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Studies with artificial extrachromosomal elements in trypanosomatids: could specificity in the initiation of DNA replication be linked to that in transcription?

Historically, artificial replicons have served as useful models for the definition of regulatory elements involved in chromosomal replication and transmission in yeast and DNA replication in bacteria. Here, Pradeep Patnaik examines what we have learnt so far from the replicative behaviour of various artificial extrachromosomal elements available for trypanosomatids. He highlights the involvement of transcription regulatory elements in virtually every eukaryotic origin of replication analysed in detail and, by drawing upon the extensive literature supporting a close association between DNA replication and transcription, he speculates that the nature and organization of origins of replication on a chromosome also may hold clues to the manner by which an organism regulates gene expression.

Journal Article↗

Deregulated expression of c-Myc in a translocation-negative plasmacytoma on extrachromosomal elements that carry IgH and myc genes.

The induced expression of c-Myc in plasmacytomas in BALB/c mice is regularly associated with nonrandom chromosomal translocations that juxtapose the c-myc gene to one of the Ig loci on chromosome 12 (IgH), 6 (IgK), or 16 (IgL). The DCPC21 plasmacytoma belongs to a small group of plasmacytomas that are unusual in that they appear to be translocation-negative. In this paper, we show the absence of any c-myc-activating chromosomal translocation for the DCPC21 by using fluorescent in situ hybridization, chromosome painting, and spectral karyotyping. We find that DCPC21 harbors c-myc and IgH genes on extrachromosomal elements (EEs) from which c-myc is transcribed, as shown by c-myc mRNA tracks and extrachromosomal gene transfer experiments. The transcriptional activity of these EEs is supported further by the presence of the transcription-associated phosphorylation of histone H3 (H3P) on the EEs. Thus, our data suggest that in this plasmacytoma, c-Myc expression is achieved by an alternative mechanism. The expression of the c-Myc oncoprotein is initiated outside the chromosomal locations of the c-myc gene, i.e., from EEs, which can be considered functional genetic units. Our data also imply that other "translocation-negative" experimental and human tumors with fusion transcripts or oncogenic activation may indeed carry translocation(s), however, in an extrachromosomal form.

Animals↗

[Isolation and preliminary characteristics of the extrachromosomal elements of Bacillus thuringiensis DNA].

Centrifugation of Bacillus thuringiensis 351 DNA in CsCl-ethidium bromide density gradient has revealed, besides the main band of chromosomal DNA (p = 1.56 g/cm2), some additional bands with higher density, which usually correspond cicle superhelix DNAs, are revealed. 6 discrete bands are observed under electrophoresis of total fraction of minor DNA bands, which suggests, that the preparation contains cicle DNAs of different size. The treatment with DNAse results in the appearance of 3 bands corresponding to opened cicle DNA forms, their molecular weight being 12-10(6)--4-10(6) daltons. Cicle super-helix and opened cicle DNA forms are found in minor fraction by means of electrone microscopy. Statistical analysis has revealed the presence of at least three types of cicle molecules of different size. The variant 351-10, free of extrachromosomal elements, is isolated after ethidium bromide treatment at high temperature. Possible mechanisms of cicle DNAs formation are discussed. Biological function of DNA extrachromosomes fragments in Bacillus thuringiensis is still obscure.

Bacillus thuringiensis↗

Genomics of clostridial pathogens: implication of extrachromosomal elements in pathogenicity.

The recently decoded genomes of the major clostridial toxin-producing pathogens Clostridium perfringens, Clostridium tetani, Clostridium botulinum and Clostridium difficile have provided a huge amount of new sequence data. Recent studies have focused on the identification and investigation of pathogenic determinants and the regulatory events governing their expression. The sequence data revealed also the genomic background of virulence genes, as well as the contribution of extrachromosomal elements to a pathogenic phenotype. This has generated new insights in clostridial pathogenesis - and will continue to do so in the future - and has deepened our understanding of the anaerobic lifestyle of clostridial species.

Clostridioides difficile↗

Stable transformation of Leptomonas seymouri by circular extrachromosomal elements.

To define the cis-acting sequences necessary for gene expression and DNA replication in trypanosomatids, we have developed a selectable vector that can be grown in Escherichia coli and maintained stably in the insect trypanosomatid Leptomonas seymouri. The vector is relatively small (6 kilobase pairs) and contains a portion of the L. seymouri alpha-tubulin gene positioned in-frame with a truncated neomycin phosphotransferase gene that confers resistance to the aminoglycoside G418. This construct is maintained in cells as a high-copy-number circular extrachromosomal element containing several head-to-tail copies of the transforming plasmid. In L. seymouri, alpha-tubulin-neomycin phosphotransferase fusion RNAs are polyadenylylated and possess a trans-spliced mini-exon. Additional DNA sequences can be inserted into the vector, propagated, and expressed in transformed cells.

Animals↗

Extrachromosomal elements in a super-suppression system of yeast. III. Enhanced detection of weak suppressors in certain non-suppressed psi-strains.

Individual colonies of an adenine-requiring strain of the genotype ade 2-1, SUPQ5, [psi minus] exhibit high and uniform frequencies of ADE+ revertants (5-1 times 10-5). The frequencies for strains of the genotypes ade 2-1, SUPQ5+, [psi+] and ade 2-1 SUPQ5+, [psi minus] are by contrast 1-2 times 10-7 and 0-5 times 10-7 respectively. Mutation from [psi minus] to [psi+] can occur. This suggests that the original mutation from [psi+] to [psi minus] is not necessarily a physical loss of, or extensive deletion, the extrachromosomal elements. Although mutation from [psi minus] to [psi+] occurs, the majority of ADE+ revertants from ade 2-1, SUPQ5, [psi minus] strains contain other types of mutation. Crosses between most ADE+ revertants and ade 2-1, SUPO5+, [psi minus] strains yield adenine-requiring diploids and tetrad segregations of 0:4, 1:3 and 2:2 ADE+ :ade minus. It is argued that most mutations give rise to weak recessive suppressors (SUPX) which are themselves incapable of suppressing the ochre allele ade 2-1, unless the allele SUPQ5 is also present. Tests with two suppressors, S-theta and S-zeta, isolated by Dr R. A. Gilmore, which are themselves incapable of suppressing ade 2-1, show that they do not do so even in the presence of SUPQ5. Thus the set of suppressors isolated by us is a special class which can perhaps only be observed in the genetic background ade 2-1, SUPQ5, [psi minus]. There is evidence that some of the new suppressors are capable, in the absence of SUPQ5, of weakly suppressing trp 5-48 and perhaps can 1-100. All ochre alleles are, however, suppressed by the combination SUPQ5, SUPX. SUPQ5 is able to suppress trp 5-48, his 5-2, lys 1-1 and can 1-100 in a [psi minus] background, although it does so variably. It is incapable of suppressing ade 2-1 in such a background. It is argued that the alteration in the [psi] determinant results in a lowered efficiency of suppression by SUPQ5 rather than a changed specificity.

Adenine↗

Novel submicroscopic extrachromosomal elements containing amplified genes in human cells.

In previous work, several methotrexate (MTX)-resistant variants were isolated frm the human cell line HeLa BU25, which exhibited a high degree of dihydrofolate (DHFR) gene amplification (estimated to be 250- to 300-fold). These variants did not contain any chromosome with a homogeneously staining region (HSR) and exhibited only a small average number of minute chromosomes per cell: these two types of karyotypic abnormalities generally accompany selective gene amplification. We now report that structures containing amplified DHFR genes in one of these variants (HeLa BU25-10B3) can be isolated by pulsed-field gradient or field-inversion gel electrophoresis as homogeneous DNA molecules of approximately 650 kilobases (kb). Electron microscopy of metaphase spreads from these cells reveals chromatin fibres with a similar DNA content, which are probably related to the above elements. These represent a novel type of extrachromosomal structures in mammalian cells.

Drug Resistance↗

Unstable amplification of two extrachromosomal elements in alpha-difluoromethylornithine-resistant Leishmania donovani.

We describe the first example of unstable gene amplification consisting of linear extrachromosomal DNAs in drug-resistant eukaryotic cells. alpha-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an amplified ornithine decarboxylase (ODC) gene copy number contained two new extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a preexisting wild-type chromosome, probably by a conservative amplification mechanism. Both elements were unstable in the absence of DFMO, and their disappearance coincided with a decrease in ODC activity and an increase in DFMO growth sensitivity. These results suggest the possibility that ODC70-C may play a role in DFMO resistance. These data expand the diversity of known amplification mechanisms in eukaryotes to include the simultaneous unstable amplification of both linear and circular DNAs. Further characterization of these molecules will provide insights into the molecular mechanisms underlying gene amplification, including the ability of linear amplified DNAs to acquire telomeres and the determinants of chromosomal stability.

Animals↗