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.     

Spontaneous Amplification of the Adh4 Gene in Saccharomyces Cerevisiae

Five spontaneous amplifications of the ADH4 gene were identified among 1,894 antimycin A-resistant mutants isolated from a diploid strain after growth at 15 degrees. Four of these amplifications are approximately 40-kb linear extrachromosomal palindromes carrying telomere homologous sequences at each end similar to a previously isolated amplification. ADH4 is located at the extreme left end of chromosome VII, and the extrachromosomal fragments appear to be the fusion of two copies of the end of this chromosome. The fifth amplification is a chromosomal amplification carrying an extra copy of ADH4 on both homologs of chromosome VII. These results suggest that the ADH system can be used to study amplification in Saccharomyces cerevisiae.     

Recombination mediates production of an extrachromosomal circular DNA containing a transposon-like human element, THE-1

An abundant class of HeLa extrachromosomal circular DNA containing the transposon-like element, THE-1, is shown to arise via site specific recombination. The chromosomal locus from which these circles are derived, however, is single-copy. Northern blot analysis detects homology to two polyadenylated RNAs in HeLa cells. The possible presence of an origin of replication and its role in generating these small polydisperse circles is discussed.     

Localization of a bidirectional DNA replication origin in the native locus and in episomally amplified murine adenosine deaminase loci.

Gene amplification is frequently mediated by the initial production of acentric, autonomously replicating extrachromosomal elements. The 4,000 extrachromosomal copies of the mouse adenosine deaminase (ADA) amplicon in B-1/50 cells initiate their replication remarkably synchronously in early S phase and at approximately the same time as the single-copy chromosomal locus from which they were derived. The abundance of ADA sequences and favorable replication timing characteristics in this system led us to determine whether DNA replication initiates in ADA episomes within a preferred region and whether this region is the same as that used at the corresponding chromosomal locus prior to amplification. This study reports the detection and localization of a discrete set of DNA fragments in the ADA amplicon which label soon after release of synchronized B-1/50 cells into S phase. A switch in template strand complementarity of Okazaki fragments, indicative of the initiation of bidirectional DNA replication, was found to lie within the same region. This putative replication origin is located approximately 28.5 kbp upstream of the 5' end of the ADA gene. The same region initiated DNA replication in the single-copy ADA locus of the parental cells. These analyses provide the first evidence that the replication of episomal intermediates involved in gene amplification initiates within a preferred region and that the same region is used to initiate DNA synthesis within the native locus.     

Analysis of IS1236-mediated gene amplification events in Acinetobacter baylyi ADP1

Recombination between insertion sequence copies can cause genetic deletion, inversion, or duplication. However, it is difficult to assess the fraction of all genomic rearrangements that involve insertion sequences. In previous gene duplication and amplification studies of Acinetobacter baylyi ADP1, an insertion sequence was evident in approximately 2% of the characterized duplication sites. Gene amplification occurs frequently in all organisms and has a significant impact on evolution, adaptation, drug resistance, cancer, and various disorders. To understand the molecular details of this important process, a previously developed system was used to analyze gene amplification in selected mutants. The current study focused on amplification events in two chromosomal regions that are near one of six copies of the only transposable element in ADP1, IS1236 (an IS3 family member). Twenty-one independent mutants were analyzed, and in contrast to previous studies of a different chromosomal region, IS1236 was involved in 86% of these events. IS1236-mediated amplification could occur through homologous recombination between insertion sequences on both sides of a duplicated region. However, this mechanism presupposes that transposition generates an appropriately positioned additional copy of IS1236. To evaluate this possibility, PCR and Southern hybridization were used to determine the chromosomal configurations of amplification mutants involving IS1236. Surprisingly, the genomic patterns were inconsistent with the hypothesis that intramolecular homologous recombination occurred between insertion sequences following an initial transposition event. These results raise a novel possibility that the gene amplification events near the IS1236 elements arise from illegitimate recombination involving transposase-mediated DNA cleavage.     

Unexpected loss of genomic DNA from agarose gel plugs

Intact chromosomal DNAs are routinely prepared by embedding cells in agarose plugs before lysis. The large sizes of the genomic DNAs cause their retention while other macromolecules diffuse into and out of the gel matrix during lysis, washing and restriction cleavage incubations. However, in an analysis of agarose-embedded chromosomal DNAs cleaved with restriction enzymes, fragments larger than 30 kilobases were found to have eluted from the gel plugs. Since loss of fragments from gel plugs may affect qualitative and quantitative interpretations of electrophoretic patterns, an analysis of the diffusion of DNA segments from agarose plugs was performed. The two variables monitored were the time dependence and the DNA fragment size dependence of the diffusion process. The results indicate that small fragments (less than or equal to 2 kilobases) are quickly lost from 1% agarose gel plugs; moreover, significant amounts of large DNA segments (i.e., the 48.5-kilobase lambda phage chromosome) are also lost. In addition to urging caution in the analysis of restriction cleavage data, these observations suggest that intact small organelle genomes and extrachromosomal DNAs also may be lost from genomic DNAs prepared in agarose gel plugs.     

Gene amplification in cancer

Gene amplification is a copy number increase of a restricted region of a chromosome arm. It is prevalent in some tumors and is associated with overexpression of the amplified gene(s). Amplified DNA can be organized as extrachromosomal elements, as repeated units at a single locus or scattered throughout the genome. Common chromosomal fragile sites, defects in DNA replication or telomere dysfunction might promote amplification. Some regions of amplification are complex, yet elements of the pattern are reproduced in different tumor types. A genetic basis for amplification is suggested by its relative frequency in some tumor subtypes, and its occurrence in "early" preneoplastic lesions. Clinically, amplification has prognostic and diagnostic usefulness, and is a mechanism of acquired drug resistance.     

Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major.

We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major (S. M. Beverley, J. A. Coderre, D. V. Santi, and R. T. Schimke, Cell 38:431-439, 1984). Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-head configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.     

DNA metabolism during maturation of male germ cells. II. Amplification structures in the nuclei of rat germ and somatic cells

Amplification structures have been found in preparations of histone-depleted somatic (liver) and sex (spermatogonia, spermatocytes 1) rat cells. Multi-forked chromosomal (2-4 replicative forks originating from a single strand of DNA) and extrachromosomal circular amplification structures have been detected in the nuclei of sex cells. All the circular molecules of DNA detected belong, according to size, to the class of small nuclear polydispersed circular DNAs. Chromosomal amplification structures (eye-in-eye or several replicative forks originating from one DNA strand) have been only detected in the nuclei of somatic cells.     

CpG island mapping of a mouse double-minute chromosome.

The development of double-minute chromosomes (DMs) and subsequent gene amplification are important genomic alterations resulting in increased oncogene expression in a variety of tumors. The molecular mechanisms mediating the development of these acentric extrachromosomal elements have not been completely defined. To elucidate the mechanisms involved in DM formation, we have developed strategies to map amplified circular DM DNA. In this study, we present a long-range restriction map of a 980-kb DM. A cell line cloned from mouse EMT-6 cells was developed by stepwise selection for resistance to methotrexate. This cloned cell line contains multiple copies of the 980-kb DM carrying the dihydrofolate reductase (DHFR) gene. A long-range restriction map was developed in which a hypomethylated CpG-rich region near the DHFR gene served as a landmark. This strategy was combined with plasmid-like analysis of ethidium bromide-stained pulsed-field gels and indicated that a single copy of the DHFR gene was located near a hypomethylated region containing SsII and NotI sites. At least 490 kb of this DM appears to be composed of unrearranged chromosomal DNA.     

Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes.

A macrorestriction map representing the complete physical map of the Rhodobacter sphaeroides 2.4.1 chromosomes has been constructed by ordering the chromosomal DNA fragments from total genomic DNA digested with the restriction endonucleases AseI, SpeI, DraI, and SnaBI. Junction fragments and multiple restriction endonuclease digestions of the chromosomal DNAs derived from wild-type and various mutant strains, in conjunction with Southern hybridization analysis, have been used to order all of the chromosomal DNA fragments. Our results indicate that R. sphaeroides 2.4.1 carries two different circular chromosomes of 3,046 +/- 95 and 914 +/- 17 kilobases (kb). Both chromosome I (3,046 kb) and chromosome II (914 kb) contain rRNA cistrons. It appears that only a single copy of the rRNA genes is contained on chromosome I (rrnA) and that two copies are present on chromosome II (rrnB, rrnC). Additionally, genes for glyceraldehyde 3-phosphate dehydrogenase (gapB) and delta-aminolevulinic acid synthase (hemT) are found on chromosome II. In each instance, there appears to be a second copy of each of these genes on chromosome I, but the extent of the DNA homology is very low. Genes giving rise to enzymes involved in CO2 fixation and linked to the gene encoding the form I enzyme (i.e., the form I region) are on chromosome I, whereas those genes representing the form II region are on chromosome II. The complete physical and partial genetic maps for each chromosome are presented.     

The pattern of gene amplification is determined by the chromosomal location of hairpin-capped breaks

DNA palindromes often colocalize in cancer cells with chromosomal regions that are predisposed to gene amplification. The molecular mechanisms by which palindromes can cause gene amplification are largely unknown. Using yeast as a model system, we found that hairpin-capped double-strand breaks (DSBs) occurring at the location of human Alu-quasipalindromes lead to the formation of intrachromosomal amplicons with large inverted repeats (equivalent to homogeneously staining regions in mammalian chromosomes) or extrachromosomal palindromic molecules (equivalent to double minutes [DM] in mammalian cells). We demonstrate that the specific outcomes of gene amplification depend on the applied selection, the nature of the break, and the chromosomal location of the amplified gene relative to the site of the hairpin-capped DSB. The rules for the palindrome-dependent pathway of gene amplification defined in yeast may operate during the formation of amplicons in human tumors.     

DNA amplification in arsenite-resistant Leishmania

Arsenite-resistant variants of a trypanosomatid protozoan, Leishmania mexicana amazonensis, were selected in vitro by stepwise increases of sodium arsenite concentrations up to 30 microM in the culture medium. These variants were found to contain amplified DNA as extrachromosomal supercoiled molecules of about 69 kb. They originate from a single chromosome in the wild-type cells. There is evidence of chromosomal changes in these cells associated with the selection for arsenite resistance. The apparent absence of these circular molecules in the wild type and their loss from the drug-sensitive revertants suggest amplification of chromosomal DNA into these extrachromosomal circles as the mechanism of arsenite resistance.     

Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells.

To test the validity of various models for recombination between extrachromosomal DNAs in mammalian cells, we measured recombination between a plasmid containing a herpesvirus thymidine kinase (tk) gene with an internal BamHI linker insertion mutation (ptkB8) and a tk gene deleted at both ends (tk delta 3' delta 5'). The two DNAs shared 885 base pairs of perfect tk homology except for the interruption at the linker insertion site. Recombination events that restored the mutated insertion site to wild type were monitored by the generation of hypoxanthine-aminopterine-thymidine-resistant colonies after cotransformation of Ltk- cells with the two DNAs. We found that cleavage of the ptkB8 DNA at the linker insertion site was essential for gene restoration. If the tk delta 3' delta 5' DNA was ligated into mp10 vector DNA, then recombination with the cleaved ptkB8 DNA was inefficient. In contrast, if it was excised from that vector by cleavage at flanking restriction sites, then recombination was stimulated about 150-fold. Using restriction site polymorphisms, we showed that most of the recombination events leading to restoration of the tk gene with the excised tk delta 3' delta 5' fragment involved three double-strand duplexes: two ptkB8 DNAs and one tk delta 3' delta 5' fragment. These results are much more readily explained by the single-strand annealing model of recombination than by the double-strand break repair model, and they suggest that the deficiency of the latter pathway for extrachromosomal mammalian recombination may be due, at least in part, to the obligate tripartite nature of the reaction. Finally, we measured the effect of DNA homology on the efficiency of the ptkB8-tk delta 3' delta 5' reaction. Our results showed a near-linear relationship between the efficiency of recombination and the amount of homology flanking either side of the linker insertion site. Moreover, we could detect thymidine kinase-positive transformants with as little as 10 base pairs of homology.     

Functional copy-number alterations in cancer

Understanding the molecular basis of cancer requires characterization of its genetic defects. DNA microarray technologies can provide detailed raw data about chromosomal aberrations in tumor samples. Computational analysis is needed (1) to deduce from raw array data actual amplification or deletion events for chromosomal fragments and (2) to distinguish causal chromosomal alterations from functionally neutral ones. We present a comprehensive computational approach, RAE, designed to robustly map chromosomal alterations in tumor samples and assess their functional importance in cancer. To demonstrate the methodology, we experimentally profile copy number changes in a clinically aggressive subtype of soft-tissue sarcoma, pleomorphic liposarcoma, and computationally derive a portrait of candidate oncogenic alterations and their target genes. Many affected genes are known to be involved in sarcomagenesis; others are novel, including mediators of adipocyte differentiation, and may include valuable therapeutic targets. Taken together, we present a statistically robust methodology applicable to high-resolution genomic data to assess the extent and function of copy-number alterations in cancer.     

Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe

In order to elucidate the mechanisms of illegitimate recombination in eukaryotes, we have studied the structure of DNA fragments integrated by illegitimate recombination into the genome of fission yeast. Nonhomologous recombination was rarely identified when a long region of homology with the chromosomal leu1 ⁺ gene was present in the introduced leu1::ura4 ⁺ DNA fragment; but a decrease in length of homology leads to an increase in the ratio of nonhomologous to homologous recombination events. The introduced DNA fragments were integrated into different sites in the chromosomes by nonhomologous recombination. The results suggested that there are multiple modes of integration; most events simply involve both ends of the fragments, while in other cases, fragments were integrated in a more complicated manner, probably via circularization or multimerization. To analyze the mechanism of the major type of integration, DNA fragments containing the recombination junctions of three recombinants were amplified by inverted polymerase chain reaction (IPCR) and their nucleotide sequences were determined. There was no obvious homology between introduced DNA and chromosomal DNA at these recombination sites. Furthermore it was found that each terminal region of the introduced DNA was deleted, but that there were no or very small deletions in the target sites of chromosomal DNA. Two models are proposed to explain the mechanism of nonhomologous integration.     

Recombination of homologous DNA fragments transfected into mammalian cells occurs predominantly by terminal pairing.

The mechanism by which double-strand cleavages stimulate the joining of plasmid DNA fragments introduced into cultured mammalian cells was investigated by cotransfecting pairs of plasmids encoding deletion mutations in a dominant selectable gene into LMtk- cells. Plasmid recombination substrates were produced by creating deletions of different sizes within the neo coding region of the pSV2neo plasmid. Complementing pairs of deleted plasmid DNAs were linearized at specific unique sites before cotransfection into mouse LMtk- cells by the calcium phosphate precipitation method. Cleaving one donor plasmid produced a 4- to 10-fold stimulation in the production of colonies able to survive in medium containing G-418. The linearization of the second plasmid further increased the efficiency by another factor of 6 to 15 when the cut was made on the opposite side of the homology, approximately equidistant from the center of the overlap. Fifty-seven individual G-418-resistant colonies representing the products of individual crosses were isolated, and the genomic DNAs containing the presumably integrated, functional recombinant neo genes were analyzed on Southern blots. A band consistent with the exchange of markers flanking the neo gene was present in 90% of the DNAs examined. In only one case was the pattern indicative of either a double crossover or a gene conversion event. These results support the idea that homologous extrachromosomal DNA fragments are joined through annealing of overlapping single-stranded ends. This DNA-joining phenomenon may represent the activity of cellular DNA repair enzymes; its relationship to genetic recombination occurring at the chromosomal level remains to be determined.     

Homologous recombination between direct repeat sequences yields P-glycoprotein containing amplicons in arsenite resistant Leishmania.

The protozoan parasite Leishmania often responds to drug pressure by amplifying part of its genome. At least two loci derived from the same 800 kb chromosome were amplified either as extrachromosomal circles or linear fragments after sodium arsenite selection. A 50 kb linear amplicon was detected in six independent arsenite mutants and revertants grown in absence of arsenite rapidly lost the amplicon and part of their resistance. The circular extrachromosomal amplicons, all derived from the H locus of Leishmania, were characterized more extensively. In all cases, direct repeated sequences appeared to be involved in the formation of circular amplicons. Most amplicons were generated after homologous recombination between two linked P-glycoprotein genes. This recombination event was, in two cases, associated with the loss of one allele of the chromosomal copy. A novel rearrangement point was found in a mutant where the amplicon was created by recombination between two 541 bp direct repeats surrounding the P-glycoprotein gene present at the H locus. It is also at one of these repeats that an H circle with large inverted duplications was formed. We propose that the presence of repeated sequences in the H locus facilitates the amplification of the drug resistance genes concentrated in this locus.     

Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin ( oriA ). Transformants were recovered only with the plasmid containing oriA , and all transformants contained an integrated plasmid copy at oriA , suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.