a putative transposase of 415 amino acids which displays sequence homology to the Staphylococcus aureus IS256 family. Members of this class of elements have been described in the genus Mycobacterium-for example IS1081 is present in the M. tuberculosis complex, IS1245-IS1311 in M. avium, and IS6120 in M. smegmatis; these elements exhibit an 89%, 45% and 16% amino acid identity with IS1395, respectively. Investigation of the host range of IS1395 by Southern blot analysis revealed additional IS1395-related repeated sequences in M. gordonae and M. celatum. Moreover, IS1395 represents a useful epidemiological tool for M. xenopi strain typing as it provides a diversity of restriction fragment length polymorphism patterns.

Amino Acid Sequence↗

PubMed2004

The functional mapping of long-range transcription control elements of the HOX11 proto-oncogene.

Mapping of transcriptional control elements normally depends on the generation of a series of deletion mutants. The consequences of particular deletions are then functionally assessed by their ability to alter gene expression. The information derived from such investigations provides a general regulatory profile of the gene of interest, as well as generating a focus for future experiments. Due to the limitations of conventional DNA cloning methods, it has previously not been possible to use such an approach to rapidly assess the role of long-range regulatory elements that frequently lie further than 20 kb away from the coding region. In order to identify regulatory elements of the proto-oncogene HOX11 that may be mutated in a subset of childhood T-cell acute lymphoblastic leukaemia specimens, we generated nested deletions from a P1 artificial chromosome (PAC). This clone contained 95 kilobases (kb) of the HOX11 locus at 10q24; including 63 kb of 5' regulatory DNA. The deletion series was produced by the use of a recombination based cloning system and clones were subsequently transfected into mammalian cells. We have identified several long-range regulatory elements that mediate transcriptional control of HOX11. This approach is simple, rapid, and inexpensive. Furthermore, it generates multiple deletion clones in a single experiment. This novel approach opens up a new avenue for investigating long-range transcription control. Additionally, by allowing analysis of these elements in the natural context of large integrants the approach does not require the use of artificial extrachromosomal elements. This methodology can be applied to any gene cloned into a PAC or BAC vector and could also be useful in identifying appropriately sized deletion mutants for functional testing in transgenic models.

Cell Line, Tumor↗

PubMed1989

Gene amplification associated with the dominant cob-354 cobalt resistance trait in Dictyostelium discoideum.

A DNA amplification is correlated with the dominant, unstable cob-354 cobalt resistance trait in the cellular slime mold, Dictyostelium discoideum. The amplified DNA is present as about 50 copies of an extrachromosomal element. Cells grown under nonselective conditions in the absence of cobalt ions lose both the cobalt resistance trait and all extrachromosomal copies of the amplified DNA. The amplified DNA is transferrable to new genetic backgrounds by parasexual genetic crosses. These results explain the inability to map the cob-354 trait to a linkage group. The chromosomal origin of the amplified DNA is group III or VI. Thus the resistance trait appears to be independent of the previously known cobalt resistance locus, cobA, which maps to group VII. A developmental defect involving the production of multiply-tipped aggregates that do not complete fruiting body formation also is correlated with the presence of the amplified DNA.

Animals↗

PubMed1990

Autonomous replication of a DNA fragment containing the chromosomal replication origin of the human c-myc gene.

The c-myc genes of HeLa cells are preferentially replicated in the transcriptional direction, from chromosomal origin sequences which display cell type-specific activity. Using a run-off replication assay involving in vitro extension of replication forks initiated in intact HeLa cells, bidirectional replication was observed to begin within a 3.5 kb domain 5' to the c-myc gene. To characterize the replication origin further a 2.4 HindIII-Xhol subfragment of the c-myc 5' flanking DNA was cloned in a selectable vector and transfected into HeLa cells. The resulting pNeo.Myc-2.4 construct persisted as a circular extrachromosomal element for more than 300 cell generations under selection, with recovery of approximately 500-1000 times the mass of plasmid initially introduced into the cells. Extrachromosomal circular pNeo.Myc-2.4 monomer was reisolated in supercoiled form, along with oligomeric and miniplasmid variants which had been generated in vivo; however, chromosomally integrated copies of the plasmid were not detectable in cultures containing extrachromosomal pNeo.Myc-2.4. The recovered pNeo.Myc-2.4 plasmid was resistant to Dpnl digestion and sensitive to Mbol digestion. After transfection with pNeo.Myc-2.4 BrUdR pulse labeling of long-term or short-term cultures demonstrated that the plasmid replicated semiconservatively, under controls similar to those imposed on chromosome replication. Bisection of the pNeo.Myc-2.4 insert suggested that c-myc 5' flanking DNA within 1.2 kb 5' to promoter P1 was sufficient to confer autonomously replicating sequence activity on the plasmid vector in transient replication assays.

Chromosomes, Human↗

PubMed1992

Genomic organization of the halophilic archaeon Haloferax mediterranei: physical map of the chromosome.

Pulsed field gel electrophoresis (PFG) has been used to study the genomic organization of the halophilic archaeon Haloferax mediterranei. Analysis of the different genomic elements as well as the restriction patterns obtained with several endonucleases revealed that this microorganism has a circular chromosome of 2.9 Mb and, at least, three extrachromosomal elements of 490, 320 and 130 kb respectively. The complete physical map of the chromosome for the endonucleases PacI and BamHI has been constructed, and several BcII, BgIII and DraI restriction fragments have been aligned on these maps. The localization of heterologous and homologous genes on the physical map, including those for rRNA, lay the ground work for the construction of a genetic map.

Chromosomes, Bacterial↗

PubMed1965

NATURE AND INTERACTIONS OF THE GENETIC ELEMENTS GOVERNING PENICILLINASE SYNTHESIS IN STAPHYLOCOCCUS AUREUS.

Novick, Richard P. (The Rockefeller Institute, New York, N.Y.), and Mark H. Richmond. Nature and interactions of the genetic elements governing penicillinase synthesis in Staphylococcus aureus. J. Bacteriol. 90:467-480. 1965.-It has been found previously that penicillinase-producing strains of Staphylococcus aureus each harbor an extrachromosomal element, or plasmid, which apparently carries all the genetic information necessary for penicillinase synthesis. These plasmids behave in a manner consistent with their being small chromosomelike structures in that they comprise linkage groups containing several markers and in that they undergo such genetic events as mutation, segregation, and recombination. There is currently no evidence for conjugal cell-cell transfer of the plasmids or for a state of stable integration into the staphylococcal chromosome. A certain amount of variability has been encountered among the penicillinase plasmids harbored by different staphylococcal strains. It has been found that: (i) there are at least three molecular variants of the enzyme itself; (ii) most, but not all, of the penicillinase plasmids carry a genetic determinant of resistance to mercuric ion; (iii) plasmids carried by a very small number of the strains bear a determinant of resistance to erythromycin; (iv) the plasmids determine the fraction of penicillinase excreted into the medium during growth, and this also varies from strain to strain. The penicillinase plasmids appear to behave as integral genetic structures. The entire known linkage group is transduced intact, and is occasionally lost completely as a spontaneous occurrence during the growth of the organisms. Rarely, the plasmid markers dissociate during transduction, resulting in transduced clones which have inherited only a part of the plasmid linkage group. Similarly, dissociation occurs as a spontaneous event during normal growth, also resulting in rare clones which appear to have lost one or more but not all of the plasmid markers. When crosses are performed between two plasmid-harboring strains, a plasmid heterozygote is formed. In most cases, this persists for only one or a few cell divisions before segregating, with or without the formation of recombinant plasmids. In two instances thus far observed, the heterozygote persists as a stable plasmid heterodiploid, in which the continued presence of both plasmids can be readily demonstrated. The fate of the heterozygote, i.e., early segregation or persistence as a heterodiploid, depends upon which particular pair of plasmids is involved in the cross. This observation has led to the hypothesis of a plasmid-linked determinant of plasmid compatibility. A pair of plasmids is considered compatible if it can form a stable heterodiploid, incompatible if it cannot.

Cell Division↗

PubMedDate not supplied

[A new planarian extrachromosomal virus-like element revealed by subtraction hybridization].

A combination of suppression subtraction hybridization (SSH) and a new technique of mirror orientation selection (MOS) was used to compare the total DNA for two, sexual (SR) and asexual (AR), races of freshwater planarian Giradia tigrina. Several race-specific DNA fragments were found. A new element termed planarian extrachromosomal virus-like element (PEVE) was revealed in AR. The PEVE genome contains two unique regions, Ul and Us, which are flanked by inverted repeats. Two variants observed for the PEVE genome differ in combination of single- and double-stranded regions corresponding to Ul and Us. The PEVE genome codes for two helicases, one homologous to the circovirus replication initiation protein (Rep) and one corresponding to the helicase domain of papillomavirus E1. PEVE is nonuniformly distributed though the planarian body and is possibly replicated only in certain parenchymal cells.

Amino Acid Sequence↗

PubMed1998

Macrorestriction analysis of Desulfurella acetivorans and Desulfurella multipotens.

The genomes of the phylogenetically and physiologically unique bacteria Desulfurella acetivorans DSM 5264T and D. multipotens DSM 8415T were characterized and compared by pulsed field gel electrophoresis (PFGE). Macrorestriction patterns made of large PFGE separated DNA fragments were generated by digesting the genomic DNAs of both strains with the rare cutting restriction endonucleases ApaI, AscI, EagI, RsrII, SacII, SalI as well as with the intron encoded endonuclease I-CeuI. The sum of calculated fragment sizes from digests of the first six enzymes yielded estimates for the chromosome sizes of D. acetivorans with a mean of 1939.0 +/- 26.0 kb and for D. multipotens with a mean of 1864.0 +/- 23.0 kb. Within the patterns obtained from EagI and RsrII cleavages the apparent differences could be attributed to DNA insertion or deletion and to point mutation. The single, circular chromosomes of the two strains contain two copies of 23S rRNA genes each. Different extrachromosomal elements were detected in both strains.

DNA, Bacterial↗

PubMed1986

Isolation of Trypanosoma cruzi DNA fragments which function as ARS elements in Saccharomyces cerevisiae.

Genomic banks from Trypanosoma cruzi total cell DNA and kinetoplast DNA were constructed in a bacterial plasmid carrying the yeast URA3 gene marker. This vector is by itself unable to be maintained in Saccharomyces cerevisiae since it can neither replicate as an extrachromosomal element, nor readily integrate into the chromosome of ura3-52 yeast hosts. Using this system, we isolated sequences from nuclear and kinetoplast DNA of T. cruzi which enabled the vector to replicate autonomously in S. cerevisiae. The cloned DNA fragments fit the description of ARS sequences previously isolated from other organisms.

Animals↗

PubMed1987

A new non-mendelian genetic element of yeast that increases cytopathology produced by M1 double-stranded RNA in ski strains.

The Saccharomyces cerevisiae SKI (superkiller) genes are repressors of replication of M, L-A, and L-BC double-stranded (ds) RNAs; ski strains have an increased M dsRNA copy number and, as a result, are cold-sensitive for growth at 8 degrees. Growth is normal, however, at higher temperatures. We have found a new cytoplasmic genetic element [D] (for disease) that makes M1 dsRNA-containing superkiller strains grow slowly at 30 degrees, not at all at 37 degrees, and only very poorly at 20 degrees. These growth defects require three factors: a chromosomal ski mutation, the presence of M1 dsRNA, and the presence of the new cytoplasmic factor, [D]. We have isolated mutants unable to maintain [D] (mad), at least one of which is due to mutation of a single chromosomal locus. Further, [D] can be cured by growth at 37-39 degrees. We present evidence that [D] is not M, L-A, L-BC or W dsRNAs or mitochondrial DNA, 2 mu DNA, or [psi], but [D] depends on L-A for its maintenance. We also show that [D] is distinct from [B], a cytoplasmic element that allows M1 dsRNA to be stably replicated and maintained in spite of defects in certain chromosomal MAK genes that would otherwise be necessary. [D] activity is blocked by the presence of another extrachromosomal element, called [DIN] (for [D] interference). [D] and [DIN] may be different natural variants of the same molecule.

Genes, Fungal↗

PubMed1991

A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration.

A CHO cell line with a single copy of the DHFR locus on chromosome Z2 was used to analyze the structure of the amplification target and products subsequent to the initial amplification event. Dramatic diversity in the number and cytogenetic characteristics of DHFR amplicons was observed as soon as eight to nine cell doublings following the initial event. Two amplicon classes were noted at this early time: Small extrachromosomal elements and closely spaced chromosomal amplicons were detected in 30-40% of metaphases in six of nine clones, whereas three of nine clones contained huge amplicons spanning greater than 50 megabases. In contrast, the incidence of metaphases containing extrachromosomal amplicons fell to 1-2% in cells analyzed at 30-35 cell doublings, and most amplicons localized to rearranged or broken derivatives of chromosome Z2 at this time. Breakage of the Z2 chromosome near the DHFR gene, and deletion of the DHFR gene and flanking DNA was also observed in cells that had undergone the amplification process. To account for these diverse cytogenetic and molecular consequences of gene amplification, we propose that chromosome breakage plays a central role in the amplification process by (1) generating intermediates that are initially acentric and lead to copy number increase primarily by unequal segregation, (2) creating atelomeric ends that are either incompletely replicated or resected by exonucleases to generate deletions, and (3) producing recombinogenic ends that provide preferred sites for amplicon relocalization.

Animals↗

PubMed1969

Defective phage and chromosome mobilization in Pseudomonas putida.

The transfer of transducing phage DNA in association with the mandelate genetic region of Pseudomonas putida strain PRS1 (termed pfdm) has been achieved by growing together mandelate-positive PpG2 cells harboring pfdm as an extrachromosomal element and mandelate-deleted PpG1 strains. This transfer is analogous to sexual conjugation in the enterobacteria. The transfer of pfdm elements is always associated with chromosome mobilization and some rare recombinants acquire genetic donor ability. We have therefore concluded that the pfdm elements are responsible for initiation of chromosome mobilization in a manner not yet fully understood.

Bacteriophages↗

PubMed1985

Genetics of clindamycin resistance in Bacteroides.

Results presented in this paper have shown that a widely distributed LM-resistance determinant is present on at least 3 distinct Bacteroides R-plasmids. In fact these plasmids bear no homology outside of the defined regions implicated in the LM resistance. This resistance determinant on pBF4, pBFTM10, and pBI136 is located within DNA segments bounded on each side by a directly repeated sequence of more than 500 bp. The intervening sequences of these 3 elements are variable, and range in size from about 3.7 kb to 7.2 kb (Fig. 7). Apart from the EcoRI/AvaI restriction sites which characterize the repeated sequence, there is a notable lack of common restriction sites within these elements. These results suggest that the elements do possess a certain degree of structural similarity but significant evolutionary divergence has occurred. The presence and location of the directly repeated sequences, their association with specific deletions, and their association with an antibiotic-resistance determinant, are features common to many antibiotic-resistance transposons described for other prokaryotes. In addition, these elements are highly mobile, being found on a number of R-plasmids. The unique relationship between pBF4, pBI106, and pBI136 described here is a clear indication of the potential for these DNA sequences to move from one molecule to another. Given the extensive dissemination and the genetic and structural characteristics described above, it seems likely that the LM-resistance determinant is carried on transposon-like elements present in pBF4, pBFTM10, and pBI136. However, further experimentation will be necessary to document the transposition event. Bacteroides strains such as B. fragilis V503, possess a transmissible LM-resistance determinant which does not appear to be associated with detectable extrachromosomal elements (5,9,10). Presently, a number of strains of this type have been found over a wide geographic area. The LM-resistance genes associated with these strains are apparently similar to the one carried on the Bacteroides R-plasmids because homology between the 2 has been observed. However, it is important to note that within the limits of Southern filter blot hybridization, neither V503 nor its transconjugants possess the directly repeated sequence found on the LM-resistance plasmids (Fig. 8). The elusive nature of the V503 LM-resistance elements presents an intriguing problem. One model that has been proposed is that these resistance determinants reside on a conjugative transposon similar to Tn916 of Streptococcus faecalis (3).(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteroides↗

PubMed1991

Hydroxyurea accelerates loss of extrachromosomally amplified genes from tumor cells.

Gene amplification is one mechanism mediating the resistance of tumor cells to antineoplastic agents and the overexpression of a variety of oncogenes in diverse tumor types. There is mounting evidence that acentric extrachromosomal elements such as double minute chromosomes are common intermediates in the amplification process. These acentric structures partition unequally at mitosis and can be lost in the absence of selection. In the present study, we used human and hamster cell lines documented to contain extrachromosomally amplified drug resistance genes to investigate the feasibility of enhancing the loss rate of the extrachromosomally amplified genes to make the cells more sensitive to drug treatment. The results show that treatment of each of these lines with hydroxyurea accelerates the loss of the extrachromosomally amplified drug resistance genes. Loss of these extrachromosomal genes was associated with a corresponding increase in the drug sensitivity in the cases examined. The mechanism of accelerated loss does not appear to involve a differential effect on the replication of extrachromosomal DNA sequences. The results suggest that hydroxyurea treatment may provide a valuable tool for the general accelerated elimination of extrachromosomally amplified genes.

Aspartate Carbamoyltransferase↗

PubMed1989

Construction of an EBNA-producing line of well-differentiated human hepatoma cells and of appropriate Epstein-Barr virus-based shuttle vectors.

Using cloned Epstein-Barr nuclear antigen 1 (EBNA) and oriP elements from the Epstein-Barr virus (EBV) in conjunction with liver-specific growth media, we have constructed an EBNA-producing line of well-differentiated human hepatoma cells (Hep-EBNA-2) and appropriate EBV-oriP vectors. These vectors, pBEDC1 and pBEUG1, were maintained as free extrachromosomal elements only in cells that expressed the trans-acting EBNA protein. They were readily rescued from transfected Hep-EBNA-2 cells upon transformation of recA- Escherichia coli with cellular low-Mr DNA. They are true shuttle vectors in that they can propagate as free closed circular elements in both human Hep-EBNA-2 cells and E. coli. Finally, we have demonstrated the vector capability of our shuttle system by inserting into the SV40 expression cassette of pBEUG1 a large full-length cDNA encoding coagulation factor VIII. Our data clearly show that EBV-oriP episomes are able to stably propagate in an hepatic background and that neither high levels of EBNA protein nor multiple copy episomes significantly interfere with the expression of the set of hepatic functions that have been analyzed. These results are discussed in terms of gene amplification and cloning of genes that program liver differentiation.

Antigens, Viral↗

PubMed1997

Comparison of a new insertion element, IS1407, with established molecular markers for the characterization of Mycobacterium celatum.

Genomic analyses of 18 Mycobacterium celatum strains obtained from different patients in three countries (United States, United Kingdom, and France) were performed; the methods used in this study were restriction fragment length polymorphism (RFLP) analysis, pulsed-field gel electrophoresis (PFGE) analysis, and PCR restriction analysis (PRA) of the hsp-65 gene. A new insertion sequence, IS1407 (GenBank accession no. X97307), belonging to the IS256 family, was identified in M. celatum type 1 strains and was characterized by sequencing. When a probe for Mycobacterium xenopi IS1395-like sequences was used, the RFLP analysis of M. celatum type 1 strains revealed that they contained three or four copies of IS1407 in identical genomic positions, while this element was absent in all M. celatum type 2 strains. PFGE performed with three different endonucleases revealed a unique large restriction fragment (LRF) pattern for M. celatum type 1 strains, whereas the LRF patterns obtained for M. celatum type 2 strains were polymorphic. Moreover, PFGE of nondigested genomic DNA revealed extrachromosomal elements in M. celatum type 2. The type strain of M. celatum type 3 could not be differentiated from M. celatum type 1 strains on the basis of the results of the RFLP analysis, the PFGE analysis, and the PRA of IS1407. In this study we confirmed that M. celatum types 1 and 2 represent distinct genomic clusters and that the molecular markers in M. celatum type 2 exhibit greater polymorphism than the molecular markers in M. celatum type 1.

Bacterial Proteins↗

PubMed1998

Snapshot of a large dynamic replicon in a halophilic archaeon: megaplasmid or minichromosome?

Extremely halophilic archaea, which flourish in hypersaline environments, are known to contain a variety of large dynamic replicons. Previously, the analysis of one such replicon, pNRC100, in Halobacterium sp. strain NRC-1, showed that it undergoes high-frequency insertion sequence (IS) element-mediated insertions and deletions, as well as inversions via recombination between 39-kb-long inverted repeats (IRs). Now, the complete sequencing of pNRC100, a 191,346-bp circle, has shown the presence of 27 IS elements representing eight families. A total of 176 ORFs or likely genes of 850-bp average size were found, 39 of which were repeated within the large IRs. More than one-half of the ORFs are likely to represent novel genes that have no known homologs in the databases. Among ORFs with previously characterized homologs, three different copies of putative plasmid replication and four copies of partitioning genes were found, suggesting that pNRC100 evolved from IS element-mediated fusions of several smaller plasmids. Consistent with this idea, putative genes typically found on plasmids, including those encoding a restriction-modification system and arsenic resistance, as well as buoyant gas-filled vesicles and a two-component regulatory system, were found on pNRC100. However, additional putative genes not expected on an extrachromosomal element, such as those encoding an electron transport chain cytochrome d oxidase, DNA nucleotide synthesis enzymes thioredoxin and thioredoxin reductase, and eukaryotic-like TATA-binding protein transcription factors and a chromosomal replication initiator protein were also found. A multi-step IS element-mediated process is proposed to account for the acquisition of these chromosomal genes. The finding of essential genes on pNRC100 and its property of resistance to curing suggest that this replicon may be evolving into a new chromosome.

Chromosome Mapping↗

PubMed1979

Deoxyribonucleic acid sequence homologies among bacterial insertion sequence elements and genomes of various organisms.

Plasmid and phage deoxyribonucleic acid (DNA) harboring bacterial insertion sequence (IS) elements IS1, IS2, and IS5 were characterized and used as probes to detect homologous sequences in various procaryotic and eucaryotic genomes. The hybridization method used permits the detection of sequences partially homologous to the elements. Hybridization of the IS-containing probes to each other revealed a region of limited homology between IS1 and IS2. Homologous sequences were then detected by computer analysis of the published IS1 and IS2 nucleotide sequences. The homologous sequence contains a tandemly repeated tetranucleotide sequence which resembles the repeated sequence at the hot spot for spontaneous mutations in the lacI gene (P. J. Farabaugh, U. Schmeissner, M. Hofer, and J. Miller, J. Mol. Biol. 126:847-863, 1978). Homology between the IS elements and various genomes was determined by hybridizing labeled DNA containing IS1, IS2, and IS5 sequences to Southern blots of chromosomal DNA cleaved with restriction endonucleases. IS1 and IS5 appear limited to the enteric bacteria, whereas IS2 sequences can also be detected in Pseudomonas putida, Pseudomonas aeruginosa, and Serratia marcescens. Bacteria which appear not to possess extrachromosomal elements, e.g., Caulobacter crescentus, did not show homology with any insertion sequences tested. In addition, sequences homologous to IS1, IS2, or IS5 were not detected in Saccharomyces cerevisiae, Dictyostelium discoideum, or calf thymus DNA.

Bacteria↗